Official programme & abstracts for the

Joint 11th International and 9th European Symposium on Purines and Pyrimidines in Man (PP’03)

9-13 June 2003

Hotel Zuiderduin, Egmond aan Zee, the Netherlands

Editor-in-chief: Prof. Dr. G.J. Peters

Published by: Drukkerij Peters in alliance with VU University Medical Center, Amsterdam, the Netherlands

May 2003

2 Contents

Welcome 4

Organising committee 5

Contact addresses 6

Support 7

Young Investigator Award 7

Registration fee 8

Venue Hotel Zuiderduin 8

Plan Hotel Zuiderduin floor 0 9

Plan Hotel Zuiderduin floor 1 10

Programme PP’03 11

Invited presentations (I01 – I24) 22

Oral presentations (O01 – 046) 50

Poster presentations (P01 – P95) 92

Author index 189

Overview International and European Symposia on Purine and Pyrimidine Metabolism 192

3 Welcome to the Netherlands

This series of congresses on Purine and Pyrimidine metabolism started 30 years ago in Tel Aviv and has since then moved around the world. It is now coming back to places which have been visited before. Three years ago the meeting was in Tel Aviv for the second time. The meeting has also been held twice in Austria. You can find an overview of the meetings on the last page of this abstract book. Twenty-one years ago the meeting was held in the most southern part of the Netherlands, Maastricht. Now we welcome the international meeting for the 2nd time in the Netherlands but at the other site with an excellent view on the sea. Usually the weather conditions permit to go to the beach. Alternatively you can take a walk through the dunes during the afternoon break.

The meeting is planned in such a way that you have some time off after the lunch, enabling to relax or to prepare for the poster sessions. For these poster sessions we will try a novel concept. Each poster presenter will have a few minutes to highlight the most important points of his/her research to encourage everybody to come and take a look at the posters, which are located close to the lecture hall. To compensate for this we continue in the evening with two exciting sessions. In the methods sessions we are very pleased that we can give you an up to date overview of analytical and molecular biological techniques. We were encouraged to organize a special Lesch-Nyhan syndrome session, because molecular biology has provided some new insights in this disease.

The meeting again provides an excellent environment for translational research. For that purpose we have a session called from “Bench to the Clinic” which is intended to give you some insight, both from a company and an investigator point of view, how drugs proceed from the test tube to the patient. These meetings on Purine and Pyrimidine metabolism always provided an excellent forum for so-called horizontal translational research: from one specialty to the other. In the past we had excellent examples, e.g. from immune deficiency to cancer, now we have examples from rheumatology to cancer to cardiology and vice versa. These interactions have made this series of meetings a success in the past and will do similarly in the future. This will be the task of a new generation. Many of the original participants and organizers retired or are retiring and a new generation is taking over, which has the challenge to keep this meeting as exciting as ever in a much more competing field. I attended the first meeting as a Ph.D. student and made friendships with others who are also here as senior investigators. Our current Ph.D. students should be enabled to proceed similarly. However, they have many other challenges outside and inside the field. For the last reason we should keep in close contact with others working on nucleosides and nucleotides, Therefore we decided to publish the proceedings in Nucleosides, Nucleotides & Nucleic Acids.

This meeting would not have been possible without the help of much support. First I have to thank my collaborators in the oncology laboratory, other colleagues at the VU University Medical Center, my colleagues from the national organizing committee for their support in planning, and the members of the scientific committee who suggested sessions and speakers and reviewed the abstracts timely enabling us to make an objective selection of the abstracts. The excellent support of the conference office was instrumental in enabling this electronic review and all other logistics. They will be around for any question you may have. As you noticed we did not do any paper announcement and proceeded entirely electronically. The generous support of the various sponsors is greatly appreciated. Without their support it would not have been possible to keep the costs of the meeting within limits. Finally I regret that several colleagues from the Far East (Taiwan, China, Japan) are not able or allowed to travel because of circumstances outside their control, such as SARS, and I sincerely hope that a next time they will be able to attend.

I wish everybody a successful meeting

Prof. Dr. Godefridus J. Peters

4 Organising Committee

Local organising committee: Board of the ESSPPMM: G.J. Peters, Amsterdam, chairman G.J. Peters (NL) President R.A. De Abreu, Nijmegen I. Sebesta (CZ) Vice-President J.W. De Jong, Rotterdam G. Van den Berghe (B) Past-President A.B.P. Van Kuilenburg, Amsterdam A.C. Skladanowski (PL) Secretary A.P. IJzerman, Leiden A. Griesmacher (A) Treasurer

International Scientific Committee: ESSPPMM Scientific Committee: J. Balzarini, Belgium J. Balzarini, Leuven J. Barankiewicz, USA E.A. Carrey, London M.A. Becker, USA R.A. De Abreu, Nijmegen R.I. Christopherson, Australia S. Eriksson, Uppsala R.A. De Abreu, The Netherlands A. Giacomello, Rome R.B. Diasio, USA M.L. Löffler, Marburg S. Eriksson, Sweden W. Makarewicz, Gdansk M. Fukushima, Japan E. Marinello, Siena V. Gandhi, USA V. Micheli, Siena N. Kamatani, Japan B. Munch-Petersen, Roskilde M. Müller, Austria M.M. Müller, Vienna D. Perrett, UK G.J. Peters, Amsterdam G.J. Peters, The Netherlands J.D. Puig, Madrid R.L. Sabina, USA I. Sebesta, Prague I. Sebesta, Czech Republic H.A. Simmonds, London -Honorary Member H.A. Simmonds, UK A.C. Skladanowski, Gdansk A.C. Skladanowski, Poland R.T. Smolenski, Harefield - Gdansk R. Smolenski, Poland O. Sperling, Tel Aviv F.F. Snyder, Canada M. Staub, Budapest O. Sperling, Israel G. Van den Berghe, Brussels M. Staub, Hungary N. Zöllner, Munich - Honorary Member L.F. Thompson, USA G. Van den Berghe, Belgium A.B.P. Van Kuilenburg, The Netherlands G. Weber, USA

5 Contact Addresses

Scientific programme Prof. Dr. G.J. Peters Dept. Medical Oncology VU University Medical Center Address: De Boelelaan 1117 1081 HV Amsterdam The Netherlands Phone: +31 20 444 2633 Fax: +31 20 444 3844 E-mail: [email protected]

Symposium secretariat Ms. S. van Geloven Dept. Medical Oncology BR232 VU University Medical Center Address: De Boelelaan 1117 1081 HV Amsterdam The Netherlands Phone: +31 20 4442630 Fax: +31 20 4443844 [email protected] E-mail: [email protected]

Registration and logistics PAOG Course and Congress Organisation VU University Medical Center MF/D-237 Address: Van der Boechorststraat 7 1081 BT Amsterdam The Netherlands Phone: +31 (0)20 444 8444 Fax: +31 (0)20 444 8445 E-mail: [email protected]

6 Support

We would like to thank the following companies/institutes for their support:

Main Sponsor

Taiho Pharmaceutical Co., Ltd. www.taiho.co.jp/english/

Sponsors

Applied Biosystems www.applera.com

Eli Lilly & Company www.lilly.com

Roche Diagnostics Nederland BV www.roche-diagnostics.com

Royal Netherlands Academy of Arts and Sciences www.knaw.nl/english/index.html

Sanofi-Synthélabo www.sanofi-synthelabo.com

Shire BioChem Inc. www.shire.com

Contributor

Thermo Electron BV

Young Investigator Award

The Young Investigator Award has been awarded to: C. Carnrot, Uppsala, Sweden, Z. Horvath, Vienna, Austria, Z. Khalpey, Harefield, UK, S. Mani, Calgary, Canada, A. Yuen, Harefield, UK.

The Young Investigator Award has been made possible by generous contributions from PP91 and ESSPPMM '93.

7 Registration fee

The full symposium fee includes the following: • admission to all sessions • programme & abstract book • welcome drinks on Monday, June 9 • coffee and tea during breaks • lunch from Tuesday, June 10 to Friday, June 13 • dinner at Zuiderduin on Tuesday, June 10 & Wednesday, June 11 (including 1 drink and 1 coffee) • excursion to Enkhuizen & the Zuiderzee Museum on Wednesday afternoon, June 11 • barbecue at Grand Café Lido on Thursday evening, June 12 • symposium proceedings

Venue Hotel Zuiderduin

Hotel Zuiderduin is perfectly situated within hundred metres of the beach and the town centre of Egmond aan Zee.

The symposium will be held at the 0-floor of the hotel (see floor plan on the next page). On Monday, June 9, you are all invited to come to the Van Speykzaal for the welcome reception at 18:00 hrs. From Tuesday, June 10, on all oral presentations will take place at the Zuiderduinzaal. The poster area is situated in the Foyer and the Le Regal room with are both adjacent to the Zuiderduinzaal. During the symposium, coffee and tea will be served in Le Regal, and lunch and dinners will take place at the Restaurant of Zuiderduin

Hotel rooms All rooms are fully equipped with a colour television, telephone, alarm clock and bathroom. The room rates are including breakfast and taxes. Check-in is from 15:00 hrs and check-out before 10:30 hrs.

Facilities The hotel is accommodated with a wide range of leisure facilities, including a heated indoor swimming pool and massage whirlpool (free of charge). For a service charge the following facilities are available: saunas, Turkish and Japanese baths, solarium, beauty parlour, hairdresser and squash courts. Hotel guests can also use outdoor tennis courts.

After symposium hours you can relax and have a drink at one of the hotel bars.

Internet facilities are available at Lounge 1 on the 1st floor. The charges are € 1,- per 8 minutes

Finally the hotel has a large parking area, including an indoor parking for cars and bikes.

Opening hours Restaurant: • breakfast from 07:00 – 10:30 hrs • lunch from 12:30 – 14:00 hrs • dinner from 18:00 – 21:00 hrs

Swimming pool: • 07:00 – 19:00 hrs

Sauna: • 10:00 – 23:30

8 Plan Hotel Zuiderduin floor 0

9 Plan Hotel Zuiderduin floor 1

10 Programme PP’03

Monday, June 9 15:00 - 18:00 registration (hotel reception) 18:00 - 19:00 welcome reception (Van Speykzaal)

Tuesday, June 10 08:30 - 08:45 Welcome (Zuiderduinzaal)

Session 1: Antimetabolites in the treatment of arthritis (Zuiderduinzaal) Chairs: Kamatani, N; Becker, M.

08:45 - 09:15 I01 Dijkmans, B.A.C.: Antimetabolites in the treatment of arthritis: Current status of the use of antimetabolites . 09:15 - 09:30 O01 Urano, W.U.: The efficacy and the toxicity of methotrexate in rheumatoid arthritis patients are associated with polymorphisms in the methylenetetrahydrofolate reductase gene 09:30 - 09:45 O02 Becker, M.A.: Febuxostat, a selective non-purine uric acid production inhibitor, is safe and decreases serum urate in healthy volunteers 09:45 - 10:15 I02a Aarden, L.: Immunosuppressive activities of methotrexate and mycophenolic acid 10:15 - 10:30 O04 Koshiba, M.: Modification of cytokine milieu by signaling through A2A adenosine receptors in rheumatoid arthritis

10:30 - 11:00 coffee and tea break (Le Regal)

Session 2: From bench to the clinic (1) (Zuiderduinzaal) Chairs: Gandhi, V.; Balzarini, J.

11:00 - 11:30 I03 Gandhi, V.: Clofarabine in Acute Leukemias: Clinical success and pharmacokinetics 11:30 - 12:00 I04 Schramm, V. L.: Immucillins as Antibiotics for T-Cell Proliferation and Malaria 12:00 - 12:30 I05 Nguyen, B.: ALIMTA (Pemetrexed) from lab bench to clinic.

12:30 - 15:00 lunch break (Restaurant) and free time

Session 3: Poster (presentation) session (Zuiderduinzaal & Le Regal/Foyer) Chairs: Peters, G.J.; Griesmacher, A.

15:00 - 16:30 Poster presentation session (short oral presentations): P30-P37 and P61-P95

16:30 - 17:30 poster session with coffee, tea and soft drinks (all posters)

11 Session 4: From bench to the clinic (2) (Zuiderduinzaal) Chairs: IJzerman, A.; Balzarini, J.

17:30 - 18:00 I02b Pieters, R.: Prevention of hyperuricemia with rasburicase 18:00 - 18:30 I06a Koomen, G.J.: New (1-DEAZA)Purine Derivatives via Efficient C-2 Nitration of the (1-DEAZA)Purine Ring 18:30 - 18:45 O05 Balzarini, J.: 6-[2-(Phosphonomethoxy)alkoxy]pyrimidines: a new class of acyclic pyrimidine nucleoside phosphonates with antiviral activity 18:45 - 19:00 O06 Bergman, A.M.: Antiproliferative activity and mechanism of action of fatty acid derivatives of gemcitabine in leukemia and solid tumor cell lines and xenografts

19:00 – 20:00 dinner (Restaurant)

Session 5: Methodology (Zuiderduinzaal) Chairs: Perrett, D.; Van Kuilenburg, A.B.P.

20:00 - 20:30 I06b Svoboda, M.: LC-MS techniques in clinical applications 20:30 - 20:45 O07 Smolenski, R.T.: Nucleotide analysis by liquid chromatography/mass spectrometry 20:45 - 21:15 I07 Lutz, V.: Quantitative real-time PCR for thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase with the Light-Cycler 21:15 - 21:45 I08 Wicki, R.: New Levels in Gene Expression Profiling 21:45 - 22:00 O08 Hooijberg, J.H.: Online fluorescent method to assess BCRP activity as a function of cellular folate homeostasis

12 Wednesday, June 11

Session 6: Purines in the cardiovascular system (Zuiderduinzaal) Chairs: Smolenski, R.T.; De Jong, J.W.

08:30 - 09:00 I09 Blackburn, R.: Pulmonary Consequences of Adenosine Overload: Lessons from Adenosine Deaminase Deficient Mice 09:00 - 09:30 I10 Headrick, J.: Species and age-dependent differences in purine metabolism, adenosinergic signalling, and ischemic tolerance 09:30 - 10:00 I11 Struthers, A.: Allopurinol: Will it reduce cardiovascular events in patients? 10:00 - 10:15 O09 Gutensohn, W.: Coordinate Regulation of the Expression of Ecto-5'-Nucleotidase (CD73) and the A2a Adenosine Receptor in a Human B-Cell Line 10:15 - 10:30 O10 Yuen, A.H.Y.: Prevention of adriamycin induced heart failure by increase in endogenous adenosine production

10:30 - 11:00 coffee and tea break (Le Regal)

Session 7: Chemotherapy in solid and hematological malignancies (Zuiderduinzaal) Chairs: Kaspers, G.J.L.; De Abreu, R.A.

11:00 - 11:15 O11 De Bruin, M.: Role of Platelet derived endothelial cell growth factor / thymidine phosphorylase in fluoropyrimidine sensitivity and potential role of deoxyribose-1- phosphate 11:15 - 11:30 O12 Williams, M.V.: Down-Regulation of Human Deoxyuridine Triphosphate Nucleotidohydrolase (dUTPase) using Small Interfering RNA (siRNA) 11:30 - 11:45 O13 Saiko, Ph.: Cytotoxic and apoptotic effects of novel heterodinucleoside dimers consisting of 5-fluorodeoxyuridine and Ara-c in human cancer cell lines 11:45 - 12:00 O14 Mauritz, R.: Increased thymidylate synthase (TS) but unaltered dihydropyrimidine dehydrogenase (DPD) mRNA levels after administration of 5-fluorouracil (5-FU) to patients with colon cancer 12:00 - 12:15 O15 Cardoen, S.: Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in human leukemia CCRF-CEM and EHEB cell lines 12:15 - 12:30 O16 Verschuur, A.C.: Increased cytotoxicity of 2’,2’-difluoro-2’-deoxycytidine in human leukemic cell-lines after a preincubation with cyclopentenyl cytosine 12:30 - 12:45 O17 Iven, H.: Can whole blood substitute washed red blood cells for the analysis of TPMT activity and 6-mercaptopurine metabolites? 12:45 - 13:00 O18 Giacomello, A.: Potential role of mycophenolate mofetil in the management of neuroblastoma patients

13:00 - 13:15 delegates should gather in front of the hotel reception for the buses to Enkhuizen and the Zuiderzee Museum; lunch bags will be handed out.

13:15 - 18:00 excursion to Enkhuizen and the Zuiderzee Museum (open air museum)

18:00 - 19:00 dinner (Restaurant)

13 Session 8: Lesch-Nyhan syndrome (Zuiderduinzaal) Chairs: Puig, J.G.; Sperling, O.

19:00 - 19:05 Simmonds, A.H.: Tribute to Françoise Roch-Ramel 19:05 - 19:30 I12 McCarthy, G.T.: Medical Diagnosis, Management and Treatment of Lesch Nyhan Disease 19:30 - 19:55 I13 Marinaki, A.M.: Lesch-Nyhan Disease 19:55 - 20:20 I14 Jinnah, H.A.: Mutations of the HPRT Gene in Lesch-Nyhan Disease and its Variants 20:20 - 20:30 O19 Torres, R.J. : Adenosine transport in HPRT deficient lymphocytes from Lesch- Nyhan patients. 20:30 - 20:40 O20 Becker, B.F.: Urate Oxidation in CSF and Blood of Patients With Inflammatory CNS Disorders 20:40 - 20:50 O21 Carrey, E.A.: An unusual pyridine nucleotide accumulating in erythrocytes: its identity, and positive correlation with degree of renal failure

14 Thursday, June 12

Session 9: New inborn errors; role of mitochondria (Zuiderduinzaal) Chairs: Eriksson, S.; Loeffler, M.

08:30 - 09:00 I15 Saada-Reisch, A.: New inborn errors; role of mitochondria; Deoxyribonucleoside kinases in mitochondrial DNA depletion. 09:00 - 09:30 I16 Hirano, M.H.: Thymidine Phosphorylase Deficiency Causes MNGIE: An Autosomal Recessive Mitochondrial Disorder 09:30 - 09:45 O23 Bianchi, V.B.: Metabolism of thymidine in mitochondria: role of dNT2, the mitochondrial deoxyribonucleotidase. 09:45 - 10:00 O24 Gattermann, N.: Severe impairment of nucleotide synthesis through inhibition of mitochondrial respiration 10:00 - 10:15 O25 Gojkovic, Z.G.: New class of suicide genes for cancer gene therapy 10:15 - 10:30 O26 Snyder, F.F.: Production and Properties of Polyethylene-Glycol Conjugated Adenosine Phosphorylase: Administration to PNP deficient mice

10:30 - 11:00 coffee and tea break (Le Regal)

Session 10: Membrane targets (Zuiderduinzaal) Chairs: Jansen, G.; Stone T.W.

11:00 - 11:30 I17 Cass, C.: Nucleoside transporters 11:30 - 11:45 O27 Casado, F.J.: Nucleoside transporters in proliferation in normal and transformed cells 11:45 - 12:15 I18 Borst, P.: Role of Multidrug-resistance associated proteins in resistance to nucleoside analogs 12:15 - 12:30 O29 Gorodeski, I.: Estrogen Attenuates P2X7-R - Mediated Apoptosis of Uterine Cervical Cells by Blocking Calcium Influx 12:30 - 12:45 O30 Snyder, F.F.: Mitochondrial function dependent proliferation assay for the diagnosis of mitochondrial disorders in human fibroblasts

12:45 - 15:00 lunch break (Restaurant) and free time

Session 11: Poster (presentation) session (Zuiderduinzaal & Le Regal/Foyer) Chairs: Sebesta, I; Micheli, V.

15:00 - 16:30 Poster presentation session (short oral presentations): P1-P29 and P38-P60

16:30 - 17:30 Poster session with coffee, tea and soft drinks (all posters)

15 Session 12: Antimetabolites: parasites and viruses (Zuiderduinzaal) Chairs: Van den Berghe, G.; Christopherson, R.I.

17:30 - 18:00 I19 Christopherson, R.I.: Cloning and expression of malarial pyrimidine enzymes 18:00 - 18:15 O33 De Koning, H.P.: Rational design of purine and pyrimidine drugs against protozoan infections through selective uptake 18:15 - 18:30 O34 Deville-Bonne, D.: Improving NDP Kinase for Antiviral Nucleotide Analogs Activation 18:30 - 18:45 O35 McLennan, A.G.: Regulation of dinucleoside polyphosphates by the YgdP and ApaH diadenosine polyphosphate hydrolases is essential for intracellular invasion by Salmonella enterica serovar Typhimurium 18:45 - 19:00 O36 Slominska, E.M.: The Effect of N-Methyl-2-Pyridone-5-Carboxamide - A Nicotinamide Catabolite on Poly ADP-Ribosylation and Oxidative Stress Injury in Endothelial Cells

19:30 - 22:30 Barbecue at the beach (Grand Café Lido, Westeinde 1, Egmond aan Zee; approximately 250 metres walking distance from Hotel Zuiderduin)

16 Friday, June 13

Session 13: Enzymology; Signal transduction (Zuiderduinzaal) Chairs: Staub, M. ; Sabina R.I.

08:30 - 09:00 I20 Piskur, J.: Diversity and origins of deoxyribonucleoside kinases 09:00 - 09:30 I21 Graves, M. : Regulation of Nucleoside Uptake by Protein Kinase Inhibitors and Mitogenic Signaling. 09:30 - 09:45 O37 Sasvari-Szekely, M.: Selective increase of dATP pools upon activation of deoxycytidine kinase in lymphocytes: implications in apoptosis 09:45 - 10:00 O38 Sabina, R.L.: Multi-level regulation of human AMP deaminase isoform E (AMPD3) by calmodulin 10:00 - 10:15 O40 Mani, R.S. Biophysical Studies of ATP, Deoxycytidine and Gemcitabine Binding to Human Recombinant Deoxycytidine Kinase 10:15 - 10:30 O31 Allegrini, S.: Functional studies reveal that cytosolic "High Km" 5'-Nucleotidase (cN-II) active site has a structure similar to that of HAD superfamily

10:30 - 11:00 coffee and tea break (Le Regal)

Session 14: Genetic polymorphisms (1) (Zuiderduinzaal) Chairs: Peters, G.J; Snyder, F.F.

11:00 - 11:30 I22 Nishiyama, M.: Transcriptional regulation of DPYD gene expression and aberrant methylation in the 5'flanking region 11:30 - 12:00 I23 Van Kuilenburg, A.B.P.: Pharmacogenetic and clinical aspects of a dihydropyrimidine dehydrogenase deficiency 12:00 - 12:15 O42 Kamatani, N: Identification of 4 unique mutations in exon 4 of uromodulin (UMOD) gene in 4 separate families with familial juvenile hyperuricemic nephropathy (FJHN). 12:15 – 12:30 O43 Peterson, C.: What role do methylated metabolites play for the cellular effects of thiopurines?

12:30 - 14:00 lunch break (Restaurant)

Session 15: Genetic polymorphisms (2) (Zuiderduinzaal) Chair: Marinaki A.M.; Nishiyama, M.

14:00 - 14:30 I24 Coulthard, S.: The Clinical Impact of Thiopurine Methyltransferase Polymorphisms on Thiopurine Treatment 14:30 - 14:45 O44 Shobowale-Bakre, E.A.: Pre-treatment TPMT PHE/genotyping: a safe guide for initial thiopurines drugdosing for specific patient groups 14:45 - 15:15 O41 De Jonge, R.: Clinical consequences of polymorphisms in the methylenetetrahydrofolate reductase gene 15:15 - 15:30 O45 Marinaki, A.M.: A mutation in the ITPA gene predicts intolerance to azathioprine. 15:30 - 15:45 O46 Taniguchi, A.T.: Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis 15:45 - 16:00 O32 Spychala, J.: The association of ecto-5'-nucleotidase (eN), integrin b1, EGFR and vimentin with invasive breast carcinoma: The role of eN in invasive phenotype

16:00 - 16:30 Business Meeting ESSPPMM and closing session (Zuiderduinzaal)

17 Posters

Subject: Gout P01 Sperling, O.: Modulation of Glycogen Phosphorylase activity affects 5 Phophoribosyl-1- Pyrophosphate availability in Rat Hepatocyte Cultures P02 Stone, T.W.: Purine levels and cytokine release with diuretic therapy in rheumatoid arthritis P03 Puig, J.G.: The Pathophysiology of Hyperuricemia in Essential Hypertension: A pilot study P04 Tsutsumi, Z.T.: Serum Adiponectin Concentration in Patients with Primary Gout P05 Ka, K.: Effect of low purine-containing low malt beer (happo-shu) and regular low malt beer on purine bases P06 Ichida, K.I.: SLC22A12 mutation is responsible for most renal hypouricemia in Japan P07 Yamada, I.Y.: Pharmacokinetics/Pharmacodynamics of Y-700, A novel Xanthine Oxidase Inhibitor, in Rats and Man P08 Hoshide, S.H.: PK/PD and safety of a single dose of TMX-67 (febuxostat) in subjects with mild and moderate renal impairment P09 Komoriya, K.K.: Pharmacodynamics & pharmacokinetics of TMX-67 (febuxostat), a novel xanthine oxidase / xanthine dehydrogenase (XOD) inhibitor, in patients with hyperuricemia

Subject: Lesch-Nyhan syndrome P10 Zoref-Shani, E.: A novel point Mutation (I137t) in the Conserved 5-Phosphoribosyl-1- Pyrophosphate Binding Motif of HPRT (HPRTJERUSALEM) in a LNS Variant P11 Mizunuma, M.M.: Disruption in the hypoxanthine phosphoribosyl-transferase gene caused by translocation in a patient with Lesch-Nyhan syndrome P12a Yamada, Y.: Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in Asian HPRT deficient families P12b Sebesta, I.: Highly skewed X-inactivation pattern as a cause of female gout P13 Micheli, V. : Are allopurinol and metabolites found in HPRT deficient erythrocytes responsible for increased NAD synthesis? P14 Torres, R.J.: Effects of hypoxanthine on adenosine transport in human lymphocytes. Implications in the pathogenesis of Lesch-Nyhan syndrome P15 Marinello, E.: EPR spin trapping of a radical intermediate in the urate oxidase reaction P16 Verdu, A.: Cognitive abilities in a series of patients with Hypoxanthine guanine phosphoribosyltransferase deficiency P17 Visser, J.E.: The motor features of Lesch-Nyhan disease P20a McCarthy, G.T.: Lesch Nyhan Disease, essentials of diagnosis and management - a European perspective P20b Simmonds, H.A.: Nucleotide degradation products in cerebrospinal fluid (CSF) in inherited and acquired pathologies

Subject: Inborn errors P21 Ceballos-Picot, I.C.P.: Genotype and clinical features of adenine phosphoribosyltransferase (APRT) deficiency in French families P22 Race, V.: Adenylosuccinate lyase deficiency: study of physiopathologic mechanism(s) P23 Marinaki, A.M.: Adenylosuccinate lyase deficiency - first UK case P24 Laurence, A.D.J.: Elevated erythrocyte CDP-choline levels associated with b-thalassaemia in patients with transfusion independent anaemia P25 Pawa, S.P.: Nucleotides can afford protection in cirrhosis induced by thioacetamide P26 Marinello, E.: Serum Folate Level in Mozambique's Children P27 Marinaki, A.M.: Deoxyuridine accumulation in urine in thymidine phosphorylase deficiency (MNGIE) P28 Escuredo, E.; The Genetic Basis of the Interaction Between Pyrimidine 5' Nucleotidase deficiency and Hemoglobin E P29 Marinaki, A.M.: Purine nucleoside phosphorylase deficiency: Is there a mitochondrial pathology?

18 Subject: Gene polymorphisms P30a Salerno, C.: Urinary methylxanthine and autistic disorder: absence of previously reported correlation P30b Grunebaum, E.G.: Novel mutations and a 'hot-spot' in Purine Nucleoside Phosphorylase deficient patients P31 Taniguchi, A.T.: The frequency and diversity of the haplotypes near adenine phosphoribosyltransferase (APRT) locus are different between patients with APRT deficiency and normal controls P32 Haglund, S.: Pyrosequencing of Thiopurine S-methyltransferase (TPMT) alleles in a Swedish general population and in IBD-patients P33a Marinaki, A.M.: Polymorphism in the MTHFR gene affects thiopurine methyltransferase activity P33b Marinaki, A.M.: Allele frequency of inosine triphosphate pyrophosphohydrolase gene polymorphisms in a Japanese population P34 Arenas, M.: Genetic determinants of the thiopurine methyltransferase intermediate activity phenotype P35 Calascibetta, A.: Association between Thymidylate Synthase polymorphism with the microsatellite instability in colorectal cancer P36 Mauritz, R: Polymorphic tandem repeats in the thymidylate synthase gene and TS protein and mRNA levels in different tissues of colorectal cancer patients

Subject: Metabolism and enzymology P38a Loeffler, M.L.: Dihydroorotate dehydrogenase expression analysis in normal and drug-resistant cells and tissues P38b Usova, E.V.: Phosphorylitic cleavage of deoxyribonucleosides catalysed by human deoxycytidine kinase – a side reaction or a function. Preliminary Biochemical and NMR Studies P39 Staub, M.: The Effect of Signalling Pathway Modulations on the Activity of the Deoxycytidine Kinase P40 Krawiec, K.: Unusual phosphate donors for human deoxyrybonucleoside kinases. P41 Munch-Petersen, B.M.P.: The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster can phosphorylate glycerol P42 Munch-Petersen, B.M.P.: Amino acid residues determining the substrate specificity of deoxyribonucleoside kinases P43 Wang, L.W.: Mitochondrial deoxyguanosine kinase/thymidine kinase mutations and mitochondrial DNA depletion syndrome P44 Al-Madhoun, A. S.: Biochemical and Biological Evaluation of 3-(Carboranylalkyl) thymidines as Substrates for Human Thymidine Kinases 1 and 2 P45 Carnrot, C.M.E.: Cloning and biochemical characterisation of thymidine kinase in Ureaplasma urealyticum P46 Torres, R.J.: Hypoxanthine effects on cyclic amp levels in human lymphocytes P47 Kovari, J.: Mechanistic and physiological studies of dUTPases P48 Rinaldo-Matthis, A.R.M.: Crystal structure of a human deoxyribonucleotidase P49 Rylova, S.N.: Differential activity of pyrimidine nucleoside kinases in proliferating, resting and adipocyte differentiated 3T3-L1 cells P50 Karlsson, S.: Cloning of a mitochondrial UMP/CMP kinase from Drosophila melanogaster P51 Ipata, P.L.: Purine and Pyrimidine Salvage in Whole Rat Brain: Utilization of ATP-derived Ribose- 1 Phosphate and 5-phosphoribosyl-1-Pyrophosphate Generated in Experiments with Dialyzed Cell- free Extracts P52 Higgins, M.J.H.: Colocalization of Human Cytidine Triphosphate Synthetase 1 with Microtubules P53 Snyder, F.F.: Identification of binuclear zinc coordination for human guanine deaminase by site directed mutagenesis P55 Carrey, E.A.: GTP Concentrations are Elevated in Erythrocytes of Renal Transplant Recipients when Conventional Immunosuppression is replaced by the Inosine Monophosphate Dehydrogenase Inhibitor Mycophenolic Acid Mofetil (MMF) P56 Di Pietrantonio, F.: Cyclase and Phosphodiesterase Activity on Pre-T Lymphoid Human Cells, treated with Dimethyl Sulfoxide (DMSO)

19 P57 Tozzi, M.G. Identification of the 5'-nucleotidase activity altered in neurological syndromes P58 Khalpey, Z.: Exposure to Human Blood Decreases Swine Endothelial ECTO-5'-Nucleotidase Activity P59 Marinello, E.: Liver Transplant: Adenosine Metabolism and Apoptosis P60 Szydlowska, M.: The immunological and kinetical characteristic of AMP-deaminase from normal and Hepatocellular carcinoma (HCC) human liver

Subject: Purine metabolism in cardiovascular disease P61a Salerno, C.: Biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency P61b Kalsi, K.K. : AMPD1 C34T Mutation Selectively affects AMP-deaminase activity in the Heart P62 Yuen, A.H.Y.: Association of improved cardiac function in donors with C34T mutation of the AMP Deaminase 1 gene P63 Smolenski, R.T.: Purine Metabolism in Pigs and Humans and its Implications for Xenotransplantation P64 Osborne, F.: Overexpression of Human ECTO 5' Nucleotidase in Pig Endothelial Cells and its Implication for Adenosine Production and Xenotransplantation P65 Kalsi, K.K.: Lidoflazine combined with nucleotide precursors increases ATP content and Adenosine production in cardiomyocytes

Subject: Gene therapy P66 Solaroli, S.N.: Investigation of substrate recognition of Drosophila melanogaster nucleoside kinase by site directed mutagenesis P67 Marinello, E.: Evaluation of ADA Gene Expression and Transduction Efficiency in ADA/SCID Patients Underwent Gene Therapy

Subject: Chemotherapy of malignancies P68 Marinello, E.: Metabolism of Adenosine in Human Colorectal Tumor P70 Smal, C.: New evidences for regulation of deoxycytidine kinase activity by reversible phosphorylation P71 Van Kuilenburg, A.B.P.: Cyclopentenyl cytosine sensitises SK-N-BE(2)c neuroblastoma cells to cladribine P72 Van Kuilenburg, A.B.P.: Gemcitabine and cyclopentenyl cytosine: a promising combination for the treatment of neuroblastoma P73 Hubeek, I.: Modulation of cytarabine resistance in childhood acute myeloid leukemia P74 Bergman, A.M.: Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines P75 Wehelie, R.: Pyrimidine deoxynucleoside salvage in Ureaplasma urealyticum: nucleoside analogues acted as potent inhibitors of mycoplasma growth P76 Horvath, Z.: Synergistic effects of BCNU and Didox combination chemotherapy in 9L glioma cells P77 Bauer, W.B.: Amidox, an inhibitor of ribonucleotide reductase, potentiates the action of Ara-C in HL-60 human promyelocytic leukemia cells P78 Temmink, O.H.: Mechanism of action of trifluorothymidine (TFT) in combination with a thymidine phosphorylase inhibitor (TPI) P79 Smid, K.: The effect of different fluoropyrimidines with or without a thymidine phosphorylase inhibitor (TPI) on the expression of platelet derived endothelial cell growth factor / thymidine phosphorylase (PD-ECGF/TP) P80 Temmink, O.H.: Combination studies of trifluorothymidine (TFT) with antifolates and the DNA synthesis inhibitors irinotecan, oxaliplatin and gemcitabine P81 Sigmond, J.: Combination studies of 5-fluorouracil with UCN-01 or staurosporine P82 Razmara, M.: 5’-nucleotidases levels measured in peripheral blood cells from patients with chronic and acute leukemia

20 P83 Spoto, G.: Cyclic guanosine monophosphate role in human carcinoma pathogenesis P84 Giacomello, A.: Cyclic nucleotides and neuroblastoma differentiation P85a Honma, Y.: Differentiation-inducing Activity of Purine and Pyrimidine Derivatives on Myelogenous Leukemia Cells: The most suitable analog depends on the leukemia type P85b Gutsche, S.: 6-mercaptopurine (6-MP) metabolism in man and its fingerprints in erythrocytes (RBC) P86a Kulikowski, T.: ATP-site directed potential inhibitors of nucleoside triphosphatases (NTPase)/helicases and polymerases of hepatitis C and other selected Flaviviridae viruses P86b Shobowale-Bakre, E.A.: Pre-treatment TPMT phenotyping/genotyping : an excellent guide for initial thiopurine drug dosing

Subject: Methodology P87 Kotalczyk, A.: A time saving and rapid HPLC-method for the determination of 6-thioguanine and 6 thioguanine (-nucleotides) P88 Lindqvist, M.: Real time RT-PCR methodology for quantification of TPMT gene expression P89 Sigmond, J.: Quantitative detection of deoxycytidine kinase by real time light cycler PCR in comparison with conventional competitive template RT-PCR P90 Hubeek, I.: Immunocytochemical detection of deoxycytidine kinase P91 Van Kuilenburg, A.B.P.: Determination of the deoxycytidine kinase activity in cell homogenates with a non radiochemical assay using reversed-phase HPLC. Identification of a novel metabolite of 2-Chlorodeoxyadenosine P93 Slominska, E.M.: Application of liquid chromatography - mass spectrometry for identification and quantitation of metabolic alterations in chronic renal failure P94 Kaneko, K.: Detection of prothrombin and osteopontin in a renal stone found in a hyperuricemic patient using 2D-PAGE and LCMS analysis P95 Noordhuis, P.: A non-radioactive sensitive assay to measure 5-fluorouracil incorporation into DNA of solid tumors

21

I01

Antimetabolites in the treatment of arthritis: Current status of the use of antimetabolites

B.A.C. Dijkmans, G. Jansen VU University Medical Center, AMSTERDAM, The Netherlands

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a chronic inflammation of the synovial joints and infiltration of blood-derived cells. Effective drug treatment of RA patients is aimed at diminishing the release of pro-inflammatory cytokines (e.g. TNF-α) that play a major role in the pathophysiology of RA. In daily practise rheumatologists use the antimetabolites methotrexate (MTX) and leflunomide for the treatment of patients with arthritis. Onother antimetabolite, mycophenolate mofetil, has only a modest place. The current clinical status (efficacy/toxicity) of these 3 antimetabolites in the treatment of RA will be discussed. Methotrexate: Despite its clinical use for many decades, the exact mechanism(s) by which MTX exerts immunosuppressive or anti-inflammatory effects has not been fully clarified. Most likely a combination of antifolate effects and anti-purine effects contribute to the therapeutic profile of MTX. At present MTX is considered as anchor-drug for treatment of RA patients. MTX is used as monotherapy or in combination with other anti-rheumatics, e.g. sulphasalazine, (hydroxy)chloroquine and corticosteroids. Recently, combinations of MTX with novel TNF-α antibodies (infliximab, etanercept) demonstrated markedly improved therapeutic effects for RA patients. MTX treatment can be associated with toxic side effects, including gastrointestinal, hematological, liver and pulmonary toxicity, but rheumatologists have learned to monitor and influence (e.g. by folic acid/leucovorin supplementation) toxicity to acceptable proportions. Leflunomide: Leflunomide is an inhibitor of pyrimidine biosynthesis via inhibition of dihydroorotate dehydrogenase. Immunosuppressive and antiproliferative effects of leflunomide are illustrated by inhibition of T-cell proliferation in response to cytokines (IL-2) and inhibition of antibody synthesis. The current position of leflunomide is that of an established anti-rheumatic drug that is administered after MTX and sulphasalazine. Adverse effects of leflunomide (diarrhoea, hypertension, rash and increased liver enzymes) are generally mild and relatively infrequent. Mycophenolate mofetil (MM): MM is a prodrug, the inactive 2-morpholino ester of mycophenolic acid, which is an inhibitor of IMP dehydrogenase. The immunosuppressive effects of MM have found widespread application in the field of organ transplantation. At present some trials are underway in patients with RA and systemic lupus erythematosus. Collectively, antimetabolites as single agent or in combinations have proven efficacy for treatment of RA patients. Further clinical exploitation of combinations of antimetabolites with novel TNF-α blockers is a challenging option for further clinical evaluation in arthritis treatment.

22 I02a

Immunosuppressive activities of methotrexate and mycophenolic acid

S. De Lathouder1, A.H. Gerards2, E.R. De Groot1, M.G. Valkhof1, L.A. Aarden1 1Sanquin Research at CLB, AMSTERDAM, The Netherlands 2VU University Medical Center, AMSTERDAM, The Netherlands

Methotrexate (MTX) and mycophenolic acid (MPA) are immunosuppressive drugs used for the treatment of various immunological disorders. MTX is widely used in the treatment of rheumatoid arthritis (RA). MPA is used to prevent graft rejection after transplantation and is now experimentally used in RA. MPA is an inhibitor of inosine monophosphate-dehydrogenase and MTX is a folate antagonist that inhibits tetrahydrofolate-reductase. Both drugs inhibit the production of several cytokines (such as IL-4, IFN-γ, TNF-α and GM-CSF) after T-cell stimulation with anti-CD3/anti-CD28. The mechanism by which inhibition is achieved is different for both drugs. When T cells are activated in the presence of MTX, the cells enter the cell cycle and progress to S phase and mitosis. At that point, the cells die by apoptosis. Resting T-cells are not affected. The mechanism of MPA is different. When resting T-cells are activated in the presence of MPA, some activation markers (CD69 and CD25) are upregulated, but the cells stay small and do not enter S phase. Addition of guanosine and adenosine can overcome this cell cycle arrest, even after several days. The observation that MTX cannot prevent T cell activation but induces apoptosis in activated T cells and that MPA reversibly prevents activation of T cells could explain the immunosuppressive effects both drugs.

23 I02b

Prevention of hyperuricemia with rasburicase

J.M. de Bont, R. Pieters Erasmus MC - Sophia Children's Hospital, ROTTERDAM, the Netherlands

Tumour lysis syndrome (TLS) is a potentially life-threatening complication in patients with haematological malignancies1. Massive lysis of tumour cells can lead to hyperuricemia, hyperkalemia, hyperphosphatemia and hypocalcemia1,2. This can result in renal failure, because of the precipitation of uric acid crystals and calcium phosphate salts in the kidney1 Until recently the standard prophylaxis and treatment of hyperuricemia consisted of decreasing uric acid production with allopurinol and facilitating its excretion by urinary alkalinisation and hyperhydration1,2,3. By inhibiting the enzyme xanthine oxidase, allopurinol blocks the conversion of hypoxanthine and xanthine into uric acid. An alternative treatment is the use of urate oxidase, an enzyme which oxidates uric acid into allantoin. Allantoin is ten times more soluble in urine than uric acid and is therefore excreted easily. Rasburicase, the recombinant form of urate oxidase has shown to be very effective and is in contrast with the non- recombinant form, uricozyme, associated with a low incidence of hypersensitivity reactions2,3. Besides the rapid onset of action rasburicase has several other beneficial features compared to allopurinol2: • No accumulation of hypoxanthine and xanthine, preventing xanthinenephropathy. • No interactions with chemotherapeutic agents such as 6-mercaptopurine and cyclophosphamide. • No further need for urinary alkalinisation, reducing the risk of precipitation of calcium phosphate salts. • Because the mean plasma terminal half-life of rasburicase is approximately 17-20 hrs, it is sufficient to administer it once a day. Compassionate-use programme Rotterdam In 2001 the Erasmus MC-Sophia Children's Hospital in Rotterdam participated in a multi-centre compassionate-use programme for rasburicase. The major purpose of this programme was to review efficacy and safety of rasburicase. Fourteen oncological patients with a high risk of hyperuricemia/TLS (ALL n=11, ANLL n=2, Wilms tumour stage IV n=1) were given rasburicase (0,2 mg/kg/day) during the first few days of their induction chemotherapy. Before therapy was started, 12-24 hrs after the first dose of rasburicase and subsequently daily LDH, WBC, uric acid, potassium, phosphate, calcium and creatinine were measured. Before starting therapy none of the patients had obvious clinical signs of TLS. 13 out of 14 patients had a very good and fast response to rasburicase, leading to levels of uric acid below 0,01 mmol/l within a day. None of the patients developed metabolic disturbances or acute renal failure during the first phase of chemotherapy. To review the safety of rasburicase all adverse events were registered Three adverse events occurred, but they were most likely complications of the administered chemotherapy and not of rasburicase. No allergic reactions to rasburicase were seen. Review of literature Pui et al.5 administered rasburicase intravenously at 0,15 mg or 0,20 mg/kg/day to 131 children, adolescents and young adults with newly diagnosed leukaemia or lymphoma. They found a decrease in uric acid levels of 95-99% 4 hours after administration of rasburicase. None of the patients required dialysis. Treatment with rasburicase was well tolerated, with none of the adverse events having a clear relationship to the administration of rasburicase. In a compassionate-use trial6 173 children and 72 adults were treated with rasburicase (0,20 mg/kg/day). Rasburicase produced a dramatic decrease in uric acid levels in all patients, whether they received it for prophylaxis (n=79) or treatment (n=166). Uric acid levels had a median decline from 9,7 to 0,6 mg/dL in children and 11,9 to 0,7 mg/dL in adults. In contrast to the previous (above mentioned) study5, four children and six adults required hemodialysis, as a result of hyperphosphatemia (n=2), azotemia (n=5) or both (n=3). The adverse events registered ranged from mild vomiting to skin reactions, fever, myalgia, headache and oedema. Goldman et al.3 included 52 paediatric patients with leukaemia or lymphoma in a randomised controlled trial comparing allopurinol (10 mg/kg/day) to rasburicase (0,20 mg/kg/day). Within four hours after administration of rasburicase uric acid levels dropped by 86% in the rasburicase arm and by 12% in the allopurinol arm (p<0,0001). One patient in the allopurinol group required assisted renal support. The adverse

24 events that occurred (fever, pain, mucositis) were mild and most likely secondary to the disease and chemotherapy. Rasburicase appears to be superior to allopurinol, but is also far more expensive than therapy with allopurinol, urinary alkalinisation and hyperhydration. Annemans et al.7,8 conducted an economic evaluation to assess cost-effectiveness (costs per life-year saved), assuming that rasburicase reduces 100% of the costs associated with TLS and 90% of the costs associated with hyperuricemia. They concluded that prevention of hyperuricemia/TLS with rasburicase is highly cost-effective in adults and children and that treatment of hyperuricemia/TLS with rasburicase is highly cost-effective in adults and cost saving in children. They calculated that even if the reduction of costs would be only 60%, prevention and treatment of hyperuricemia/TLS with rasburicase remains cost-effective7. This makes rasburicase not only of great clinical benefit, but also an economically attractive new option in the management of hyperuricemia. References 1. Jeha S. Tumor lysis syndrome. Seminars in hematology 2001; 38(4) suppl 10: 4-8 2. Brant JM. Rasburicase: An innovative new treatment for hyperuricemia associated with tumor lysis syndrome. Clinical journal of oncology nursing 2002; 6(1): 12-16 3. Goldman SC, Holcenberg JC, Finklestein JZ et al. Blood 2001; 97(10): 2998-3003 4. Easton J, Noble S, Jarvis B. Rasburicase. Paediatric drugs 2001; 3(6): 433-439 5. Pui CH, Mahmoud HH, Wiley JM et al. Journal of clinical oncology 2001; 19(3): 697-704 6. Pui CH, Jeha S, Irwin D et al. Recombinant urate oxidase (rasburicase) in the prevention and treatment of malignancy-associated hyperuricemia in pediatric and adult patients: results of a compassionate-use trial. Leukemia 2001; 15: 1505-1509 7. Annemans L, Moeremans K, Lamotte M et al. Pan-European multicentre economic evaluation of recombinant urate oxidase (rasburicase) in prevention and treatment of hyperuricaemia and tumour lysis syndrome in haematological cancer patients. Support Care Cancer 2003; 11:249-257 8. Annemans L, Moeremans K, Lamotte M et al. Incidence, medical resource utilisation and costs of hyperuricemia and tumour lysis syndrome in patients with acute leukaemia and non-Hodgkin's lymphoma in four European countries. Leuk Lymphoma 2003;44(1): 77-83

25 I03

Clofarabine in Acute Leukemias: Clinical Success and Pharmacokinetics

V. Gandhi MD Anderson Cancer Center, HOUSTON, TX, United States of America

Two analogues of deoxyadenosine active in hematologic malignancies, fludarabine and cladribine, differ in their mechanisms of action and in their spectrum of clinical responses. A hybrid of these drugs, clofarabine, retains the 2-chloroadenine aglycone of cladribine. Reminiscent of fludarabine, clofarabine is further derivatized with a fluorine molecule in the arabinosyl configuration at the critical 2'-position of the carbohydrate. The 2'-fluoro group greatly inhibits cleavage of the glycosidic linkage, a mechanism of clearance for the parent drugs which also generates potentially toxic halogenated nucleobases. Laboratory investigations of clofarabine demonstrated that it accumulates as the clofarabine triphosphate after phosphorylation by deoxycytidine kinase. The triphosphate of the drug is an effective substrate for replicative DNA polymerases and competes well with normal deoxyadenosine triphosphate, and once incorporated into DNA disrupts further synthesis. In addition, clofarabine triphosphate inhibits ribonucleotide reductase, decreasing cellular deoxynucleotides.

Recent phase I dose-escalation clinical investigations identified 40 mg/m2/d as the maximum tolerated dose of clofarabine when administered daily for 5 days by intravenous one hour infusions to patients with acute leukemias. In contrast, for patients with solid tumors and indolent leukemias, the maximum tolerated dose was 2 and 3 mg/m2/d, respectively. For acute leukemias, hepatotoxicity was dose limiting while myelosuppression was common in solid tumor and indolent leukemias (Ref. 1). Plasma pharmacology studies done in 25 acute leukemia patients indicate a linear increase in the plasma clofarabine concentration with increasing doses. At 40 mg/m2, the median plasma clofarabine level was 1.5 µM (range 0.42 to 3.2 µM; n = 7). Cellular pharmacokinetic studies done at the end of the first clofarabine infusion in 26 patients appeared dose-proportional, but showed a wide variation in the concentrations of clofarabine triphosphate. At the maximum tolerated dose, the concentration was a median 19 µM (range 3 - 52 µM). In the majority of cases, more than 50% of the analog triphosphate was present at 24 hr after infusion. Compared to clofarabine triphosphate concentration, the endogenous level of dATP was low, resulting in a favorable ratio of analog triphosphate to normal dNTP for incorporation into DNA. In association with the accumulation of triphosphate there was a decrease in DNA synthesis. At 40 and 55 mg/m2 doses, the inhibition of DNA synthesis was maintained to 24 hr (Ref. 2). Consistent with this observation, at the MTD, after 5 days of therapy, there was a consistent cytoreduction in the WBC count in the peripheral blood of all these patients (n = 12) with a median decline of 97% (range 68 to 99%). Among 32 patients with acute leukemias in this phase I study, 2 achieved complete remission and 3 had a marrow CR without platelet recovery. This encouraging outcome resulted in a phase II study exclusively for patients with acute leukemias.

Sixty-two patients with acute leukemias (including myelogenous leukemia N=31, myelodysplastic syndrome, MDS; N=8, chronic myeloid leukemia in blastic phase; N=11 and acute lymphocytic leukemia; N=12) were treated with clofarabine 40 mg/m2 IV over 1 hour daily x 5, every 3 to 6 weeks. Overall, 30 patients achieved complete or partial remission with an overall response rate of 48% (Ref. 3). At the MTD, clofarabine plasma level was 1.5 µM, which resulted in a median 15 µM triphosphate concentration in the cells. This intracellular level of clofarabine triphosphate was greater in the circulating leukemia blasts of responders (median, 18 µM) compared to non-responders (median, 10 µM; p=0.03). Furthermore, this value increased only in responders (median, 1.8-fold; p=0.008) after the second clofarabine infusion. We conclude that clofarabine is active in acute leukemias and MDS, and has an acceptable toxicity. The cellular pharmacokinetic profile indicates a potential for prognostic significance.

References.

1. Kantarjian, H. Gandhi, V., Kozuch, P., et al. Phase I clinical and pharmacology study of clofarabine (chloro-2’-fluoro-deoxy-9-beta-D-arabinofuranosyladenine) in patients with solid and hematologic cancers. J Clin. Oncol, 21:1167-1173, 2003.

26 2. Gandhi V, Kantarjian HM, Faderl S, et al. Cellular pharmacokinetics and pharmacodynamics of clofarabine (2-chloro-2’-fluoro-arabinosyladenine) in acute leukemia blasts during a phase I clinical trial. Proc. AACR Abstract # LB124. Presented at the 93rd Annual Meeting of the AACR. April 6-10, 2002. San Francisco.

3. Kantarjian, H., Gandhi, V., Cortes J., et al. Phase II clinical and pharmacology study of clofarabine in patients with refractory or relapsed acute leukemias. Blood, in press, 2003.

27 I04

Immucillins as Antibiotics for T-Cell Proliferation and Malaria

V.L. Schramm Albert Einstein College of Medicine, BRONX, NY, United States of America

Human genetic deficiency of purine nucleoside phosphorylase (PNP) causes a specific T-cell immunodeficiency. Deoxyguanosine (dGuo) accumulates in blood and is converted to toxic levels of dGTP in T-cells. Control of undesirable T-cell proliferation might be achieved by inhibitors of PNP sufficient to cause whole-body inhibition. Kinetic isotope effects were used to solve the transition state structure of bovine PNP. Chemically stable analogues were designed to resemble the transition state and were synthesized. The Immucills are the most powerful inhibitors known for PNP. Immucillin-H (ImmH) induces apoptosis in rapidly dividing human T-cells in the presence of dGuo. ImmH is orally available in mice and is effective in xenogeneic graft-vs-host disease. ImmH has entered human phase I/II clinical trails against T-cell leukemia, and causes elevated dGuo in humans. Plasmodium falciparum is a purine auxotroph and known pathways of human and malarial purine salvage all involve PNP. P. falciparum PNP is inhibited by ImmH and parasites cultured in human RBC without added hypoxanthine are killed by ImmH. Added hypoxanthine but not nucleosides rescue cultured P. falciparum from ImmH toxicity.

28 I05

ALIMTA (Pemetrexed) from lab bench to clinic

B. Nguyen Eli Lilly and Company, INDIANAPOLIS, IN, United States of America

Alimta, which inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase, has shown broad activity in a variety of tumors including thoracic, colorectal, pancreas and breast cancers. The development of antifolates has been challenging because of severe hematological and gastrointestinal toxicities. Analyses have shown that patients with folate deficiency, as indicated by homocysteine and methylmalonic acid levels, were at greater risk for Alimta toxicity. Such observations led to the programmatic intervention in late 1999 for the overall development of Alimta in which all patients received folic acid and vitamin B12 supplementation.

In a phase III study of Alimta plus cisplatin versus single-agent cisplatin in patients with malignant pleural mesothelioma, median survivals were 12.1 months versus 9.3 months, respectively (log-rank p=0.020). Median time-to-progressive disease was significantly longer in the Alimta/cisplatin arm as compared to the single-agent cisplatin arm (5.7 months vs. 3.9 months, p=0.001), and tumor response rates were 41.3% versus 16.7%, respectively. The addition of folic acid and vitamin B12 resulted in a significant reduction in toxicities in the Alimta/cisplatin arm with no detrimental effect on the efficacy of the combination regimen. A summary of the results from a phase III study of single-agent Alimta versus Taxotere in patients who had previously failed chemotherapy will also be presented at the meeting. Overall, Alimta showed a significantly better benefit-risk profile than Taxotere.

29 I06a

New (1-DEAZA)Purine Derivatives via Efficient C-2 Nitration of the (1-DEAZA)Purine Ring

G.J. Koomen University of Amsterdam, AMSTERDAM, The Netherlands

A couple of years ago, a new selective nitration reaction of substituted purine and deazapurines at the 2- position was developed in our lab.1) The resulting nitro groups could be easily exchanged via aromatic nucleophilic substitution reactions. The nitration reaction, which could also be carried out with purine nucleosides on solid support 2) turned out to be a combination of an electrophilic and a radical process, as could be established by 1H, 13C and 15N NMR spectra. Recently, the proposed mechanism was unequivocally established by observing CIDNP effects during the reaction at low temperature. Reduction of the nitro group produced the nitroso derivative, which could also be used for a variety of structural modifications, partly via hetero Diels-Alder reactions.3) The synthetic approach developed for temozolomide 4) could be used for the development of substi- tuted benzoyl triazenes with different t1/2 values as precursors for the in vivo production of both diazomethane (as by temozolomide) and 6-benzylguanosine (O6-BG) as an O6 alkyl-guanine-DNA --transferase (AGT) inhibitor. Biological data of the different analogues on Adenosine receptors5), on Plasmodium Falciparum and on Cancer cell lines will be presented.

NHR1 HN NHR1 N N X N O N X N N N O2N N 2 N N N N N X = CH, N R HN N H ribose H ribose ribose

X = N

1 NHR 1 Cl NHR N N N N X N O N N N N N N O2N N N ribose ribose O ribose

X = N

OCH2Ph NHR1 NHR1

N N HO N O N N N N N N N N N N N H N N N N H ribose ribose CH3 Y

1. P.Y.F. Deghati, M.J. Wanner and G.J. Koomen, Tetrahedron Letters, 1291-1295 (2000) 2. B. Rodenko, M. J. Wanner and G.J. Koomen, J. Chem. Soc. Perkin I, 1245-1252 (2002) 3. M.J. Wanner and G.J. Koomen, J. Chem. Soc. Perkin I, 1908-1915 (2001) 4. M.J. Wanner and G.J. Koomen, J. Chem. Soc. Perkin I, 1877-1880 (2002) 5. M. W. Beukers, M.J. Wanner, J.K. Von Frijtag Drabbe Künzel, E.C. Klaasse, A.P IJzerman and G.J. Koomen, J. Med. Chem. (2003)

30 I06b

LC-MS techniques in clinical applications

M. Svoboda Applied Biosystems, DARMSTADT, Germany

LC/MS techniques using combinations of quadrupole(s) and/or time-of-flight analyzers emerged as an extremely powerful tool for analytical challanges in the clinical arena in the last years. In combination with soft ionization techniques like Ionspray, Atmospheric Pressure Chemical Ionization and – most recently – PhotoSpray LC/MS is becoming an indispensable tool for applications like neonatal screening, metabolomics and drug monitoring. An overview of the current LC/MS instrumentation as well as examples of these applications will be presented in this talk.

31 I07

Quantitative RT-PCR using the LightCycler System for specific detection of TP (Thymidine Phosphorylase), DPD (Dihydropyrimidine Dehydrogenase) and TS (Thymidylate Synthase) from fresh and formalin-fixed, paraffin-embedded tissue

V. Lutz, M. Poignée, T. Ullrich, G. Maass, Roche Diagnostics GmbH, PENZBERG, Germany

The enzymes thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) metabolize pyrimidines in cells under both normal as well as pathological conditions. In particular, TP, DPD and TS play a major role in the metabolism of fluorouracil-based chemotherapeutics, which are widely used as anticancer therapy e.g. for colorectal cancer (CRC) and breast cancer (BC). Based on preclinical data, the variant levels of TP, DPD and TS in tumor tissue have an impact on the individual drug response to Xeloda, an oral fluoropyrimidine carbamate with antineoplastic activity, or other fluoropyrimidines, respectively, but due to various study protocols and methods examining either RNA, DNA, protein or enzyme activity, the role of TP, DPD and TS in cancer is still unclear. One important aspect in understanding the molecular basis of carcinogenesis is the quantitation of differential gene expression of normal and neoplastic cells. However, the availability of fresh pathological tissue is limited since optimal collection conditions are not easily realizable in clinical routine. In contrast, most pathological specimens are routinely formalin fixed and paraffin-embedded prior to histological evaluation. We have therefore developed a reliable method for RNA isolation from paraffin-embedded samples, and a standardized and quantitative method to assess TP, DPD and TS mRNA expression levels in microdissected, formalin-fixed, paraffin-embedded tissues using the LightCycler System. The feasibility of the method has been demonstrated by determining the expression levels and ratios for TP, DPD, and TS relative to the housekeeping gene G6PDH in several tumor tissues. Therefore, the development of a reliable method for mRNA isolation from paraffin-embedded samples with subsequent RT-PCR analysis is a major breakthrough for the acceptance and usefulness of RT-PCR technology for the analysis of biomarkers in research based and clinical studies. The aim of this project is to provide in the future specific tests for molecularly targeted cancer drugs, enabling the more precise prediction of individual clinical outcomes (drug efficacy and safety) and thus more efficient cancer therapies.

32 I08

New Levels in Gene Expression Profiling

R. Wicki Applied Biosystems, ROTKREUZ, Switzerland

Gene Expression analysis is a complex mixture of technologies and knowledge that aim at understanding the mechanisms driving cell life. Improvement of technologies for quantitative measuring of nucleic acid, such as Real Time PCR, and integration of experimental results with the knowledge about genes, proteins and functions is the way we are following to take Gene Expression to a new level.

A collection of ready to use Gene Expression Assays-on-Demand(TM) for more than 18.000 human genes and 9000 mouse genes is available on-line. These assays eliminate the major bottlenecks in using Real time PCR for Gene Expression studies involving many genes. Features and search possibilities for those assays will be demonstrated. We will also present our Assays-by-Design(SM) Service.

The new Micro Fluidic Card accessory allows for highly parallel Gene Expression analysis in a low density array format. From 12 and up to 384 genes can be analysed in parallel on a single run, with only two pipetting steps per sample. With a total reaction volume of 2ul, more information can be gathered from very small amounts of samples.

The Celera Discovery System (CDS) is a research platform that provides scientists with simplified online access to the most comprehensive, up-to-date set of integrated biological data available, incorporated into one annotated assembly, along with sophisticated visualization and analysis tools. Some of the many powerful capabilities of CDS for human or mouse Gene Expression analysis will be mentioned.

33 I09

Pulmonary Consequenses of Adenosine Overload: Lessons from Adenosine Deaminase Deficient Mice

R. Blackburn University of Houston Medical School, HOUSTON, TX, United States of America

Chronic lung diseases are associated with persistent lung inflammation and damage that lead to the progressive loss of lung function. In contrast to most injury and repair responses, the inflammation seen in these disorders is chronic and may last throughout the life of the afflicted individual. The mechanisms that are responsible for the intensity and chronicity of these disorders have not been elucidated. Adenosine is a signaling nucleoside that can elicit a variety of cellular functions by engaging cell surface adenosine receptors. Many of the actions of adenosine are cytoprotective or anti-inflammatory in nature, however, studies have shown that adenosine levels are elevated in the lungs of patients with chronic lung disease suggesting this signaling pathway might be involved in the exacerbation of these disorders. Support for this hypothesis comes from recent studies in mice deficient in the enzyme adenosine deaminase (ADA) that controls the levels of adenosine in tissues and cells. Adenosine levels accumulate in the lungs of these mice and are associated with increased lung inflammation and histopathologies seen in asthma and chronic obstructive pulmonary disease. Furthermore, lowering adenosine levels or antagonizing adenosine receptors can reverse many of the lung phenotypes seen. In addition, pharmacologic and genetic experiments suggest that adenoisne receptor signaling on various cell types in the inflamed lung play an important role in phenotypes seen in the lungs of ADA deficient mice. These findings suggest that chronic elevations in adenosine can access signaling pathways that can mediate aspects of chronic lung disease.

34 I10

Species and age-dependent differences in purine metabolism, adenosinergic signalling, and ischemic tolerance

J.P. Headrick Griffith University, SOUTHPORT Q, Australia

Adenine nucleotide and adenosine metabolism are crucial in determining ischaemic tolerance, through modulating energy state, radical generation, and receptor -mediated cardioprotective actions. Investigating myocardial 5'-AMP metabolism, adenosine salvage and adenosine responses in hearts from mice, rats, and guinea pigs, we document increased net myocardial 5'-AMP dephosphorylation as mass-specific metabolic rate rises or body mass declines (normoxic rates of ~4, 9, and 11 nmol/min/g in guinea pig, rat and mouse, respectively). Nonetheless, coronary venous adenosine levels are maintained at similar levels in all species (50-70 nM). Preserved extracellular adenosine levels are accomplished in part by increased salvage vs. deamination in mouse vs. larger species. Cardiovascular sensitivity to adenosine also differed between species, with myocardial A1 receptor sensitivity greatest in mouse and coronary A2 sensitivity greatest in guinea pig. Interestingly, although post-ischaemic loss of adenosine and its catabolites is relatively low in the mouse vs. other species, myocardial tolerance to ischaemia is poor. Inhibition of either adenosine deamination (20 µM EHNA) or phosphorylation (5 µM iodotubercidin) enhances ischaemic tolerance in both mouse and rat. These effects are receptor-dependent. However, simultaneous blockade of both pathways substantially limits post-ischaemic recovery in both species, revealing a key role for paths of adenosine salvage in ischaemic tolerance. Blockade of adenosine kinase also reveals an important (receptor- independent) role for phosphorylation in the anti-ischaemic effects of exogenously applied adenosine. Unfortunately, whilst these metabolic and adenosinergic manipulations prove beneficial in young tissue, aged myocardium most likely to suffer ischaemic events is insensitive to adenosine-based strategies. Recent studies in mouse reveal the ischaemia and adenosine intolerant phenotype is manifest well prior to senescence (being evident within the first 12 months of life), is unrelated to changes in A1 adenosine receptor transcription or expression, and may involve changes in intracellular signaling and catabolism of nucleotides and adenosine. Evidence is presented supporting a role for the decline in adenosine-mediated protection in ischaemic intolerance in aged mammalian myocardium.

35 I11

Allopurinol: Will it reduce cardiovascular events in patients?

A. Struthers, DUNDEE, United Kingdom

36 I12

Medical Diagnosis, Management and Treatment of Lesch Nyhan Disease

G.T. McCarthy Chailey Heritage Clinical Services, NR LEWES, EAST SUSSEX, United Kingdom

1. Introduction and aims of presentation. The aim of this presentation is to inform about LND from the point of view of the affected boys and their families living with the condition from day to day AND to show the importance of research in treating and managing the disease. 2. What is LND? Outline enzyme defect, incidence, genetic implications, and importance of family history. History of investigation into LND. The genetic defect in LND lies in the deficiency (at <2% of normal) of an enzyme of purine metabolism Hypoxanthine - Guanine Phosphoribosyl Transferase - abbreviated as HPRT. Not all boys with HPRT deficiency have LND, there are 3 intermediate forms called LND variants, all of which have varying but milder neurological symptoms. The fifth is called PARTIAL HPRT deficiency or Kelley-Seegmiller syndrome (KSS) where no neurological symptoms are found. However, uric acid overproduction occurs in all five categories. 3. Diagnosis a) Clinical pictureÆ in the baby -- neurological signs - developmental delay, hypotonia, irritability, sand in diaper. Sometimes kidney failure precipitated by infection. Æ in the older child -- 'athetoid CP' irritability, self injury - biting lips, fingers b) Biochemical Investigations Æ importance of metabolic investigations - uric acid in blood and urine. Vital to know that the kidneys of children clear their uric acid better than adults but urine will show grossly elevated levels. Ratio of uric acid and creatinine is raised 2-4 fold. HPRT activity in both disrupted and intact blood cells and sometimes from fibroblasts cultured from skin biopsy. c) Radiological Æ ultrasound of kidneys may give the first clue to diagnosis of kidney failure in baby boys with HPRT deficiency. 4. Early diagnosis is important Æ appropriate management starts from an early age. This benefits the baby and parents and may prevent the birth of another affected child by promoting genetic counselling for family members. 5. Management of LND MOTOR DISORDER Æ evolution from hypotonia to dystonia, rigidity, hypertonia AFFECTS Æ trunk, limbs, oral muscles swallowing and speech. Torsion dystonia causes hiatus hernia and feeding problems - vomiting, pain, bleeding, failure to thrive. Practical management of posture in lying sitting and standing. Daily living activities of feeding hand function, dressing and toiletting, play and educational activities. Speech and communication. 6. SELF INJURIOUS BEHAVIOUR. SIB Æ description of behaviour, how it differs from SIB in children with severe/profound intellectual disabilities and management of SIB. 7. BEHAVIOUR & COGNITIVE PROBLEMS an outline of investigation of cognitive ability in boys and men with HPRT deficiency. Other behavioural problems. 8. Drug treatment in LND i. Allopurinol. ii. Muscle relaxants Baclofen, Diazepam etc. iii. Anxyolytics Carbamazepine, Valproate, Gabapentin. iv. L dopa and others. 9. Growth and puberty. 10. Life expectancy. The Future.

37 I13

Lesch-Nyhan Disease

A.M. Marinaki1, L.D. Fairbanks1, D. Perrett2, H.A. Simmonds1 1Guy's Hospital, LONDON, United Kingdom 2St. Bartholomew's Hospital, LONDON, United Kingdom

The relationship between hypoxanthine-guanine phosphoribosyltransferase (HPRT) and the severe neurological manifestations of Lesch-Nyhan disease (LND) remains an enigma nearly 40 years on since the first case was reported by Lesch and Nyhan in 1964. Whilst complete deficiency HPRT deficiency is axiomatic for LND the fact that LND can present neonatally in acute renal to paediatric rather than neurological units can mask diagnosis initially. At the other end of the spectrum are LND variants presenting in their teens or early adulthood only with gout or kidney stones (‘partial’ HPRT deficiency, or Kelley-Seegmiller syndrome described in 1967). Treatment is also problematical. Allopurinol reduces the grossly elevated uric acid in blood and urine in consequence of lack of feed-back inhibition of purine synthesis in all types of HPRT deficiency (hence the tendency to gout), but replaces uric acid with the even more nephrotoxic xanthine if the dose is too high The dystonia, choreathetosis, spasticity and compulsive self-injurious behaviour chractersitic of LND have been related to a deficit of dopaminergic neurones in the striatum and will be discussed by Dr McCarthy. GTP depletion in erythrocytes has been demonstrated which led to the proposals that restriction in supply for GTP cyclohydrolase in brain could mimic the symptoms in hereditary progressive dystonia. However, such a link between altered nucleotide metabolism and the observed neuropathology remains speculative. Recent studies have shown decreased purine and pyridine nucleotide pools in nucleated cells and have related these findings to altered metabolism of poly-ADP-ribose affecting DNA repair and GTP-dependent neurotransmitter metabolism. Conversely accumulation of AICA riboside, an intermediate of de novo purine synthesis, has been proposed to promote apoptosis in a neuroblastoma cell line model. Analysis of global gene expression profiles of fibroblasts derived from LND patients may provide clues to other mechanisms

38 I14

Mutations of the HPRT Gene in Lesch-Nyhan Disease and its Variants

H.A. Jinnah1, L. DeGregorio1, J.C. Harris1, W.L. Nyhan2, J.P. O'Neill3 1Johns Hopkins Hospital, BALTIMORE, MD, United States of America 2UCSD School of Medicine, LA JOLLA, CA, United States of America 3University of Vermont, BURLINGTON, VT, United States of America

Introduction. Inherited mutations in the gene encoding hypoxanthine-guanine phosphoribosyl transferase (HPRT) result in Lesch-Nyhan disease (LND), which is characterized by hyperuricemia combined with devastating neurological and behavioral abnormalities. Mutations in the HPRT gene may also be associated with less severe Lesch-Nyhan variants (LNV) that include hyperuricemia with or without varying degrees of neurological or behavioral dysfunction. The purpose of the current studies was to compare the mutations in these two populations.

Methods. New and previously reported mutations causing HPRT deficiency were compiled into a database and grouped into two categories. The first category included those resulting in LND and the second category included those resulting in LNV. The less severe phenotypes could not be further differentiated because of inadequate clinical information in many prior reports. Genotype-phenotype correlations were made based on the locations and types of mutations.

Results.

Mutation LND (n=206) LNV (n=64) NA (n=8) Total (n=278)

Single base substitution Missense 64 48 4 115 Nonsense 23 1 1 25 Splice error 28 7 0 31

Deletion Coding sequences 58 2 3 61 Splice error 4 0 0 4

Insertion Coding sequences 18 1 0 19 Splice error 1 0 0 1

Others Duplication 3 3 0 6 Substitutions 2 0 0 2 Females 5 0 0 5 Double 0 2 0 2

Conclusions. There was no apparent correlation for clinical phenotype and mutation location. However, there was a rough correlation for clinical phenotype and the type of mutation. Specifically, those mutations predicted to result in severe disruption of enzyme function were more often associated with LND rather than LNV. This correlation becomes even more striking after excluding unstable and potentially revertible mutations (splice site and duplication mutations). A detailed examination of the exceptions to this correlation revealed in most cases unusual molecular mechanisms allowing residual enzyme activity. These results are consistent with prior enzyme studies showing a correlation between residual activity and disease severity. Analysis of HPRT mutations may therefore provide some predictive value for early-detected cases.

39 I15

New inborn errors; role of mitochondria; Deoxyribonucleoside kinases in mitochondrial DNA depletion.

A. Saada-Reisch Shaare Zedek Medical Center, JERUSALEM, Israel

Mitochondrial encephalomyopathies include a heterogeneous group of diseases expressing dysfunction of the mitochondrial respiratory chain (RC).They are caused by mutations in either the mitochondrial (mtDNA) or the nuclear genome with maternal or Mendelian inheritance respectively. Many point mutations and single large-scale deletions in the mitochondrial genome, and several point mutations in the nuclear genome, involving specific RC complexes have been characterized. Lately attention has focused on a group of Mendelian disorders represented by impaired intergenomic communication resulting in qualitative (multiple deletions) and/or quantitative (depletion) defects in mtDNA. Mutations in the genes of mitochondrial adenine nucleotide translocator, polymerase gamma and helicase have been associated with progressive ophtalmoplegia with multiple mtDNA deletions. The discovery of mutant thymidine phosphorylase, in mitochondrial neurogastrointestinal encephalomyopathy associated with multiple deletions and mtDNA depletion, indicated that imbalances in the mitochondrial deoxynucleotidetriphosphate (dNTP) pools might affect mtDNA integrity. This was further emphasized by the recent finding, of two enzymes involved the mitochondrial deoxyribonucleotide salvage pathway, which are mutated in tissue specific forms of mtDNA depletion; deoxyguanosine kinase (dGK) ,in the hepatoencephalopatic form and thymidine kinase 2 (TK2) in myopathic form. The pathogenicity of these mutations was confirmed by western blot for dGK and by enzymatic assays and cloning for TK2. These findings, together the observation that nucleoside antiviral therapy cause secondary mtDNA depletion, emphasize the importance of mitochondrial nucleotide metabolism and salvage pathway in the maintenance and integrity of mtDNA. Many questions remain open including understanding of the mechanism trough with the defective genes perturb mtDNA replication, why some tissues are spared while others are severely affected, searching for additional genes in unexplained cases of mtDNA depletion and exploring the possibility of intervention of mitochondrial nucleotide metabolism as possible treatment.

40 I16

Thymidine Phosphorylase Deficiency Causes MNGIE: An Autosomal Recessive Mitochondrial Disorder

M.H. Hirano, Y. Nishigaki, R. Marti Columbia University, NEW YORK, NY, United States of America

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TPase). The disease is characterized clinically by onset between the second to fifth decades, ptosis, progressive external ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy and leukoencephalopathy. Clinical diagnostic tests typically reveal leukoencephalopathy, and lactic acidosis whileskeletal muscle biopsies show neurogenic changes, together with mitochondrial abnormalities such as ragged-red-fibers, ultrastructurally abnormal mitochondria and decreased cytochrome c oxidase (COX) activity, either in isolation or in association with multiple respiratory chain enzyme defects. In addition to these signs of mitochondrial impairment, studies of mtDNA from skeletal muscle have shown depletion and multiple deletions. In MNGIE patients, TPase activity in buffy coat samples are drastically reduced (2±5 nmol/mg-protein/hour [mean±standard deviation], n=27) compared to controls (667±205[n=19]). TPase is a cytosolic enzyme required to maintain nucleoside homeostasis, therefore, loss of TPase function cannot account directly for the mitochondrial pathology. In addition, skeletal muscle does not express TPase and paradoxically shows mitochondrial alterations providing further evidence that loss of TPase does not directly alter mtDNA. As a consequence of loss of TPase function in MNGIE patients, mean plasma levels of thymidine (8.6µM ±3.4 [mean±standard deviation, n=25]) and deoxyuridine (14.2µM ±4.4, n=25) are elevated more than 100-fold above control values (thymidine <0.05µM, n=23 and deoxyuridine <0.05µM, n=20). We have hypothesized that loss of TP function leads to increased levels of circulating and intracellular thymidine and deoxyuridine which cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities. In cultured fibroblasts and non-skeletal muscle tissues of MNGIE patients, we identified respiratory chain defects without depletion or multiple deletions of mtDNA. Therefore, we sequenced mtDNA and demonstrated the presence of site-specific somatic mtDNA point mutations in tissues and cultured cells from MNGIE patients. MNGIE was the first human disorder of mitochondrial nucleoside/nucleotide metabolism to be molecularly characterized.

41 I17

Nucleoside transporters

C.E. Cass Cross Cancer Institute, EDMONTON, Canada

42 I18

Role of Multidrug-resistance associated proteins in resistance to nucleoside analogs

P. Borst Netherlands Cancer Institute, AMSTERDAM, the Netherlands

43 I19

Cloning and expression of malarial pyrimidine enzymes

R.I. Christopherson1, R.I. Menz2, O. Cinquin1, M. Shojaei1 1University of Sydney, SYDNEY, Australia 2Flinders University, ADELAIDE, Australia

We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) are targets for development of antimalarial drugs which would be active against drug-resistant parasites. These genes have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli. We found that the recombinant malarial OMP decarboxylase was toxic to E. coli and expressed at low levels prior to induction with IPTG. To overcome this problem and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used in the expression vector, and extra copies of rare tRNAs. Transformation of E. coli BL21 (DE3) with pMICO and pET-ODC resulted in high level expression of OMP decarboxylase when induced with IPTG (250 µM). Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA. The enzyme has a turn-over number of 5/s and a Mr of 38,158 by mass spectroscopy, consistent with removal of the start methionine. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms. Using our knowledge of the catalytic mechanism of dihydroorotase, we have designed and synthesized several potent inhibitors, TDHO and HDDP, which have Ki values of less than 1 µM for interaction with the target enzyme, and IC50 values of less than 10 µM for inhibition of the growth of P. falciparum in erythrocytic culture. The antimalarial drug, atovaquone, blocks electron transport in the parasite and indirectly inhibits reaction 4 of the pyrimidine pathway catalyzed by dihydroorotate dehydrogenase. We have shown that atovaquone induces accumulation of pyrimidine precursors before this step, and there is a major depletion of dTTP but not dCTP, precursors of DNA.

44 I20

Diversity and origins of deoxyribonucleoside kinases

J. Piskur1, M.P.B. Sandrini1, A.K.L. Joergensen1, W. Knecht1, B. Munch-Petersen2 1Technical University of Denmark, LYNGBY, Denmark 2Roskilde University, ROSKILDE, Denmark

Deoxyribonucleoside triphosphates, precursors for DNA replication in the cell, are provided by the de novo and salvage pathways. The key salvage enzymes are deoxyribonucleoside kinases, which phosphorylate deoxyribonucleosides to the corresponding deoxyribonucleoside monophosphates. In mammals four enzymes, TK1, TK2, dCK and dGK, with overlapping substrate specificites and different regulation of gene expression and cellular localisation, can be found. In contrast, insects, like the fruit fly and mosquito, have only a single TK2-like enzyme, dNK, with a broad substrate specificity. Several other organisms have recently been examined in our laboratory for the presence of deoxyribonucleoside kinases, the corresponding genes sub- cloned and the recombinant enzymes analysed for their kinetic parameters, substrate specificity and structure- function relationship. These findings provide a background to understand the evolutionary history of deoxyribonucleoside kinases. Apparently, the modern eukaryote TK1 originates from an ancient TK1 enzyme, which was likely to be present already in the progenitor of bacteria and eukaryotes. On the other hand, in the eukaryote lineage at least two gene duplication rounds, followed by further specialisation, have taken place to generate the modern mammalian enzymes, TK2, dCK and dGK, from the common progenitor, TK2/dCK/dGK kinase. The recently characterised novel kinases are also a valuable tool for development of novel suicide genes for gene therapy.

45 I21

Regulation of Nucleoside Uptake by Protein Kinase Inhibitors and Mitogenic Signaling

L. Graves, B.S. Mitchell, M. Huang University of North Carolina, CHAPEL HILL, NC, United States of America

Our laboratory has been studying the regulation of pyrimidine synthesis by protein kinase signaling. The uptake of pyrimidine nucleosides for salvage synthesis requires specific equilibrative or concentrative nucleoside transporters. We recently observed that the uptake of pyrimidine nucleosides and nucleoside analogs (ARA-C, CPEC) in human leukemic K562 cells was prevented by inhibitors of p38 MAPK, EGFR, BCR-ABL, PKC and CDK's. Comparison of inactive analogs of inhibitors p38 MAPK and PKC suggested that inhibition of transport occurred independently of kinase inhibition and through direct effects on the equilibrative transporter ENT1. By contrast, exposure of myeloid 32D or mouse embryo fibroblast cells to inhibitors of BCR-ABL or SRC suggested that the activity of these kinases regulated the expression and activity of both the ENT1,2 nucleoside transporters. The ability of protein kinase inhibitors to prevent nucleoside uptake and the current status of the regulation of pyrimidine nucleotide transporters by protein kinases will be discussed.

46 I22

Transcriptional regulation of DPYD gene expressoin and aberrant methylation in the 5'flanking region

M. Nishiyama1, K. Ukon1, T. Noguchi1, K. Tanimoto1, K. Hiyama1, M. Fukushima2 1Hiroshima University, HIROSHIMA, Japan 2Taiho Pharmaceuticals, HANNO-SHI, Japan

Dihydropyrimidine dehydrogenase (DPD) is the initial key enzyme in 5-fluorouracil (5-FU) catabolism, and over 80-90% of an administered dose of 5-FU is inactivated via the catabolic pathway. DPD demonstrates considerable interindividual variations and the activity relates to 5-FU response. Much attention has been focused on DPD as a predictive marker of individual 5-FU response, and recently a transcriptional mechanism is considered to be very important to understand DPD phenotype, since DPD activity closely correlates with the mRNA levels. Though, the detailed mechanisms remain unclear. We cloned a 3kb (- 2918~+78bp) 5’ flanking region of DPD gene (DPYD) from human placenta genome library, studied the functions, and found that aberrant methylation of DPYD promotor region strongly influenced cellular sensitivity to 5-FU through the regulation of DPYD expression. The full length of the cloned region contains CpG islands as reported by Diasio et al (BBA, 2000), and showed strong promoter activities in MKN45, HSC42, COLO320DM, 293T, and HepG2 cells. A series of mutants of the region deleted from 5’ site suggested that several up- and down-regulation sites might exist in the -1154~-64 region in addition to the previously reported 2 transcriptional regulatory elements just close to the transcription start site. Deletions from -1154 to -674 and from -453 to -338 resulted in approximately 40~60% of increase of the activity, whereas deletions from -674 to 453 and from -338 to -64 resulted in approximately 50% loss of the activity. The promoter activities closely correlated with mRNA expression levels in the cell lines except HepG2. HepG2 showed significantly high promoter activity, but the DPYD expression was almost undetectable, suggesting the existence of genetic variation of the region. Nevertheless, no difference was observed in the nucleotide sequence of the region in genome DNA. We found that methylation of the region played an important role in the expression regulatory mechanisms. Treatment of HepG2 cells with 5’aza Cytidine for 15 days increased remarkably both the DPYD expression and their sensitivity to 5-FU. Transcriptional mechanisms controls DPD phenotype at least in part, and aberrant methylation in DPD promotor region can play an important role in the transcriptional regulation.

47 I23

Pharmacogenetic and clinical aspects of a dihydropyrimidine dehydrogenase deficiency

A.B.P. Van Kuilenburg1, R. Meinsma1, A.H. Van Gennip2 1Academic Medical Center, AMSTERDAM, The Netherlands 2Academic Hospital Maastricht, MAASTRICHT, The Netherlands

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases and it catalyzes the NADPH-dependent reduction of uracil and thymine to 5,6-dihydrouracil and 5,6-dihydrothymine, respectively. DPD is also responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU). The catabolic route plays a significant role as more than 80% of the administered 5-fluorouracil is catabolized by DPD. In this light, a pharmacogenetic disorder has been described concerning cancer patients with a complete or partial deficiency of DPD suffering from severe toxicity, including death, following the administration of 5FU. Owing to the pharmacogenetic consequences associated with a DPD deficiency, we have performed an extensive study of the molecular aspects of patients with a DPD deficiency. The importance of a DPD deficiency in the etiology of unexpected severe 5FU toxicity is demonstrated by the high prevalence (61%) of the cases in which a decreased DPD activity can be detected in peripheral blood mononuclear cells. Patients with a partial DPD deficiency appeared to have a 3.4 fold higher risk of developing grade IV neutropenia compared to patients with a normal DPD activity. Furthermore, in patients with a low DPD activity, the onset of toxicity occurred, on average, twice as fast compared to patients with a normal DPD activity. In patients suffering from severe 5FU-associated toxicity, 12 mutations have been identified in DPYD including one splice site mutation (IVS14+1G>A); two nonsense mutations (R21X, E386X); 4 missense mutations (M166V, V335L, I560S and D949V) and 5 polymorphisms (C29R, R21Q, S534N, I543V, V732I). The IVS14+1G>A mutation proved to be the most common one and could be detected in 28% of all patients suffering from severe 5FU associated-toxicity. Considering the common use of 5FU in the treatment of cancer patients, the severe 5FU-related toxicities in patients with a low activity of DPD and the high prevalence of the IVS14+1G>A mutation, analysis of the DPD activity in PBM cells or screening for the IVS14+1G>A mutation should be routinely carried out prior to the commencement of the 5FU treatment.

48 I24

The Clinical Impact of Thiopurine Methyltransferase Polymorphisms on Thiopurine Treatment

S. Coulthard Northern Institute For Cancer Research, NEWCASTLE UPON TYNE, United Kingdom

Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood. Although current treatment results in long term survival in over 70% of cases there is evidence that as many as 50% could have been cured using a less complex regimen with a lower incidence of long term side effects. In previous studies it has been found that thiopurines given as part of continuing therapy are key agents in preventing relapse. However optimal administration during continuing therapy is often not achieved. Variation in the level of thiopurine methyltransferase (TPMT) activity appears to be a major molecular determinant of the extent of thiopurine metabolism. TPMT activity shows a trimodal distribution pattern. A lack of activity is found in approximately one in 300 Caucasians; approximately 11% have intermediate activity and the remaining 89% high activity. Congenital loss of activity is associated with grossly elevated levels of active drug and profound myelosuppression on exposure to thiopurine drugs. This loss of activity has been attributed to single nucleotide polymorphisms (SNPs) within the TPMT gene. The frequency of SNPs is related to ethnicity, with the most common in Caucasians being TPMT*3A which is characterized by a G to A transition at position 460 with a substitution of alanine for tyrosine at amino acid 154 (A154Y) and a transition of A to G at nucleotide 719 resulting in a change of tyrosine to cysteine at position 240 (Y240C). Polymorphisms have also been identified within the 5’ flanking promoter region of the TPMT gene due to a variable number of tandem repeats (VNTR*3-*8). An overview of the polymorphisms identified to date, their implication on the metabolism of the thiopurine drugs and therapeutic importance will be discussed.

49 O01

The efficacy and the toxicity of methotrexate in rheumatoid arthritis patients are asociated with polymorphisms in the methylenetetrahydrofolate reductase gene

W. Urano, A. Taniguchi, H. Yamanaka, E. Tanaka, H. Nakajima, Y. Matsuda, H. Akama, Y. Kitamura, N. Kamatani Tokyo Women's Medical University, TOKYO, Japan

A key enzyme involved in folate metabolism, 5,10-Methylenetetrahydrofolate reductase (MTHFR), has two common polymorphisms that affect enzyme activity. The objective of this study was to examine whether there was a correlation between the genotype or haplotype of the MTHFR gene and the efficacy or toxicity of methotrexate (MTX) in the treatment of rheumatoid arthritis (RA). 106 patients with RA who have been treated with MTX were randomly selected and used for a retrospective study to examine the correlation between genotypes or haplotypes concerning polymorphisms of the MTHFR gene, and the efficacy or toxicity of MTX. Estimation of the haplotype frequencies was performed by maximum likelihood estimation based on expectation maximization (EM) algorithm. Single locus analysis examining each locus separately showed that a higher rate of overall MTX toxicity was observed in patients with 677T than those without it (p<0.05, RR=1.25, 95%CI=1.05-1.49), while patients with 1298C were receiving significantly lower doses of MTX compared with patients without it (p<0.05, RR=2.18, 95%CI=1.17-4.06). An estimation of haplotype frequencies showed that there was no 677T-1298C haplotype in the population. Posterior distribution of the diplotype configuration for each individual was concentrated on a single configuration. Patients with 677T-1298A had a higher frequency of side effects from MTX (p<0.05, RR=1.42, 95%CI=1.11-1.82), while subjects with the 677C-1298C haplotype were receiving lower doses of MTX than those without it (p<0.05, RR=2.14, 95%CI=1.13-4.07). Both single locus and haplotype analyses have suggested that polymorphisms within the MTHFR gene are associated with both the efficacy and toxicity of MTX in RA patients.

50 O02

Febuxostat, a selective non-purine uric acid production inhibitor, is safe and decreases serum urate in healthy volunteers

M.A. Becker1, J.C. Kisicki2, R. Khosravan3, J. Wu3, D. Mulford3, B. Hunt3, P. MacDonald3, N. Joseph-Ridge3 1The University of Chicago Hospital, CHICAGO, IL, United States of America 2MDS Harris, LINCOLN, NE, United States of America 3TAP Pharmaceutical Products Inc., LAKE FOREST, IL, United States of America

Introduction: Febuxostat is a selective non-purine uric acid production inhibitor of the xanthine oxidase/xanthine dehydrogenase (XOD) enzyme. Febuxostat is under evaluation for the treatment of hyperuricemia associated with gout. In in vivo animal studies, febuxostat was more potent than allopurinol in lowering serum urate and urine uric acid concentrations. A Phase I dose-escalation clinical trial was conducted to assess the safety of febuxostat, and to determine pharmacokinetic and pharmacodynamic profiles of febuxostat during oral administration of repeated daily doses over a range of doses and regimens (e.g., QD and/or BID) to healthy subjects. Methods: This was a double blind, placebo-controlled, dose escalation study conducted in 154 healthy subjects, ages 19 to 54 years. In each dose group, 12 subjects were randomized in a 5:1 ratio to febuxostat or placebo. Subjects received orally administered febuxostat, with doses ranging from 10 to 240 mg QD and 30 mg BID. Results: Febuxostat effectively decreases serum urate concentration in a nearly dose-proportional manner up to 120 mg/day and with little further decrease noted between 120 and 240 mg/day. The reduction of serum urate concentration was accompanied by corresponding increases in serum xanthine and hypoxanthine concentrations, confirming that febuxostat lowers serum urate concentrations by means of inhibition of XOD activity. Febuxostat was safe and well tolerated. The most frequently reported adverse events included headache, nausea, dizziness, vasodilatation (flushing, feeling of warmth) and abdominal pain. No clear dose- dependence was noted for the incidence of most adverse events. However, incidence of vasodilatation (flushing, feeling of warmth) was higher at the 160, 180 and 240 mg QD doses (40-50%) as compared to the lower dose groups (0-25%). The majority of adverse events were mild in severity and self-limiting. There were no significant changes in laboratory values, electrocardiograms or physical examinations. There were nine early terminations due to adverse events and no serious adverse events or deaths reported. No dose- limiting toxicity was observed. Conclusions: Treatment with febuxostat is safe, well tolerated, and effectively decreases serum urate concentrations in a nearly dose-proportional manner with once a day dosing in healthy volunteers.

51 O04

Modification of cytokine milieu by signaling through A2A adenosine receptors in rheumatoid arthritis

M. Koshiba, Y. Nakamachi, H. Kosaka, T. Nakazawa, G. Tsuji, S. Kumagai Kobe University, KOBE, Japan

Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF) are considered to be responsible with various human diseases including rheumatoid arthritis and endotoxin shock. The anti-rheumatic effect of methotrexate is reported to be mediated by the increase in the extracellular adenosine at the inflammatory sites, but the detailed mechanisms of action of adenosine are yet to be clarified. We investigated the effects of A2A adenosine receptor and beta-adrenergic receptor signaling on the cytokine production of LPS- stimulated human CD14+ cells (monocytes/macrophages) derived from peripheral blood and rheumatoid synovial fluids. TNF production of LPS-stimulated peripheral CD14+ cells was inhibited in a concentration dependent manner by A2A receptor agonist. Co-administration of beta2-adrenergic agonist enhanced the + inhibition of the TNF production by A2A agonist. Similarly on LPS-stimulated CD14 cells from rheumatoid synovial fluids, observed was the concentration-dependent inhibition of TNF and IL-12 but not IL-1beta and IL-6 production via A2A signaling. These results suggest that the anti-rheumatic effect of extracellular adenosine is, at least in part, mediated via A2A receptor signaling by the modification and normalization of Th1-shifted cytokine milieu in the rheumatoid arthritis.

52 O05

6-[2-(Phosphonomethoxy)alkoxy]pyrimidines: a new class of acyclic pyrimidine nucleoside phosphonates with antiviral activity

J. Balzarini1, C. Pannecouque1, L. Naesens1, G. Andrei1, R. Snoeck1, E. De Clercq1, S. Aquaro2, C.-F. Perno2, H. Egberink3, I. Votruba4, A. Holý4 1Rega Institute for Medical Research, LEUVEN, Belgium 2University of Rome 'Tor Vergata', ROME, Italy 3Institute of Virology, UTRECHT, The Netherlands 4Institute Org. Chem. Biochem., PRAGUE, Czech Republic

A new class of acyclic pyrimidine nucleoside phosphonate analogues have been designed, in which the base consists of a pyrimidine containing an amino or hydroxy group at C-2 and C-4 and a 2- (phosphonomethoxy)ethoxy (PMEO) or a 2-(phosphonomethoxy)propoxy (PMPO) group at C-6. The 6- PMEO-2,4-diaminopyrimidine (PMEO-DAPY), the 6-PMEO-2-amino-4-hydroxypyrimidine (PMEA-AHPY) and the 6-PMPO-2,4-diaminopyrimidine (PMPO-DAPY, R-enantiomer) were inhibitory against DNA viruses [i.e. herpes simplex virus type 1 (HSV-1), HSV-2 and varicella-zoster virus (VZV)], but also against retroviruses [i.e. human immunodeficiency virus type 1 (HIV-1), HIV-2, feline immunodeficiency virus (FIV) and Moloney murine sarcoma virus (MSV)] in cell culture (50% effective concentration between 0.002 µg/ml and 1.9 µg/ml, depending the virus strain and cell line investigated). PMEO-DAPY and PMPO-DAPY (R- enantiomer) were also highly effective against MSV-induced tumor formation in newborn NMRI mice. In this respect they were equally active as PMEA (adefovir) and (R)-PMPA (tenofovir), which are now approved for treatment of hepatitis B virus and HIV infection, respectively. Substitution of a bromine at the 5-position of the pyrimidine ring further increased the selectivity. The novel 6-PMEO and 6-PMPO pyrimidine derivatives closely mimic the corresponding 2,6-diaminopurine nucleoside phosphonates in that the 2,4-diamino- substituted pyrimidine ring may be considered as analogous to the pyrimidine ring in the acyclic 2,6- diaminopurine nucleoside phosphonate derivatives. Hence, these novel PMEO and PMPO pyrimidine derivatives retain the most important part of the purine base for recognition by the phosphorylating (activating) cellular enzymes, with the viral reverse transcriptase being the most likely target enzyme for their antiretroviral activity. The antiviral activity of PMEO-DAPY, unlike that of and PMPO-DAPY, unlike that of zidovudine (AZT) and lamivudine (3TC), cannot be reversed by natural nucleosides or nucleobases. The PMPO enantiospecificity (preference of (R) over (S) in the PMPO-series) has also been found for the PMPA series of drugs, again pointing to a striking similarity in the activation pattern and mechanism of anti(retro)viral action between the new pyrimidine derivatives and the established purine derivatives. The molecular bases of the antiretroviral activity and intracellular metabolism of the PMEO and PMPO derivatives is under current investigation.

53 O06

Antiproliferative activity and mechanism of action of fatty acid derivatives of gemcitabine in leukemia and solid tumor cell lines and xenografts

A.M. Bergman1, P. Noordhuis1, E.M. Comijn1, J. Balzarini2, F. Myhren3, R. Tuen4, Ø. Fodstad4, H.R. Hendriks5, M.L. Sandvold3, G.J. Peters1 1VU University Medical Center, AMSTERDAM, The Netherlands 2Rega Institute for Medical Research, LEUVEN, Belgium 3Clavis Pharma, PORSGRUNN, Norway 4The Norwegian Radium Hospital, OSLO, Norway 5EORTC-NDDO, AMSTERDAM, The Netherlands

Gemcitabine is a deoxycytidine (dCyd) analog, with antitumour activity in solid tumours and some hematologic malignancies. Gemcitabine requires phosphorylation by deoxycytidine kinase (dCK) to allow formation of its active phosphate dFdCTP. It can be deaminated by deoxycytidine deaminase (dCDA). In addition to dCK deficiency altered membrane transport over the cell membrane is a mechanism of drug resistance for gemcitabine. In order to facilitate gemcitabine uptake and prolong retention in the cell, lipophillic pro-drugs were synthesized. An elaidic fatty acid group was esterified at the 5´ position on the sugar moiety of gemcitabine (CP-4126), or acylated on the 4-amino group (CP-4125), which might reduce deamination. The compounds were tested in four pairs of either ara-C (the murine leukemia L5 and L4A6 and the rat leukemia BCLO and Bara-C) or gemcitabine (the human ovarian cancer A2780 and AG6000 and murine colon C26-A and C26-G) resistant cell lines. L4A6, Bara-C and AG6000 have varying degrees of decreased dCK activity. In the parent cell lines gemcitabine was more active, but in the various resistant variants the prodrugs were more active, while the degree of cross-resistance varied. No difference in activity of the 4-amino acylated CP-4125 or the 5´ esterfied CP-4126 was found. In the malignant melanoma THX cells the nucleoside transport inhibitors NBMPR and dipyridamole increased the IC50 for gemcitabine 56 and 115-fold, respectively, while the IC50 for CP-4126 was increased 1.6 and 1.3-fold, respectively. Similar results were found in the human T-cell leukemia MOLT4 cells suggesting a nucleoside transport independent passage of CP-4126 over the cell membrane. In contrast to dFdCTP formed after gemcitabine exposure, dFdCTP pools after CP-4125 exposure continued to increase after drug removal in C26-A cells. CP-4125 induced inhibition of DNA synthesis effectively in C26-A and C26-G cells. The retention of inhibition was longer for CP-4125 than for gemcitabine in C26A cells. In vivo evaluation of a panel of 3 xenografts showed a similar activity for gemcitabine and CP4125 and CP4126. In conclusion, based on in vitro and in vivo data, cytotoxic and metabolic effects of CP-4125 and CP-4126 are different and longer lasting than for the parent drugs. Both CP-4125 and CP-4126 seem to be promising new prodrugs of gemcitabine.

54 O07 Nucleotide analysis by liquid chromatography/mass spectrometry

R.T. Smolenski1, E.M. Slominska2, H. Dziewit2, E.A. Carrey3, H.A. Simmonds3, M.H. Yacoub1 1Imperial College, London, United Kingdom 2Medical University of Gdansk, Gdansk, Poland 3Guy’s Hospital, London, United Kingdom

High Performance Liquid Chromatography with UV detection has its limitations, especially with identification of unknown metabolites and quantitative analysis of chromatographically unresolved peaks, even using in-line diode array detectors. Application of mass detection with HPLC has the potential for not only positive identification and quantitation of chromatographically unresolved peaks, but also for retrieval of structural information of unknown metabolites and isotopomer analysis. However, before it can become a routine technique for nucleotide analysis, some difficulties have to be resolved such as matching optimal chromatographic separation with optimal conditions for ionization. We developed an anion exchange chromatographic system using an amino column whereby high inorganic salt concentration in the mobile phase has been replaced by an ion-pairing agent at moderately elevated pH to elute highly polar compounds such as triphosphates. This system has been found to be superior to ion pairing due to lack of sample effect on separation and better definition of compound class retained on the column, to the anion exchange system with alkaline elution due to less in source fragmentation of triphosphates to diphosphates and to reversed-phase separation due to better chromatographic separation and efficiency of ionization. With our procedure we have been able to separate chromatographically and quantitate di- and triphosphates of adenosine, guanosine, uridine and cytidine. Perchloric acid and trichloroacetic acid extracts of heart or other cells have been run with separation quality identical to standards. Analysis of red blood cell extracts from patients with renal failure in addition to the presence of known nucleotides allowed identification of hitherto unidentified components such as 2-pyridone-5-carboxamide ribonucleoside triphosphate. Analysis of heart extracts allowed identification and quantitation of diadenosine polyphosphates. In conclusion, application of mass spectrometry in nucleotide analysis opens new opportunities for studies of nucleotide metabolism. It may not only be limited to analysis of static concentration but also for analysis of metabolic fluxes by isotopomer analysis, but practical evaluation of this possibility requires further studies.

55 O08

Online fluorescent method to assess BCRP activity as a function of cellular folate homeostasis

J.H. Hooijberg1, G.J. Peters1, G.J.L. Kaspers1, N. De Vries1, I. Kathmann1, R. Pieters2, G. Jansen1 1VU University Medical Center, AMSTERDAM, The Netherlands 2Sophia Childrens Hospital, ROTTERDAM, The Netherlands

The use of the antifolate methotrexate (MTX) plays an important role in the treatment of acute lymphoblastic childhood leukemia (ALL), and several other diseases. Recently, the Breast Cancer Resistance Protein (ABCG2/MXR) was reported to confer cellular resistance to MTX. This observation opens the possibility of a role for BCRP in the homeostasis of natural folates as well. In theory, BCRP mediated transport of cellular folates might competitively inhibit MTX efflux, and thus influence cellular sensitivity to MTX. In this study we report effects of differences in cellular folate status on the transport activity of BCRP. We measured the cellular efflux of the BCRP substrate Hoechst 33342 in the human myeloid cell line RPMI 8226(s) and its mitoxantrone resistant and BCRP overexpressing subline 8226/MR. A new method was developed by which the cellular efflux of fluorescent compounds from suspension cells can be monitored online. This method uses a two-compartment cuvette system, divided by a semi-permeable membrane, and can be used with a relatively small amount of cells (~ 50.000). The influence of cellular folate status on BCRP mediated efflux of Hoechst 33342 was determined by culturing cells under folate-rich (2.3 microM folic acid, i.e. standard culture medium), or folate-restricted conditions (i.e. in folate free medium). When grown in medium containing 2.3 microM folic acid, the efflux rate of Hoechst 33342 in 8226/MR cells was 2-fold higher (~ 0.64 pmol/min/106 cells) than in wild type 8226(s) cells (~ 0.30 pmol/min/106 cells). When 8226/MR cells were cultured for 48 hours in folate-free medium Hoechst 33342 efflux was decreased to a comparable level as in 8226(s) cells (~ 0.28 pmol/min/106 cells). As determined by Western blotting the expression of BCRP in both these cell lines did not alter upon changes in cellular folate status. In conclusion, cellular folate homeostasis appears to influence BCRP mediated transport activity. These results are relevant to understand the role of BCRP in drug resistance. Supported by the Dutch Cancer Society, grant VU-2000-2237.

56 O09

Coordinate Regulation of the Expression of Ecto-5'-Nucleotidase (CD73) and the A2a Adenosine Receptor in a Human B-Cell Line

W. Gutensohn, R. Napieralski, B. Kempkes University of Munich, Munich, Germany

A human B-cell line (P493-6) was used as a model system to study the regulation of the expression ecto-5´- nucleotidase (CD73) and one of the adenosine receptors. In P493-6 cells the activity of two different mitogenic signals, the Epstein-Barr virus nuclear antigen 2 (EBNA2) and myc, can be independently regulated. A stepwise shut off of EBNA2 and myc leads to a change from a more lymphoblastoid phenotype to a resting state. Shut off of EBNA2 – either in the presence or absence of myc activity – causes a significant increase in enzymatic activity and surface expression of CD73 as well as an increased adenosine receptor response in cyclicAMP formation. The adenosine receptor in question is identified as the A2a subtype by using specific agonists and inhibitors and by measuring mRNA levels. Shut off of myc has an additional positive effect on CD73 expression but not on the adenosine receptor. Upon turn on of EBNA2 and myc both CD73 and the A2a receptor are repressed. Induction and repression of both components follow a rather slow time course in the order of days. The signaling pathways involved in this regulation are unknown sofar and a direct connection with regulatory elements described in the promotor region of the human CD73 gene is not evident. A coordinated up-regulation of the ecto-enzyme and an A2 adenosine receptor could reflect the establishment of a positive feed back loop for autocrine stimulation in the presence of extracellular nucleotides.

57 O10

Prevention of adriamycin induced heart failure by increase in endogenous adenosine production

A.H. Yuen1, M. Boscoe1, R. Lango2, K. Suzuki1, E.M. Slominska2, M.H. Yacoub1, R.T. Smolenski1 1Imperial College, LONDON, United Kingdom 2Medical University of Gdansk, GDANSK, Poland

Adenosine (Ado) triggers several protective mechanisms that may attenuate development of heart failure. We developed a procedure allowing sustained increase in endogenous Ado production by the combined application of Ado metabolism inhibitors and nucleotide precursors. This study aimed to evaluate whether this intervention will attenuate development of experimentally induced heart failure.

Blood adenosine and other purine metabolites concentration was evaluated following administration of inhibitor/substrate solution containing Ado deaminase inhibitor - deoxycoformycin, Ado kinase inhibitor - 5'- aminoadenosine and nucleotide precursors - adenine and ribose. After these preliminary studies, in two experimental groups (AC, AT), heart failure was induced by repeated intraperitoneal injections of adriamycin for two weeks while in two other groups saline was injected (CC, CT). Subsequently in the following four weeks an inhibitors/substrate solution was administered in treated groups (AT, CT) in the form of daily intraperitoneal injections while saline was given to controls (AC, CC). Cardiac function was monitored using transthoracic echocardiography at weekly intervals and is expressed as left ventricular ejection fraction (EF±S.E.M).

Administration of inhibitors/substrates induced prolonged elevation of blood adenosine concentration for up to 6 h with its peak, about 1 µM after 30 min following administration. Some other changes such as elevated hypoxanthine or uric acid concentrations were also observed. There were no differences in cardiac function at the beginning of the experiment between all four groups (EF=68.2±1.9, 71.4±1.1, 72.4±1.4 and 73.4±1.9% for AC, AT, CC and CT respectively). At the end of the experiment, after four weeks of treatment with inhibitors/substrates cardiac function was singnificantly improved in adriamycin and inhibitors/substrates group (AT, EF=68.2±3.5%) as compared to adriamycin group (AC, EF=58.3±1.8, p=0.022). There were no differences in cardiac function between CC and CT (EF=78.7±1.1 and 79.3±1.2% in CC and CT respectively).

Increase in endogenous Ado production by inhibition of Ado metabolism and nucleotide precursor supply resulted in attenuation of development of heart failure induced by adriamycin. Regulation of adenosine production is protective not only during ischemia/reperfusion injury or allograft rejection as we have previously shown, but this may also be applicable in heart failure.

58 O11

Role of Platelet derived endothelial cell growth factor / thymidine phosphorylase in fluoropyrimidine sensitivity and potential role of deoxyribose-1-phosphate

M. De Bruin1, T. Van Capel1, A.C. Laan1, K. Smid1, M. Fukushima2, K. Hoekman1, H.M. Pinedo1, G.J. Peters1 1VU University Medical Center, AMSTERDAM, The Netherlands 2Taiho Pharmaceuticals, HANNO-SHI, Japan

Platelet derived endothelial cell growth factor (PD-ECGF) / thymidine phosphorylase (TP) catalyses the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate and is involved the metabolism of fluoropyrimidines. It can activate 5'-deoxyfluorouridine (5’DFUR) and possibly 5-fluorouracil (5FU) but inactivates trifluorothymidine (TFT). We studied the contribution of TP activity to the sensitivity for these fluoropyrimidines by modulating its activity and/or expression level in colon and lung cancer cells using a specific inhibitor of TP (TPI) or via stable transfection of TP. Expression was analysed using competitive template-RT-PCR (CT-RT-PCR), western blot and an activity assay. To determine the role of TP in the sensitivity to 5FU, 5'DFUR and TFT, cells were cultured with the various fluoropyrimidines with or without TPI and differences in IC50s were established. TPI modulated 5’DFUR, increasing the IC50s 2.5 to 1396-fold in WiDR and Colo320TP1, respectively. 5FU could be modulated by inhibiting TP but to a lesser extent than 5'DFUR: IC50s increased 1.9 to 14.7-fold for WiDR and Colo320TP1, respectively. There was no effect on TFT. There appears to be a threshold level of TP activity to influence the 5'DFUR and 5FU sensitivity, which is higher for 5FU. In the metabolism of fluoropyrimidines the levels of dR-1-P might play an important role eiter as inhibitor of the activation of 5’DFUR or as co-substrate for activation of 5FU. We therefore determined the levels of dR-1-P, using a purine nucleoside phosphorylase based assay and suprisingly it was found that TdR incubation did not increase dR-1-P accumulation in Colo320 and its PD-ECGF/TP transfected variant Colo320TP1. There was a huge discrepancy between thymine production and the measured dR-1-P level, 0.05% of the expected value for dR-1-P was found, indicating that there was a rapid disappearance of dR-1-P. However, in cellular extracts, TdR incubation increased dR-1-P, measurable by trapping, which was inhibited by a TPI. dR-1-P directly added to cellular extracts, disappeared. In conclusion, large amounts of dR-1-P are produced by Colo320TP1 cells, which rapidly disappear thus not resulting in a net accumulation of dR-1-P in these cells. dR-1-P appears to be the limiting factor in 5FU activation by PD-ECGF/TP.

59 O12

Down-Regulation of Human Deoxyuridine Triphosphate Nucleotidohydrolase (dUTPase) using Small Interfering RNA (siRNA)

M.V. Williams, A.W. Studebaker The Ohio State University, COLUMBUS, OHIO, United States of America

Human dUTPase is a member of the homotrimeric family of dUTPases that include enzymes from most eukaryotes, prokaryotes and RNA viruses. dUTPase is the enzyme responsible for hydrolyzing dUTP to dUMP and pyrophosphate, which prevents dUTP from being incorporated into DNA and simultaneously provides a precursor for dTMP synthesis. It has been suggested that dUTPases could be used as a target for the development of specific agents useful for treatment of infections caused by certain viruses and also for cancer chemotherapy, however, it has not been possible to develop agents that selectively inhibit dUTPase activity in vivo. Furthermore, studies to determine the role(s) of dUTPase in cellular metabolism in human cells have been hampered because cells deficient in dUTPase activity have not been isolated or constructed. Structural and functional studies have demonstrated that homotrimeric and monomeric dUTPases contain five conserved amino acid domains that contribute to the formation of the catalytic site. Since recent studies have demonstrated that gene expression can be down-regulated using small interfering RNA (siRNA). To determine whether such an approach could be used to decrease dUTPase activity, we synthesized a double- stranded siRNA targeted against domain 3 (nucleotides 339 to 357) of human dUTPase and used this siRNA to determine its effect on dUTPase activity in SW620, HT29 and HeLa cells. Cells were transfected with 1 to 4 µg of siRNAdut3 and examined 48 hrs after transfection for residual dUTPase activity. There was a significant decrease in dUTPase activity in cells transfected with siRNAdut3 when compared to non- transfected controls. dUTPase activity was decreased approximately 50 ± 0.9% in HeLa and HT29 cells and approximately 27 ± 11% in SW620 cells. This difference probably reflects not only differences in rate of transcription of dut gene, and stability of dUTPase specific mRNA in the cell lines but also differences in transfection efficiencies of the cells. There was no effect on uracil-DNA glycosylase activities in transfected cells when compared to non-transfected controls. These results demonstrate that a siRNA can be used to decrease expression of dUTPase in human cells and that such an approach could be useful either alone or in combination with established therapies for anti-viral and/or cancer chemotherapy.

60 O13

Cytotoxic and apoptotic effects of novel heterodinucleoside dimers consisting of 5-fluorodeoxyuridine and ara-c in human cancer cell lines

Ph. Saiko1, W. Bauer1, Z. Horvath1, Th. Hoechtl1, M. Grusch1, G. Krupitza1, R. Mader1, H. Schott2, M. Fritzer- Szekeres1, T. Szekeres1 1University of Vienna - General Hospital, VIENNA, Austria 2University of Tuebingen, TUEBINGEN, Germany

5-fluorinated pyrimidines have become very useful in the treatment of human solid tumors, including breast cancer, gastrointestinal adenocarcinomas and squamous cell carcinomas arising in the head and neck. Moreover, they have invoked interest because of their synergistic interaction with other antitumor agents. One of them, ara-C, has only been used in the treatment of leukemia so far, since the active form of the drug does not reach solid tumors. In search for possible alternative chemotherapeutic agents we tested a number of recently synthesized heterodinucleoside phosphate dimers consisting of 5-FdU and ara-C, which had already exhibited cytotoxic activity in leukemia and prostate cancer cell lines. These dimers enter the tumor cells due to their lipophilic character which also enables the transport of ara-C to the tumor site. After cellular uptake, the 5´-phosphate- derivatives of 5-FdUrd and ara-C can be released via enzymatic cleavage. In our present study, we investigated the cytotoxic effects of several 5-FdU-ara-C-Dimers in HT29 colon carcinoma cells as well as in ara-C sensitive and resistant H9 lymphoma cells. Moreover, we analyzed the induction of apoptosis in HT29 cells. Cytotoxic activity was determined by growth inhibition and clonogenic assays, while induction of apoptosis was detected employing a cell staining method using hoechst/propidium iodide. We can show that all dimers tested inhibited the number of colonies of HT29 human colon cancer cells with IC50 values ranging from 35 to 100nM. While examining the two different H9 cell lines, no significant difference in cytotoxicity could be observed between ara-C sensitive and resistant cells, indicating that these compounds might be used in the treatment of ara-C resistant tumors. Growth inhibition assays resulted in IC50 values ranging from 250 to 300nM. Furthermore, the investigated dimers induced dose and time dependent apoptosis in HT29 cells. We therefore conclude that these novel heterodinucleoside dimers exhibit in vitro cytotoxic activity against human colorectal cancer cells and human lymphoma cells. They are able to circumvent ara-C resistance in H9 cells and might therefore offer an additional option for the treatment of human tumors.

61 O14

Increased thymidylate synthase (TS) but unaltered dihydropyrimidine dehydrogenase (DPD) mRNA levels after administration of 5-fluorouracil (5-FU) to patients with colon cancer

R. Mauritz, C.J. Van Groeningen, K. Smid, C.H. Smorenburg, H.M. Pinedo, G.J. Peters VU University Medical Center, AMSTERDAM, The Netherlands

Clinical resistance to 5-FU is associated with upregulation of its target enzyme, TS or the rate-limiting catabolic enzyme DPD. In vitro studies have demonstrated that increased TS enzyme levels following exposure to 5-FU result from a disrupted autoregulation of TS mRNA translation. This study explores the effect of 5-FU exposure on TS mRNA levels in primary tumors and metastases of colon cancer patients. TS mRNA levels were determined in biopsy specimens from patients who were either not exposed to 5-FU or given one pre- surgery bolus of 5-FU (500 mg/m2). Tumor biopsy specimens were obtained 1 to 48 hours after 5-FU administration. TS mRNA expression was measured by competitive template RT-PCR using ß-actin as an internal standard. In both patient groups a wide variation in intratumoral TS mRNA levels was observed. No difference was observed between TS mRNA levels in colon tumors (n = 19; median TS/ß-actin 0.96 × 10-3, range 0.11 – 46.4 × 10-3) and in liver metastases (n = 53; median TS/ß-actin 1.01 × 10-3, range 0.068 – 16.8 × 10-3) of unexposed patients. However, TS mRNA levels were significantly higher both in colon tumors and in liver metastases of patients that were pretreated with 5-FU compared to samples of patients not exposed to 5- FU: median TS mRNA expression in colon tumors of exposed patients (n = 14; median TS/ß-actin 2.87 × 10-3, range 1.03 – 69.8 × 10-3) was 3.0-fold higher than in unexposed patients (p = 0.01), while in liver metastases of exposed patients (n = 16; median TS/ß-actin 5.38 × 10-3, range 0.67 – 38.5 × 10-3) TS mRNA levels were 5.3- fold higher than in unexposed patients (p < 0.001). DPD mRNA levels did not differ significantly between colon tumors and liver metastases. In addition, no effect of 5-FU exposure on DPD mRNA expression was observed in both patient groups. In conclusion, 5-FU administration did not alter DPD mRNA levels but was associated with increased TS mRNA expression both in primary colon tumors and in liver metastases. (Supported by the Netherlands Organization for Scientific Research).

62 O15

Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in human leukemia CCRF-CEM and EHEB cell lines

S. Cardoen1, E. Van den Neste1, C. Smal1, J.-F. Rosier2, A. Ferrant3, G. Van den Berghe1, F. Bontemps1 1ICP and Catholic University of Louvain, BRUSSELS, Belgium 2C.H. Jolimont-Lobbes, LA LOUVIèRE, Belgium 3Clinic St. Luc, BRUSSELS, Belgium

The human leukemia cell line EHEB, established from a patient with B-chronic lymphocytic leukemia, was previously found to be less sensitive (10- to 1000-fold) to the nucleoside analogue 2-chloro-2’-deoxyadenosine (CdA) than other human lymphoblastic cell lines. Moreover, DNA synthesis, measured by thymidine incorporation into DNA, was unexpectedly increased in EHEB cells, up to 2-fold, after a 24 h-incubation with 10 µM CdA (Cardoen et al., Clin. Cancer Res. 7, 3559-66, 2001). Analysis by flow cytometry, using double labelling with propidium iodide and bromodeoxyuridine, showed that CdA provoked an increase in the proportion of cells in S phase, synthesizing actively DNA. These results contrast with those reported in other leukemic cell lines sensitive to CdA, like CCRF-CEM cells, in which CdA inhibits DNA synthesis and provokes an accumulation of most cells in either early S phase or at the G1-S border. Kinetics and synchronisation experiments showed that CdA stimulates the progression of EHEB cells from G1 to S phase, rather than blocking them in S phase. This led us to study the effect of CdA on proteins regulating the G1/S checkpoint of the cell cycle, and firstly on the phosphorylation of the retinoblastoma (Rb) protein, which is increased during the G1/S transition. We observed that CdA enhances the phosphorylation of Rb in EHEB cells, whereas it decreases it in CCRF-CEM cells. The p53 status of this cell line was determined and found unmutated. Additional preliminary experiments showed that CdA decreases p21 expression in EHEB cells. In conclusion, we show a new mode of cellular response to CdA, implying modification of the cell cycle regulation leading to enhanced DNA synthesis. We propose that this peculiar effect might be implied in some types of yet unexplained resistance of leukemic cells to CdA.

63 O16

Increased cytotoxicity of 2’,2’-difluoro-2’-deoxycytidine in human leukemic cell-lines after a preincubation with cyclopentenyl cytosine

A.C. Verschuur1, A.H. Van Gennip2, R. Leen3, A.B.P. Van Kuilenburg3 1Emma Children's Hospital, AMSTERDAM, The Netherlands 2Academic Hospital Maastricht, MAASTRICHT, The Netherlands 3Academic Medical Center, AMSTERDAM, The Netherlands

Introduction The cytotoxicity of 2’,2’-difluoro-2’-deoxycytidine (dFdC, gemcitabine) depends amongst others on the phosphorylation into dFdCTP and on the incorporation of dFdCTP into DNA. dCTP may inhibit the phosphorylation of dFdC by feedback inhibition of deoxycytidine kinase and may decrease the incorporation of dFdCTP into DNA by competition for DNA polymerase. Cyclopentenyl cytosine (CPEC) inhibits CTP synthetase inducing a depletion of CTP and dCTP. We hypothesized that in leukemic cell-lines the cytotoxicity of dFdC could be enhanced by a preincubation with CPEC. Materials and methods MOLT-3 and HL-60 cells were preincubated for 24h with 6.25 and 12.5 nM of CPEC (kindly provided by the NCI, U.S.A.). Subsequently, 0-50 nM [3H]dFdC was added for 4h and [3H]dFdC metabolites were quantified by anionic exchange HPLC and in DNA. Apoptosis and necrosis were assessed by flowcytometry with FITC- labeled-Annexin V and propidium iodide. Results In MOLT-3 cells no alterations were observed in concentrations of dFdCMP, dFdCDP or dFdCTP after a preincubation with CPEC. However, the incorporation of dFdCTP into DNA was significantly increased in the CPEC pretreated samples by 99 ± 4% (mean relative increase ± SD) and 57 ± 2% using 3 and 32 nM of dFdC, respectively. The percentage of apoptotic and necrotic cells increased relatively by 17, 34 and 27% after 37.5 nM of CPEC and 3, 6 and 12 nM of dFdC respectively compared to the cumulative effect of both single drugs. In HL-60 increased concentrations of dFdCMP, dFdCDP and dFdCTP of 65, 42 and 43% were observed in the samples (pre)treated with 12.5 nM of CPEC and 3 nM of dFdC as compared with dFdC treated samples. The incorporation of dFdCTP into DNA was significantly increased by 773 ± 38%. The percentage of apoptotic and/or necrotic cells increased relatively by 278 and 67% after a preincubation with 12.5 nM of CPEC using 6 and 12 nM of dFdC. Discussion A preincubation with low concentrations of CPEC increases the incorporation of dFdCTP into DNA in T- lymphocytic or myeloid leukemic cells, which is paralleled by an increase of apoptosis and/or necrosis.

64 O17

Can whole blood substitute washed red blood cells for the analysis of TPMT activity and 6- mercaptopurine metabolites?

H. Iven, A. Kotalczyk, S. Gutsche Medical University of Luebeck, LUEBECK, Germany

6-Mercaptopurine (6-MP) and its prodrug azathioprine are used to treat numerous diseases including acute lymphoblastic leukaemia (ALL) and chronic inflammatory bowel disease (CIBD). 6-MP undergoes three competing metabolic pathways of which one is linked to the enzyme Thiopurine S-methyltransferase ( TPMT ) for which a genetic polymorphism was demonstrated. Numerous groups have tried to link clinical response and/or toxicity to the concentrations of 6-thioguanine-nucleotides (6-TG), 6-methylmercaptopurine- nucleotides (6-MMP) and inversely to TPMT activity determined in peripheral washed red blood cells (RBC). Recently Pike et al. (J Chromatog B 757 (2001), 1 – 9) showed that whole blood could substitute RBC to monitor 6-TG and 6-MMP concentrations. Since this conclusion is based on results obtained from 8 blood samples from patients with CIBD, we reevaluated this proposal and extended it to TPMT and 6- methylthioguanine-nucleotides (6-MTG). Methods: From 95 consecutive, unselected EDTA blood samples from children with ALL and patients with CIBD, 0.3 ml were diluted and haemolysed with 0.9 ml dest. water. The rest of the sample was centrifuged, packed RBC washed once with ice-cold saline and then haemolysed with 5 volumes of dest. water. The haemoglobin concentration in the haemolysed samples was determined and they were kept at –80° C until analysis. TPMT activity was measured in duplicate, 6-TG, 6-MMP, and 6-MTG after acid hydrolysis as single determinations by established HPLC methods. Both, RBC and whole blood samples were always analysed in parallel in the same series. Intraindividual differences and the % difference from RBC activity/concentrations were calculated and used for statistics. Results: TPMT activity in RBC ranged between 0.1 and 75.8 ng MTG/gHb/h (mean: 47.6). TPMT activity in whole blood was 9.3 % higher (range –25,2 to +46.5; p<0.0001). One patient with TPMT deficiency could be equally well identified. In RBC, 6-TG ranged between 0 (i.e. TPMT before treatment) and 9533 ng/gHb (i.e. ALL patient on 6-TG treatment). In whole blood 6-TG was 8.3% higher ( range –100 to +107; p=0.0272, n=71). Major differences occurred at low concentrations. 6-MMP in RBC ranged between 0 and 162402 ng/gHb. In whole blood of some patients with CIBD 6-MMP could not be quantified because of an interfering compound. In 58 sample pairs the difference was +1.74 % (range –71.2 to + 46.5; p = 0.5748). 6-MTG in RBC ranged between 0 and 7229 ng/gHb. The mean difference in whole blood was +9.5% (range –54.1 to + 61.9; p=0.0003). Conclusions: Whole blood can substitute RBC for monitoring TPMT activity and the concentrations of 6- TG, 6-MTG, and 6-MMP, giving results approx. 10% higher than in RBC. Department of Pharmacology, *Center for Pediatrics , Medical University of Luebeck, Ratzeburger Allee 160. D-23538 Luebeck, FRG .

These studies were supported by the Deutsche José Carreras Leukaemie Stiftung.

Note: To convert ng/gHb into pmol/8x108 RBC divide by 7 for 6-TG and 6-MMP and 7.59 for 6-MTG (based on a MCH of 30 pg).

65 O18

Potential role of mycophenolate mofetil in the management of neuroblastoma patients

A. Giacomello, E. Messina, L. Barile, F. Lupi University of Rome 'La Sapienza', ROME, Italy

In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA), at concentrations (10-7 - 10-6M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept), induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated pathway (up-regulation of p53, p21 and bax and down-regulation of bcl-2 and survivin). MPA-induced apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy, induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide depletion. MPA-induced differentiation is not associated to p27 accumulation as occurs using retinoic acid. At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the former (about 10 µM) leading to competition on HPRT-mediated salvage of guanine. At hypoxanthine or oxypurinol concentrations higher than 1 µM (up to 100µM) no further enhancement of MPA effects was obtained, in agreement with the recently described safety of the allopurinol-mycophenolate mofetil combination in the treatment of hyperuricemia of kidney transplant recipients. The effect of the UDP- glucuronosyltransferases (UGT, having a major role in MPA inactivation) inhibitors curcumin and niflumic acid (NA) on MPA-induced apoptosis was studied in the neuroblastoma cell lines. A slight increase of cytotoxic activity was obtained with 10 mM NA. At concentrations higher than 1 µM curcumin was cytotoxic even when employed in the absence of MPA leading to cell death by apoptosis (almost all cells died after 24 h of incubation in the presence of 30 µM curcumin). In conclusion : 1. Cellcept, at the doses employed in immunosuppressive regimen, appears to have a potential role in the management of neuroblastoma. 2. The apoptotic and differentiating effects of his active metabolite MPA cannot be increased in patients by combination with allopurinol. 3. The natural compound curcumin is a strong apoptosis- inducing agent in neuroblastoma cells and could improve therapy and assist in the control of this common and aggressive childhood solid tumor.

66 O19

Adenosine transport in HPRT deficient lymphocytes from Lesch-Nyhan patients

R.J. Torres, I. De Antonio, C. Prior, J.G. Puig La Paz Hospital, MADRID, Spain

In previous studies, we have found that elevated concentrations of hypoxanthine reduce adenosine transport in human lymphocytes in culture (PBL-C). The aim of the present study is to analyse adenosine transport in HPRT deficient lymphocytes (PBL-LN) obtained from Lesch-Nyhan patients. Methods: [2-3H] adenosine transport, and [3H] NBTI binding assays were determined in basal conditions and after 24h incubation with 25 uM hypoxanthine. Results: 1. Adenosine transport displays a sigmoid curve in PBLLN versus the hyperbolic normal curve. Adenosine transport was significantly decreased in PBLLN with respect to PBLC at adenosine concentrations of 5 and 7 uM. 2. NBTI binding sites were significantly decreased in PBLLN versus PBLC at 10 and 15uM NBTI concentrations. The binding of [3H] NBTI quantified a number of 6,782 +- 395 high affinity sites per cell (Bmax) in PBLLN, versus 9,500 +- 9 in PBLC; p< 0.05. A dissociation constant (Kd) of 3,7+- 0,2 nM in PBLLN was observed versus a Kd of 1,25+- 0,11 nM in PBLC; p<0.05. 3. After 24 h incubation with 25 uM hypoxanthine, adenosine transport is decreased in PBLLN with respect to basal transport, and the sigmoidal kinetics changes to Michaelis Menten saturation kinetics. However, NBTI binding in PBLLN was not decreased with hypoxanthine addition. Conclusions: This study shows that adenosine transport is markedly abnormal in HPRT deficient lymphocytes, and that hypoxanthine concentration influences adenosine transport in these cells. Further studies are necessary to fully characterize adenosine transport in HPRT deficient cells.

67 O20

Urate Oxidation in CSF and Blood of Patients With Inflammatory CNS Disorders

B.F. Becker1, S. Kastenbauer2, U. Koedel2, D. Kiesl2, H.-W. Pfister2 1University of Munich, MUNICH, Germany 2LMU, MUNICH, Germany

Several disorders of the central nervous system (CNS), including bacterial meningitis, Guillain-Barré Syndrome (GBS) and multiple sclerosis (MS), are linked to inflammatory processes. Inflammation should give rise to enhanced oxidative stress, changing the levels of antioxidants and characteristic degradation products in the cerebro-spinal fluid (CSF). Urate is considered to be an important neuroprotective antioxidant in man, a specific product of oxidation being allantoin. To evaluate cerebral oxidative stress, the levels of urate and allantoin were compared quantitatively by HPLC in CSF and serum of controls (39), and patients suffering from meningitis (15), GBS (18) or MS (18). The latter has been associated with low levels of urate. Both meningitis and GBS are accompanied by disturbances of the blood-brain barrier (BBB). The distribution of urate between serum and CSF was distinctive: low CSF values in controls and MS (about 20µM, i.e., 10-15% of the plasma levels), moderately elevated levels in GBS (about 40µM), and very high levels, almost at equillibrium with serum, in meningitis. However, 5/15 patients with meningitis had CSF urate levels exceeding those of serum. Thus, at least some of the CSF urate had to be of central origin. This is supported by the absence of any correlation between CSF urate and serum urate in all groups, which, in meningitis and GBS, also demonstrates that disruption of the BBB alone is insufficient to cause increase of CSF urate. Neither the serum nor CSF urate level of MS patients was lower than in controls. Significantly higher levels of allantoin were found in serum and CSF of patients with meningitis (53 and 41µM, resp.) than in the three other groups (serum 20-30µM, CSF 5-10µM). The distribution ratio of allantoin between CSF and serum increased significantly from 0.35 (controls) to 0.61 in meningitis, suggesting greater intracerebral enhancement of oxidative processes. In conclusion, the brain relies relatively little on uric acid to protect itself from oxidative stress under normal conditions. In the special case of meningitis, and perhaps also GBS, additional uric acid is recruited to subserve as antioxidant. We were unable to substantiate the occurrence of low urate levels in MS.

68 O21

An unusual pyridine nucleotide accumulating in erythrocytes: its identity, and positive correlation with degree of renal failure

E.A. Carrey1, R.T. Smolenski2, S.M. Edbury1, A.D.J. Laurence3, A.M. Marinaki1, J.A. Duley1, L. Zhu4, D.J.A. Goldsmith1, H.A. Simmonds1 1Guy's Hospital, LONDON, United Kingdom 2University of Gdansk, GDANSK, Poland 3University College Hospital London, LONDON, United Kingdom 4London Metropolitan University, LONDON, United Kingdom

We report the identification of an unusual nucleotide that accumulates, with precursors, in the erythrocytes of patients in uraemia. The nucleotide was identified, using liquid chromatography and mass spectrometry, as 2-pyridone-5-carboxamide ribonucleoside triphosphate (2PyTP). It is related chemically to the NAD breakdown product, N1-methyl-2-pyridone-5-carboxamide (M2Py), found in high concentrations in the plasma of uraemic patients. Concentrations of 2PyTP and M2Py were measured in four categories of patients: mild renal failure (CRF), end-stage renal failure (ESRF), haemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD); also in healthy controls, and patients after successful kidney transplantation (Post-Tx). In the patient groups, mean 2PyTP concentrations are lowest in CRF (21.8 µmol/l), increasing with degree of renal failure through ESRF (55.1 µmol/l) and HD patients (70.9 µmol/l), being highest in CAPD patients (216.7 µmol/l). The greatly elevated concentrations of 2PyTP in CAPD patients reflect accumulation during a longer half-life for erythrocytes than in HD patients. Since plasma concentrations of M2Py correlate with 2PyTP in the erythrocytes in all renal failure groups, and concentrations of both fall to the control range following successful transplantation, we infer that either M2Py or 2PyTP may be a toxin implicated in the diverse symptoms of uraemia.

69 O23

Metabolism of thymidine in mitochondria: role of dNT2, the mitochondrial deoxyribonucleotidase

V.B. Bianchi, P. Ferraro, L. Gallinaro, G. Pontarin, C. Rampazzo, K. Crovatto, P. Reichard University of Padova, PADOVA, Italy

Maintenance of mitochondrial (mt) DNA requires an appropriate supply of thymidine triphosphate (dTTP). Which is the origin of mitochondrial dTTP and how is its concentration regulated to support accurate mtDNA synthesis? Mitochondria contain a constitutive thymidine kinase, TK2, that phosphorylates thymidine imported from the cytoplasm also in terminally differentiated cells. The importance of TK2 is demonstrated by the mtDNA depletion phenotype associated with its inactivation by mutation. Depletion and multiple deletions of mtDNA arise when TK2 is presented with excess thymidine substrate as in the case of MNGIE, a mitochondrial pathology caused by mutation of cytosolic thymidine phosphorylase. Normally the phosphorylation of thymidine by TK2 is counterbalanced by the dephosphorylation of dTMP by dNT2, the mitochondrial deoxyribonucleotidase discovered in our laboratory. Its 3D structure, recently solved, accounts for its specificity for dUMP and dTMP. dNT2 together with TK2 gives rise to a substrate cycle that mirrors the cytosolic TK1/dNT1 cycle regulating thymidine phosphates in the cytosol. To experimentally assess the role of dNT2 in the control of mitochondrial dTTP, we have constructed inducible cell lines that overproduce dNT2 in graded amounts. Some of them lack cytosolic TK1 and phosphorylate thymidine exclusively by mitochondrial TK2. In this system we measure the specific activity of the cytosolic and mitochondrial dTTP pools by incubating whole cells with tritiated thymidine. Alternatively, we run similar experiments with isolated mitochondria. The results obtained so far clearly show that the dTTP produced by mitochondrial phosphorylation of thymidine reaches a steady-state within mitochondria, is incorporated into mtDNA and is continually exported to the cytosol from which it feeds nuclear DNA synthesis. In pulse-chase experiments the labeled mitochondrial dTTP pool is replaced by unlabeled dTTP. In cells overproducing dNT2 incorporation of radioactive thymidine is decreased resulting in lower specific activity of the dTTP pool. At high level of dNT2 overproduction the size of this pool is also decreased. We are currently working at the downregulation of dNT2 by RNA interference with the aim of studying the effects of reduced dNT2 activity on thymidine phosphorylation by TK2. (Supported by the European Commission, Telethon and AIRC)

70 O24

Severe impairment of nucleotide synthesis through inhibition of mitochondrial respiration

N. Gattermann1, G. Hofhaus2, M. Dadak1, M. Wulfert1, M. Berneburg1, M.L. Loeffler3, H.A. Simmonds4 1Heinrich-Heine University, DüSSELDORF, Germany 2Institute of Biochemistry, DüSSELDORF, Germany 3Philipps-University Marburg, MARBURG, Germany 4Guy's Hospital, LONDON, United Kingdom

Leflunomide and Brequinar inhibit de novo pyrimidine synthesis at the level of dihydroorotic-acid dehydrogenase (DHODH) which is located in the inner mitochondrial membrane, and is coupled to the mitochondrial respiratory chain (RC). DHODH uses ubiquinone as the proximal and cytochrome c oxidase as the ultimate electron transfer system. Therefore, RC dysfunction should impair pyrimidine biosynthesis. This is supported by the fact that rho-0-cells, lacking mitochondrial DNA and thus a functioning respiratory chain, are pyrimidine auxotrophs requiring uridine for growth. Our interest in the consequences of RC dysfunction stems from the finding of acquired clonal mitochondrial DNA mutations in bone marrow of patients with myelodysplastic syndromes (MDS, preleukemias). Thus mitochondrial defects, with concomitant impairment of nucleotide biosynthesis, may shed some light on a hitherto cryptic pathophysiology, which includes megaloblastic changes. To investigate this, we used specific RC inhibitors, Antimycin A and Rotenone, to treat primary human keratinocytes and fibroblasts in culture. This resulted in severe impairment of nucleotide synthesis. Interestingly, the effects were not restricted to pyrimidine synthesis, but concerned purine synthesis, too. The effect of Rotenone did not differ significantly from that of Antimycin A. This was surprising since Rotenone inhibits complex I, which is upstream of ubiquinone where DHODH interacts with the respiratory chain. In order to avoid any unspecific effects of Antimycin A or Rotenone, we examined the consequences of two mitochondrial DNA point mutations, each causing a specific defect of complex I. These mutations were introduced into rho-0 cells and compared with wildtype mtDNA. Our results indicate that drastic inhibition of mitochondrial RC activity severely impairs biosynthesis of purine as well as pyrimidine nucleotides. The lack of specificity for de novo pyrimidine synthesis indicates that diminished ATP regeneration may play a major role. We are currently investigating whether more subtle RC defects in MDS lead to an unbalanced production of pyrimidine and purine nucleotides.

References: N Gattermann (2000) From sideroblastic anemia to the role of mitochondrial DNA mutations in myelodysplastic syndromes. Leukemia Res. 24:141-151. M Löffler et al. (1997) Dihydroorotate-ubiquinone oxidoreductase links mitochondria in the biosynthesis of pyrimidine nucleotides. Mol Cell Biochem 174: 125-129.

71 O25

New class of suicide genes for cancer gene therapy

Z. Gojkovic1, M.P.B. Sandrini1, M. Willer2, P. Kristoffersen2 1Technical University of Denmark, LYNGBY, Denmark 2ZGene A/S, LYNGBY, Denmark

Suicide genes code for enzymes that convert nontoxic prodrugs into toxic products. Among more than 30 different suicide genes described, so far the most used is Herpes simplex virus thymidine kinase (HSV-TK) in combination with the nucleoside analog gancyclovir. Nucleoside analogs require activation by phosphorylation. This activation, catalyzed by deoxyribonucleoside kinase, is the rate limiting process and severely inhibits the potential of many drugs in cancer chemotherapy.

We have isolated and tested a variety of deoxyribonucleoside kinases with the aim to develop a wide range of potent suicide systems. This approach, called ZAS (ZGene Activation System) can benefit potential patients either by significantly increasing the therapeutic effect and shortening the course of the therapy (because of faster activation) or eliminating side effects and toxicity (because of the lower drug dose which can be used). The corresponding genes from different organisms were subcloned and tested for phosphorylation activity on different, FDA approved, anti-cancer and anti-viral nucleoside analogs. ZAS suicide genes exhibited much higher activation rates than HSV-TK and were able to phosphorylate a large number of analogs. ZAS was tested in several cancer cell lines and showed an increased activation of Cladribine, AZT, Gemcitabine, Fludarabine, Cytarabine and Vidarabine. This synergy between the ZAS gene therapy and chemotherapy opens novel possibilities for enhanced therapy.

The major obstacle of currently used suicide cancer therapies is that prodrug has to be forced to different cancers and cell types. Prodrugs exhibit great differences in affinity, biodistrubution or stability depending on cell types. Therefore, our approach to develop activator genes and adapt them to drugs, which are already effective against certain cancer types, is fundamentally different from the existing suicide methods. Because of the fairly small size (550-1100 bp), the ZAS genes are applicable for a wide range of delivery systems. The size of the genes and multidrug activation makes it possible to use cocktail treatment regimes and even further improve the therapy. All this makes ZAS genes both powerful safety mechanism and suicide system for cancer gene therapy.

72 O26

Production and Properties of Polyethylene-Glycol Conjugated Adenosine Phosphorylase: Administration to PNP deficient mice

F.F. Snyder, L.J. Brewerton, E. Fung University of Calgary, CALGARY, ALBERTA, Canada

Purine nucleoside phosphorylase was engineered to accept 6-amino substituted purine nucleosides by two active site substitution, Asn243Asp;Lys244Gln. The enzyme preferentially accepts adenosine nucleosides and is referred to as adenosine phosphorylase (AP). Recombinant AP has been conjugated to branched polyethylene glycol polymers of approximately 42.5 kDa. SDS PAGE and matrix assisted laser desorption/ionization characterization of the product indicated the conjugation of as many as 4 PEG molecules per AP subunit. The PEG conjugated enzyme retained greater than 90% of the native catalytic activity. Purine nucleoside phosphorylase deficient B6-NPG, mice were injected intraperitoneally with a single bolus of native and PEG-AP enzyme. Peak plasma activities were obtained at 4 and 8 hours for the native and PEG- conjugated enzymes and were 10-fold greater for PEG-AP as opposed to native AP injected mice. The native AP activity in plasma declined quickly and was essentially negligible by 10 hours having a half-life of 68 minutes. The PEG-AP enzyme persisted in plasma for greater than 5 days having a half-life of 65 hours. Tissues from PEG-AP treated mice showed only minimal levels of activity and there was no evidence of loss of AP activity in urine samples. Although engineered AP has a preference for adenosine, substantial activity has been retained with inosine. As hypouricemia is a recognized feature of PNP deficiency in man, the consequence of PEG-AP administration on urate levels of PNP deficient mice was examined. Urine urate was increased 2.7-fold for voids between 22 and 58 hours and 1.8-fold between 94 and 154 hours for PEG-AP treated mice providing evidence for in vivo function of the administered enzyme. The ability of AP to protect human CEM lymphoblasts in culture from the toxic effects of deoxyadenosine, 0.010 mM, plus the adenosine deaminase inhibitor, EHNA, was demonstrated by the addition of AP to the culture media. These studies provide evidence for consideration of PEG-AP as an alternative enzyme therapy for the inherited deficiency of adenosine deaminase. Supported by the Canadian Institutes of Health Research.

73 O27

Nucleoside transporters in proliferation in normal and transformed cells

F.J. Casado, R. Valdés, S. Duflot, M. Molina, S. Fernández-Veledo, M. Pastor-Anglada University of Barcelona, BARCELONA, Spain

The concentrative nucleoside transport systems of the CNT family are tightly regulated by the differentiation and the proliferation status of the cell. We showed that in two different experimental hepatocarcinoma models, the expression of rCNT1 and rCNT2 are lost in the early phases of tumor progression. The activity of these transporters is related to cell cycle progression, apparently as a consequence of changes in nucleotide metabolism, since ribonucleotide reductase inhibition leads to rCNT1 induction. The relationship between proliferation, differentiation and nucleoside transporter expression has been recently elucidated using primary cultures of murine bone marrow macrophages. MCSF-induced proliferation only up-regulated the equilibrative nucleoside transporter ENT1, while activation mediated by IFN-gamma decreased ENT1 expression and induced CNT1 and CNT2 by a STAT-1 independent mechanism. All these observations may be relevant to the pharmacological properties of these transporters, since most drugs currently used in antitumoral and antiviral therapy are structurally related to nucleosides. Evidence suggests that transporter mRNA levels may not reflect protein expression. Indeed, cells from chronic lymphocytic leukemia patients showed no clear relationship between NT mRNA amounts and the ex-vivo fludarabine citotoxicity, whereas a significant correlation was found between equilibrative transport activity and fludarabine cytotoxic action. Although for some NT proteins, as we recently reported for hCNT1, intracelullar location is also found, the analysis of nucleoside transporter patterns in human biopsies may require immunohistochemistry. With this aim antibodies suitable for immunohistochemistry analysis on paraffin embedded tissues were raised and characterized. Recently a tissue array of gynaecological tumors was screened for hCNT1, hENT1 and hENT2 expression. According to what the cell biology of these transporters could anticipate, almost all tumor samples retained a certain expression of equilibrative transporters but a much higher variability in hCNT1 expression was found.

74 O29

Estrogen Attenuates P2X7-R - Mediated Apoptosis of Uterine Cervical Cells by Blocking Calcium Influx

I. Gorodeski CWRU School of Medicine, CLEVELAND, OH, United States of America

We have previously shown that estrogen blocks apoptosis of cultured human ectocervical cells (hECE), but the mechanism of estrogen action is unknown. Activation of P2X7-R in hECE cells stimulates Cai increase and augments apoptosis. In this study we determined the degree of which estrogen can modulate P2X7-R – related apoptosis. The first experiment studied the effect of P2X7-R – induced Cai-increase on apoptosis. The experiment utilized cultured hECE cells on filters. Cells were bathed either in normal Cao (1.2 mM) or low Cao (<0.1 mM), and treated with 100 mM BzATP (P2X7-R – agonist) for up to 9 hrs. Changes in Cai and influx of ethidium bromide (EB) were determined fluoroscopically in attached cells; apoptosis was determined as DNA fragmentation in [3H]thymidine-labeled cells, and was calculated as % solubilized DNA. In cells bathed in normal Cao BzATP induced time-dependent increase in Cai and EB influx, and augmented apoptosis. Incubation in low Cao blocked BzATP-induced Cai-increase and apoptosis, but not EB influx (i.e. P2X7 pore formation), suggesting BzATP-induced apoptosis is mediated by augmented calcium influx. The second experiment studied the degree of which estrogen can regulate calcium influx via activated P2X7-R – pores. Experiments utilized steroid-deprived hECE cells treated with either the vehicle or with 10 nM 17b-estradiol. Fura-2 – loaded cells attached on filters were shifted to low Cao and treated for 30 min with 100 mM BzATP. Addition of CaCl2 (to increase Cao to 1.2 mM) resulted in a rapid increase in Cai, and the effect was smaller in estrogen-treated cells than in estrogen-deprived cells. In parallel experiments pre-treatment with estrogen attenuated BzATP-augmented apoptosis, suggesting that the apoptosis-sparing effect of estrogen is mediated in part by blocking Cai increase. Since ATP is present in the extracellular medium at concentrations that could activate P2X7-R – pores, the present results suggest a physiological role for estrogen in the cervix as an anti- apoptotic factor. Support: NIH AG15955.

75 O30

Mitochondrial function dependent proliferation assay for the diagnosis of mitochondrial disorders in human fibroblasts

F.F. Snyder, S.D. Hodges University of Calgary, CALGARY, ALBERTA, Canada

Mitochondrial disorders are difficult to diagnose due to confounding factors including maternal transmission, heteroplasmy and the contribution of nuclear and mitochondrial gene products to organelle function. A fibroblast proliferation assay has been developed which compares cellular proliferation in a mitochondrial function sparing media versus a media, which requires mitochondrial function. Fibroblasts are harvested and plated at equal density in replicate flasks in Dulbecco's minimum essential media with 10% serum for comparison. The mitochondrial function sparing media contains glucose, pyruvate, glutamine and uridine. Uridine relieves the pyrimidine de novo synthetic requirement, which encompass the mitochondrial enzyme step catalyzed by dihydroorotate dehydrogenase. The mitochondrial function dependent media lacks glucose and uridine. Glutamine is provided as an energy source, which is dependent upon oxidative phosphorylation. Inosine is provided as a required pentose phosphate source precursor. Cells are photographed daily during a 5 to 8 day proliferation phase. Cells with normal mitochondrial function show essentially normal rates of proliferation in both media. A number of cell lines with known mitochondrial defects have been shown to proliferate to confluence in the mitochondrial function sparing media whereas they exhibit severely restricted proliferation and/or cell death in the mitochondrial function dependent media. These include cell lines with the following defects: complex I deficiency, complex IV deficiency, cytochrome C oxidase deficiency, NARP 8993 (T-G), and Surf. Putative evidence for fibroblast populations exhibiting heteroplasmy have also been observed where the major fraction of the population is unable to proliferate while a subset shows outgrowth indicative of a clonal population. We propose the mitochondrial dependent proliferation assay to be a useful tool in screening fibroblast lines from patients for whom a mitochondrial disorder is being considered as part of the differential diagnosis. Supported by the Canadian Institutes of Health Research and Alberta Children's Hospital Foundation

76 O31

Functional studies reveal that cytosolic "High Km" 5'-Nucleotidase (cN-II) active site has a structure similar to that of HAD superfamily

S. Allegrini1, M.G. Careddu1, G. Cuccu2, R. Pesi2, A. Scaloni3, M.G. Tozzi2 1University of Sassari, SASSARI, Italy 2University of Pisa, PISA, Italy 3IABBAM - CNR, NAPELS, Italy cN-II is an ubiquitous cytosolic mononucleotide phosphatase, acting also as phosphotransferase. Even though its physiological role is not yet well understood, it is generally believed that the enzyme participates, along with other six 5’-nucleotidases, one membrane bound, one mitochondrial and four cytosolic, in the nucleotide pools regulation. cN-II activity seems to play its major role regulating the intracellular IMP concentration. cN-II is also involved in the metabolism of purine prodrugs, furthermore a cN-II hyperactivity has been related to severe paediatric neurological syndromes, including Lesch-Nyhan syndrome (1). cN-II acts through the formation of a phosphorylated enzyme intermediate. The phosphorylation occurs on Asp52 (2) that is part of a conserved motif – DXDX(T/V) - common to a big family of phosphotransferases named HAD superfamily. This motif is present in at least four among the other 5’-nucleotidases. Recently the structure of mitochondrial deoxyribonucleotidase (dNT-2) has been published (3). From a comparison with the structure of phosphoserine phosphatase, another member of HAD superfamily, the authors could show the high homology between their active site (composed of three separate motives). Computer alignments of cN-II with other members of HAD superfamily, included the four cytosolic 5’-nucleotidases and dNT-2, allowed us to identify a number of amino acids which could constitute cN-II active site. Point mutants of these amino acids, T249, S251, K292, D351, D356 were prepared. For each of them, both a conservative and a non-conservative mutation were obtained trying, in this second case, to keep the physical dimension of the mutated amino acid as similar as possible to that present in the wild type protein. Modification of the kinetic parameters of cN-II caused by the substitutions are in good agreement with those described for other enzymes of this superfamily (4), suggesting a similar role in the catalytic activity. Other mutants were prepared for M53 and T56 ( motif I ) and the modification of their kinetic properties were determined. 1.Pesi et al. (2000) NeuroReport 11, 1827-1831 2.Allegrini et al. (2001) JBC 276 (36), 33526-33532 3.Matthis et al. (2002) Nat. Stuct. Biol. 9 (10), 779-787 4.Collet et al. (1999) JBC 274 (48), 33985-33990

77 O32

The association of ecto-5'-nucleotidase (eN), integrin b1, EGFR and vimentin with invasive breast carcinoma: The role of eN in invasive phenotype

J. Spychala1, A. Ostapkowicz1, N. Hironobu2, N. Sakon2, F. Masaru2, K. Inai2 1University of North Carolina, CHAPEL HILL, NC, United States of America 2Fukui Medical University, FUKUI, Japan

Breast carcinoma is a heterogeneous disease and expression profiling has identified several subtypes that may be useful in determining more effective individualized treatment regimens in the clinic. Previously we have found ecto-5'-nucleotidase overexpression in ER negative breast cancer. In order to associate the expression of eN with specific cell phenotype, we have performed expression profiling at the level of protein in several breast cancer cell lines. Our focused analysis of membrane and cytoskeletal proteins revealed that eN is specifically co-expressed with a number of membrane proteins, such as EGFR, CD44, N-cadherin, OB-cadherin, caveolin, integrin b1 and integrin a5. Among cytoskeletal proteins eN co-expresses with vimentin, merlin, fascin, moesin and spectrin. Also several signaling molecules, such as tyrosine kinase Lyn, trimeric Gai and PKC tightly co- express with eN. This expression profile is characteristic for normal fibroblasts and was also found in more aggressive and tumorigenic breast cancer cell lines that have undergone Epithelial to Mesenchymal Transdifferentiation (EMT). Since eN and several indentified membrane proteins, that either co-express or have exclusive expression pattern, are components of lipid rafts, these results suggests that there is a significant remodeling of this membrane microdomain in EMT. The co-expression of eN with vimentin also correlated with the responsiveness to ConcanavalinA in more aggressive breast cancer cell lines suggesting functional association. Preliminary survey of clinical samples from breast cancer patients show coexpression of eN with Integrin b1 and vimentin at the invasive edge of the tumor. Thus, these data suggest that elevated expression of eN and increased potential to generate extracellular adenosine may have specific functions related to cell migration and strengthen the significance of this protein as a novel marker for invasive and metastatic breast carcinoma.

78 O33

Rational design of purine and pyrimidine drugs against protozoan infections through selective uptake

H.P. De Koning, L.J.M. Wallace, M.I. Al-Salabi, D. Candlish, J.S. Burchmore, S.A. Baldwin University of Glasgow, GLASGOW, United Kingdom

Purine and pyrimidine analogues have been hugely successful as antiviral and antineoplastic agents, but no coherent strategy has been developed for exploiting this class of compounds for the treatment of parasitic disease. A major challenge in any such effort is the potential for toxic side-effects. By comparing the structural basis for substrate recognition of nucleoside and nucleobase transporters in protozoan and mammalian cells, analogues can be selected which will be efficiently accumulated by the parasite but not by the host cells. We have developed a technique for the construction of quantitative models of substrate-transporter interactions. Using this approach, models were constructed of the substrate binding by purine and pyrimidine transporters, including Trypanosoma brucei hypoxanthine transporters H2 and H4, and nucleoside transporters P1 and P2, Leishmania major nucleobase transporter NBT1 (responsible for allopurinol uptake), Toxoplasma gondii nucleoside transporter TgAT2, and the common human facilitative nucleobase transporter hFNT1 (characterised in erythrocytes). We have shown that these models have predictive value for the selective uptake of purine analogues. In addition, these models also provide important insights in the structural basis for substrate recognition by solute transporters and highlight putative structural similarities between transporters of related species. It was shown that hFNT1 binds the purine ring in a very different way from the protozoan nucleobase transporters, but hat, in particular, T. brucei H2 and L. major NBT1 form almost the same interactions with purines, and of similar strength. Likewise, the T. brucei P1 purine nucleoside transporter bound nucleosides in a very similar way as T. gondii AT2, which displays an equally high affinity for most pyrimidine nucleosides as for purine nucleosides. We are currently proceeding to identify the amino acids responsible for particular bonds, using functional mutants, site-directed mutagenesis and modelling of protein folding. The genes encoding the T. b. brucei nucleoside transporter have been described and we recently cloned the first protozoan nucleobase transporter gene, TbNBT1, which encodes the H4 transporter. This gene was expressed in yeast and Xenopus oocytes, and encodes a high affinity purine nucleobase transporter of the ENT family, with low affinity for inosine and adenosine.

79 O34

Improving NDP Kinase for Antiviral Nucleotide Analogs Activation

D. Deville-Bonne1, S. Gallois-Montbrun1, B. Schneider1, V. Giacomoni-Fernandes1, M. Véron1, Y. Chen2, S. Morera2, J. Janin2 1Pasteur Institute, PARIS, France 2LEBS, GIF-SUR-YVETTE, France

Nucleoside analogs (ddC, AZT, d4T) used in anti-AIDS therapies are devoid of the 3'OH in the ribose moiety preventing viral DNA elongation. Nucleoside analogs are phosphorylated into triphospho-derivatives by cellular kinases of the salvage pathway with Nucleoside Diphosphate (NDP) kinase as the last enzyme. The phosphorylation efficiency of antiviral drugs by human NDP kinase is drastically reduced. Our previous work demonstrated a crucial role for the 3’OH of natural nucleotides involved in a H-bond network with the substrate phosphate and two active site residues. In order to improve the processing of nucleoside prodrugs, we have engineered a NDP kinase variant with an enhanced activity for nucleotide analogs. An hydroxyl group was introduced in the enzyme, substituting the missing 3'OH of the analogs. The X-ray structure of the N115S mutant complexed with a triphosphate derivative of AZT indicates that the increased activity reflects an improved geometry of binding. The N115S and L55H mutations were combined in NDPK. The catalytic efficiency of the L55H-N115S double mutant was more than 80 fold improved for the triphospho form of AZT, d4T, ddT and acyclovir derivatives, whilst it was 5 times decreased for natural nucleotides as compared to the wild type enzyme. Such a mutant could be used as a suicide enzyme in cell therapies to improve the concentration of activated nucleoside analogs.

80 O35

Regulation of dinucleoside polyphosphates by the YgdP and ApaH diadenosine polyphosphate hydrolases is essential for intracellular invasion by Salmonella enterica serovar Typhimurium

A.G. McLennan, T.M. Ismail, C.A. Hart University of Liverpool, LIVERPOOL, United Kingdom

Salmonella enterica is a common and potentially dangerous pathogen, causing a variety of illnesses from food poisoning to septicaemia and typhoid fever. It has the ability to avoid host defence systems by invading and multiplying within host cells, such as the intestinal epithelium. The spread of antibiotic resistance requires the development of new anti-virulence drugs. The ygdP and apaH genes of Salmonella enterica serovar Typhimurium encode asymmetrically- and symmetrically-cleaving dinucleoside polyphosphate (NpnN) hydrolases respectively. For example, YgdP, a Nudix hydrolase, cleaves diadenosine tetraphosphate (Ap4A) producing AMP and ATP, with Km and kcat values of 18 µM and 18 s-1,while the unrelated ApaH cleaves Ap4A producing 2ADP, with Km and kcat values of 37 µM and 37 s-1. Disruption of ygdP reduced intracellular invasion of human Hep-2 epithelial cells by S. Typhimurium by 9-fold, while disruption of apaH reduced invasion 250-fold and resulted in filamentous growth. Disruption of both genes led to a 3000-fold reduction in invasion. Adhesion of the mutants to both HEp-2 and U-937 macrophage-like cells was greatly reduced compared to the wild type. Invasive capacity of both single mutants was restored by transcomplementation by a plasmid expressing the ygdP gene, suggesting that loss of invasion was due to increased intracellular NpnN. The wild type level of 3 µM adenylated NpnN (ApnN) was increased 1.5-, 3.5- and 10-fold in the ygdP, apaH and double mutants respectively. Expression of the putative ptsP virulence gene immediately downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences between the mutants and wild type. It is proposed that the increase in intracellular NpnN in the mutants leads to changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian cells. These enzymes may represent new targets for anti-virulence drugs. Preliminary data with effective synthetic Ap4A analogues will be presented.

81 O36

The Effect of N-Methyl-2-Pyridone-5-Carboxamide - A Nicotinamide Catabolite on Poly ADP- Rybosylation and Oxidative Stress Injury in Endothelial Cells

E.M. Slominska1, R.T. Smolenski2, F. Osborne2, H. Dziewit1, J. Swierczynski1, M.H. Yacoub2 1Medical University of Gdansk, GDANSK, Poland 2Imperial College, LONDON, United Kingdom

The poly(ADP-ribosyl)-ation is essential mechanism of DNA repair but if excessively stimulated may impair cellular viability by depletion of NAD pool and induction of apoptosis. Therefore some studies demonstrated deleterious effects of inhibitiors of poly(ADP-ribose) polymerase (PARP) while the other have shown that it may be protective during clinical conditions associated with ischemia reperfusion injury. We studied whether 2PY (N-methyl-2-pyridone-5-carboxamide) which is a product of nicotinamide metabolism excessively accumulating in chronic renal failure (CRF) could inhibit PARP. Subsequently, we have analysed the effect of 2PY on oxidative stress injury in endothelial cells. Effect of 2PY and nicotinamide (NA) on poly(ADP- ribosyl)-ation have been evaluated with isolated PARP and endothelial cell lysates. Cellular effects were studied with cultured human endothelial line (PIEC) exposed to 100 uM peroxynitrite for 60 min followed by measurement of cellular ATP, NAD and lactate dehydrogenase release. 2PY or NA was added at 0.3, 1 and 3 mM concentration while 3 mM imidazole was given to controls. Half-maximal concentrations for the enzyme inhibitory effect of 2PY was 0.53 mM and at 0.75 mM with the isolated PARP and endothelial cell homogenate, respectively. Cultured endothelial cells ATP decreased from 16.1±2.1 to 2.3±0.6 nmol/mg cellular protein after peroxynitrite exposure in controls. Addition of 2PY or NA protected from peroxynitrite induced ATP decrease already at 0.3 mM concentration. Maximal effect was observed at 3 mM 2PY or NA, with ATP maintained at 10.2±1.7 and 13.1±1.5 in cells treated with 2PY and nicotinamide respectively (p<0.001 vs. control). 2PY and NA attenuated peroxynitrite induced endothelial lactate dehydrogenase release and NAD pool decrease. Conclusion: 2PY inhibits poly(ADP-ribose) polymerase in sub-milimolar concentrations. Endogenous concentration in healthy subjects (about 1 µM) may be too low to exert any effects but inhibition of PARP is likely in CRF where 2PY concentration is elevated up to 20 times. 2PY effectively prevented oxidative stress injury in endothelial cells pointing out to important beneficial effect of transient elevation of 2PY concentration. However, analysis whether prolonged elevation of 2PY concentration which occurs in CRF is beneficial or deleterious require further studies.

82 O37

Selective increase of dATP pools upon activation of deoxycytidine kinase in lymphocytes: implications in apoptosis

M. Sasvári-Székely, G. Keszler, T. Spasokoukotskaja, Z.C. Csapó, S. Virga, M. Staub Semmelweis University, BUDAPEST, Hungary

Stimulation of the activity of deoxycytidine kinase (dCK), the principal nucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction - presumably by supplying dATP or its analogues for the apoptosome deserves consideration. Two-hour exposure of human tonsillar lymphocytes to 2-chloro-deoxyadenosine (CdA), to the DNA polymerase inhibitor aphidicolin as well as to the topoisomerase II inhibitor etoposide led to a 2 - 2.5 fold activation of dCK. When the dNTP pools were determined, both deoxypyrimidine triphosphate pools were reduced after the treatments, the dGTP levels were roughly constant, while dATP levels elevated by 60 %, 100 % and 50 % in the CdA, aphidicolin and etoposide-treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria. As far as the molecular basis of dCK activation is concerned, phosphorylation of the dCK protein by PKC or downstream members of the PI(3)K signalling pathway has previously been suggested. However, neither the PKC activator PMA and the PKC inhibitor bisindolyl-maleimide, nor the PI(3)K inhibitor wortmannin influenced either basal or stimulated dCK activity. Results of native western blotts clearly indicated parallel changes between the enzyme activity and protein levels of dCK. Moreover, the more intensive bands corresponding to CdA- and aphidicolin-treated samples exhibited a bit higher mobility as compared to the less intensive bands of the untreated cell extracts, probably due to the presence of negatively charged group(s) or to other changes of aminoacid residues resulting from post-translational modification during enzyme activation. These results show a conformational change of the dCK protein upon activation that probably renders the C- terminal epitope more accessible to the antibody.

This work was supported by the following grants: Hungarian National Grant ETT 273/2001, OTKA T035203 and EC-BMH-CT98/3079.

83 O38

Multi-level regulation of human AMP deaminase isoform E (AMPD3) by calmodulin

R.L. Sabina, D.K. Mahnke-Zizelman Medical College of Wisconsin, MILWAUKEE, WISCONSIN, United States of America

A recently proposed regulatory mechanism for human AMP deaminase isoform E (AMPD3) involves its reversible association with the cytoplasmic membrane (J Biol Chem 277:42654, 2002). Membrane-bound isoform E is relatively inactive due to potent noncompetitive inhibition by phosphatidylinositol 4,5- bisphosphate (J Biol Chem 274:25701, 1999). Protonation of histidine residues in the AMPD3 polypeptide promotes membrane recruitment of isoform E under conditions of metabolic acidosis. Herein, we describe a Ca2+-dependent protein-protein interaction with calmodulin (CaM) that contributes to this mechanism for isoform E regulation. Ca2+-CaM has several effects on enzyme behavior, including: 1) displacement of membrane-bound enzyme, 2) antagonism of membrane binding by the soluble enzyme, and 3) modest activation of the soluble enzyme through a Km effect (Km [-ATP], mM [n=3]: control, 1.8±0.1; Ca2+-CaM, 1.0±0.2*; CaM only, 1.6±0.3; Ca2+ only, 1.7±0.2. *, p<0.05 when compared to other conditions in two-tailed t- tests). We now further propose that the association between CaM and isoform E plays a counter-regulatory role to the previously described enzyme-lipid interaction. This working model may also be related to the indirect stimulatory effect that calcium has on erythrocyte AMPD (FEBS Lett 244:417, 1989). Currently, we are attempting to identify a CaM-binding domain in isoform E and have preliminary evidence that critical determinants are located within residues 65-89 of the AMPD3 polypeptide.

84 O40

Biophysical Studies of ATP, Deoxycytidine and Gemcitabine Binding to Human Recombinant Deoxycytidine Kinase

R.S. Mani1, E.V. Usova2, S. Eriksson2, C.E. Cass3 1University of Alberta, EDMONTON, ALBERTA, Canada 2Swedish Univ. of Agricultural Sciences, UPPSALA, Sweden 3Cross Cancer Institute, EDMONTON, Canada

Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, activates a number of medically important nucleoside analogs by their 5’-phosphorylation. The enzyme exhibits different affinities for various substrates, implying that it can assume different conformational states. For this reason, in this study, the binding of phosphate donors (ATP and UTP), and the phosphate acceptors (deoxycytidine (dCyd) and gemcitabine (Gem), a drug often used in the treatment of solid tumors) to dCK was studied by spectroscopic methods. Biophysical studies were conducted with purified recombinant human dCK. The Mr determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 ± 1,000, indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with ellipticity values of –11,900 ± 300 and –12,500 ± 300 deg.cm2.dmol-1, respectively, indicating the presence of approximately 40% α-helix and 50% β-structure. Circular Dichroism studies in the aromatic and far- ultraviolet range and UV difference spectroscopy indicated that binding of ATP, UTP, dCyd and Gem to dCK reduced its α-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence demonstrated that dCyd (or Gem) and ATP bound to different sites on dCK and fluorescence quenching revealed bimodal binding of dCyd and Gem and unimodal binding of ATP and UTP. We have also demonstrated the formation of a ternary complex involving dCK, dCyd (or Gem), and ATP (or UTP). Spectroscopic studies revealed changes in the secondary and tertiary structures of dCK upon binding of substrates. The nature and extent of these changes differed with the substrate, with the result that dCK exhibited different affinities for different substrates.

85 O41

Clinical consequences of polymorphisms in the methylenetetrahydrofolate reductase gene

R. De Jonge Erasmus MC, ROTTERDAM, The Netherlands

The enzyme 5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the major circulatory form of folate. The enzyme plays a pivotal role in cellular folate metabolism because it is located at a metabolic branch point directing one-carbon fragments towards homocysteine remethylation at the expense of purine and pyrimidine synthesis. Severe MTHFR deficiency results from rare mutations and patients present in infancy or adolescence with homocystinuria and neurological and vascular complications. Mild MTHFR deficiency results from two common polymorphisms in the MTHFR gene (allele frequencies ∼ 33%): 677 C>T and 1298 A>C. The central role of MTHFR in cellular folate distribution is demonstrated by the observation that reduced MTHFR activity (e.g., MTHFR 677 TT subjects) alters normal intracellular folate distribution: folates committed to purine and pyrimidine synthesis accumulate at the expense of 5-methyltetrahydrofolate (the most abundant form in wild type MTHFR 677 CC subjects). The relationship between these polymorphisms and disease might be explained by several mechanisms. First, the MTHFR 677 C>T and 1298 A>C polymorphisms in interaction with poor B-vitamin and folate status result in mildly elevated plasma homocysteine levels. Hyperhomocysteinemia is a well- established independent risk factor for cardiovascular disease and has been linked to other diseases such as pregnancy complications, Down’s syndrome and Alzheimer’s disease. Second, reduced MTHFR activity leads to reduced availability of 5-methyltetrahydrofolate required for S-adenosylmethionine (SAM) biosynthesis and consequently results in DNA hypomethylation. Third, low availability of folates for pyrimidine and purine synthesis may lead to uracil misincorporation into DNA leading to double-strand breaks and chromosomal damage. DNA hypomethylation and uracil misincorporation are considered important factors in carcinogenesis. Thus, MTHFR 677 TT subjects have a decreased risk of colorectal cancer possibly due to increased folate availability for RNA and DNA synthesis. Furthermore, both the MTHFR 677 C>T and 1298 A>C polymorphisms have been linked to a reduced risk of lymphoid leukemias but not myeloid leukemias. In this presentation, the correlation between MTHFR 677 C>T and 1298 A>C polymorphisms and several disease entities such as osteoporosis, acute lymphoid leukemia and pre-eclampsia will be discussed.

86 O42

Identification of 4 unique mutations in exon 4 of uromodulin (UMOD) gene in 4 separate families with familial juvenile hyperuricemic nephropathy (FJHN)

N. Kamatani1, E. Kudo2, M. Moritani2, T. Yamaoka2, A. Taniguchi1, H. Yamanaka1, M. Itakura2 1Tokyo Women's Medical University, TOKYO, Japan 2The University of Tokushima, TOKUSHIMA, Japan

Scolari et al. reported a locus for medullary cystic kidney disease (MCKD2) on chromosome 16p12. Our independent genome-wide linkage study of a large Japanese family identified a locus for familial juvenile hyperuricemic nephropathy (FJHN) at the same region. Subsequent studies have suggested that the 2 diseases (MCKD2 and FJHN) could be allelic variants. Recently, Hart et al. for the first time found that mutations in uromodulin (UMOD) gene were responsible for the 2 conditions. They found 4 independent mutations in 3 families with FJHN and one family with MCKD2. We examined DNA from 5 separate families whose diagnosis is compatible with that of FJHN. Including the large family we reported previously, 4 of the 5 families had different unique missense mutations in exon 4 of UMOD gene. The mutations segregated along with the FJHN phenotype. In 3 of the 4 families, other amino acids were substituted for conserved cysteine residues (Cys). In the large family, we found a missense mutation that substituted leucine for conserved proline residue. Our findings further confirmed that UMOD gene is responsible for most of the families with FJHN. The facts that 3 of the 4 families of us and 2 of the 3 families of Hart et al. had missence mutations involving Cys residue strongly suggest that structural changes of UMOD molecule due to impaired formation of intra- or intermolecular disulphide bond or substitution of proline residue may be responsible for the 2 disorders. The domain encoded by exon 4 probably has critical importance in functions of UMOD molecule, because all 8 mutations found in UMOD gene were in exon 4. The fact that 8 separate families so far reported with MCKD2/FJHN had different responsible mutations is compatible with the theories of population genetics. Thus, diseases with autosomal dominant inheritance, with high penetrances and with considerable disability are not likely to be caused by common mutations with common origins.

87 O43

What role do methylated metabolites play for the cellular effects of thiopurines?

C. Peterson, M. Lindqvist, S. Rahman, S. Almer University of Linköping, LINKÖPING, Sweden

Thiopurines (azathioprine, 6-mercaptopurine, 6-thioguanine) are old drugs but still cornerstones in the treatment of inflammatory bowel disease and childhood leukemia. The drugs undergo deamination, methylation and phosphorylation to form identical terminal thioguanine nucleotides. Weinshilboum showed many years ago that thiopurinemethyltransferase (TPMT) is a polymorphic enzyme. Several mutations have been identified and about 10% of Caucasians have reduced enzyme activity. Methylation and phosphorylation can be regarded as competing metabolic pathways. Me-MP lacks cytotoxicity and evidence has been presented for a correlation between the formation of thioguanine nucleotides (TGN) and clinical effects. We have determined erythrocyte TGN and me-TIMP concentrations in more than 2500 blood samples from IBD-patients and confirmed previous results showing marked differences in metabolite pattern after treatment with TG and AZA or 6-MP at doses equitoxic to the bone marrow. TGN levels are severalfold higher after TG, and after 6-MP the concentration of metylated TIMP is much higher than that of TGN. Aim: To elucidate the cellular effects of thiopurine metabolites, we compared the cytotoxic effects of TG and 6-MP and metabolites in a human T-cell acute lymphoblastic cell line (Molt-4) and in sublines resistant to 6- MP and TG. Methods: Resistant sublines were produced by stepwise increase in 6-MP and TG concentrations to the Molt-4 cells during several months. Cytotoxicity was determined by the colorimetric MTT-assay of viable cells. The drug concentration causing a 50% reduction in the number of viable cells during exposure for 72 hours was determined (IC50). Results: In the parental cell line, the cytotoxicties of 6-MP, TG and TIMP were similar with IC50 values of 7.2, 7.7 and 7.9 nM, respectively. Me-TIMP and me-MPR were more cytotoxic with IC-50-values of 1.7 and 1.4 nM whereas me-MP and me-TG were non-toxic. As expected, the resistant cell lines showed low sensitivity to the parent compounds. However, me-TIMP and me-MPR had markedly higher cytotoxicity on the resistant cell lines as compared to the parental line with IC50-values of 0.3 and 0.4 nM. Conclusion: Certain methylated thiopurine metabolites have the potential to contribute to the therapeutic and toxic effects of thiopurines.

88 O44

Pre-treatment TPMT PHE/genotyping: a safe guide for initial thiopurines drugdosing for specific patient groups

E.A. Shobowale-Bakre, A. Ansari, A.M. Marinaki, L.D. Fairbanks, J.A. Duley, J.D. Sanderson Guy's Hospital, LONDON, United Kingdom

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme. Deficiency of the enzyme is predictive of thiopurine drugs (i.e. azathioprine, mercaptopurine and thioguanine)intolerance. In Caucasian and African populations, 11% are carriers while 0.3% of the individuals are totally TPMT deficient. Two-thirds of these carriers have a high risk of drug induced side-effects on a full dose and are typically withdrawn from therapy when bone marrow depression sets in. To test the hypothesis that carriers should tolerate half the normal dose, we identified 28 TPMT carriers who had pre-therapy TPMT phenotyping done in our laboratory mainly from gastroenterology and dermatology clinics(2 patients had autoimmune hepatitis, 6 skin and or oral ulceration and 3 eczema). Each patient was given azathioprine(aza) at a reduced dose of 1mg/kg (initially 0.5mg/kg) as a steroid-sparring agent according to departmental guidelines. Clinical notes were reviewed after one year of treatment for adverse effects and clinical response (defined as withdrawal of steroids :- complete, partial or non-response). Phenotype results were confirmed for all patients by genotyping for the three most common TPMT allelic variants-: TPMT *3A (G460A- Ala154Thr and A719G-Tyr240Cys; 10% in Caucasians), *3B (G460A-Ala154Thr),*3C (A719G- tyr240Cys; 10-14% in Africans and 2.5% in South Asians) considering the inter-ethnic variations of TPMT genotypes and the racial mix of the UK population. 6 of the 28 patients experienced side- effects requiring drug withdrawal (2 pacreatitis, 4 nausea/malaise). The 4 patients that experienced nausea on aza were switched onto low dose mercaptopurine. No additional episodes of myelotoxicity or other side effects were observed over the treatment period. 24 of the 26 carriers who remained on drug therapy responded favourably. Others factors must be considered for the 2 patients that did not respond to therapy. In Conclusion, pre-therapy TPMT testing is a very cost- effective and useful tool for guiding against drug over-dosing in TPMT carriers.

89 O45

A mutation in the ITPA gene predicts intolerance to azathioprine

A.M. Marinaki1, J.A. Duley2, M. Arenas1, S. Sumi1, A. Ansari1, J.D. Sanderson1, C.M. Lewis1, E.A. Shobowale-Bakre1, L.D. Fairbanks1 1Guy's Hospital, LONDON, United Kingdom 2University of Queensland, QUEENSLAND, Australia

Introduction. Adverse drug reactions to AZA or 6-mercaptopurine (6-MP) occurs in 20-30% of inflammatory bowel disease (IBD) patients and often necessitates withdrawal of therapy. Polymorphism in the TPMT gene predicts intolerance in a minority of these patients. Inosine triphosphate pyrophosphatase (ITPase) deficiency is a benign polymorphism in Caucasian populations and is characterized by accumulation of the nucleotide ITP in erythrocytes. As 6-MP is activated through a thio-IMP intermediate, we predicted that the abnormal nucleotide thio-ITP would accumulate in ITPase deficient patients treated with thiopurine drugs, resulting in toxicity. We have reported (Hum Genet (2002) 111 :360–367) the genetic basis of ITPase deficiency; a 94C>A missense mutation (Pro32 to Thr) in the ITPA gene results in complete deficiency in homozygotes and approximately 25% activity in heterozygotes. Homozygotes for an intron 2 mutation (IVS2+21A>C) averaged 60% of controls. Methods. ITPA and TPMT genotypes and TPMT phenotypes were determined retrospectively in 62 IBD patients with adverse drug reactions to AZA therapy. The adverse drug reaction cohort was compared to 68 patients who did not experience an adverse reaction to AZA therapy. Results. Variant TPMT genotype was not significantly associated with adverse drug reactions overall, but the association of TPMT heterozygosity with side effects was significant in a subset of 14 patients with nausea and vomiting (OR 5.5, CI 1.4-21.3, p=0.0206). Overall, the ITPA 94C>A mutation was significantly associated with adverse drug reactions (OR 4.2, CI 1.6-11.5, p=0.0033) which sub-divided into: flu-like symptoms (OR 4.7, CI 1.2-18.1, p=0.0308), pancreatitis (OR 6.2, CI 1.1-32.6, p=0.0485) and rash (OR 10.3, CI 4.7-62.9, p=0.0190). Bone marrow suppression, myalgia and hepatitis were not predicted by ITPA nor TPMT genotypes. The ITPA intron mutation did not predict adverse drug reactions. Conclusion. The ITPA 94C>A mutation predicts intolerance to AZA. Thioguanine therapy may benefit AZA intolerant patients with ITPase deficiency.

90 O46

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8- dihydroxyadenine urolithiasis

A. Taniguchi, H. Yamanaka, S. Tsuchida, S. Kuno, N. Kamatani Tokyo Women's Medical University, TOKYO, Japan

Adenine phosphoribosyltransferase deficiency is associated with 2.8-dihydroxyadenine (DHA) urolithiasis. The largest number of patients has been reported in Japanese. So far, four mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese. The most common mutation is M136T. Three other mutations, a nonsense (W98X), a frameshift caused by CCGA insertion in exon 3 and a termination failure at stop codon (X186S). We now report two new mutations in patients with 2,8- dihydroxyadenine urolithiasis. We examined 3 patients (patient A, B, C) with 2.8-dihydroxyadenine urolithiasis. The five exons and splice-donor sites were amplified with several sets of primers and the sequence analysis of PCR products were performed on automated DNA sequencer. The patient A is homozygous for G1359A leading to the amino acid substitution R67Q which have been already reported in USA. The patient B was shown to be homozygous for a single base substitution G1831A leading to amino acid substitution G133D. Patient C was found to be compound heterozygous for two mutations: a W98X which is one of the common mutations in Japanese population and a G1409A transition which is a new missense mutation leading V to M substitution in codon 84. These mutations were confirmed by restriction endonucleases. Based on the structure of the enzyme from Leishmania donovani, Saccharomyces cerevisiae and Giardia lumblia, R67 and G133 are located in the PRPP binding site and V 84 are supposed to be located at the dimer interface, suggesting these mutation are expected to affect the enzymatic activity.

91 P01

Modulation of Glycogen Phosphorylase activity affects 5-Phophoribosyl-1-Pyrophosphate availability in Rat Hepatocyte Cultures

P. Boer, O. Sperling Tel-Aviv University, TEL-AVIV, Israel

The effect of modulation of the rate of glycogenolysis on the availability of 5-phosphoribosyl-1- pyrophosphate (PRPP) was investigated in rat hepatocyte cultures. dbcAMP, forskolin and glucagon, activating glycogen phosphorylase through activation of protein kinase A (PKA), were found to raise PRPP availability by 44%-56%. Arg-vasopressin and phenylephrine, activating glycogen phosphorylase through the phosphoinositide cascade, and the protein kinase C (PKC) activators 1,2-dioctanoyl-sn-glycerol (DOG) and phorbol 12-myristate β-acetate (PMA), did not affect PRPP availability. dbcAMP, but not phenylephrine, was also shown to increase the degradation of pre labeled glycogen in these cells by 57%. Caffeine and CP-91149, inhibitors of glycogen phosphorylase, decreased PRPP availability by 33% and 43%, respectively. The finding that induction of glycogenolysis enhances, and inhibition of glycogenolysis decelerates PRPP generation suggests that glycogenolysis is a major contributor to PRPP generation in liver tissue in the basal (postabsorptive) state.

92 P02

Purine levels and cytokine release with diuretic therapy in rheumatoid arthritis

T.W. Stone1, C.M. Forrest1, G. Harman2, R.B. McMillan1, C. Rana1, S. Shaw2, N. Stoy2, L.G. Darlington2 1University of Glasgow, GLASGOW, United Kingdom 2Epsom General Hospital, EPSOM, United Kingdom

Treatment with diuretics has been reported to increase blood urate levels, and urate is a potent free radical scavanger. Since free radicals are implicated in rheumatoid arthritis and since it is rare to find a patient with both gout and RA, we examined the effects of treating rheumatoid arthritic patients with bumetanide to try to improve their arthritic control. We measured blood levels of urate, other major purine metabolites, lipid peroxidation products and the cytokines tumour necrosis factor-a (TNFa), interleukin-1b (IL-1b) and interleukin-6 (IL-6). In addition, we examined the ability of the adenosine receptor agonist 5'N- ethylcarboxamido-adenosine (NECA) to modify cytokine release in cultures of whole blood taken from the patients. Seventy patients, aged 18-75 years, were recruited from routine rheumatology clinics and rheumatoid arthritis was diagnosed using the American Rheumatism Association revised criteria. Patients were excluded if they were taking any drug which would affect uric acid levels in the blood. Patients were randomised to receive bumetanide 4mg/day or placebo. Results showed increased levels of urate, but not of other purines, in the blood of drug-treated patients compared with placebo-treated controls. There were no significant changes in clinical measurements of disease activity or in ESR or CRP levels. There were no over all differences in the blood levels of the cytokines, nor in the basal or stimulated production of cytokines from the blood cultures. However, while NECA depressed the release of TNFa, it failed to depress the release of IL-1b or IL-6, a difference from earlier studies of healthy control subjects and, thus, a difference which may contribute to the disease activity.

93 P03

The Pathophysiology of Hyperuricemia in Essential Hypertension: A pilot study

J.G. Puig1, R.J. Torres1, L.M. Ruilope2, C. Campo2, T. Sancho1, I. Bernardino1 1La Paz Hospital, MADRID, Spain 2Hospital 12 de Octubre, MADRID, Spain

Background: Hyperuricemia in essential hypertension has been related to renal uric acid underexcretion. This impaired uric acid excretion may be linked to an increased tubular sodium reabsorption mediated by insulin. In this pilot study we examined whether insulin influences the renal handling of urate. Patients and Methods: Ten non-obese (body mass index, 28,22 Kg/m2), non-diabetic male patients with essential hypertension (mean age+-SD, 54+-7 years) and normal glomerular filtration rate were submitted to and oral glucose tolerance test (OGTT, 75 g). Serum glucose, insulin, urate and urinary uric acid to creatinine ratio were measured at baseline and 120 min following the OGTT. Results: Two patients had impaired fasting glucose (>110 and <126 mg/dL) and only one patient showed glucose intolerance (141 mg/dL, 120 min after the OGTT). Serum insulin increased from a mean baseline value of 16 mcU/mL to 72 mcU/mL (mean increase, 4.5 fold). Mean baseline glucose was 95 mg/dL and 120 min after the OGTT, 105 mg/dL. Mean serum urate remained stable (8.1 mg/dL). Urinary uric acid to creatinine ratio decreased from a mean baseline value of 0.44+-0.18 mg/mg to 0.28+-0.12 mg/mg (p<0.05) after the OGTT. In 5 patients in whom urinary sodium to creatinine ratio was determined, sodium excretion was markedly reduced (from 1.23+-0.61 at baseline to 0.70+-0.26 mmol/mg creatinine following the OGTT; p<0.05). Conclusion: This pilot study shows that acute endogenous insulin secretion is accompanied by a reduction in uric acid excretion that may be mediated by the sodium-retaining effect of insulin.

94 P04

Serum Adiponectin Concentration in Patients with Primary Gout

Z. Tsutsumi, T. Yamamoto, S. Takahashi, Y. Moriwaki, T. Ka, H. Toshikazu Hyogo College of Medicine, NISHINOMIYA, Japan

Objectives: Adiponectin is an adipose tissue derived protein. It was suggested that decreased adiponectin concentration in serum is a possible risk factor for atherosclerosis. Clinical studies have shown that obesity is closely related to the decreased adiponectin concentration in serum. Atherosclerosis and obesity have been frequently observed in patients with gout. Therefore, we investigated adiponectin concentration in serum, together with abdominal fat area, serum insulin level, urinary excretion of c-peptide, as well as markers of urate metabolism in patients with gout. Subjects and Methods: Age- and body mass index-matched male patients with primary gout (n=88) and healthy adult male (n=58) were included in the study. Primary gout was diagnosed on the basis of criteria advocated by ARA. Adiponectin was measured by a radio-immunoassay method using a commercially available kit (Human adiponectin RIA, Linco Research Inc. MO, USA). Abdominal fat area was measured by a computed tomography at the level of umbilicus . Other biochemical parameters were measured in our laboratory. Results: Serum adiponectin concentration was not different between gout patients and control subjects (9.8 +/- 1.4 vs 8.3+/- 0.5 microg/mL). Serum adiponectin level was negatively correlated with body mass index, visceral fat area, subcutaneous fat area, serum insulin level, and urinary excretion of c-peptide, while it was positively correlated with serum HDL-cholesterol level. However, there were no significant relationships between adiponectin and any markers of urate metabolism. Multivariate analysis demonstrated that the strongest contributor to serum adiponectin concentration was body mass index, followed by subcutaneous fat area. Conclusion: Obesity contributes to decreased serum concentration of adiponectin, irrespective of gout or not. However, decreased serum concentration of adiponectin was correlated with various elements of metabolic syndrome. Further investigations on the relationship between adiponectin and atherosclerosis in gout will be required.

95 P05

Effect of low purine-containing low malt beer (happo-shu) and regular low malt beer on purine bases

K. Ka, T. Yamamoto, Y. Moriwaki, S. Takahashi, Z. Tsutsumi, M. Fukuchi, H. Toshikazu Hyogo College of Medicine, NISHINOMIYA, Japan

Background: Regular beer contains large amounts of purines, which may increase the plasma concentration of uric acid. Low malt beer also contains considerable amounts of purines, though low molt beer less contains purines than regular beer. On the other hand, purines in low purines-containing low malt beer are less than 10% of purines in regular beer. Therefore, we tested regular and low purine-containing low malt beer in order to determine whether they caused an increase in plasma concentration and urinary excretion of purine bases (hypoxanthine, xanthine, and uric acid). Subjects and Methods: Five healthy males were given regular low malt beer (10 ml/kg of body weight) and low purine-containing low malt beer (10 ml/kg of body weight). Blood and urine sampled were collected before and after regular beer and low purine-containing low malt beer. Results: The plasma concentrations of uric acid, hypoxanthine, and xanthine increased by 1.10-fold (P<0.05), 4.23- (P<0.05), and 9.723-fold (P<0.05), respectively, 90 minutes after beginning regular beer ingestion. On the other hand, the plasma concentrations of xanthine increased by 5.77-fold (P<0.05), respectively, 90 minutes after beginning low purine-containing low malt beer ingestion, though those of uric acid or hypoxanthine did not increase. The 1-hour urinary excretion of hypoxanthine and xanthine increased by 4.31- (P<0.01), and 11.15-fold (P<0.01), respectively, when measured 2 hours after beginning regular happo-shu ingestion, though that of uric acid or uridine did not increase. On the other hand, the 1-hour urinary excretion of xanthine increased by 4.52-fold (P<0.05), when measured 2 hours after beginning purine-free happo-shu ingestion, though that of uric acid, hypoxanthine, or uridine did not increase, when measured 2 hours after ingestion. Conclusions: These results suggest that the purines contained in regular happo-shu contribute to an increase in the plasma concentration of uric acid due to purine degradation and the increase in plasma concentration of uric acid may be significantly less with purine-free happo-shu than with regular happo-shu. In addition, uridine contained in regular happo-shu seems to contribute to an increase in the plasma concentration of uridine along with ethanol.

96 P06

SLC22A12 mutation is responsible for most renal hypouricemia in Japan

K. Ichida1, M. Hosoyamada2, I. Hisatome3, H.E. Endou2, T. Hosoya1 1Jikei University School of Medicine, TOKYO, Japan 2Kyorin University School of Medicine, TOKYO, Japan 3Tottori University, YONAGO, Japan

Renal hypouricemia is an inherited and heterogeneous disorder characterized by increased urate clearance (CUA). We recently established that urate was reabsorbed via URAT1 on the apical membrane in the proximal tubules, and that mutations in SLC22A12 encoding URAT1 cause renal hypouricemia. We elucidated a URAT1 function for renal urate handling in vivo by examining 32 unrelated patients with idiopathic renal hypouricemia (18 males and 14 females). Serum urate levels of all the patients were below 2 mg/dl and averaged 0.93 ± 0.49 mg/dl (n=32). Serum urate levels of the male and female patients were 0.77± 0.31 mg/dl (n=18) and 1.14 ± 0.61 mg/dl (n=14), respectively. Mean CUA/Ccr (0.584 ± 0.264; n=32) and urinary urate excretion (704.6 ± 233.0 mg/day; n=31) were increased. CUA/Ccr and urinary urate excretion of the male and those of female patients were 0.668 ± 0.202 (n=18) and 747.7 ± 242.1 mg/day (n=17), and 0.476 ± 0.301 (n=14) and 655.9 ± 226.9 mg/day (n=12), respectively. Three male patients had exercise-induced acute renal failure (9.4%) and 4 patients two of whom were male urolithiasis (12.5%). The frequency of exercise-induced acute renal failure in patients with renal hypouricemia was higher than initially perceived. We identified 8 new mutations and 2 previous reported mutations that result in loss of function. Thirty patients had SLC22A12 mutations, 24 homozygotes and 6 heterozygotes. Mutation G774A dominated SLC22A12 mutations (74.1 % in 54 alleles). Serum urate levels were significantly lower and CUA/Ccr significantly higher in heterozygotes as compared to healthy subjects, and these changes were even more significant in homozygotes. These CUA/Ccr relations demonstrated a gene dosage effect, corresponding with the difference in serum urate levels. In contrast to healthy subjects, the CUA/CCr of patients with homozygous SLC22A12 mutations was unaffected by the anti-uricosuric drug pyrazinamide and the uricosuric drugs benzbromarone and probenecid. Our findings indicate that URAT1 is the main reabsorptive urate transporter and SLC22A12 was responsible for most renal hypouricemia. Furthermore, we established that pyrazinamide, benzbromarone, and probenecid exert anti-uricosuric and uricosuric effects by acting on URAT1 in vivo.

97 P07

Pharmacokinetics/Pharmacodynamics of Y-700, A novel Xanthine Oxidase Inhibitor, in Rats and Man

I. Yamada*, A. Fukunari*, T. Osajima, M. Kamezawa, H. Mori, J. Iwane Mitsubishi Pharma Corporation, CHIBA, Japan

Objectives Y-700, 1-(3-Cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid, has been introduced as a novel xanthine oxidase inhibitor, which bears no structural relationship to the known inhibitor, allopurinol (1). The present study was conducted to evaluate the pharmacokinetics and pharmacological actions of Y-700 in rats and healthy adult male volunteers. Design and Methods Male Sprague-Dawley rats were used for animal study. A rat model of hyperuricemia was established by repeated treatment of the animal with the uricase inhibitor, potassium oxonate (250 mg/kg, s.c.) . Plasma uric acid (UA) levels were determined by the phosphotungstic acid colorimetry. 14C - labelled Y-700 (504.9 kBq/mg) was used for the study of absorption, metabolism and excretion in rats, and the concentrations of radioactivity in samples were determined by LSC or HPLC equipped with a radio-detector. In a clinical study, single doses (5, 20 or 80 mg) of Y-700 or placebo was administered orally to healthy Japanese volunteers. Serum UA levels were determined by the uricase method. Plasma and urinary concentrations of Y-700 and urinary amounts of xanthine and hypoxanthine were detected by a validated HPLC method. Results Y-700 was absorbed rapidly in both species and was eliminated with t1/2 of 2.7-5.0 h for rats and 27.6-40.2 h for humans. In rats and humans, Cmax and AUC of oral Y-700 were increased dose-dependently. Bioavailability was 84.1% (1 mg/kg) in rats. Only Y-700 was detected in rats and humans plasma. Total radioactivity in urine and feces accounted for 21.8% and 79.8% of the dose, respectively. Urinary excretions of Y-700 in rats and humans were 1.1% and 1.5%, respectively. In hyperuricemic rats, oral Y-700 (0.3 – 3 mg/kg) showed hypouricemic action in a dose-dependent manner, and was more potent and a longer lasting than allopurinol. In humans, hypouricemic action at doses of 20 and 80 mg were statistically significantly different from the placebo. Emax of change in serum UA level at doses 20 and 80 mg were -1.01 and 2.66 mg/dL, respectively. No direct relationship between tmax(2.2-3.0 h) and Etmax(18.7-36.0 h) was recognized. The hypouricemic action of Y-700 was maintained throughout the post dose. Urinary excretion (0-72 h) of xanthine and hypoxanthine were significantly increased compared with placebo group. Discussion The present study demonstrated that Y-700 is a new effective inhibitor of XO with a potent and a long- lasting hypouricemic action in rats and healthy human volunteers. The data suggest that Y-700 is likely to possess clinical efficacy in the patients. Another important finding was that Y-700 is a drug of non-renal excretion type. The distinctive elimination route of Y-700 is expected to provide a beneficial property as a new drug for the treatment of gout and hyperuricemia. In other words, unlike the case for allopurinol, it may not be necessary to adjust the dosage of Y-700 in patients according to their degree of renal function.

(1) Ishibuchi, S.; Morimoto, H.; Oe, T.; Ikebe, T.; Inoue, H.; Fukunari, A.; Kamezawa, M.; Yamada, I.; Naka, Y. Synthesis and structure-activity relationships of 1-phenylpyrazoles as xanthine oxidase inhibitors. Bioorg. Med. Chem. Lett. 2001, 11, 879-882.

* These authors equally contributed to this work.

98 P08

PK/PD and safety of a single dose of TMX-67 (febuxostat) in subjects with mild and moderate renal impairment

S. Hoshide1, Y. Takahashi1, T. Ishikawa1, J. Kubo1, M. Tsuchimoto1, K. Komoriya1, I. Ohno2, T. Hosoya2 1Teijin Limited, TOKYO, Japan 2Jikei University School of Medicine, TOKYO, Japan

Objective: Decreased renal urate clearance associated with renal impairment causes enhancement of hyperuricemia. In patients with decreased renal clearance, it is well recognized that plasma oxypurinol, an active metabolite of allopurinol, shows a prolonged half-life due to its purine-like structure. To investigate the effects of renal impairment on PK/PD and safety of TMX-67, a novel XOD inhibitor with non-purine structure, an open label, single dose clinical study was conducted in normal or renally compromised subjects. Methods: Fifteen subjects (13 males/2 females) were assigned to three groups of 5 subjects based on creatinine clearance (Ccr, mL/min); group 1: Ccr>80; group 2: 50

99 P09

Pharmacodynamics & pharmacokinetics of TMX-67 (febuxostat), a novel xanthine oxidase / xanthine dehydrogenase (XOD) inhibitor, in patients with hyperuricemia

K. Komoriya1, K. Takeda1, H. Kobayashi1, J. Kubo1, M. Tsuchimoto1, T. Nakachi2, H. Yamanaka3, N. Kamatani3 1Teijin Limited, TOKYO, Japan 2TOCROM, TOKYO, Japan 3Tokyo Women's Medical University, TOKYO, Japan

Introduction and Objective: TMX-67, a novel XOD inhibitor, is a drug under clinical development for the treatment of hyperuricemia and gout. A study was conducted to investigate the pharmacokinetics (PK) and pharmacodynamics (PD) as diurnal changes in the serum urate (sUA) level before and after repeated administration of TMX-67 to hyperuricemic patients. Methods: Ten hyperuricemic patients (sUA level 8.0 – 13.7 mg/dL) received a 10 mg once daily (QD) dose of TMX-67 for 2 wks, followed by a 20 mg QD dose for 4 wks. On the day before TMX-67 administration (Day -1) and the day of the last administration (Day 41), each patient was admitted to the hospital for laboratory tests and PK/PD evaluation. Serum urate levels were determined at 0, 2, 4, 6, 8, 12, and 24 hrs. The time course of plasma drug concentrations was also determined on the day of the last administration. Results: Before TMX-67 administration (Day -1), diurnal changes of sUA were small (range of 8.4-9.1 mg/dL) and the mean sUA concentration for 24 hrs (sUA0-24) was 8.7 mg/dL. After the last TMX-67 administration (Day 41), a small mean sUA decrease from 6.0 (0 hr) to 5.5 mg/dL (6 and 8 hrs) was observed and then the mean sUA gradually elevated to 6.4 mg/dL (24 hrs). The mean sUA0-24 was 5.8 mg/dL which corresponds to the mean sUA value approximately 2 hrs after administration, respectively. The pharmacokinetic profiles for TMX-67 and its oxidative metabolites were nearly the same as that in healthy volunteers in a previous phase I study. Adverse events were minimal. Conclusions: The pharmacodynamic profile after repeated oral administration of TMX-67 suggests that the mean sUA 2 hrs after drug administration may accurately represent the sUA for the clinical evaluation of efficacy of TMX-67 at a 20 mg QD dose.

100 P10

A novel point Mutation (I137t) in the Conserved 5-Phosphoribosyl-1-Pyrophosphate Binding Motif of HPRT (HPRTJERUSALEM) in a LNS Variant

E. Zoref-Shani1, O. Sperling1, Y. Bromberg1, J. Hirsch1, S. Feinstein2, Y. Frishberg2 1Tel-Aviv University, TEL-AVIV, Israel 2Shaare Zedek Medical Center, JERUSALEM, Israel

We have reported before a case of partial HPRT deficiency, characterized (in fibroblast lysates) by reduced affinity of HPRT for 5-phosphoribosyl-1-pyrophosphate (PRPP), manifested in low activity at low (physiological), but not at high PRPP concentrations, and with normal Km for hypoxanthine [1]. Here we report that we identified in this patient a novel point mutation (I137T) in the gene encoding HPRT. The mutation, ATT to ACT, resulting in substitution of isoleucine to threonine, occurred at codon 137 (exon 6), which is within the polypeptide segment (130-141) encoding the binding site for PRPP. The structure of this sequence is comprised of beta strand β6-β6’ and a turn, which is followed immediately by the beginning of alpha helix α5. Residue 137, an isoleucine in the wild type protein, stabilizes the kinked beta strand β6-β6’ and turn that have direct interaction with PRPP. The trajectory and stereochemistry of the main chain in this area and its stability is clearly essential to the binding of the 5’ phosphate moiety of the PRPP. We suggest that perturbation by substitution to threonine, a polar residue versus the apolar and bulky isoleucine, may destabilize the trajectory of the polypeptide affecting the affinity to PRPP. Possible effects on the binding of the base, guanine or hypoxanthine, might well be masked due to the sequential nature of substrate binding, since it appears that the PRPP plays a role in forming the binding pocket of the base.

[1] Zoref-Shani, E., Feinstein, S., Frishberg, Y., Bromberg, Y. and Sperling, O., Kelley-Seegmiller syndrome due to a unique variant of hypoxanthine-guanine phosphoribosyltransferase: reduced affinity for 5-phosphoribosyl-1-pyrophosphate manifested only at low, physiological substrate concentrations. BBA, 1500, 197-203, 2000

101 P11

Disruption in the hypoxanthine phosphoribosyl-transferase gene caused by translocation in a patient with Lesch-Nyhan syndrome

M.M. Mizunuma1, Y. Yamada2, K. Yamada2, S. Sonta2, N. Wakamatsu2, K. Kaneko3, N. Ogasawara2, S. Fujimori3 1Laboratory of Purine Metabolism, TOKYO, Japan 2Àichi Human Service Center, AICHI, Japan 3Teikyo University School of Medicine, TOKYO, Japan

Germline mutations at hypoxanthine phosphoribosyltransferase gene (HPRT1) cause HPRT deficiency responsible for heritable Lesch-Nyhan (LN) syndrome. More than 200 different mutations associated with LN syndrome have been reported. Most of the mutations (~85%) are single point mutations causing missense, nonsense and abnormal splicing, and the remaining of the mutations are concerned with large genomic alterations such as total or partial gene deletions and duplications. However the structure of bareakpoint junctions in large genomic alterations have been defined for only 11 mutations. In this study, we have identified the structure of the large alteration mutation in the HPRT1 in LN patient (HRT-25). Since the full-length HPRT1 cDNA product of HRT-25 was not amplified by RT-PCR, we attempted to amplify cDNA using 3?RACE method. Sequencing of the PCR products by 3?RACE revealed intron 3 (137-bp) and unknown sequence (1008-bp) in the exon 3 downstream region of HRT-25 cDNA. We next examined for this unknown sequence using the genome database, it was shown that unknown sequence was in agreement with the sequences 0.5 - 0.6 Mb telomere domain away from HPRT1 gene. This result suggests that the HPRT was disrupted in the region surrounding exon 4 by translocation or inversion between HPRT1 allele and different allele in X chromosomes. This translocation mutation is the first report for the mutant HPRT1 associated with LNS. It is thought that the characterization of the other macro-alterations will complete the picture of how these types of germinal mutations occur in human.

102 P12a

Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in Asian HPRT deficient families

Y. Yamada, K. Yamada, S. Sonta, N. Wakamatsu, N. Ogasawara Àichi Human Service Center, AICHI, Japan

Inherited mutation of a purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT, MIM308000), gives rise to Lesch-Nyhan syndrome (MIM300322) or HPRT-related gout (MIM300323). We have identified 33 mutations in 27 Japanese, 7 Korean, and 1 Indian families with the patients manifesting different clinical phenotypes, including two rare cases in female subjects, by the analysis of all nine exons of HPRT gene (HPRT1) from the genomic DNA and reverse transcribed mRNA using polymerase chain reaction (PCR) technique coupled with direct sequencing. Moreover, prenatal gene diagnoses were carried out in 12 fetuses in 9 families by PCR-RFLP method and direct sequencing using both mRNA and genomic DNA from chorionic villi or amniotic fluid cells. In the two partial HPRT deficient patients with hyperuricemia and gout, two missense mutations (V188A and Y195C) in the exon 8 of HPRT1 were identified. Three independent missense mutations (Y72C, L147P, and L159E) were detected from the patient manifested with hyperuricemia and mild neurological symptoms, such as ataxia and spasticity. In a severe form of partial HPRT deficiency referred as Lesch-Nyan syndrome without self-mutilation, a single nucleotide substitution (532+2T>C) resulted in splicing error and markedly decreased expression of normal mRNA. By the analysis of HPRT1 from classical Lesch-Nyhan patients, we have defined lots of mutations, 2 nonsense mutations (R51X in 3 families and Q109X in a family), 4 missense mutations (A64P, L65P, G70E and L78Q), 6 single base substitutions causing splicing error (27+1G>T, 28-1G>C, 318-1G>T, 403-2A>G, 533-9T>A, and 610-1G>A), 3 insertions (212insG, 330insA, and 435insTTTG), 9 deletions (50delA, 631delA, 289delGT, 316delGT, 318delAATG, 532+1delGT, 609-16del74-bp, 1-368del353-bp, and a total gene deletion), a translocation, and an abnormal methylation. Furthermore, we have identified recently a large genomic deletion (~15 kb) including the promoter region and the whole exon 1 of HPRT1, by the Southern analysis and PCR methods referring the nucleotide sequences from human genome database.

103 P12b

Highly skewed X-inactivation pattern as a cause of female gout

I. Sebesta1, L. Dvorakova1, J. Hujova1, R. Dobrovolny1, L. Stolnaja1, E. Tietzeova1, M. Hrebicek1, J.A. Duley2 1Charles University, PRAGUE, Czech Republic 2Guy's Hospital, LONDON, United Kingdom

Gouty arthritis in females is unusual and patients with such clinical presentation are suspicious for purine genetic defects. Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT), described as Kelley-Seegmiller syndrome is an X-linked inborn error of purine metabolism associated with gout and urolithiasis and has been reported exclusively in males. Female carriers have somatic cell mosaicism of HPRT activity, and are healthy. Genetic studies were performed in a girl suffering from gouty arthritis of big toe. Hyperuricemia and affected joint were found at the age of 9 years, when therapy with allopurinol started. No evidence of neurological involvement was found. The absence of HPRT activity, together with raised activity of adenine phosphoribosyltransferase in lysed erythrocytes and raised concentration of nicotinamide adenine dinucleotide in erythrocytes confirmed partial HPRT deficiency at the age of 16 years. Deficient HPRT activity was found also in the patient´s father, who presented with renal colic and gout since the age of 18 years. The sister of the proband is healthy and her HPRT activity is within normal limits. Mutation analysis using direct sequencing revealed that both sisters inherited from their father a previously described mutation in the 3rd exon of HPRT gene, c.158>C (V53A). The extreme skewing of the X- inactivation was found. The symptomatic girl has predominantly active the paternal mutated chromosome, while her asymptomatic sister has almost exclusively active maternal non-affected chromosome. In conclusion: the point mutation and skewed X-inactivation ratio in favour of mutant allele were responsible as genetic basis for clinical expression in this first described female patient with partial HPRT deficiency. This condition should be taken to differential diagnosis of unexplained hyperuricemia in girls or women. (Supported by grants MSM-11110005 of the Czech Ministry of Education and NE 6557-3-01 of the Czech Ministry Health ).

104 P13

Are allopurinol and metabolites found in HPRT deficient erythrocytes responsible for increased NAD synthesis?

V. Micheli, G. Jacomelli, S. Sestini, L. Notarantonio, B. Cerboni, L. Peruzzi, G. Pompucci University of Siena, SIENA, Italy

INTRODUCTION: Inborn deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) causes hyperuricemia, gout and different degrees of neurological disturbances, the most serious of which is Lesch- Nyhan Syndrome [1]. HPRT-deficient erythrocytes present associated biochemical alterations: GTP depletion, increased concentrations of ZTP, phosphoribosylpyrophosphate (PPribP), NAD and UDP sugars [2], increased activity of adenine phosphoribosyltransferase (APRT), IMPdehydrogenase, UMPsynthetase [3], 5’nucleotidase [4], nicotinate phosphoribosyltransferase (NAPRT) and NAD synthetase (NADs) [5,6]. Aim of this study was to ascertain whether allopurinol, usually administered to such patients, and its metabolite oxypurinol, or metabolites abnormally increased in HPRT deficient erythrocytes (NAD, PPribP) could directly affect NAPRT and NADs in normal erythrocytes and thus be responsible for their increase in patients. Since allopurinol is known to affect orotate phosphoribosyltransferase (OPRT)- OMPdecarboxylase (ODC) activities [3], these enzymes were used as control. MATERIALS AND METHODS: Specific activities of NAPRT, NADs, APRT, HPRT, OPRT and ODC were measured in erythrocyte lysates by previously described HPLC-linked methods [5,6] in experimental conditions mimicking the metabolic situation of HPRT deficient erythrocytes. Conditions included addition of allopurinol, oxypurinol, NAD, PPribP to the assay mixtures; incubation of erythrocytes before lysis with PPribP generating medium either alone or plus nicotinate (increasing NAD production), plus allopurinol or oxypurinol. The endogenous nucleotide pattern was also measured in pre-incubated erythrocytes. RESULTS: No remarkable increase of NAPRT and NADs activities in the above conditions mimiking HPRT deficient erythrocytes was found. Direct effect by the mentioned metabolites was ruled out (only a mild protection of PPribP on NAPRT activity). Nucleotide pattern did not show any alteration following incubations (except for NAD increase with NA, and for respective nucleoside monophosphates appearing with allopurinol or oxypurinol), nor were enzyme activities increased, except OPRT and ODC as expected after allopurinol and oxypurinol incubation. Data were confirmed in allopurinol-treated patients. 1) M.Lesch , W.L. Nyhan, Am. J. Med., 36 561-570 (1970) 2) H.A. Simmonds et al., Clin. Chim Acta 171 197-210 (1988) 3) T.D. Beardmore et al. J.Lab.Clin. Med., 81 43-52 (1973) 4) R.Pesi et al. Neuroreports, 11,9, 173-177 (2000) 5) V.Micheli et al., Life Sci, 64,26,2479-2487 (1999) 6) V.Micheli et al., Bioch.Biophys Acta,1587, 45-52 (2002)

105 P14

Effects of hypoxanthine on adenosine transport in human lymphocytes. Implications in the pathogenesis of Lesch-Nyhan syndrome

R.J. Torres, I. De Antonio, C. Prior, J.G. Puig La Paz Hospital, MADRID, Spain

Introduction: We postulate that hypoxanthine excess could be implicated in the pathogenesis of the neurological symptoms of Lesch-Nyhan patients. This hypoxanthine excess could affect adenosine function by altering adenosine transport or adenosine receptor binding. The purpose of this study is to examine the effect of hypoxanthine on adenosine transport and receptor- mediated adenosine action in human cells. To this approach, we have studied the effect of hypoxanthine on adenosine transport in peripheral blood lymphocytes (PBL) cultures. Methods: [2-3H] adenosine transport, and [3H] NBTI binding assays were determined in basal conditions and after 24h incubation with hypoxanthine at different concentrations. Results: 1. Hypoxanthine at concentrations ranging from 1 to 50 uM concentrations caused a dose-dependent decrease on adenosine transport after 24 hours incubation-time. 2. Hypoxanthine at 25 uM originates a significant decrease, versus control cells, of the Vmax for the adenosine transport in PBL cultures (9 +- 0,11 vs. 19 +- 0,5 pmol/106 cells/min; p<0.001). 3. Hypoxanthine originates a dose-dependent reduction, in the number of [3H] NBTI binding sites per cell in PBL cultures. 4. Hypoxanthine at 25 uM originates a significant decrease of the Bmax for NBTI with respect to their respective control cells (7,880 +- 322 vs. 9873 +- 404 high affinity sites per cell; p<0.001) in PBL cultures. Conclusions: This study shows that an excess of hypoxanthine reduces adenosine transport in cultured PBL.

106 P15

EPR spin trapping of a radical intermediate in the urate oxidase reaction

E. Marinello, E. Busi, L. Terzuoli, R. Basosi, B. Porcelli University of Siena, SIENA, Italy

Urate oxidase is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin, occupying a pivotal position in the chain of enzymes responsible for the metabolism of purines. The end product of purine metabolism varies from species to species. In bacteria and some marine invertebrates, purines are degraded via uric acid, allantoin, allantoic acid, and urea, and then to ammonia and carbon dioxide. With the phylogenetic evolution, the chain of reactions necessary to convert purines to their final metabolic products has become progressively truncated through successive loss of urease, allantoicase, allantoinase, and urate oxidase. Most mammals are known to contain urate oxidase and thus excrete allantoin as the end product of purine metabolism, whereas man, arthropoid apes, and some genera of New World monkeys remain the only mammals which lack this enzyme, and as a consequence excrete uric acid as the end product of purine metabolism. Uric acid is also the end product of purine metabolism in birds and some reptiles. The uricase has often been classified as a copper-dependent enzyme, but in bacteria, yeast, fungi and bovine liver is free of copper. Transitory intermediates have been reported to occur during the uricase reaction. The proposed mechanism assumes an oxidation by O2 during the course of the enzyme-catalysed reaction and the evolution of CO2 during the decay of the intermediate. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR- spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (hog liver, porcine liver, Bacillus Fastidiosus, Candida Utilitis), was performed in the presence of DEPMPO (5- diethoxyphosphoryl-5-methyl-pyrroline-N-oxide) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. The generation of an urate radical, using Fe(II) and H2O2, in the presence of DEPMPO, gave the same radical adduct. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.

107 P16

Cognitive abilities in a series of patients with Hypoxanthine guanine phosphoribosyltransferase deficiency

A. Verdu1, R.J. Torres2, I. De Antonio2, J.G. Puig2 1Hospital Virgen de La Salud, TOLEDO, Spain 2La Paz Hospital, MADRID, Spain

OBJECTIVE To estimate cognitive abilities in a series of 12 children with deficiency of hypoxanthine guanine phsophoribosyltranseferase

METHODS Estimates of some aspects of cognition by the use of 1. Standardized IQ test (Kaufman Brief Intelligence Test,K-BIT), 2. Standardized Test of Non Verbal Intelligence based in abstrac/visual reasoning (TONI-2), 3. A questionnaire designed to systematize caregiver observations. Age range form 7 years 2 months to 17 years 6 months. Each subject tested in 3 sessions of 30-40 minutes.

RESULTS The mean estimated IQ (K-BIT) was 53.4 (range 43-78). The mean IQ as measured by TONI-2 was 83.7 (range 72-110). Questionnaire:-Good range of self-awareness, sociability, alertness, orientation, emotional reactions and memory for past events, -Mild to moderate impairment for short term memory, -Moderate impairment for attention/concentration.

CONCLUSIONS 1. Most of these subjects show a mild to moderate cognitive impairment. There seems to be a typical pattern of weakness and strenghts (verbal reasoning more affected than visual/abstract reasoning). 2. Mental retardation is not invariable as some subjects show a low average intelligence. 3. All subjects showed deficient attention/concentration and self-abusive tendencies that interfered with the testing process. 4. The social and emotional behaviour of patients with Lesch-Nyhan disease suggests that cognitive abilities may be better than showed by standardized tests.

108 P17

The motor features of Lesch-Nyhan disease

J.E. Visser1, S.G. Reich2, J.C. Harris2, G. Barabas3, G.E. Eddey3, H.A. Jinnah2 1UMC St Radboud, NIJMEGEN, The Netherlands 2Johns Hopkins Hospital, BALTIMORE, MD, United States of America 3Matheny School and Hospital, PEAPACK, NJ, United States of America

Introduction. Deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND), characterized by hyperuricemia and a neurobehavioral phenotype including self-injurious behavior, cognitive dysfunction, and motor disability. Although the genetic, metabolic and behavioral aspects of LND have been addressed in several studies, the nature of the motor disorder has received less attention. Many reports describe LND as choreoathetosis with spasticity. However, other reports describe dystonia with hypotonia. These marked differences in the published literature might arise from phenotypic variability, temporal variability due to disease progression, or varied use of descriptive terminology. To distinguish among these possibilities, we conducted a prospective evaluation of the motor disorder in our patients.

Methods. The patient population consisted of 19 cases ranging from 6-40 years of age. The diagnosis was based on HPRT enzyme activity <1% with typical hyperuricemia, self-injurious behavior, and motor disability. Variant cases with higher levels of residual HPRT and those without self-injury were excluded. A detailed neurological examination was conducted by a specialist in motor disorders and videotaped for review by a second examiner. Descriptive terminology was applied according to established guidelines, with a 4-point severity scale (0=absent, 1=mild, 2=moderate, 3=severe).

Results. Dystonia was the most common and most severe motor feature in all 19 cases (average score, 3.0±0.0). Chorea was observed in 13 cases and it was generally not severe (0.8±0.7). Ballismus was observed in 7 cases, again not severe (0.4±0.6). Hypotonia was evident in 16 cases. Spasticity occurred in only 5 cases and was generally mild (03±0.6). In most of these cases, spasticity of the legs or one arm was combined with hypotonia elsewhere. Mild rigidity occurred in 2 cases. True cerebellar ataxia was not observed.

Conclusions. The motor syndrome of LND is best described as an action dyastonia superimposed upon hypotonia. Although other extrapyramidal signs such as chorea and ballismus may occur, these are less frequent and less severe than dystonia. True spasticity is not common; when it occurs it is often asymmetric or limited to the legs in a pattern suggestive of myelopathy. This motor syndrome is consistent with dysfunction of the basal ganglia.

109 P20a

Lesch Nyhan Disease, essentials of diagnosis and management - a European perspective

G.T. McCarthy1, H.A. Simmonds2, L.D. Fairbanks2 1Chailey Heritage Clinical Services, NR LEWES, EAST SUSSEX, United Kingdom 2Guy's Hospital, LONDON, United Kingdom

The purposes of this presentation are to describe the essential diagnostic features and investigations of this very rare condition; to share methods of management, which have been found to be effective; to promote the need for support between families; and to outline research in progress; finally to promote the benefits of pooling data across Europe via the internet. WHAT IS LESCH NYHAN DISEASE? A description of LND will introduce the poster followed by presentation of the condition over time DIAGNOSIS the essential diagnostic investigations will be explained and problems which can mask the diagnosis GENETIC INFORMATION This includes X linkage, carrier detection using DNA probes, pre-natal diagnosis. genetic counselling MANAGEMENT OF THIS COMPLEX CONDITION Motor Disorder Speech and Communication Management of self-injurious behaviour - psychological management including parental understanding of stress management; intellectual development Restraints Medical Problems associated– swallowing, hiatus hernia, nutrition, seizures, apnoeic attacks; dietary management.

Medical Treatment Allopurinol medication and the need for regular metabolic and renal monitoring will be explained. RECENT RESEARCH ADVANCES: i) imaging techniques, ii) investigation of neuronal development in HPRT deficiency, iii) studies of self-injurious behaviour in young children with LNS EUROPEAN PERSPECTIVE Princess Margaret Nucleotide Metabolism Centre Why is a collaborative approach essential? FAMILY SUPPORT NETWORKS: - sharing information across Europe i). The Purine Metabolic Patients Association (PUMPA) in the United Kingdom; ii) Baschirotti Institute Italy, Paola Cargiolli; iii) LNS Francais.

110 P20b

Nucleotide degradation products in cerebrospinal fluid (CSF) in inherited and acquired pathologies

H.A. Simmonds1, L.D. Fairbanks1, J.A. Harris2, J.A. Duley1 1Guy's Hospital, LONDON, United Kingdom 2Johns Hopkins Hospital, BALTIMORE, MD, United States of America

CSF nucleotide degradation products have been reported in neurologiclal disorders, meningitis, renal failure and hypoxia. The present study, using RPLC, compares concentrations in patients genetically deficient in adenylosuccinase (ADSL), purine nucleoside phosphorylase (PNP), Lesch-Nyhan Disease (LND: hypoxanthinephosphoriboxyltransferase deficiency HPRT) and its variants (the majority on allopurinol), with neurological controls and in tuberculous meningitis. The results a) confirm grossly elevated concentrations of SAICAR 314µmol/l (control mean <0.01) and SAdo 401µmol/l (control 0.9) in ADSL, but normal hypoxanthine and xanthine; b) demonstrate the absence of purine bases in PNP, inosine replacing hypoxanthine in concentrations comparable to hypopcanthine in LND, but concentrations of guanosine are 3 fold that of xanthine in untreated LND; c) confirm hypoxanthine alone is grossly elevated in LND not on allopurinol. Although hypoxanthine and xanthine are elevated by allopurinol, concentrations are not different between classic LND and partial variants and oxipurinol is present in all on allopurinol; d) show grossly elevated uric acid and its precursors (xanthine, hypoxanthine and inosine) in tuberculous meningitis. Although CSF purines in these genetic disorders are elevated with the defect, as in plasma, concentrations of uric acid are uniformally low compared with plasma. (except PNP - virtually absent in both). This finding, together with the presence of oxipurinol in allopurinol-trested LND patients, indicates a limited ability of plasma components to cross the normal blood-brain barrier and confirms the absence of functional xanthine dehydrogenase in human brain. The elevated hypoxanthine relative to xanthine in untreated LND illustrates the normal recycling of hypoxanthine via HPRT to ATP in human brain. The higher CSF guanosine in PNP contrasts with the inosine/guanosine ratio in plasma and suggests the turnover of GTP is high. However, the normal xanthine in LND argues against this. The finding of detectable amounts of SAICAR as well as SAdo only in LND, accords with some increased activity of the synthetic route in the absence of hypoxanthine salvage via HPRT. The elevated uric acid plus precursor purines in tuberculous meningitis confirms severe damage to the normal blood-brain barrier.

111 P21

Genotype and clinical features of adenine phosphoribosyltransferase (APRT) deficiency in French families

I. Ceballos-Picot, S. Chevaliez, H. Lassal, D.M. Daudon, P. Perignon University of Paris, PARIS, France

The purpose of our study was to characterize the clinical, diagnostic, and pronostic features of complete adenine phosphoribosyltransferase (APRT) deficiency in french patients, as well as to determine their genotype. Twenty one individuals with complete APRT deficiency (type I), (diagnosed by enzymatic APRT activity determination in erythrocytes and analysis of urinary calculi or crystals by infrared spectroscopy) were identified in 18 families from 1990 to 2003. There were 13 females and 8 males, and the median age at diagnosis was 28 years (range 1 to 72 years). 60% of cases were adults, suggesting that total APRT deficiency is not essentially a disease of children as was once thought. However the time from onset to correct diagnosis was usually much longer in adults. Eighteen patients were index cases and three asymptomatic adult patients were diagnosed during screening of first-degree relatives. This screening is important because an effective treatment with allopurinol is available if the condition is diagnosed early and the prognosis depends on renal function remaining at the time of diagnosis. The eighteen index cases had symptomatic disease. 2,8- dihydroxyadenine (DHA) urolithiasis led to mild and moderate renal insufficiency for 15 patients; 3 patients had advanced renal failure. The analysis of genomic alteration performed on ten patients, by polymerase chain reaction-based mutation analysis, revealed for six patients a T insertion at the exon4/intron4 junction (1833insT) resulting in the lack of exon 4 in the APRT mRNA or a A/T substitution at this junction (1833+1A- T. The other mutations are located in exon 2 (295C-G), exon 3 (1422G-A and 1444delCT) and exon 5 (2079insA; 2087T-C; 2176delTTC). These results showed a mutational hot spot at the intron 4 splice donor site (probably resulting in the lack of exon 4 in the APRT mRNA) in French patients which was also detected in a variety of Caucasian patients throughout Europe.

Supported by EC project BMH4-CT98-3079

112 P22

Adenylosuccinate lyase deficiency: study of physiopathologic mechanism(s)

V. Race, S. Marie, G. Van den Berghe, M.F. Vincent ICP and Catholic University of Louvain, BRUSSELS, Belgium

Adenylosuccinate lyase (ADSL) is an enzyme catalysing two distinct reactions in purine nucleotide synthesis. ADSL deficiency, first described in 1984, is characterised by the accumulation, in cerebrospinal fluid (CSF) and urine, of succinylaminoimidazolecarboxamide (SAICA) riboside and succinyladenosine (S-ado), the dephosphorylated derivatives of the two substrates of the enzyme, SAICA-ribotide and adenylosuccinate (SAMP). The symptoms of the disease are exclusively neurologic and the affected children display variable but mostly profound psychomotor retardation, often accompanied by epilepsy and autism. This work aims at unraveling the physiopathologic mechanism(s) leading to the selectively neurological symptoms. We investigated two major hypotheses: (i) a deficiency in the terminal products of the de novo pathway and (ii) a toxic effect of the succinylpurines (SAICAriboside and S-ado) which accumulate in huge amounts in CSF. To test the first hypothesis, we measured intracellular nucleotide concentrations in skin fibroblasts from 7 severely and 1 mildly retarded patients. Since these fibroblasts still exhibit a residual ADSL activity, we rendered them more dependent on de novo synthesis by abolishing the purine salvage pathway. The intracellular concentrations of adenylic, guanylic and uridylic nucleotides were measured by HPLC: no significant differences could be detected as compared to control cells. An exception was an accumulation of SAMP which was only detected in the fibroblasts from the patient with a near complete loss of activity toward SAMP, who is paradoxically mildly retarded. We next considered a possible neurotoxic effect of the succinylpurines. This effect was evaluated on the viability of rat embryonic cortical neurones using the MTT test. Neither SAICAriboside nor S-ado affected cell viability. The possibility of an interaction between the succinylpurines and the ionotropic glutamate NMDA receptor was excluded by the facts that they did not affect (i) glutamate toxicity (ii) binding of MK801 on the NMDA receptor (iii) calcium movements inside the cell. Until now, our results show that an explanation regarding the cause of the neurological manifestations in ADSL deficiency remains elusive.

113 P23

Adenylosuccinate lyase deficiency - first UK case

A.M. Marinaki1, M. Champion1, M.A. Kurian1, H.A. Simmonds1, S. Marie2, M.F. Vincent2, G. Van den Berghe2, J.A. Duley3, L.D. Fairbanks1 1Guy's Hospital, LONDON, United Kingdom 2ICP and Catholic University of Louvain, BRUSSELS, Belgium 3University of Queensland, QUEENSLAND, Australia

Adenylosuccinate lyase (ASDL) catalyses two steps in purine synthesis - the 8th step in the de novo pathway: conversion of SAICAR (succinylaminoimidazole-carboxamide ribotide) to AICAR (aminoimidazole- carboxamide ribotide), and conversion of S-AMP (adenylosuccinate) to AMP (adenylate) in the purine nucleotide cycle. SAICAriboside (SAICAr) and S-Ado(succinyladenosine) accumulation occurs when this enzyme is deficient. Clinical presentations vary from mild psychomotor retardation to epilepsy with convulsions starting within the newborn period. The lower the ratio of S-Ado/SAICAr in body fluids the more severe the clinical presentation.

The female patient presented on day 14 with a progressive neonatal encephalopathy and seizures (myclonus and generalised tonic clonic events). Neurological examination revealed marked axial and peripheral hypotonia, MRI showed widespread white matter changes. She died at 4 weeks of age.

Analysis of urine by HPLC showed SAICAr and S-Ado present in large quantities (S-Ado 0.925mmol/l : SAICAr 1.325mmol/l (ratio 0.7), correlating with the severe clinical picture in the patient. Further investigation detected these compounds in the plasma (34µmol/l/27µmol/l) and CSF(921µmol/l/477µmol/l) respectively.

Fibroblast cultures were grown, and the molecular analysis undertaken. cDNA analysis showed an 9G>C transition resulting in a A3P amino acid substitution. Sequencing of genomic DNA showed the patient was heterozygous for this mutation. The patient is thus a compound heterozygote with this novel 9G>C mutation on one allele and a second, still unknown, mutation on the other. It is possible that mRNA expressed from the second allele is unstable.

This is the first case of ADSL to be reported in the UK. In a previous study 10,000 urines, from a selected population of UK children with undiagnosed neurological deficits, were screened for SAICAr and S-Ado and no cases were detected. Thus, ADSL deficiency is very rare in the UK.

114 P24

Elevated erythrocyte CDP-choline levels associated with b-thalassaemia in patients with transfusion independent anaemia

A.D.J. Laurence1, M. Layton2, J.A. Duley3, H.A. Simmonds3 1University College Hospital London, LONDON, United Kingdom 2Imperial College, LONDON, United Kingdom 3Guy's Hospital, LONDON, United Kingdom

Haemolytic anaemia with basophilic stippling is characteristic of pyrimidine 5’-nucleotodase (UMPH1) deficiency (1). The selective accumulation of CDP-choline in high concentrations in the erythrocytes of a patient with haemolytic anaemia and a normal UMPH1 activity was first reported by Paglia et al (1). A defect in CDP-choline phosphotransferase which catalyses the last stage of lecithin biosynthesis was suggested. The selective accumulation of CDP-choline is thought to occur in the erythroblast as CDP-choline phosphotransferase has been shown to be inactive in the mature erythrocyte. The accumulation of CDP- ethanolamine as well as CDP-choline in a small cohort of patients with normal UMPH1 activity and either unexplained haemolytic anaemia, or secondary to chronic renal failure, led us to postulate the existence of a defect in both phosphotransferases. Correction of the haematological profile following successful renal transplantation in one renal failure patient implied possible heterozygosity for this defect (2)

Here we report a series of ten patients with transfusion independent b-thalassaemia; eight heterozygote and two heterozygote with Hb E, which also show elevated levels of both CDP-choline (86.4uM +/- 48µM) and CDP- ethanolamine (34.6µM +/- 34.5µM). This compares with levels of less than 3µM in healthy controls. In contrast to the patients previously reported with raised CDP-choline/ethanolamine associated with no other cause of their anaemia, the patients in this report all had CDP-choline levels in excess of their CDP- ethanolamine (p=0.002).

In summary elevated CDP-choline in patients with no defined cause for their haemolytic anaemia. has been suggested as a possible indicator of CDP-choline phosphotransferase deficiency Here we associate it with transfusion independent b-thalassaemia.

(1) Paglia DE, Valentine WN, Nakatani M, Rauth B (1983) Proc Natl Acad Sci 80;3081-3085. (2) Laurence A, Duley JA, Simmonds HA (1998) Adv Exp Med Biol 155-159

115 P25

Nucleotides can afford protection in cirrhosis induced by thioacetamide

S. Pawa Jamia Hamdard University, NEW DELHI, India

The present work was intended to study nucleotides as anti-hepatotoxic agent against thioacetamide (TAA)- induced cirrhosis in Wistar rats. The liver pathology of the Wistar rats treated chronically with TAA closely mimics that of human disease Cirrhosis was induced by oral intake of TAA in drinking water (300 mg/l) for four months. Nucleotides pretreatment significantly decreased lipid peroxidation, which is an important index of oxidative stress. Thioacetamide caused decrease in the activities of peroxide-metabolizing enzymes (glutathione peroxidase, and catalase), and a noteworthy increase in the activity of xanthine oxidase (XO). XO catalyzes the hydroxylation of a wide variety of purines, pyrimidines, and nitrogenous compounds and generates reactive oxygen species as by-product. Pretreatment with nucleotides resulted in attenuation of biochemical parameters associated with TAA-induced cirrhosis. Appreciable regeneration of the injured liver was also seen in nucleotide-pretreated group. The mechanism by which these nucleotides influence tissue repair is unclear, however, it can be hypothesized that it may manifest its effect via xanthine oxidase pathway. Increase in the activity of XO by TAA results in significant depletion in purines and pyrimidines, thereby aggravating tissue damage. Supplementation with nucleotides replenishes the loss caused by TAA, thereby acting as potential hepatoprotectant.

116 P26

Serum Folate Level in Mozambique's Children

E. Marinello1, R. Leoncini1, R. Guerranti1, G. Cinci1, A. Tabucchi1, F. Carlucci1, D. Vannoni1, A.B. Agostinho2 1University of Siena, SIENA, Italy 2National Institute of Saude, MAPUTO, Mozambique

Folate and vitamin B12 deficiencies are two unequivocal nutritional anemias in man. Folate is essential for normal development of red blood cells and is essential, as coenzyme, in nucleotide metabolism, DNA and RNA synthesis. It is involved in protein metabolism as well as tissue growth and cell function. It has been used in the prevention and treatment of folic acid anemia. Measurements of folate levels constitutes a direct and reliable means of determining the existence of folate deficiency. Vitamin B12 is essential for normal folic acid metabolism; it helps the formation of red blood cells and the maintenance of central nervous system. The deficiency or incapacity of absorption caused the disease known as pernicious anemia. We have made a study on seric Folate/Vitamin B12 content in children frequenting schools of Maputo (Mozambique). The children were healthy and subjected to complete blood count and many different tests (glycemia, cholesterol, Ca, Mg, total protein, albumin, Fe); also weight and height were registered. The subjects were 173 (age ranging between 6 and 16 years) subdivided in 80 males and 93 females. No analytical parameter showed statistical difference related to sex for this reason we have studied the children as an unique group. The values that we have found for serum folate and vitamin B12 are respectively: 6.32pg/ml ± 3.47 and 782.70ng/ml ± 573.07. All the data have passed the Kolmogorov-Smirnov test for normal distribution. The analysis of correlation between various analyte has shown an interesting relationship between folate and platelet content. The equation describing this relationship is: [folate]= 0.01573[platelet]+3.574 with p<0.0001 (extremely significant). Also MVC and HCT show an extremely significant positive correlation with folate content. These data are not underlined in previous papers in literature and could reflect a stimulating and specific effect of folate on cell proliferation and could be an interesting approach to study the nucleotide metabolism in these subjects, and specifically in their megakaryocytes. The same study could be replicated in Italian scholar population.

117 P27

Deoxyuridine accumulation in urine in thymidine phosphorylase deficiency (MNGIE)

A.M. Marinaki1, J.A. Duley2, E.A. Carrey1, S.R. Hammans3, L.D. Fairbanks1 1Guy's Hospital, LONDON, United Kingdom 2University of Queensland, QUEENSLAND, Australia 3Southampton General Hospital, SOUTHAMPTON, United Kingdom

INTRODUCTION. The autosomal recessive condition mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by a deficiency of thymidine phosphorylase (TP) and is characterised by mtDNA multiple deletions and depletion. TP catalyses the catabolic conversion of thymidine to thymine. Thymidine accumulates in the urine of TP deficient patients and is a diagnostic marker for the disorder. We report the presence of the unusual nucleoside deoxyuridine in the urine of a patient with MNGIE due to TP deficiency. RESULTS. A random urine specimen from a previously described female MNGIE patient now 38 years old (Science 1999, 283: 689-92) was fractionated by anion exchange LC prior to reversed phase HPLC. An unknown peak co-eluting with uric acid, visible after fractionation or incubation with uricase, was identified as deoxyuridine (150 µmol/mmol creat, normally not detected), by retention time, shared spectral characteristics to an authentic deoxyuridine standard and co-elution of the peak with an added standard. Thymidine (125 µmol/mmol creat, normally not detected), uracil (77 µmol/mmol creat, control range 1.4-15.4 µmol/mmol creat), and thymine (48 µmol/mmol creat, normally not detected) were also elevated. Interestingly, uric acid production on a creatinine basis was grossly raised (0.8 µmmol/mmol creat, normal range 0.3+/- 0.05), but may be a consequence of cachexia since the patient studied was severely underweight. DISCUSSION. Our results confirm that thymidine is a useful marker in screening for TP deficiency. However, raised uracil and thymine are also found in dihydropyrimidine dehydrogenase and dihydropyrimidinase deficiencies. The presence of deoxyuridine in urine of TP deficient patients thus provides a second unique metabolic marker for MNGIE. We propose a mechanism of increased intracellular thymidine pools leading to the synthesis and accumulation of the nucleotide TMP. Thymidylate synthetase is inhibited by raised levels of TMP, resulting in the accumulation of dUMP. We suggest that dUMP then has two metabolic fates: degradation by nucleotidase to deoxyuridine and excretion in the urine; or phosphorylation to dUTP. Incorporation of dUTP into mtDNA would be consistent with the pattern of mtDNA deletion and depletion characteristic of TP deficiency.

118 P28

The Genetic Basis of the Interaction Between Pyrimidine 5' Nucleotidase deficiency and Hemoglobin E

E. Escuredo1, J.A. Duley2, D.C. Rees3, A.M. Marinaki1 1Guy's Hospital, LONDON, United Kingdom 2University of Queensland, QUEENSLAND, Australia 3King's College Hospital, LONDON, United Kingdom

INTRODUCTION. Hemoglobin E is a hemoglobin variant, occurring with a polymorphic prevalence throughout Southeast Asia and Northeast India. We have previously described a family in which an interaction between a biochemical deficiency of pyrimidine 5’ nucleotidase (P5’N-1) deficiency and HbE resulted in an unexpectedly severe anemia with features of hemolysis and dyserythropoiesis. In this study we explored the genetic basis of the severe clinical phenotype. RESULTS. A novel P5’N-1 splice site mutation (IVS8+1-2 delGT) was found in the family by genomic sequencing of the P5’N-1 gene, and is predicted to result in skipping of exon 8 and premature termination of the protein. A homozygous IVS8+1-2 delGT genotype correlated with a severe phenotype in the propositus when a homozygous HbE genotype was co-inherited. However, the phenotype of HbE trait appeared to be unchanged by the co-inheritance of a single P5’N-1 mutant allele. The ratio of P5’N-1 activity to that of deoxypyrimidine nucleotidase (P5’N-2) in HbE heterozygous and homozygous patients with a normal P5’N-1 genotype, were decreased (P5’N-1/P5’N-2 0.24-0.55, controls >0.7). Pyrimidine metabolism was also studied in the erythrocytes of 6 patients with HbE/beta-thalassemia. The compound heterozygous genotype, HbE/beta-thalassemia is an important cause of significant thalassemia, resulting in a variable, often severe condition. The variation in clinical phenotype is largely unexplained and it is possible hemoglobin instability may be exacerbated by acquired or genetic deficiency of P5'N-1. Inhibition of P5’N-1 relative to P5’N-2 was variable (range 0.39-0.97). Although there was a trend towards lower enzyme ratios with decreasing Hb and an increasing percentage of HbF, this was not significant. CONCLUSION. The severe phenotype seen in the propositus in this study consequent on the two genetic defects raises the possibility that acquired or inherited variation in P5'N-1 activity may modulate the severity of HbE and other beta-thalassemia syndromes.

119 P29

Purine nucleoside phosphorylase deficiency: Is there a mitochondrial pathology?

A.M. Marinaki1, J.A. Duley2, A.R. Gennery3, L.D. Fairbanks1 1Guy's Hospital, LONDON, United Kingdom 2University of Queensland, QUEENSLAND, Australia 3Newcastle Upon Tyne NHS Trust, NEWCASTLE, United Kingdom

INTRODUCTION. Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive disorder of purine metabolism. PNP-deficient children present early with a range of neuromuscular problems including spastic diplegia, tetraparesis, ataxia, hypertonia or hypotonia, tremor, behavioural problems, varying degrees of mental retardation, and are typically diagnosed as cerebral palsy. Neurological symptoms often precede immunodeficiency due to severe T-cell depletion and dysfunction. B-cell dysfunction may lead to autoimmunity. Biochemically, the disorder is characterised by low plasma uric acid and gross purine overproduction in the form of (deoxy-)nucleosides in the urine. Red cell nucleotide abnormalities include accumulation of dGTP with low GTP, and raised NAD. FAMILY STUDY: The first child (male) presented with an autoimmune haemolytic anaemia and was diagnosed as PNP deficient at 2 years. He died post-BMT of gut graft-versus-host disease. The propositus (female) was the second child in the family. A prenatal diagnosis of an affected child was made at 12 weeks, but the mother elected to continue with the pregnancy as a suitable bone marrow donor had been identified. The child was transplanted at 2 months and received immunosuppressive therapy (CyA and high dose methyl- prednisolone). Neurologically, the child appears well and developmentally normal, active with eye contact and smiling. She is mildly floppy but without clear evidence of neuromuscular disorder as at 8 months age. However, at 3 months post-BMT she developed a large pericardial effusion and hypertrophic obstructive cardiomyopathy (HOCM), very rare in a child this age, was diagnosed. This has been accompanied by pneumonitis, refluxing and aspiration difficulties which will require fundoplication. MOLECULAR STUDIES. Sequencing of PNP cDNA showed a homozygous 383A>G transition, heterozygous in the parents, resulting in a Asp128 to Gly substitution. This mutation has been previously reported in PNP deficiency CONCLUSION. Studies of a PNP deficient mouse model suggest that the accumulation of dGTP in mitochondria results in abnormal mtDNA repair. This raises the intriguing possibility that the cardiomyopathy in the propositis may have a mitochondrial pathogenesis linked to PNP deficiency through an imbalance in nucleotide pools.

120 P30a

Urinary methylxanthine and autistic disorder: absence of previously reported correlation

C. Salerno University of Rome 'La Sapienza', ROME, Italy

Although the exact prevalence of biochemical defects in autism is unknown, inherited disorders on purine biosynthesis and interconversion, such as adenylosuccinate lyase deficiency and 5'-nucleotidase superactivity, have been often associated with autistic symptoms, focusing the attention on the metabolic fate of endogenous and exogenous purines in patients with behaviour abnormalities. Great expectations in patient's families arose from recent reports [1, 2] that 7-methylxanthine levels in urine samples are greatly decreased in a significant fraction (47%) of autistic children, while urinary xanthine levels are increased. Consequently, it was proposed that determination of methylxanthine and xanthine in the urine has a diagnostic value for autism and that methylxanthine could be used to treat individuals exhibiting symptoms of autistic disorder. We developed a HPLC method for separation of urinary uric acid, xanthine, hypoxanthine, and most of the methylxantine metabolites (caffeine, theophylline, theobromine, 1- methylxanthine, 1-methylurate, 3-methylxanthine, 3-methylurate, 7-methylxanthine, 7-methylurate). Urinary levels of methyxanthines were strictly dependent on the diet. Theobromine, 7-methylxanthine, 7- methylurate, and 2-methylxanthine were identified in the urine upon administration of chocolate. Comparison of data obtained with 59 autistic patients and 64 age- and sex-matched normal volunteers did not reveal any difference in the levels of xanthine and methylxanthine derivatives in the two groups. In light of these findings, it appears that abnormalities in methylxanthine and xanthine excretion cannot be used to discriminate autistic disorders and represent distinctly uncommon symptoms in autism.

[1] Zhang J, Rivers G, Peyser J, Zack B, Horvath K, Rushe J (2000) Analysis of compounds in the urine of autistic children with HPLC and mass spectrometry. World Congress of Pediatric Gastroenterology, Boston [2] Rusche JR, Zhang J (2002) Methylxanthines in the diagnosis and treatment of autistic disorder. US Patent Application 20020019407 A1

121 P30b

Novel mutations and a 'hot-spot' in Purine Nucleoside Phosphorylase deficient patients

E.G. Grunebaum, J. Zhang, C.M. Roifman Hospital for Sick Children, TORONTO, Canada

The lack of purine nucleoside phosphorylase (PNP) enzyme activity in human and in mice results in thymus abnormalities and T lymphocyte dysfunction. Patients with PNP deficiency typically suffer from recurrent infections, neurologic impairment and autoimmune phenomena. Bone marrow transplantation is available for some of these patients and ongoing research in gene therapy may provide an alternative approach, particularly if the transformed cells have a survival advantage over the patient’s naïve cells. The laboratory findings of lymphopenia, reduced serum uric acid and abnormal PNP enzyme activity assist in the diagnosis of PNP-deficient patients, which is further confirmed by genetic analysis. Since the identification of the gene coding for the PNP protein, several mutations have been found. Here we report 3 novel mutations in 3 unrelated patients recently diagnosed at the Canadian Center for Primary Immunodeficiency Diseases as suffering from PNP deficiency. These include a G348A, C700T and C769G, resulting in putative Ala117Thr, His257Asp and Arg234Stop, respectively. Two of these patients also had a C to T transition at codon 172, which leads to a premature stop at amino acid 57. Combined with mutations previously described in the literature, the C172T change appears to be particularly common among patients with PNP deficiency. It was found in unrelated families originating from Asia, North-America and Europe thereby suggesting that this is a specific “hot spot” in the PNP gene. The C172T abolishes the only Taq1 restriction site in exon 2, enabling rapid diagnosis of this mutation. The identification of mutations in the PNP gene may allow better genetic counseling as well as early intrauterine or even pre-implantation confirmation of PNP-deficiency. Restriction site analysis for the more frequent mutations enables quicker and easier diagnosis.

122 P31

The frequency and diversity of the haplotypes near adenine phosphoribosyltransferase (APRT) locus are different between patients with APRT deficiency and normal controls

A. Taniguchi Tokyo Women's Medical University, TOKYO, Japan

The susceptible genes for common diseases are supposed to be common origin among different families (common disease common variant hypothesis). Based on this hypothesis, the haplotype near a susceptible gene among patients should be different from normal people, suggesting a genome-wide association studies utilizing haplotypes of SNP are powerful method to search susceptible genes for common diseases. The purpose of this study is to examine the potential of association studies utilizing haplotypes for detection of common variant genes. The incidence of the adenine phosphoribosyltransferase (APRT) deficiency is very high in Japanese. The carrier frequency of the most frequent mutant allele (M136T), designated as APRT*J, was estimated to be 0.73%, which showed a founder effect for APRT*J. We genotyped 80 Japanese cases with APRT deficiency including 42 cases with APRT*J/APRT*J and 93 cases with controls (APRT*1/APRT*1), for 23 SNP markers that spanned about 2000 kb. An expectation-maximization algorithm estimated 21 haplotypes (frequency > 2) from these data. Haplotype block defined by D’ (the standard coefficient of linkage disequilibrium), revealed 40 to 50 kb in APRT*1/APRT*1. The length of haplotype block of APRT*J/APRT*J were much longer than that of APRT*1/APRT*1, showing APRT*J originated from a common ancestors. The haplotype frequencies of APRT*J/APRT*J or patients with APRT deficiency including APRT*J/APRT*J are extremely different from APRT*1/APRT*1. The result of this study showed the genome-wide association studies utilizing haplotypes are valuable as a means to detect disease susceptibility genes for common diseases.

123 P32

Pyrosequencing of Thiopurine S-methyltransferase (TPMT) alleles in a Swedish general population and in IBD-patients

S. Haglund1, M. Lindqvist2, S. Almer2, C. Peterson2, J. Taipalensuu1 1County Hospital Jonköping, JONKÖPING, Sweden 2Linköping University, LINKÖPING, Sweden

BACKGROUND: The thiopurine drugs; 6-mercaptopurine, 6-thioguanine and azathioprine, are used in the treatment of leukaemia, inflammatory bowel disease (IBD) and to prevent organ transplant rejection. About 30- 40% of IBD-patients fails to benefit from this treatment. One important factor that may explain interindividual differences in therapeutic efficacy and development of adverse reactions is genetic variations in the enzymes metabolising these drugs. The therapeutic efficiency and presence of adverse reactions partly depend on the levels of thiopurine S-methyltransferase enzymatic activity. The TPMT enzymatic activity has been associated with genetic polymorphism at the gene locus for TPMT on chromosome 6. AIMS: The aims of this study were to 1) develop pyrosequencing as a tool to genotype TPMT for nine single nucleotide polymorphism’s (SNPs) known to reduce TPMT activity, 2) examine a Swedish general population (n = 800) for the most frequently reported TPMT alleles (TPMT*3A, TPMT*3B, TPMT*3C and TPMT*2) and 3) investigate the relationship between TPMT genotype and phenotype in a group of IBD-patients (n = 24) and healthy volunteers (n = 6), selected because of low, intermediate or normal TPMT enzymatic activity. In this latter group all nine SNPs were analysed. METHOD: We established a PCR and pyrosequencing based method to screen for all known SNPs resulting in reduced TPMT enzymatic activity, i.e. nine SNPs in all. RESULTS: Genotypes found in the general population were TPMT*1/*3A (*3A allelic frequency 3,8%), TPMT*1/*3C (*3C allelic frequency 0,44%), TPMT *1/*3B (*3B allelic frequency 0,13%) and TPMT*1/*2 (*2 allelic frequency 0,06%). None of the nine individuals judged as normal metabolisers from their enzymatic activity were carrier of any of the investigated SNPs. Eight individuals with low activity were genotyped as either TPMT*3A/*3A (n=4), TPMT*3A/*3C (n=2) or TPMT*1/*3A (n=2), whereas 13 individuals with intermediate enzymatic activity were genotyped as either TPMT*1/*3A (n=12) or TPMT*1/*2 (n=1). CONCLUSION: Pyrosequencing is a rapid and reliable method for precise and parallel identification of multiple sequence variants. Next to wild type, the most frequent alleles in Sweden are TPMT*3A and TPMT*3C. There was a close correlation between genotype and phenotypic expression, i.e. enzymatic activity.

124 P33a

Polymorphism in the MTHFR gene affects thiopurine methyltransferase activity

A.M. Marinaki1, M. Arenas1, J.A. Duley2, E.A. Shobowale-Bakre1, A. Ansari1, J.D. Sanderson1, L.D. Fairbanks1 1Guy's Hospital, LONDON, United Kingdom 2University of Queensland, QUEENSLAND, Australia

In this study, we investigate the effect of common genetic polymorphism in the MTHFR (methylenetetrahydrofolate reductase) gene on erythrocyte activity of TPMT (thiopurine methyltransferase). S- Adenosyl-methionine (SAM) is the methyl donor for the reaction catalysed by TPMT, and is converted to S- adenosyl-homocysteine (SAH). The adenosyl moiety of SAH is subsequently cleaved and homocysteine remethylated to methionine. A common polymorphism in the MTHFR gene, the thermolabile 677C>T variant, is associated with decreased MTHFR enzyme activity and impaired remethylation of homocysteine to methionine leading to mild homocysteinaemia. We proposed that the MTHFR 677C>T variant may indirectly affect TPMT activity.

The frequency of the MTFHR 677TT homozygous genotype was compared between 52 patients with intermediate TPMT activity (4-8U) and a wild type TPMT genotype, and 55 patients with normal TPMT activity and genotype (in the range 12-15U). The MTHFR 677TT genotype was significantly under-represented in the normal activity group (p=0.0296). In order to establish whether MTHFR genotype was associated with adverse drug reactions to AZA, MTHFR 677TT genotype frequencies in a cohort of 61 IBD patients who had experienced adverse drug reactions to AZA therapy were compared to 68 patients who had no side effects. The MTHFR 677TT genotype was not significantly associated with adverse drug reactions to AZA. Although the MTHFR 677C>T allele and 677TT genotype appeared to be under-represented in a subset of 16 patients with hepatitic, pancreatic or abdominal pain side effects, the association was not quite significant.

The accumulation of methylated thio-nucleotides in patients has been suggested as a cause of pancreatic and hepatic side effects. Our results suggest that MTFHR genotype indirectly influences TPMT activity, and imply that a wild type MTHFR genotype may be necessary for a high methylator phenotype. More patients with pancreatic or hepatitic side effects should be genotyped for MTHFR to establish the role of folate metabolism in methylated thionucleotide toxicity.

125 P33b

Allele frequency of inosine triphosphate pyrophosphohydrolase gene polymorphisms in a Japanese population

A.M. Marinaki1, S. Sumi1, M. Arenas1, L.D. Fairbanks1, S. Harihara2, K. Shimizu3, A. Ueta4, J.A. Duley1 1Guy's Hospital, LONDON, United Kingdom 2Tokyo University Japan, TOKYO, Japan 3Naruto University, NARUTO, Japan 4Nagoya City University, NAGOYA, Japan

The enzyme inosine triphosphate pyrophosphohydrolase deficiency (ITPase) catalyses the pyrophosphohydrolysis of inosine triphosphate (ITP) to inosine monophosphate (IMP). ITPase is present in many human tissues as well as RBCs and in addition to ITP, can also utilise other purine compounds as substrates. ITPase deficiency is a clinically benign autosomal recessive condition characterised by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. A deficiency of ITPase may predict adverse reactions to therapy with the thiopurine drug 6-mercaptopurine and its prodrug azathioprine. We have reported the structure of the ITPA gene and identified five SNPs in 8 families with ITPase deficiency. One missense mutation (94C>A) and one intron mutation (IV2+21 A>C) affected enzyme activities. Homozygotes for the 94C>A (Pro32 to Thr) mutation had zero erythrocyte ITPase activity while 9 heterozygotes averaged 22.5% of the control mean. ITPase activity of IV2+21 A>C homozygotes averaged about 60% of the control mean. Compound heterozygotes 94C>A/IV2+21 A>C were 10% of the control mean. In addition, three silent polymorphisms (138G>A, 561G>A and 708G>A) were found. In this study, we examine the frequencies of ITPA polymorphisms in 100 healthy Japanese individuals. DNA was extracted from blood samples after informed consent had been obtained. The 5 SNPs in ITPA gene were determined using PCR-RFLP methods. In the Japanese sample, the allele frequency of the 94C>A mutation was 0.135, twice the frequency in Caucasians (0.06). The IV2+21 A>C was not found in Japanese, although it occurred with a frequency of 0.130 in Caucasians. Allele frequencies of the 138G>A, 561G>A and 708G>A were 0.57, 0.18 and 0.06 in the Japanese population, and with the exception of the 138G>A polymorphism similar to allele frequencies in Caucasians (0.31, 0.12 and 0.05 respectively). Polymorphism in the TPMT gene are associated with adverse drug reactions to thiopurine drugs. However, the frequency of TPMT deficiency-associated alleles is reported to be <1% in Japanese populations. It is thus possible that polymorphism in the ITPA gene may explain adverse drug reactions in Japanese patients treated with thiopurine drugs.

126

P34

Genetic determinants of the thiopurine methyltransferase intermediate activity phenotype

M. Arenas1, J.A. Duley2, C.M. Lewis1, E.A. Shobowale-Bakre1, E. Escuredo1, A. Ansari1, J.D. Sanderson1, L.D. Fairbanks1, A.M. Marinaki1 1Guy's Hospital, LONDON, United Kingdom 2University of Queensland, QUEENSLAND, Australia

INTRODUCTION. Polymorphism in the TPMT gene is associated with reduced TPMT activity. Variable number tandem repeats (VNTR*3 to VNTR*9) in the promoter region of the TPMT gene are also reported to modulate the TPMT activity phenotype. In this study we investigate concordance between TPMT activity in the intermediate activity range and open reading frame (ORF) and VNTR genotypes. METHODS. Patient were selected for non-overlapping TPMT activities within the intermediate activity range (4 - 8 U, 108 patients) and normal range (12 - 15 U, 53 patients), and were genotyped for VNTR type, and TPMT*3A, TPMT*3C and TPMT*2 ORF polymorphisms. RESULTS. 41% of patients from the intermediate TPMT activity group were heterozygous for a TPMT ORF polymorphism. Marked linkage disequilibrium was noted between VNTR*6b - TPMT*3A (D’=1), VNTR*4b - TPMT*3C (D’=0.67) and VNTR*6a - TPMT*1 (D’=1) alleles. To investigate the influence of the number of VNTR repeat units on TPMT activity, the number of Type A, B or total repeat units summed for both alleles in the ORF heterozygous, wildtype intermediate and normal activity groups and were compared in an analysis of variance. For Type A and total repeats, significant differences (p<0.05) in the median number of repeats were found between the heterozygous intermediate activity group and the wild type intermediate or normal activity groups. There was no significant difference in the median Type A, B or total repeat number between the two wild type groups. CONCLUSION. Our results suggest there is poor concordance between TPMT ORF genotype and activity in the carrier range and that VNTRs do not significantly modulate basal TPMT enzyme activity.

127 P35

Association between Thymidylate Synthase polymorphism with the microsatellite instability in colorectal cancer

A. Calascibetta1, L. Rausa1, L. Gullotti1, R. Buettner2, R. Sanguedolce1 1University of Palermo, PALERMO, Italy 2University of Bonn, BONN, Germany

5-Fluorouracil (5Fu) is the main drug used for the treatment of colorectal cancer and Thymidylate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5’ untranslated region, that are polymorfic in tumours and depending on ethnic factor; more over this gene has an other polymorphism located in the 3' untraslated region, characterised by the absence of a 6 bps olygonucleotide. These polimorfisms have been reported to influence the TS-mRNA expression and the TS levels. Colorectal cancer often shows an other kind of genomic instability called Microsatellite Instability (MSI), that at present time is considered one of the most important marker of tumour genomic instability. High colorectal cancer MSI frequency is associated with a significant better survival and with a lower incidence rate of metastasis. While 5’ and 3' UTR polymorphisms are related with a particular genomic instability, the aim of the study is to try to find any relationship between the 5’ with the 3' UTR polymorphisms, the MSI, the TSmRNA expression, with the TS levels and with the prognosis and drug treatment, in 54 paraffin embedded primary colorectal cancer samples using an ABI PRISM sequencer. Our data show that the genotype 2R / 2R is more frequently associated with MSI + and lower TS levels. More over we did not find any significant association between the 2R / 3R and 3R / 3R genotypes with MSI + and MSI –, while these genotypes were associated with a higher TS expression. In the same group of patients we evaluated the presence of polymorphism in the 3' UTR: we found in only 5 patients the presence of the -6bp/-6bp genotype that it was not related with MSI, the 5' UTR polymorphism and with the TS levels. As consequence we can hypothesise that patients bearing colorectal cancer with MSI+, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant.

128 P36

Polymorphic tandem repeats in the thmidylate synthase gene and TS protein and mRNA levels in different tissues of colorectal cancer patients

R. Mauritz, I.J. Beumer, K. Smid, C.J. Van Groeningen, G.J. Peters VU University Medical Center, AMSTERDAM, The Netherlands

High expression of thymidylate synthase (TS) has been related to poor response of colorectal cancer patients to treatment with 5-fluorouracil (5-FU). Recent studies suggest that a tandem repeat polymorphism within the enhancer region of the TS gene promotor influences TS expression: the triple repeat (3R/3R) genotype might be associated with higher TS levels than the double repeat (2R/2R) or heterozygous (2R/3R) genotype. Most studies thus far focussed on the relation between TS genotype and survival of patients treated with 5-FU-based chemotherapy. Only few studies explored the relation between TS genotype and in vivo TS expression and although all reports suggest an increase in TS activity with the number of tandem repeats, conflicting data are reported concerning TS mRNA levels in relation to the TS genotype. We are currently undertaking a retrospective analysis of the TS polymorphism in normal and malignant tissues of patients with advanced colorectal cancer. DNA is obtained from either freshly frozen tissue, paraffin slides or frozen tissue sections. Subsequently the polymorphic status is assessed by PCR amplification. By now, 61 samples from 46 patients were analysed. We found 20 patients with the 2R/3R genotype, 14 with the 3R/3R and 12 with the 2R/2R variant. Conform previous studies we found a high level of homology between the TS polymorphic status of normal and malignant tissues from one patient. The TS genotypes will be correlated to TS enzyme and mRNA expression levels in addition to survival data.

129 P38a

Dihydroorotate dehydrogenase expression analysis in normal and drug-resistant cells and tissues

M.L. Loeffler1, M. Hayek-Ouassini1, K. Steger1, A. Klein2, T. Monses3, W. Knecht2 1Philipps-University Marburg, MARBURG, Germany 2Institute Physiological Chemistry, MARBURG, Germany 3Hautklinik, GIESSEN, Germany

Analysis of the temporal or permanent and spatial expression of proteins is a crucial step towards understanding gene function and regulation and elucidating physiology and pathophysiology of enzymes in metabolic pathways. Here, we present our approach and establishment of methods to follow the expression of the fourth enzyme of pyrimidine biosynthesis, dihydroorotate dehydrogenase (DHODH) protein and mRNA in various cells and tissues as precedent for further investigation. (1) Polyclonal immunoglobulins raised against DHODH, as described in Adv. Exp. Med. Biol. 431: 507-513 (1998), were applied for immunochemical quantification of the enzyme in two lines of mouse B-lymphocytes by Western blotting/ECL. The parental cells were sensitive to leflunomide (Arava), a potent inhibitor of DHODH, while adapted cells exhibited a 40-fold tolerance to the drug. The corresponding overexpression of DHODH protein was demonstrated with homogenates of theses cells. (2) The detection of DHODH mRNA expression in homogenates of tissue and cultered cells from the rat reproductive tract by means of RT-PCR was achieved. (3) By non-radioactive hybridization technique the DHODH mRNA distribution was examined in paraffin sections of the appropriate rat tissue and kidney. (4) Immunocytochemical proof of DHODH protein in sections from the same tissues verified that the pattern of expressed RNA corresponded to the distribution of the encoded protein. (5) The occurrence of protein correlated to catalytic enzyme histochemistry of DHODH that was visualized by application of the nitroblue tetrazolium/formazan technique for dehydrogenase activity in cryostat sections previously (Histochemistry Cell Biol. 105:119-128 (1996). Support: Marburg-Giessen DFG Graduiertenkolleg and P.E. Kempkes-Stiftung FB Medizin Marburg.

130 P38b

Phosphorylitic cleavage of deoxyribonucleosides catalysed by human deoxycytidine kinase - a side reaction or a function. Preliminary Biochemical and NMR Studies

E.V. Usova1, T.V. Maltseva2, S. Eriksson1 1Swedish Univ. of Agricultural Science, UPPSALA, Sweden 2Medivir AB, STOCKHOLM, Sweden

Human deoxycytidine kinase (dCK) is a key enzyme in the 5’-phosphorylation of purine and pyrimidine deoxynucleosides with dCyd as the most efficient substrate. The ability of dCK to degrade deoxyribonucleosides to free nucleobases and (most likely) deoxyribose-1-phosphate was demonstrated for the first time by 1H -31P correlation spectroscopy and by 3H-labelling methods. The new reaction was studied with regard to several factors such as concentration of enzyme and deoxyribonucleosides, temperature and pH of the buffer. The reaction depended on inorganic phosphate and all natural deoxyribonucleosides could be cleaved but the specific activity of phosphorylitic reaction compared to the kinase reaction was in most cases low, about 2-10%. However, with dThd which is very poor kinase substrate the rate of cleavage was several fold higher than its phosphorylation. The formation of free nucleobases occurred with reduced dCK because the reaction was highly dependent on the presence of reducing agents such as DTT. Thus, recombinant dCK can most likely act as a phosphorylase similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is of importance for the design of in vitro experiments with dCK and it might play a physiological role in the metabolism of some deoxyribonucleosides.

131 P39

The Effect of Signalling Pathway Modulations on the Activity of the Deoxycytidine Kinase

M. Staub, G. Keszler, Z.C. Csapó, T. Spasokoukotskaja, S. Virga, M. Sasvári-Székely Semmelweis University, BUDAPEST, Hungary

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the salvage of three natural deoxyribonucleosides as well as several therapeutic nucleoside analogues, which in turn can enhance its enzymatic activity and improve the efficacy of the cytostatic therapy, as shown earlier. The increase of dCK activity has been suggested as a compensatory mechanism of cells, induced by damage of DNA. The potentiated dCK activity is able to supply the repair of DNA with dNTPs in quiescent cells. The possible functions of signalling molecule(s) leading from DNA damage to activation of dCK have been investigated in this work. The tyrosine kinase inhibitors, Tyrphostin and Genistein, (between 10 and 300 mM) elevated dCK activity by 200% during 120 min treatment of human lymphocytes. The unspecific phosphatase inhibitor NaF (15 mM), used as an inhibitor of the small G-protein GTPase activity, can also elevate the dCK activity to 200% of the untreated activity. Calyculin A, the inhibitor of Protein Phosphatase 2A (PP2A), also increased the dCK activity in treated cells by the same range. Moreover gamma-irradiation (1-3 Grays) provoking double-strand DNA breaks, enhanced also significantly dCK activity in lymphocytes. The elevation of dCK activity induced by all treatments mentioned above, can be released by 10 mM dCyd. On the other hand, hyperosmotic stress of cells, induced by sorbitol treatment, was found to decrease the activity of dCK. Conclusion: Several signalling molecules seem to function between DNA damage and increase of dCK activity i.e. the elevated DNA repair capacity of cells.

132 P40

Unusual phosphate donors for human deoxyrybonucleoside kinases

K. Krawiec1, B. Munch-Petersen2, S. Eriksson3, D. Shugar1, B. Kierdaszuk1 1University of Warsaw, WARSAW, Poland 2Roskilde University, ROSKILDE, Denmark 3Swedish Univ. of Agricultural Sciences, UPPSALA, Sweden

Krzysztof Krawiec 1, Birgitte Munch-Petersen 2, Staffan Eriksson 3, David Shugar 1,4 and Borys Kierdaszuk 1 1 University of Warsaw, Institute of Experimental Physics, Department of Biophysics, 93 Żwirki i Wigury St., PL-02-089 Warsaw, Poland; 2 University of Roskilde, Department of Life Sciences and Chemistry, DK-4000 Roskilde, Denmark; 3 Swedish University of Agricultural Sciences, Department of Veterinary Medical Chemistry, Biomedical Centre, S-75123 Uppsala, Sweden; 4 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5a Pawińskiego St., PL-02-106 Warsaw, Poland

Inorganic tripolyphosphate (PPPi) and pyrophosphate (PPi) were examined as potential phosphate donors for all four human deoxyrybonucleoside kinases: deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), cytosolic thymidine kinase (TK1), and mitochondrial thymidine kinase (TK2); and additionally the deoxynucleoside kinase (dNK) from Drosophila melanogaster. This is the first report on the substrate activity of PPPi, which was proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not dAdo, as acceptor substrate, illustrating also dependence of donor properties on acceptor. Products of phosphorylation were shown to be 5˘-phosphates. In contrast to ATP, the phosphorylation reaction follows strict Michaelis-Menten kinetics, with Km values of 74 and 92 µM for dCK and dGK, respectively, and Vmax values 40-50% that for ATP. With the other three enzymes, as well as for dCK with dAdo as acceptor, no, or only low levels (<1 % of that for ATP) of activity were observed. PPi was inactive (<0.1%) as a phosphate donor with all enzymes, but was a competitive inhibitor vs ATP, as was PPPi in systems with no or low donor activity. Activities of the enzymes towards PPPi perfectly correlates with classification of kinase activities, which were previously suggested using their activities with analogues of 2'- and 3'- triphosphates of adenosine. Deoxyribonucleoside kinases which easily accepts all mentioned analogues can also effectively utilize PPPi as a phosphate donor, while kinases with more restricted specificity towards those analogues, are also restricted towards PPPi. The extension of mentioned investigations, including tests with longer polyphosphates and more complete set of acceptors, are in progress.

133 P41

The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster can phosphorylate glycerol

B. Munch-Petersen1, B. Stitt2, C. Zhu1, R. Browne2, J. Piskur3, C. Grubmeyer2 1Roskilde University, ROSKILDE, Denmark 2Temple University, PHILADELPHIA, United States of America 3Technical University of Denmark, LYNGBY, Denmark

Deoxyribonucleoside kinases are found in nearly all organisms except in fungi and a few bacteria. These enzymes participate in the salvage pathway providing nucleic acids precursors, and they also activate a number of medically important enzymes. In vertebrates, it seems to be a common characteristica to possess more than one kinase with overlapping specificities. Recently, in the fruitfly Drosophila melanogaster, we found that there was only a single deoxyribonucleoside kinase, Dm-dNK, but in contrast to other kinases, this enzyme is able to phosphorylate all four deoxynucleosides as well as a broad number of nucleoside analogues. In two other insects, Bombyx mori the silk butterfly, and Anopheles gambiae, the malaria mosquito, we also found a multisubstrate deoxyribonucleoside kinase with the capacity of phosphorylating all four deoxyribonucleosides. Currently, several development projects are in progress using the Dm-dNK for biotechnological production of DNA reagents and for gene therapy. We have investigated further the substrate properties of the Dm-dNK and found a surprising new property of the enzyme. In addition to nucleosides, it is able to phosphorylate glycerol. Data on this new property and activity towards other polyalcohols and sugars will be presented. Because of this new substrate property, we suggest to rename the enzyme to: Multifunctional deoxyribonucleoside kinase.

134 P42

Amino acid residues determining the substrate specificity of deoxyribonucleoside kinases

B. Munch-Petersen1, M.P.B. Sandrini2, W. Knecht2, H. Eklund3, J. Piskur2 1Roskilde University, ROSKILDE, Denmark 2Technical University of Denmark, LYNGBY, Denmark 3SLU, UPPSALA, Sweden

The multisubstrate deoxyribonucleoside kinase from insects – dNK (EC 2.7.1.145) is closely structurally related to the two human mitochondrial deoxyribonucleoside kinases, thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK). TK2 and dGK have recently been found to be essential for mitochondrial DNA integrity and mutated forms of these enzymes cause mitochondrial DNA depletion with lethal consequences. Increased knowledge about structure-function relationship of deoxyribonucleoside kinases helps to understand the relation between structure, function and substrate specificity, which is a prerequisite for the development of novel mutant enzymes for suicide gene therapy. The recently solved 3D structures of the Drosophila melanogaster dNK and human dGK are very similar. When the dNK and dGK structures were used for modelling of TK2 and dCK respectively, there were only a few differences in the residues closest to the base. Substitution of a few of these residues changed the specificity from pyrimidine to purine deoxyribonucleosides. Lately, we isolated, cloned and characterized the sole deoxyribonucleoside from Anopheles Gambia, which is also a multisubstrate kinase but in contrast to the drosophila dNK, the mosquito enzyme has essentially the same specificity towards both purine and pyrimidine deoxyribonucleosides. Since the residues in the binding pockets for the mosquito and drosophila dNK’s appears to be identical, other more distant residues must also be involved in determination of the substrate specificity.

135 P43

Mitochondrial deoxyguanosine kinase/thymidine kinase mutations and mitochondrial DNA depletion syndrome

L. Wang, S. Eriksson Swedish Univ. of Agricultural Sciences, UPPSALA, Sweden

Mitochondrial DNA (mtDNA) synthesis is irrespective of cell cycle phase and therefore a constant supply of mitochondrial DNA precursors is vital for mtDNA maintenance. Mitochondrial deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2) catalyse the initial phosphorylation of deoxynucleosides which is the rate limiting step in mtDNA precursor synthesis. Deficiency in either dGK or TK2 have been observed in patients with severe mtDNA diseases, e.g. earlier onset of progressive live failure, lactic acidosis and neurological abnormalities due to mutations in the dGK gene and reduction of respiratory function and devastating myopathy due to mutations in TK2 gene. We used site-directed mutagenesis to introduce those point mutations or base insertion, observed in patients, in the dGK and TK2 genes. The recombinant mutant enzymes were expressed in E. coli and purified and characterised with regards to substrate specificity and catalytic efficiency. Thr-77 to Met mutation of TK2 resulted in an enzyme with low catalytic efficiency as compared with wild type TK2 and also altered substrate specificity. Arg-142 to Lys mutation of dGK resulted in an inactive enzyme. The 4 bp insertion in exon 7 which introduced a stop codon and C-terminal 22 amino acids truncation of dGK protein and this C-terminal truncated dGK protein was also inactive. Glu227Lys mutant dGK enzyme showed very low residual activity. In summary mutations in dGK genes probably result in cells with dGK deficiency, which will probably starve the mtDNA synthesis with depleted dATP and dGTP pools. TK2 mutations likely resulted in imbalanced dTTP and dCTP pools in mitochondria. Insufficient supply of dNTPs may well be the cause of mtDNA depletion. These results implicate that dGK and TK2 play an important role in mtDNA precursor synthesis and the maintenance of mtDNA integrity.

136 P44

Biochemical and Biological Evaluation of 3-(Carboranylalkyl) thymidines as Substrates for Human Thymidine Kinases 1 and 2

A.S. Al-Madhoun1, W. Ji2, J. Johnsamuel2, J. Yan2, W. Tjarks2, S. Eriksson3 1Medical BioSciences, SLU, UPPSALA, Sweden 2College of Pharmacy, COLUMBUS, OHIO, United States of America 3Swedish Univ. of Agricultural Sciences, UPPSALA, Sweden

A small library consisting of two series of thymidine derivatives containing o-carboranylalkyl groups at the N-3 position was prepared. In both series, alkyl spacers of 2-7 methylene units were placed between the o- carborane cage and the thymidine scaffold. In one series, an additional dihydroxypropyl substituent was introduced at the second carbon atom of the carborane cage. In the series of N-3 substituted carboranyl thymidines without additional dihydroxypropyl substituent, three steps were required to obtain the target compounds in overall yield as high as 75%, while in the series of N-3 substituted carboranyl thymidines with additional dihydroxypropyl substituent nine to ten steps were necessary with significantly lower overall yield. All target compounds were good substrates for TK1 while they were, if at all, poor substrates for TK2. There was only a minor difference in phosphorylation rates between N-3 substituted carboranyl thymidines with additional dihydroxypropyl substituents with TK1 (range: 13 to 49% relative to thymidine) and their counterparts lacking this group (range: 11 to 57% relative to thymidine). In order to gain a better understanding of the phosphorylation by TK1, apparent Km, Vmax and Ki values for these analogues were determined. Taking together, the tether lengths of two and five methylene groups in both series were found to be good substrate for TK1 and a hypothesis for this result is proposed. The results of stability studies including incubation with human and bacterial thymidine phosphorylase and human cytosolic nucleotidases will be also presented. In vitro studies related to cytotoxicity and uptake will be included.

137 P45

Cloning and biochemical characterisation of thymidine kinase in Ureaplasma urealyticum

C.M.E. Carnrot Biomedical Center, UPPSALA, Sweden

Ureaplasma urealyticum (U.u), belonging to the mycoplasmas, is a human pathogen colonising the urogenital tract. Two genes coding for a presumed thymidine kinase (TK) and deoxynucleoside kinase (dAK) have been identified in the genome of U.u. These enzymes are most likely required for the salvage pathway in DNA precursor synthesis. Here we present the cloning, expression and biochemical characterisation of U.u TK. The enzyme is pyrimidine specific and phosphorylates thymidine, with a Km value of 5.4 mM and a Vmax of 3.6 mmol mg-1 min-1. 5’-fluorodeoxyuridine (5FdU), 3’-azidothymidine (AZT) and deoxyuridine are also good substrates, and the latter appears to have a very high Km value. 5FdU and AZT have Km values of 9.3 and 44.6 mM respectively. Their corresponding Vmax values are 4.7 and 0.6 mmol mg-1 min-1. All nucleoside triphosphates (NTPs) or dNTPs but not dTTP can act as phosphate donors. ATP is the most efficient phosphate donor with a specificity constant of 4.5 x 104 M-1s-1. dTTP acts as a feedback inhibitor with a Ki of 0.18 mM. Also thymidine inhibits TK but noncompetitively, at concentrations >10 mM. The availability of recombinant TK and knowledge of the properties of this enzyme will facilitate studies of the physiological role of TK in U.u and future possibilities to use TK as target in the development of new drugs against mycoplasma infections.

138 P46

Hypoxanthine effects on cyclic amp levels in human lymphocytes

R.J. Torres, I. De Antonio, C. Prior, J.G. Puig La Paz Hospital, MADRID, Spain

Introduction: Adenosine A2 receptor activation stimulates adenylate cyclase (increasing cAMP levels). Inhibition of adenosine uptake by hypoxanthine could lead to accumulation of extra-cellular adenosine that binds to adenosine A2A receptors and increases cAMP production. A2A receptors are highly concentrated in the basal ganglia and may modulate dopaminergic activity. Hypoxanthine excess could be implicated in the pathogenesis of the neurological symptoms of Lesch-Nyhan syndrome. The aim of this study is to evaluate the effect of hypoxanthine on receptor-mediated adenosine action in human cells using peripheral blood lymphocytes. Methods: To test hypoxanthine effect on A2A receptor activation, cells were first incubated with 5 uM or 25 uM CGS-21680, or without agonist and then different hypoxanthine concentrations (0, 5, 25 or 50 uM) were added and cells incubated for 60 min at 37oC. Results: We determined the effect of 5uM (close to mean normal concentration of hypoxanthine in cerebrospinal fluid) vs. 25 uM (near Lesch-Nyhan concentration 17,5 +- 2,8) extra-cellular concentrations of hypoxanthine on basal and CGS 21680-stimulated levels of cAMP in PBL cultures. Basal cAMP levels were significantly increased with the addition of 25 uM and 50 uM hypoxanthine versus 5 uM hypoxanthine. cAMP levels in PBL stimulated with 5 uM CGS 21680 were significantly increased with the addition of 25 uM and 50 uM hypoxanthine versus 5 uM hypoxanthine. In PBL stimulated with 25 uM CGS 21680, no significant differences were found between cAMP levels with the addition of hypoxanthine 5 uM, or without hypoxanthine. However, hypoxanthine 25 uM caused a significant increase in cAMP levels compared with basal or 5 uM hypoxanthine concentrations. Conclusions: Adenosine and dopamine receptors in the basal ganglia are coupled and adenosine is known to modulate dopaminergic neurotransmission. Thus hypoxanthine, by means of its effect on adenosine uptake and adenosine receptors, can modulate dopaminergic neurotransmission and dopamine content in the basal ganglia neurons. This may explain the dopaminergic deficit shown by Lesch-Nyhan patients and in HPRT-deficient culture cells and could give some clues on the relation between the enzyme deficiency and the dopaminergic defect.

139 P47

Mechanistic and physiological studies of dUTPases

J. Kovari1, A. Bekesi1, I. Zagyva1, A. Nagy1, O. Barabas2, B.G. Vertessy1 1Institute of Enzymology, BUDAPEST, Hungary 2Eötvös Loránd University, BUDAPEST, Hungary

The essential enzyme dUTP pyrophosphatase is responsible for preventive DNA repair via exclusion of uracil. dUTPase hydrolyses dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing dUMP for dTTP biosynthesis. Lack or inhibition of the enzyme induces thymine-less cell death in all cells and tissues performing active DNA synthesis such as neoplasia, serving therefore as an important chemotherapeutic target. Developmental regulation of the Drosophila melanogaster enzyme is suggested to be involved in thymine-less apoptosis. In addition to sequence motifs conserved amongst dUTPases, the fruit fly enzyme contains a unique C-terminal Ala-Pro-rich extension. Kinetic, biochemical and biophysical analyses of the full- length protein and a truncation mutant show that the Ala-Pro-segment is flexible and has no significant role in catalytic activity. For detailed mechanistic investigations, several point mutants of E. coli- and Drosophila melanogaster enzymes are being characterised. Formation of the catalytically competent closed enzyme conformer is induced by the product dUMP and the non-hydrolysable substrate analogues dUDP and dUPNPP in the Drosophila melanogaster dUTPase, while only by dUPNPP in the E. coli enzyme. Two dUTPase isoforms were identified in different developmental stages of the fruit fly. Protein levels are drastically reduced in larvae, arguing for a possible induction of developmental apoptosis. Interestingly, mRNA levels are high throughout development, indicating translational or post-translational control of dUTPase protein. Fruit fly dUTPase is the first eukaryotic enzyme characterized in details. Our results reveal significant differences between prototypes of eu- and prokaryotic dUTPases with respect to conformational flexibility of the active site, metal ion binding, and oligomerization. These differences are consistent with alterations of the catalytic mechanism and in hydropathy of subunit interfaces. Recently, we also succeeded in cloning the gene of nuclear isoform of human dUTPase into an expression vector, which will allow for detailed characterization of the human enzyme.

140 P48

Crystal structure of a human deoxyribonucleotidase

A. Rinaldo-Matthis Stockholm University, STOCKHOLM, Sweden

Mammalian 5’ deoxyribonucleotidases (dNT´s) is key proteins in the control of the deoxyribonucleotide pools sizes, essential for accurate DNA synthesis. They are potential important drug targets and reveals a relationship to the HAD family, with a phosphoserine phosphatase as the closest neighbor. The mitochondrial 5’ deoxyribonucleotidase (dNT-2) specifically dephosphorylates dUMP and dTMP, protecting mitochondrial DNA replication from excess dTTP. We have solved the structure of dNT-2, the first determined structure of a mammalian 5'-nucleotidase. The structure reveals a relationship to the HAD family, with a phosphoserine phosphatase as the closest neighbor. A structure-based sequence alignment of dNT-2 with other 5’-nucleotidases also suggests a common origin for these enzymes. The structure of dNT-2 has been studied in complex with bound phosphate and beryllium trifluoride plus thymidine as model for a phosphoenzyme product complex. Based on these structures, determinants for substrate specificity recognition and the catalytic action of dNT-2 have been outlined.

141 P49

Differential activity of pyrimidine nucleoside kinases in proliferating, resting and adipocyte differentiated 3T3-L1 cells

S.N. Rylova1, G. Flygh1, F. Albertioni2, S. Eriksson1 1Swedish Univ. of Argicultural Sciences, UPPSALA, Sweden 2Karolinska Institute, STOCKHOLM, Sweden

Nucleoside reverse transcriptase inhibitors (NRTI) in combination with protease inhibitors are a highly effective therapy for HIV infections. Prolonged NRTI treatment of HIV patients is associated with a number of metabolic complications. The lipodystrophy syndrome (LD), characterized by lipoatrophy (face, limbs, buttocks), fat accumulation (abdominal, neck) and dysmetabolism (hyperlipidaemia, insulin resistance and lactic acidosis) presents a serious risk for HIV patients. One of the proposed mechanisms for LD is NRTI -induced mitochondrial toxicity which results in mtDNA depletion, disruption of oxidative phosphorylation and possibly induction of apoptosis. The primary cause of NRTI associated mtDNA depletion is chain termination by NRTI triphosphates. Inhibition of cellular nucleoside kinases (NK) and altered mt dNTP pools may also be contributing factors. NRTI induced mitochondrial toxicity is found to be tissue specific and differs in activated and resting cells due to differential activities of NK’s, converting NRTI analogs to triphosphates. We used 3T3-L1 mouse preadipocyte cells - a model for white adipose tissue - to measure levels of NK’s, responsible for activation of the NRTI analogs d4T, AZT, 3TC, some of which are associated with LD. We compared cytosolic (TK1) and mitochondrial (TK2) thymidine kinases (phosphorylating d4T and AZT), deoxycytidine (dCK) kinase (phosphorylating 3TC) and thymidylate (TMPK ) kinase (limiting in AZT activation) activities in proliferating, resting and adipocyte differentiated 3T3-L1 cells and in CEM lymphoblasts. We found a 30-fold drop in TK1 activity and 3-fold decrease in dCK activities in extracts from resting 3T3- L1 compared to proliferating cells. In differentiated 3T3-L1 (9 days after adipocyte induction) there is no change in TK1 activity, a slight decrease in dCK activity, but a 90% increase in TK2 activity compared to resting preadipocytes. In contrast, CEM cells have very high activities for TK1 and dCK but below 1% for TK2. TMPK activity was 7-fold higher in proliferating preadipocytes than in CEM cells. It was 2 fold lower in resting preadipocytes versus proliferating cells, and slightly higher in differentiated 3T3-L1 adipocytes compared to resting 3T3-L1. These results show that rate limiting NRTI activating enzymes are differentially expressed during adipocyte differentiation.

142 P50

Cloning of a mitochondrial UMP/CMP kinase from Drosophila melanogaster

A. Karlsson, S. Curbo, M. Amiri, F. Foroogh, M. Johansson Karolinska Institute, STOCKHOLM, Sweden

The group of characterized UMP/CMP kinases catalyses the phosphoryl transfer from ATP to CMP, UMP and dCMP, resulting in the formation of ADP and the corresponding nucleoside diphosphates. The human UMP/CMP kinase phosphorylates structural analogs of pyrimidine nucleotides that are often administered as nucleosides in the treatment of cancer and viral infections. We have cloned a UMP/CMP kinase from Drosophila melanogaster (Dm-UMP/CMP kinase). It has similar enzymatic properties as the human UMP/CMP kinase and thus uses uridine and cytidine substrates. In contrast to the human UMP/CMP kinase that is located in the cytosol the Dm-UMP/CMP kinase is located in the mitochondria. So far no human mitochondrial UMP/CMP kinase has been discovered and it is therefore puzzling to find a mitochondrial UMP/CMP kinase in the lower species Drosophila melanogaster.

143 P51

Purine and Pyrimidine Salvage in Whole Rat Brain: Utilization of ATP-derived Ribose-1-Phosphate and 5-phosphoribosyl-1-Pyrophosphate Generated in Experiments with Dialyzed Cell-free Extracts

P.L. Ipata, C. Barsotti, M.G. Tozzi University of Pisa, PISA, Italy

The object of this work stems from our previous studies on the mechanisms responsible of ribose 1-P and 5- phosphoribosyl-1-pyrophosphate mediated nucleobase salvage and 5-fluorouracil activation in rat brain (Mascia, L. et al. (2000) Biochim. Biophys. Acta 1474, 3585-3589; Mascia, L. et al. (1999) Biochim. Biophys. Acta 1472, 93-98). Here we show that, when ATP at 'physiological concentration' is added to dialyzed extracts of rat brain, in the presence of natural nucleobases or 5-fluorouracil, adenine-, hypoxanthine-, guanine-, uracil- and 5-fluorouracil-ribonucleotides are synthesized. The molecular mechanism of this peculiar nucleotide synthesis relies on the capacity of rat brain to salvage purine and pyrimidine bases by deriving ribose 1- phosphate and 5-phosphoribosyl-1-pyrophosphate from ATP, even in the absence of added pentose or pentose phosphates. The levels of the two sugar phosphates formed are compatible with those of synthesized nucleotides. We propose that the ATP-mediated 5-phosphoribosyl-1-pyrophosphate synthesis occurs through the action of purine nucleoside phosphorylase, phosphopentomutase and 5-phosphoribosyl-1-pyrophosphate synthetase. Furthering our previous observations on the effect of ATP in the 5-phosphoribosyl-1- pyrophosphate-mediated 5-fluorouracil activation in rat liver (Mascia, L. and Ipata, P.L. (2001) Biochem. Pharmacol. 62, 213-218), we now show that the ratio [5-phosphoribosyl-1-pyrophosphate]/[ATP] plays a major role in modulating adenine salvage in rat brain. On the basis of our 'in vitro' results, we suggest that massive ATP degradation, as it occurs in brain during ischemia, might lead to an increase of the intracellular 5- phosphoribosyl-1-pyrophosphate and ribose 1-P pools, to be utilized for nucleotide resynthesis during reperfusion (Barsotti, C. et al. (2002) J. Biol. Chem. 277, 9865-9869). Finally, we also show that the pathway of ATP degradation in rat brain follows the AMP - IMP - Inosine route or the AMP - Adenosine - Inosine route at high and low Energy Charge value respectively.

144 P52

Colocalization of Human Cytidine Triphosphate Synthetase 1 with Microtubules

M.J.H. Higgins, L. Graves University of North Carolina, CHAPEL HILL, NC, United States of America

Cytidine triphosphate (CTP) has emerged as an important nucleotide regulator of cellular processes such as differentiation, phospholipid metabolism and DNA replication. The last step in the de novo synthesis of CTP is catalyzed by CTP synthetase 1 (CTPS1) which amidates UTP to form CTP. Although CTPS1 has been well characterized in bacteria and yeast, there remains relatively little information about human CTPS1 cellular biology. In this study, we characterized the cellular localization of human CTPS1 in HEK 293 cells. After confirming expression of our human CTPS1 construct in HEK 293 cells via immunoblot, we performed immunostaining of HEK 293 cells using an antibody against the N-terminal Xpress tag attached to human CTPS1. Immunostaining revealed that the localization of hCTPS was cytoskeletal; however, human CTPS1 did not colocalize with actin as determined by costaining with Alexa488-phalloiden. To investigate whether the endogenous human CTPS1 also colocalized with the cytoskeleton, we generated an antibody against the C- terminal 14 amino acids and performed immunostaining on untransfected HEK 293 cells. Endogenous human CTPS1 also localized to the cytoskeleton and, interestingly, colocalized with microtubules. While treatment of untransfected HEK 293 cells with a specific pharmacological inhibitor of CTPS, CPEC (cyclopentenyl cytosine), did not greatly affect the localization of human CTPS1, CPEC incubation was associated with an unexpected amount of microtubule formation in HEK 293 cells. Here we show for the first time that human CTPS1 localizes to microtubules and treatment with the specific inhibitor of CTPS, the anticancer drug CPEC, is associated with microtubule formation. Further investigation will include CTPS effects on tubulin polymerization as well as how tubulin affects CTPS activity. A more complete understanding of where and how hCTPS1 operates in the cell will not only benefit our basic understanding of pyrimidine metabolism but also help in efforts to develop more specific anticancer drugs.

145 P53

Identification of binuclear zinc coordination for human guanine deaminase by site directed mutagenesis

F.F. Snyder, H. Zhang, F.F. Snyder University of Calgary, CALGARY, ALBERTA, Canada

Guanine deaminase, which catalyzes the conversion of guanine to xanthine, irreversibly removes the guanine nucleobase from the cellular pool of guanine containing purines. We have previously cloned human guanine deaminase (J. Biol. Chem. 1999), characterized 5 cDNA alleles in the mouse (Genome 2002) and identified the E. coli homolog (J. Bact. 20001). Guanine deaminase shares the 9-residue motif, PGLVDTHIH, believed to be responsible for coordination of zinc, with other amino and amido-hydrolases. Recent crystallographic studies of dihydroorotase and phosphotriesterase have identified binuclear metal centers. These include coordination of the alpha-metal site through the two histidines of the above-mentioned motif, a carboxylated lysine, and an aspartate. The beta-metal site includes the same lysine and two additional carboxy-terminal side histidine residues. Exposure of guanine deaminase to the heavy metal atom chelator, 1,10-phenanthroline, results in the loss of activity. By comparative analysis of the human and E. coli primary guanine deaminase sequence we identified conserved histidine residues including the lysine residue corresponding to the putative binuclear metal center. By site directed mutagenesis we have made the following individual modifications to human guanine deaminase: His82Ala, His84Ala, Lys201Ala, His146Asn, His240Asn, and His279Ala. The specific activity and affinity with guanine was established for each of these proteins and modification of His 82, 84, 240, and 279 caused greater than 95% loss of specific activity. Substitution of Lys201 resulted in approximately a 75% decrease in activity. The kinetic findings were also correlated with zinc analysis of the purified proteins. The wild type protein had approximately 1.7 atoms of zinc per subunit, consistent with the coordination of two zinc atoms. The histidine substitutions resulted in the partial loss of zinc per subunit. In contrast replacement of His146, not predicted to be involved in the zinc coordination sites resulted in only a 50% loss of specific activity and no loss of associated zinc. These studies provide evidence for a binuclear zinc center associated with the catalytic function of human guanine deaminase. Supported by the Canadian Institute of Health Research

146 P55

GTP Concentrations are Elevated in Erythrocytes of Renal Transplant Recipients when Conventional Immunosuppression is replaced by the Inosine Monophosphate Dehydrogenase Inhibitor Mycophenolic Acid Mofetil (MMF)

E.A. Carrey, D.J.A. Goldsmith, S.M. Edbury, A.M. Marinaki, H.A. Simmonds Guy's Hospital, LONDON, United Kingdom

MMF is an effective immunosuppressant developed for use in organ transplantation. The active metabolite, mycophenolic acid, is a potent inhibitor of inosine monophosphate-dehydrogenase (IMPDH). IMPDH catalyses the first step in the synthesis of GMP from IMP derived via either the purine synthetic or salvage pathways. Elevated GTP concentrations were first noted in erythrocytes of immunodeficient children treated with another IMPDH inhibitor, Ribavirin (1); IMPDH activity was elevated in such patients compared with controls (2670pmol/h cf 8.5pmol/h per mg protein). A similar elevation of GTP was reported recently in erythrocytes of heart transplant recipients given MMF (2). In the present report we measured nucleotides by HPLC in renal failure patients’ erythrocytes before and after successful transplantation and noted a mean drop in GTP followed by an abrupt elevation in those on MMF. Since GTP concentrations are elevated in renal failure and fall after successful transplantation the latter was attributed initially to possible rejection, but subsequently related to the change from conventional triple therapy to MMF. GTP concentrations in 24 transplanted patients on triple therapy were in the normal range (mean 40µmol/l) but elevated 3 fold in 24 patients on MMF (mean 128µmol/l). Such elevation of erythrocyte GTP by IMPDH inhibitors is curious since normal human erythrocytes lack functional IMPDH, as confirmed in vitro where radiolabelled hypoxanthine in intact erythrocytes is found only in IMP. Such an effect in erythrocytes by an immunosuppressant which demonstrably inhibits lymphocyte IMPDH in vitro, inducing severe GTP depletion (3), indicates the long-term efficacy of MMF therapy in renal transplant recipients requires careful monitoring. Studies in progress comparing graft survival in these two patient cohorts have not yet revealed any differences.

1.Montero C et al. Demonstration of induction of erythrocyte IMP-dehydrogenase activity in ribavirin treated patients using an HPLC-linked method. Clin Chim Acta 1995; 238:169-178. 2. Wiegel G et al. Effect of mycophenolate mofetil therapy on inosine monophosphate dehydrogenase induction in red blood cells of heart transplant recipients. Clin Pharmacol Ther 2001;69: 137-144 3. Qiu Yet al. Mycophenolic acid-induced GTP depletion also affects ATP and pyrimidine synthesis in mitogen-stimulated primary human T-lymphocytes. Transplantation 2000; 69: 890-897

147 P56

Cyclase and Phosphodiesterase Activity on Pre-T Lymphoid Human Cells, treated with Dimethyl Sulfoxide (DMSO)

F. Di Pietrantonio, E. Di Matteo, M. Di Nicola, O. Trubiani, R. Primio, E. Serra, G. Spoto University ‘G. D'Annunzio’, CHIETI, Italy

Cellular death can take two different forms respectively called necrosis and apoptosis (PCD). These two forms differ from each other due to morphological and biochemical characteristics and biological incidence and meaning. Almost all cells can experience PCD independently from the synthesis of new proteins or from the phase of cellular cycle in which they are. In the immune system, PCD is a mechanism that is widely used to select the clones that are functionally helpful for T and B lymphocytes during differentiation process. DMSO typically promotes cellular differentiation due to its ability to arrest cellular growth at the G0-G1 phase. Nevertheless, in other experimental conditions, for example in cellular cultures of pre-t lymphoid human cells (RPMI-8402) DMSO induces apoptosis instead of a terminal differentiation. We studied cyclase and Phosphodiesterase (PDE) activity on this cellular type during apoptosis, has been demonstrated that DMSO probably induces apoptosis. Methods: the utilized cells were the lymphocyte pre-t cells (RPMI-8402) in which apoptosis was induced with reproducible and standardized DMSO. Cellular line was maintained in culture and the cells were let grow until reaching the number of 2.5 x 106 /ml. During the logarithmic phase of growth, the cells were treated with 1.5% of DMSO for a time included between 0 and 72 hours. Subsequently, the samples were washed and re-suspension in PBS and derived for the analysis in HPLC. Results: The obtained values, in the analysis of the cyclase and phosphodiesterase activity, are absolutely specular. Adenylate-cyclase and cAMP-PDE activity has demonstrated values just determinable. Guanylate- cyclase and cGMP-PDE activity has demonstrated values that were variable depending on the time of treatment with DMSO. Discussion: the data obtained from studying these activities agree with the type of the cell treatment with the DMSO. In fact, the DMSO seems to activate the cells toward a "forced differentiation" because after 24 hours there is a peak in the synthesis of the chains if the gene TCR, which then decreases in the 48th hour. The dynamics of the transcription gene was different. Its translocation from the cytoplasm to the nucleus constantly increases from 0 until 72 hour.

148 P57

Identification of the 5'-nucleotidase activity altered in neurological syndromes

M.G. Tozzi1, R. Pesi1, M. Camici1, F. Crementieri1, V. Micheli2, G. Jacomelli2, L. Notarantonio2 1University of Pisa, PISA, Italy 2University of Siena, SIENA, Italy

Nucleoside monophosphate phosphohydrolases comprise a family of enzymes that dephosphorylate nucleotides both in intracellular and extracellular compartments. Members of this family exhibit different sequence, location, substrate specificity and regulation. Besides the ectosolic 5’-nucleotidase, several cytosolic (cN-I, cN- II, pN-I, dNT-I) and one mitochondrial enzymes (dNT-II) have been described (1-5). Nevertheless, several researchers refer to as 5’-nucleotidase any AMP-dephosphorylating activity, lacking a more accurate identification. An increase of cytosolic 5’-nucleotidase activity has been associated with neurological disorders (6,7). The identification of the specific enzyme involved in these pathologies would be fundamental for the comprehension of the linkage between the alteration of enzyme activity and alteration of brain functions. We present here a relatively simple protocol which enabled us to measure the specific activity of three different nucleotidases in primary cultures of fibroblasts of patients and controls. Patients have been chosen on the base of their symptoms including autistic features, speech retardation and impaired urate/creatinine ratio in urine. Human skin fibroblasts were obtained after informed consent, and were grown at 37°C in RPMI medium supplemented with 10% foetal calf serum. We measured the activity of cN-I as the rate of radiolabelled AMP hydrolysis, cN-II as the rate of phosphotransfer from IMP to radiolabelled inosine and dNTI and II as the rate of radiolabelled dUMP hydrolysis. Our preliminary results indicate that the described neurological symptoms, in at least one case, are associated with an increase of cN-I activity. Cultured fibroblasts from patients are utilised for further metabolic studies. Combined data of metabolic aspects and enzyme activity in erythrocytes and fibroblasts will help identifying the metabolic pattern characteristic of these neurological syndromes.

1.Sala-Newby G.B. et al J. Biol. Chem. 274, 17789 (1999). 2.Allegrini S. et al J. Biol. Chem. 276, 33526 (2001). 3.Amici A. et al. Blood 96, 1596 (2000). 4.Ramazzo C. et al J. Biol. Chem 275, 5409 (2000). 5.Ramazzo C. et al PNAS 97, 8239 (2000). 6.Page T. et al PNAS 94, 11601 (1997). 7.Pesi R. et al NeuroReport 11, 1827 (2000).

149 P58

Exposure to Human Blood Decreases Swine Endothelial ECTO-5'-Nucleotidase Activity

Z. Khalpey1, K. Kalsi1, A.H. Yuen1, Z. Kochan2, J. Karbowska2, E.M. Slominska2, M. Forni3, M. Maccherini4, M.L. Bacci3, P. Batten1, M. Lavitrano5, M.H. Yacoub1, R.T. Smolenski1 1Imperial College, LONDON, United Kingdom 2Medical University of Gdansk, GDANSK, Poland 3University of Bologna, BOLOGNA, Italy 4University of Siena, SIENA, Italy 5University of Milan, MILAN, Italy

BACKGROUND: Ecto-5'-nucleotidase (E5'N or CD73) is an extracellular endothelial enzyme involved in the final step of purine nucleotide breakdown and the formation of anti-inflammatory, antiaggretory and immunosuppressive adenosine. Understanding of the regulation of expression of this enzyme may provide an insight into overcoming acute vascular rejection. We evaluated whether confrontation of pig endothelial cells with human blood induces changes in the activity of E5'N or the other enzymes of adenosine metabolism in pig heart homogenates and endothelial cells.

METHODS: Pig hearts (wild type and transgenic for human decay accelerating factor-hDAF) were perfused with fresh human blood for 4 h using a constant flow perfusion system. Pig aortic endothelial cells were used to study the effect of human plasma on E5'N activity in vitro. Cells were exposed to human plasma for up to 3h. Enzyme activity was measured in heart or cell homogenates with an HPLC based procedure.

RESULTS: Activity of E5'N was 6.60±0.33 nmol/min/mg protein in wild type pig hearts and 8.54±2.10 nmol/min/mg protein in transgenic pigs overexpressing hDAF. Ex vivo perfusion of pig heart with fresh human blood for 4 h resulted in a decrease in E5'N activity to 62% and 61% of initial in wild type and transgenic pig hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. Initial pig aortic endothelial activity of E5'N was 9.10±1.40, 9.62±1.56 and 9.15±1.87 nmol/min/mg protein in control, heat inactivated and active complement plasma groups. Activity decreased to 71% and 50% of initial after 3 h exposure to heat inactivated and active complement human plasma respectively while it remained constant at 9.62±0.88 nmol/min/mg protein (107%) in controls incubated without human plasma.

CONCLUSION: pig heart activity of CD73 decreases following exposure to human blood which may affect adenosine production and exacerbate hyperacute and acute vascular rejection. Complement may play a significant role in the observed effect but its exact clarification requires further studies.

150 P59

Liver Transplant: Adenosine Metabolism and Apoptosis

E. Marinello1, F. Carlucci1, A. Tabucchi1, D. Neri2, G. Gerunda2, R. Merenda2, F. Rosi1, F. Floccari1 1University of Siena, SIENA, Italy 2Inst. Chirurgia Gen., PADOVA, Italy

Apoptosis has been shown to play a major role in development and in the pathogenesis of numerous diseases, it has been observed in hepatocytes and sinusoidal endothelial cell following ischemia-reperfusion injury and it has been postulated as a contributing factor in ischemia-reperfusion graft dysfunction following liver transplant. To elucidate the mechanism of ischemia-mediated liver alterations, changes of purine nucleotides, adenosine catabolizing enzyme activities, glutathione antioxidant system (GSH and GSSG), caspase-3 activity and DNA fragmentation were examined in hepatic biopsy specimens from six Landrace pig which underwent experimental liver transplantation. Biopsies were obtained before explantation procedure (t1), after cold ischemia period (t2), and 30’ from reperfusion on recipient (t3). High energy phosphates (HEP) concentrations and GSH/GSSG ratio significantly decreased after ischemia, adenosine and inosine monophosphate (IMP) increased; at 30’ from reperfusion, HEP levels inverted the trend evidenced at t2. Regarding the activity of adenosine catabolizing enzymes, adenosine deaminase (ADA) and soluble AMP-ase (e-Ns) were grossly reduced both at t2 and t3, no variations were evident for purine nucleoside phosphorylase (PNP) and IMP-ase (cN-II). On apoptosis we evidenced a progressive increase of caspase-3 activity during transplant time-course. In three cases, such increase, was associated with a sustained DNA fragmentation. Data on HEP evidenced their active degradation to adenosine during ischemic time, a further degradation to inosine is however slowed down as confirmed from the reduced ADA activity. This behaviour togheder with the reduction of e-Ns activity is probably related to the preservation of AMP and adenosine, precursors of HEP via adenosine kinase. The present study is aimed at clarifying the role of ischemic insult in a liver transplantat procedure and the contribution of hepatocyte necrosis and/or apoptosis to this process. Our results suggest that, during liver transplant, the complex mixture of insults results in a cell injury which can lead to apoptotic features.

151 P60

The immunological and kinetical characteristic of AMP-deaminase from normal and Hepatocellular carcinoma (HCC) human liver

M. Szydlowska, Z. Ledziski, M. Krzyanowski, K. Kaletha Medical University of Gdansk, GDANSK, Poland

AMP-deaminase (AMP-aminohydrolase, EC 3.5.4.6) is a highly regulated enzyme, catalyzing irreversible deamination of adenylic acid (5’-AMP), located at a branchpoint of adenylate nucleotides catabolism. It plays an important role in stabilisation of adenylate energy charge (AEC) and regulation of purine nucleotides pool in the cell. The immunologic, kinetic and regulatory properties of AMP-deaminase from human liver neoplasm - hepatocellular carcinoma (HCC) purified by double phosphocellulose chromatography were investigated and compared with these obtained for the enzyme from unaffected tissue. Specific activity of the enzyme present in the cancer tissue was significantly higher from that found in the normal tissue. AMP-deaminase extracted from the cancer was less specific towards substrate analogues, and in contrast to the enzyme extracted from the normal liver hardly reacted on pH and adenylate energy charge changes tested. At physiological pH 7.0, in the absence and in the presence of important regulatory ligands, AMP-deaminase from two sources studied manifested similar sigmoid-shaped substrate saturation kinetics, with half saturation constant parameter (S0.5) being lower for the enzyme isolated from the tumor tissue. In contrast to AMP-deaminase isolated from the normal human liver, where presence of a large protein fragment corresponding to the full size of the native enzyme subunit was also detected (a result confirmed by Western blot analysis), only smaller, proteolytically degraded fragments of AMP-deaminase isolated from the cancer liver were detected in result of SDS-PAG electrophoresis. Obtained results indicate that intensive degradation processes affecting tumor tissue can be responsible for differences found.

152 P61a

Biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency

C. Salerno, C. Crif University of Rome 'La Sapienza', ROME, Italy

Adenylosuccinate lyase (ADSL) deficiency is an autosomal recessive inborn error of purine synthesis characterised by diminished levels of enzyme activity and accumulation of the dephosphorylated substrate derivatives, SAICA riboside and S-Ado. Molecular analysis of the affected families reveals the existence of about 30 different mutations in the gene, mostly in compound heterozygous form. It is widely accepted that impairment of enzyme activity is due either to remarkable susceptibility to thermal stress or to changes in the active site structure that leads to an anomalous handling of the substrates. However, the significance of protein destabilization is unclear in the case of P100A/D422Y compound heterozygosis, because the enzyme shows a Tm value that is decreased by only 8°C (to 51°C, from 59° C of control samples), while the kinetic constants are very similar to those of the wild-type protein. Analysis of the enzyme structure suggests that distorsion of amino acid chain by P100A substitution may expose Cys-98 and Cys-99 to oxidising agents. Indeed, we found that the defective enzyme is strongly inhibited by 4-hydroxy-2-nonenal, the major product of membrane peroxidation, which is believed to cause some of the tissue damage that occur in vivo under conditions of oxidative stress. Enzyme is inactivated by a mixed-type cooperative mechanism that is partially reversed in the presence of dithiotreitol. Amino acid substitution is located in domain I of the protein, in the proximity of His-86 which behaves as general acid in the catalysis. Correct geometry of this region should be essential to preserve enzyme function as it has been suggested by a comparative study of the structure of the hyperthermophilic bacterium P. aerophilum. Indeed, the latter enzyme appears to be reinforced in this point by a disulfide bridge that fixes Cys-87 (that corresponds to residue 101 in the human sequence) to the helix 3-helix 4 pair. As far as D422Y substitution is concerned, the mutation involves a cluster of helices in domain III of the protein, on the outskirts of the tetrameric enzyme and far away from the active site, which may be exposed to a noxious environmental stimulus as well.

153 P61b

AMPD1 C34T Mutation Selectively affects AMP-deaminase activity in the Heart

K. Kalsi, A.H. Yuen, P.H. Johnson, E.J. Birks, M.H. Yacoub, R.T. Smolenski Imperial College, LONDON, United Kingdom

Objectives: An improved prognosis in heart failure was described in subjects with the C34T (Glu12Stop) nonsense mutation in the AMP-deaminase 1 (AMPD1) gene. This gene is predominantly expressed in skeletal muscle and possession of this mutation leads to decreased activity of AMPD and the accumulation of cytoprotective adenosine in skeletal muscle. However, little is known about the metabolic changes within the heart, in this study we evaluated the effect of the C34T mutation on cardiac enzymes involved in adenosine metabolism. Methods: Screening for AMPD1 genotype and measurement of enzyme activities was performed on 27 patients with heart failure (HF): 16 requiring implantation of left ventricular assist device (LVAD) and 11 undergoing elective transplantation. The presence of the C34T mutation was assayed by single-stranded conformational polymorphism (SSCP) and restriction-fragment length polymorphism (RFLP) on extracted DNA. Cardiac specimens were homogenized and assayed for: AMP-deaminase (AMPD), ecto-5’-nucleotidase (E5’N), purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and adenosine kinase (AK). Enzyme activities were analyzed by measuring the conversion of substrates into product by HPLC. Results are expressed as mean activities ± SEM. Results: AMPD activity in heterozygous (C/T) individuals was 45% of that found in normal wildtype (C/C) individuals (0.59±0.02* vs 1.06±0.09 µmol/min/g wet weight, *p= 0.003). There were no significant differences in activities of any other enzymes when C/T was compared to C/C individuals, 3.96±0.41 vs 4.60±0.42 for E5’N, 1.03±0.21 vs 0.98±0.07 for PNP, 0.36±0.02 vs 0.41±0.04 for ADA, 0.018±0.002 vs 0.016±0.002 for AK. Conclusions: We have shown for the first time a correlation between AMPD1 mutation and attenuated AMPD activity in the heart therefore promoting cardioprotection in a localised manner. These changes did not affect any other enzyme involved in adenosine/nucleotide metabolism.

154 P62

Association of improved cardiac function in donors with C34T mutation of the AMP Deaminase 1 gene

A.H. Yuen, M.H. Yacoub, E.J. Birks, K. Kalsi, P.H. Johnson, R.T. Smolenski Imperial College, LONDON, United Kingdom

Background: Possession of the C34T mutation in AMP deaminase (AMPD1) gene has been shown to be associated with attenuation of the progression of heart failure and improved survival in ischemic heart disease. The aim of this study was to compare the frequency of the mutation in the heart donors, patients with heart failure and healthy controls, to evaluate if the nonsense mutation in AMPD 1 can protect against myocardial dysfunction in this critical condition.

Methods: The presence of the C34T mutation was assayed by single-stranded conformational polymorphism (SSCP) and restriction-fragment-length polymorphism (RFLP) analysis on heart biopsies. Left ventricular biopsies were collected from patients with HF due to dilated cardiomyopathy (DCM) (n=27) undergoing either heart transplantation (Tx) or LVAD implantation. Donors were assessed by transoesophageal echocardiography at the time of harvesting. Those with an ejection fraction (EF) > 40% (associated with good haemodynamics) were used for Tx (n=22) and a right ventricular biopsy taken at the time of Tx. Donors with the EF <40% (associated with poor haemodynamics) were considered unsuitable for Tx (unused donors, n=10). A small piece from the left apex was taken for analysis. Blood samples from 207 healthy volunteers were also genotyped.

Results: There was no difference in the frequency of the mutation between the patients with HF (14.8%) and healthy volunteers (13.5%). However, the frequency of the mutation in the healthy donor hearts (31.8%) was much higher when compared to the patients with HF and the healthy volunteers, and was significantly higher than the failing donor hearts (5%) (p<0.025).

Conclusion: The presence of the C34T mutation in the AMPD1 gene protect against the development of donor heart dysfunction. This suggests that attenuation of cardiac dysfunction in critically ill condition may be the mechanism of improved survival observed in the other clinical conditions.

155 P63

Purine Metabolism in Pigs and Humans and its Implications for Xenotransplantation

R.T. Smolenski1, Z. Khalpey1, H. Dziewit2, E.M. Slominska2, A.H. Yuen1, M. Forni3, M.L. Bacci3, K. Kalsi1, Z. Kochan2, J. Karbowska2, M. Maccherini4, M. Lavitrano5, M.H. Yacoub1 1Imperial College, LONDON, United Kingdom 2Medical University of Gdansk, GDANSK, Poland 3University of Bologna, BOLOGNA, Italy 4University of Siena, SIENA, Italy 5University of Milan, MILAN, Italy

Immune system is major focus of research into xenotransplantation, while metabolic compatibility of animal organs with humans which could also affect organ survival has been studied little so far. We compared concentrations of nucleotide substrates and activities of enzymes of nucleotide metabolism in pig and human blood, heart and kidney. Furthermore, we studied changes in cardiac metabolite concentration following 4h of ex vivo perfusion with human blood. Donor human heart and kidney specimens and blood of healthy subjects were used as a reference. Enzyme activities and metabolite concentrations were measured using HPLC with UV or mass detection. The most prominent difference was in ecto-5'-nucleotidase (E5'N) activity: in hearts: 1.68±0.30 nmol/min/mg wet wt in human vs. 0.46±0.02 in pigs, in kidney 0.74±0.06 nmol/min/mg wet wt in humans vs. 0.11±0.01 in pigs and in endothelial cells: 39.1±5.8 nmol/min/mg prot in human vs. 9.1±1.4 in pig cells. Baseline concentrations of ATP, GTP, UTP and CTP were similar in human and pig hearts. However, specific decrease in GTP concentration has been observed from an initial of 1.35±0.12 nmol/mg dry wt to 0.68±0.06 following perfusion with human blood. There were profound differences in blood purine and pyrimidine concentrations which for: 1) hypoxanthine, 2) inosine, 3) adenine, 4) uracil, 5) uridine and 6) uric acid were: 1) 17.7±1.5 and 0.95±0.08 µM 2) 6.45±1.42 and 0.10±0.10 µM, 3) 3.95±0.14 and 0.07±0.02 µM, 4) 17.6±0.9 and 0.35±0.09 µM, 5) 1.41±0.13 and 2.80±0.30 µM, 6) 15.0±1.9 and 290.0±16.0 µM in pig and human blood respectively. Conclusion: Low activity of E5'N activity in pigs may lead to retention of pro- inflammatory and pro-aggregatory nucleotides and attenuation of formation of anti-inflammatory and anti- aggreggatory adenosine which counteract those effects following pig to human xenotransplantation. Human blood concentration of nucleotide precursors may not be adequate to maintain guanine nucleotide pool. Overexpression of E5'N and supply of substrates for GTP synthesis or allopurinol treatment may prolong pig to human xenograft survival.

156 P64

Overexpression of Human ECTO 5' Nucleotidase in Pig Endothelial Cells and its Implication for Adenosine Production and Xenotransplantation

F. Osborne1, C. Lawson1, K. Kalsi1, M. Lavitrano2, M.H. Yacoub1, M.L. Rose1, R.T. Smolenski1 1Imperial College, HAREFIELD, United Kingdom 2University of Milan, MILAN, Italy

Ecto 5’ nucleotidase (E5’N) or CD73 is an endothelial surface enzyme involved in extracellular nucleotide breakdown leading to the formation of anti-inflammatory and immunosuppressive adenosine. Expression of E5’N in pig cells has been found to be one order of magnitude lower than in human endothelium. This is particularly important for xenotransplantation where metabolism of extracellular nucleotides into adenosine plays a crucial role in interaction of endothelial cells with inflammatory cells and platelets. The aim of this study was to establish the feasibility of over-expression of human E5’N in pig endothelial cells and see if this affects rate of adenosine production in intact cells from exogenous AMP and ATP. The pig intestinal endothelial cell line (PIEC) was transfected using Effectene with human endothelial cDNA cloned into pCDNA3.1 bearing the neomycin resistance element and cell clones with the highest activity were selected using serial dilutions. Cells used were >95% human CD73 positive by flow cytometry. E5’N activity in cell lysates and rate of degradation of AMP in intact cells were measured by HPLC. Transfected and non- transfected PIEC were compared to human umbilical vein EC (HUVEC). PIEC enzyme activity in lysed untransfected cells was 9.10±1.4 compared to 109.6±10.5 in transfected compared to 39.1±5.8 nmol/min/mg prot in HUVEC. Rate of adenosine production in intact cells from exogenous AMP was 0.099 µM/min/mg protein in non-transfected cells and 47.2 µM/min/mg protein in transfected cells. Expression of human E5’N in pigs cells caused three-fold increase in the rate of adenosine formation from extracellular ATP. In conclusion, human E5’N could be functionally expressed on the surface of pig endothelial cells. Increased production of adenosine not only from AMP but also from ATP demonstrates that E5’N activity is the rate limiting step and suggests that E5’N over-expression in transgenic pigs would be a successful strategy for xenotransplantation.

157 P65

Lidoflazine combined with nucleotide precursors increases ATP content and Adenosine production in cardiomyocytes

K. Kalsi, R.T. Smolenski, M.H. Yacoub Imperial College, LONDON, United Kingdom

Objectives: Lidoflazine (LIDO) acts not only as a calcium antagonist but also has nucleoside transport inhibitor (NTI) properties and therefore increases adenosine (ADO) concentration. Stimulation of the purine salvage pathway with nucleotide precursors increases ATP and ADO content. However the combined effect of LIDO with precursors has not been demonstrated. Our objectives were to investigate the effects of LIDO alone or combined with Adenine (Ade), Ade + Inorganic phosphate (Pi), Ade + ribose (A/R) or A/R + Pi on ADO production and ATP content in myocytes. Methods: Rat cardiomyocytes were isolated using collagenase perfusion technique. Cells were then incubated for up to 8hr as follows: (1) Control; (2) 10 microM LIDO; (3) LIDO + 100 microM Ade; (4) LIDO + Ade + 5 mM Pi; (5) LIDO + Ade + 2.5 mM Ribose and (6) LIDO + A/R +Pi. 5 microM EHNA (inhibitor of ADO deaminase) was added to all incubations. After 8hrs, extracts of myocyte suspensions were analysed by HPLC. Results are expressed as mean values ± SEM, (n=4-6). Results: ATP content was decreased after 8hr of incubation in (2) to 21.4±1.6 nmol/mg prot. from 22.1±0.9 in (1), ATP was enhanced to 24.1±1.6, 29.9±1.9, 32.5±2.5* and 35.2±2.8* respectively in (3), (4), (5) and (6) (*p<0.05). ADO was increased in (2) to 1.6±0.2, but was further increased in (3) to 2.8±0.8*, in (4) to 2.1±0.3*, in (5) to 2.8±0.8* and in (6) to 3.1±0.6* vs (1) 1.4±0.1 (*p<0.05). Conclusion: We have shown that LIDO alone decreases ATP concentration in isolated myocytes together with an increase in ADO production. The presence of a combined application of nucleotide precursors (A/R or either A/R + Pi) and LIDO stimulates adenine nucleotide resynthesis resulting in an augmentation and protection of the ATP pool and increased ADO production.

158 P66

Investigation of substrate recognition of Drosophila melanogaster nucleoside kinase by site directed mutagenesis

S.N. Solaroli1, M. Bjerke2, M. Johansson2, A. Karlsson2 1Huddinge University Hospital, HUDDINGE, Sweden 2Karolinska Institute, HUDDINGE, Sweden

Expression of herpes simplex virus thymidine kinase (HSV-TK) in tumor cells and subsequent systemic chemotherapy with the nucleoside analog ganciclovir is the prototype for combined gene/chemotherapy of malignant tumors. In search for an alternative suicide gene to HSV-TK, we have cloned and characterized a deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK). The enzyme efficiently phosphorylates several anti-viral and anti-cancer nucleoside analogs with a higher catalytic rate then other known deoxyribonucleoside kinases. Dm-dNK has broad substrate specificity but does not phosphorylate ganciclovir or other guanosine nucleoside analogs. We have performed site directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine substrates. In particular two mutants, M88R and V84A+M88R, showed activity using ganciclovir as substrate. Different truncated forms of the enzyme carrying these mutations will be presented as well as data on the expression of these mutated enzymes in cell culture system.

159 P67

Evaluation of ADA Gene Expression and Transduction Efficiency in ADA/SCID Patients Underwent Gene Therapy

E. Marinello1, F. Carlucci1, A. Tabucchi1, A. Aiuti2, F. Rosi1, F. Floccari1, R. Pagani1 1University of Siena, SIENA, Italy 2San Raffaele H. Scient. Inst., MILAN, Italy

The ADA-deficient subjects have a type of SCID caused by a gene-defect in the enzyme adenosine deaminase (ADA), that is critical for normal immune function. ADA-deficient SCID has long been considered a model disease for gene therapy trials because it's caused by a defect in a single gene. Gene transfer into hematopoietic stem cell (HSC) for adenosine deaminase (ADA)-deficient SCID has been so far limited by the low levels of engrafted transduced HSC. An improved protocol for gene transfer into autologous bone marrow (BM) CD34+ cells, combined with non-myeloablative conditioning was designed. To explore the transfection efficiency for different retroviral vector and fully evaluate the efficacy of gene therapy, the measurement of ADA activity in transduced cells is essential. A suitable capillary electophoresis (CE) method was developed for such purpose. The enzyme activity was evaluated by the quantitation of substrate disappearance and reaction product formation separated by CE. The conditions were as follow: electrolyte 20 mmol/L sodium-borate (pH 10.00), uncoated capillary 42 cm x 75 mm i.d., voltage 12 kV, analysis at 254 nm. The method was linear in the range 2-500 mmol/L (r >0.99). The intra- and inter-assay variability showed a CV <4%. Enzyme activities were linear with respect to sample amounts in the incubation mixture and respect to incubation time (both r >0.99). In healthy subjects ADA showed a value of 20.23±9.27, in patients at diagnosis it was 0.08±0.06 nmol/h/mg of protein. The method permitted a complete screening of gene therapy protocols evidencing the restoration of intracellular ADA activity in PBL, in various lymphocyte populations and the increase of ADA activity in RBC from undetectable levels to 20-30% of healthy controls, that was accomplished to the reduction of toxic deoxynucleotides. This technique is a valuable, fast and unexpensive screening tool in the evaluation of ADA activity, being useful to quickly assess the expression of ADA in hematopoietic cells of SCID patients and represents an important tool for the follow-up of patients treated with enzyme replacement therapy or in clinical gene transfer protocols.

160 P68

Metabolism of Adenosine in Human Colorectal Tumor

E. Marinello1, D. Vannoni1, A. Bernini2, F. Carlucci1, M.C. Di Pietro1, F. Floccari1, R. Leoncini1, F. Rosi1, A. Santoro1, A. Tabucchi1, G. Tanzini3 1University of Siena, SIENA, Italy 2Inst. Chirurgia Gen., SIENA, Italy 3Institute of Clin. Chirurgica Gen., SIENA, Italy

The adenosine is produced from AMP by the action of 5’nucleotidase (5’-NT) and it is converted either to inosine or back into AMP via the reactions catalysed by adenosine deaminase (ADA) and adenosine kinase (AK) respectively. The steady-state concentration of adenosine is maintained by the activities of these three enzymes. The presence of adenosine is associated with inhibition of the immune response, coronary vasodilatation, neurotransmission, hormone stimulation; the cytoprotective functions of adenosine include stimulation of angiogenesis and inhibition of inflammatory reactions. Many observations suggest that adenosine may have similar function in cancer. The aim of this work is to analyse the activities of the three enzymes involved in adenosine metabolism, in fragments of intestinal mucosa of patients affected by colorectal cancer. We analysed 20 patients 11 males and 9 females aged between 46 and 80 years. 8 of them were affected by rectal cancer, 6 by sigma cancer, 4 by right colon cancer and 2 by left colon cancer. 2 of them were T2; 14 were T3 and 4 were T4; 10 presented lymphonoid metastasis and 3 also pulmonary and liver metastasis. The grade was so distributed:2 patients were G1, 11 G2 and 6 patient G3. The activities were assayed in fragments of neoplastic and normal-appearing mucosa, surrounding the tumour in a region close (less than 3 cm) and distant (at least 10 cm) to the tumour, which were removed during the surgical resection and immediately frozen in liquid N2. The results show that the three activities are markedly higher in tumour in comparison to close or distant normal mucosa (p<0.01 for ADA and AK; p<0.05 for 5’-NT). Results suggest that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissues. The contemporary increased of ADA and 5’-NT activities might be a physiological attempt of the cancer cells to provide more substrates needed by cancer cells to accelerate the salvage pathway activity.

161 P70

New evidences for regulation of deoxycytidine kinase activity by reversible phosphorylation

C. Smal, S. Cardoen, M. Veiga-da Cunha, S. Marie, G. Van den Berghe, F. Bontemps, L. Bertrand, E. Van den Neste, A. Ferrant, V. Race ICP and Catholic University of Louvain, BRUSSELS, Belgium

Deoxycytidine kinase (dCK) is a key enzyme in the deoxynucleoside salvage pathway. Besides its natural substrates, dCK activates various nucleoside analogues used against viral infections, leukaemia and solid tumours. Knowledge of its regulation can be expected to allow optimization of the activation of these analogues, and hence to improve their clinical efficacy. In recent years, various genotoxic agents, including 2-chloro-2’-deoxyadenosine, aphidicholin and UV light, have been shown to enhance dCK activity in leukaemic cells without changing the level of dCK protein (Sasvari-Szekely et al, Biochemical Pharmacol 56, 1175-9, 1998; Van Den Neste et al, Biochemical Pharmacol 65, 573-80, 2003). These observations led to the hypothesis that dCK activity can be regulated by post-translational modification, most probably by reversible protein phosphorylation (Csapo et al, Biochemical Pharmacol 61, 191-7, 2001). In accordance with this hypothesis, we observed that dCK can be 65 % inactivated in a crude cell extract from EHEB cells, a continuous B-cell line, by treatment with protein phosphatase 2A, responsible for dephosphorylation of serine and threonine residues. Inactivation was prevented by okadaic acid. These results corroborate that dCK activity is regulated by reversible phosphorylation and indicate moreover that activation of the enzyme involves phosphorylation of serine or threonine residues. Accordingly, dCK activity was not affected by protein tyrosine phosphatase treatment. To analyse its phosphorylation in more details, dCK was expressed in human embryonic kidney (HEK)-293 cells as a His-tag fusion protein and purified by affinity chromatography on cobalt-agarose resin. After SDS-page electrophoresis and immunoblotting with anti-His antibody, dCK appeared as a doublet, possibly due to two different phosphorylation levels resulting in a mobility-shifted signal. To verify this hypothesis, purified expressed dCK was treated with λ phosphatase. This resulted in a profound decrease of dCK activity, accompanied by an increase of its electrophoretic mobility. Our results provide additional arguments in favour of a regulation of dCK activity by reversible phosphorylation.

162 P71

Cyclopentenyl cytosine sensitises SK-N-BE(2)c neuroblastoma cells to cladribine

J. Bierau1, A.B.P. Van Kuilenburg1, A.H. Van Gennip2, R. Leen1, L. Zoetekouw1, H. Caron1 1Academic Medical Center, AMSTERDAM, The Netherlands 2Academic Hospital Maastricht, MAASTRICHT, The Netherlands

Cyclopentenyl cytosine (CPEC) is a drug targeted at CTP synthetase and possesses anti-tumour activity against neuroblastoma in vitro. In neuroblastoma, incubation with CPEC causes depletion of the intracellular (deoxy)cytidine nucleotide pools, and retardation in the S-phase of the cell cycle. The effects caused by CPEC in neuroblastoma make the combination of CPEC and S-phase active deoxynucleoside analogues attractive for chemotherapy. Cladribine (CdA) is an analogue of deoxyadenosine and is an effective drug in the treatment of haematological malignancies such as hairy cell leukaemia and acute myeloid leukaemia. However, no anti-tumour activity of CdA against solid tumours has been observed in clinical trials. CdA is an inhibitor of DNA synthesis as well as DNA repair and a synergistic effect was obtained when this drug was combined with DNA damaging agents. Although CdA is a purine analogue, the first and rate-limiting step in its activation is catalysed by deoxycytidine kinase (dCK). The depletion of dCTP achieved via inhibition of CTP synthetase by CPEC may thus lead to an enhanced uptake and anabolism of CdA. In this study, it is demonstrated that CPEC sensitised the CdA-resistant human neuroblastoma cell line SK-N- BE(2)c (ED50 >> 1.5 µM) towards CdA. Pre-treatment with 100 or 250 nM CPEC sensitised SK-N-BE(2)c cells to cladribine the 96-hr ED50 values being 419 ± 125 nM and 70 ± 30 nM, respectively. Preincubation with 100 nM CPEC for 24 hr increased the amount of intracellular CdA-nucleotides 1.2 to 4.7-fold, CdAMP being the major metabolite to accumulate. DNA synthesis was inhibited by 45 % by 100 nM CPEC and > 95 % by the combination of CPEC and CdA. CdA itself did not inhibit DNA synthesis. Our results demonstrate that the cytotoxic effects of CdA can be enhanced by inhibition of CTP synthetase and suggest that CAMP may be metabolite causing the increased cytotoxicity observed.

163 P72

Gemcitabine and cyclopentenyl cytosine: a promising combination for the treatment of neuroblastoma

J. Bierau1, A.B.P. Van Kuilenburg1, A.H. Van Gennip2, R. Leen1, R. Meinsma1, H. Caron1 1Academic Medical Center, AMSTERDAM, The Netherlands 2Academic Hospital Maastricht, MAASTRICHT, The Netherlands

Neuroblastoma is the most common solid malignancy of childhood. Despite intensive chemotherapeutic regimens, the survival rate of children suffering from metastasized neuroblastoma remains very poor. Poor prognosis is often associated with amplification of the MYCN-oncogene. We have observed that 2',2'- difluorodeoxycytidine (Gemcitabine, dFdC) has potent anti-tumor activity against neuroblastoma in vitro. dFdC is a pro-drug that is activated by phosphorylation to its nucleotides, of which deoxycytidine kinase (dCK) catalyzes the first and rate-limiting step. dFdC was tested on a panel of human neuroblastoma cell lines consisting of MYCN-amplified and MYCN-single copy cell lines. In both types of cell lines, low ED50 values (nM range) were observed. Despite the fact that the specific deoxycytidine kinase (dCK) activity was 60% higher in MYCN-amplified cell lines than in MYCN-single copy cell lines, this did not correlate with the ED50 values observed. However, while treatment with dFdC induced cell death in MYCN-amplified cell lines, MYCN-single copy cell lines underwent neuronal differentiation. Shep21N cells, in which MYCN-expression is controlled by tetracyclin sensitive promotor, underwent cell death after incubation with dFdC when MYCN was expressed, but no cytotoxicity was observed when MYCN was not expressed. This may be caused by the fact that the dCK activity was higher in MYCN expressing Shep21N cells than in Shep 21 cells that did not express MYCN. Pre-incubation with the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC) significantly lowered the ED50 values of 13 out of 15 cell lines. In SK-N-BE(2)c cells that had been incubated with 100 nM CPEC for 1-4 days, the anabolism of dFdC was increased to 6-44 times as compared to untreated control cells, as measured by metabolic labeling experiments. This was paralleled by an significant increase in the expression of dCK-mRNA, dCK proteins and increase of dCK activity. We feel that the combination of dFdC and CPEC may hold promise for the treatment of high-risk neuroblastoma

164 P73

Modulation of cytarabine resistance in childhood acute myeloid leukemia

I. Hubeek1, G.J. Peters1, A.J.F. Broekhuizen1, Ch.M. Zwaan1, U. Creutzig2, G.J.L. Kaspers1 1VU University Medical Center, AMSTERDAM, The Netherlands 2AML-BFM Study Group, MÜNSTER, Germany

Cytarabine is an important drug in the treatment of childhood acute leukemia. Resistance to this drug is therefore a clinically relevant problem. To modulate cytarabine resistance we combined cytarabine with 6 potential resistance modifiers: cladribine and gemcitabine (nucleoside analogs), decitabine (hypomethylating agent), aphidicolin (inhibitor of DNA polymerases alpha, delta and epsilon), and flavopiridol and UCN01 (inhibitors of cyclin-dependent kinases). The single agents and their combinations were studied in the leukemic cell line HL60 and ex vivo in blast cells isolated from 10 children with de novo AML using the MTT assay. In the HL60 cell line cytarabine was combined with the different modulators at the ratios of their respective IC50s. In the patient samples cytarabine was tested in 6 different concentrations, the modulators were tested at a low concentration (± 10% cell kill as a single agent) and a higher concentration (± 30% cell kill as a single agent). Drug interactions were investigated by computer-assisted analysis using the Calcusyn software (median drug effect method, Chou & Talalay), by calculation of the combination index ((CI); additivity ± 1; synergistism <1; antagonistism >1). In the HL60 cell line we observed moderate synergism between cytarabine and aphidicolin (CI=0.81), cladribine (CI= 0.76), flavopiridol (CI=0.77) and gemcitabine (CI=0.82). The interaction between cytarabine and decitabine was synergistic (CI=0.3). In contrast, co-incubation of cytarabine with UCN01 resulted in antagonism (CI=1.47) In the AML patient samples co-incubation of cytarabine with aphidicolin, in both the low and the high concentration, showed strong synergism (median CI= 0.28 and 0.18 respectively, (n=9)). The combinations of cytarabine/cladribine (median CI= 0.82 and 0.51 (n=10)) and cytarabine/gemcitabine (median CI= 0.5 (=10)) were all synergistic. Nearly additive and moderately synergistic interactions were observed between cytarabine/flavopiridol (median CI=0.93 (n=10) and 0.82 (n=8)) and cytarabine/UCN01 (median CI= 0.82 and 0.74 (n=9)). However, the combination of cytarabine with decitabine was antagonistic (median CI>10 (n=6) and CI=5.92 (n=3)). In conclusion, additive or synergistic interactions of cytarabine with aphidicolin, cladribine, gemcitabine, flavopiridol and UCN01 were observed in this ex vivo model testing pediatric AML samples, supporting the future evaluation of these combinations in clinical trials with AML patients.

165 P74

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines

A.M. Bergman1, C.M. Kuiper1, P. Noordhuis1, K. Smid1, D.A. Voorn1, E.M. Comijn1, G.J. Peters1, F. Myhren2, M.L. Sandvold2, H.R. Hendriks3 1VU University Medical Center, AMSTERDAM, The Netherlands 2Clavis Pharma, PORSGRUNN, Norway 3EORTC-NDDO, AMSTERDAM, The Netherlands

Ara-C is a deoxycytidine (dCyd) analog with activity in leukemia, which requires phosphorylation by deoxycytidine kinase (dCK) to its active phosphate ara-CTP, but can be deaminated by deoxycytidine deaminase (dCDA). In addition to dCK deficiency, altered membrane transport of deoxyribonucleosides over the cell membrane is a mechanism of drug resistance. In order to facilitate ara-C uptake and prolong retention in the cell, lipophillic pro-drugs were synthesized. Fatty acid groups with a varying acyl chain length and number of double bonds were esterified at the 5´ position on the sugar moiety of ara-C. The compounds were tested in two pairs of ara-C resistant cells (the murine leukemia L5 and L4A6 and the rat leukemia BCLO and Bara-C) and two pairs of cell lines with a resistance to gemcitabine, a deoxycytidine analog with a comparable mechanism of action as ara-C (the human ovarian cancer A2780 and AG6000 and murine colon C26-A and C26-G). L4A6, Bara-C and AG6000 have varying degrees of decreased dCK activity, while the mechanism for C26-G is not yet clear. In the parent cell lines ara-C was more active, but in the various resistant variants several of the analogs were more active, while the degree of cross-resistance varied. In AG6000 with a total dCK deficiency, all compounds were inactive. Structure activity relation analysis showed that ara-C derivatives with shorter acyl chains and more double bonds were more active in the parental and drug resistant cells. Further mechanistic studies were performed with the elaidic acid derivative of ara-C (CP-4055). CP-4055 inhibited deamination of dCyd partly and induced DNA synthesis inhibition effectively in C26-A and C26-G cells, but the retention of inhibition was much longer for CP-4055 than for ara-C. In contrast to ara-C, CP-4055 inhibited RNA synthesis for 60% after drug exposure. In conclusion, CP-4055 seems to be a promising prodrug, whose effects were different and longer lasting than for the parent drug.

166 P75

Pyrimidine deoxynucleoside salvage in Ureaplasma urealyticum: nucleoside analogues acted as potent inhibitors of mycoplasma growth

R. Wehelie1, S. Eriksson2, G. Bölske3, L. Wang2 1SLU, UPPSALA, Sweden 2Swedish Univ. of Agricultural Sciences, UPPSALA, Sweden 3National Veterinary Institute, UPPSALA, Sweden

Ureaplasma urealyticum (U. urealyticum) and Mycoplasma pneumoniae(M. pneumoniae) are pathogens of both genital and respiratory tract. Radiolabeled nucleosides and nucleoside analogues were used to study the biosynthesis, interconversion, and degradation of pyrimidine nucleosides/nucleotides in U. urealyticum. [methyl- 3H]-deoxythymidine (3H-dThd) and [5- 3H]-deoxycytidine (3H-dCyd) were metabolised to mono- and diphosphates, and no triphosphates were detectable. 3H-dCyd was also converted to deoxyuridine monophosphate due to the deamination by cytidine deaminase. Subsequently incoperation of these radiolabeled precursors into DNA were also detected. Small amount of monophosphate was detected from cell cultures grown in the presence of either [6- 3H]-5-Fluorodeoxyuridine (5-FdUrd) or [6- 3H]-5-Fluorouracil (5-FUra) indicating the uptake and salvage of these nucleoside analogues, but there was no incoperation of these precursors into the DNA. The levels of deoxynucleoside kinases and nucleoside monophosphate kinases in U. urealyticum were determined and we found that deoxynucleoside kinases especially thymidine kinase activity was upregulated upon the addition of either dUrd or dTHU in the growth medium while nucleoside monophosphate kinases such as thymidylate kinase activity were not affacted. A number of pyrimidine and purine nucleoside analogues were found to inhibit the growth of U. urealyticum and M. pneumoniae and Among those tested analogues fluoropyrimidines were the most potent inhibitors. 5-FdUrd, 5-FdCyd and 5- FUra significantly inhibited U. urealyticum growth with IC50 of 0.8, 3 and 40 mM at 104 cfu/ml respectively, and 5-FdUrd inhibited M. pneumoniae growth with IC50 of 6.2 µM. The inhibitory effect of these analogues could be reversed by the addition of either dThd, dUrd or dTHU, suggesting that the mechanism of inhibition was due to the inhibition of thymidylate biosynthesis.

167 P76

Synergistic effects of BCNU and Didox combination chemotherapy in 9L glioma cells

Z. Horvath1, W. Bauer1, Th. Hoechtl1, Ph.S. Saiko1, M. Fritzer-Szekeres1, T. Tihan2, T. Szekeres1 1University of Vienna - General Hospital, VIENNA, Austria 2Johns Hopkins Hospital, BALTIMORE, United States of America

A significant number of malignant gliomas develop resistance to systemic and local 1,3-bis (2-chloroethyl)-1- nitrosourea, (BCNU) therapy, a first-choice drug in adjuvant chemotherapy. Increases in DNA repair and synthesis are implicated as the main causes of BCNU resistance. Compounds inhibiting DNA synthesis and repair are expected to act synergistically with BCNU and improve the efficacy of BCNU-therapy. Inhibitors of the enzyme ribonucleotide reductase might be used as additional agents. Ribonucleotide reductase (EC1.17.4.1; RR) catalyzes the rate-limiting step in DNA synthesis and plays a critical role in maintaining substrates for DNA repair. Ist increased rate of expression in cancer cells makes RR a critical target in cancer chemotherapy. Didox (3,4-dihydroxybenzohydroxamic acid) is a potent inhibitor of RR, and has been proven to induce apoptosis in vitro and in vivo. We studied the effects of Didox on 9L glioma cells in combination with BCNU. We analyzed intracellular deoxynucleosidetriphosphate (dNTP) pools and found that Didox depleted significantly the intracellular dNTP concentrations. After incubation with 15 and 30 µM Didox, the intracellular concentrations of dGTP fell to 34 and 16,7% of control values, respectively. The dCTP and dTTP pools also showed significant decrease (66,9 and 61,3% of control for dCTP and 84,9 and 67,4% of control for dTTP, respectively). The intracellular dATP pools have experienced an increase of 186,3 and 154,8%. Experiments using cytotoxicity, growth inhibition and clonogenicity assays showed significant synergism of Didox and BCNU. In chrystal violet assay, Didox as a single agent yielded an IC50 value of 400µM, whereas BCNU reached an IC50 value of 500µM. After combined treatment using equimolar concentrations of the two drugs, a combination index<1 was reached in concentrations between 50 and 500µM, demonstrating synergistic effects. In growth inhibition and colony formation assays with concentrations ranging from 20 to 100µM, the synergism between Didox and BCNU was also confirmed. We have also identified increased gene expressions in a number of DNA-repair related enzymes using large-scale cDNA-arrays after BCNU incubation. These effects could partially be circumvented after Didox treatment. These results introduce the combination of Didox and BCNU as an additional option for the treatment of gliomas.

168 P77

Amidox, an inhibitor of ribonucleotide reductase, potentiates the action of Ara-C in HL-60 human promyelocytic leukemia cells

W. Bauer, Z. Horvath, Th. Hoechtl, D. Karl, Ph.S. Saiko, M. Fritzer-Szekeres, T. Szekeres University of Vienna - General Hospital, VIENNA, Austria

Ribonucleotidereductase (RR) catalyzes the rate limiting step in the de-novo synthesis of deoxyribonucleotidetriphosphates (dNTPs). Since its activity was shown to be correlated to the proliferation rate in tumor cells, it is considered to be an excellent target for cancer chemotherapy. Amidox (3,4- dihydroxybenzamidoxime), a polyhydroxy-substituted benzoic acid derivative, inhibits RR by interfering with the iron binding subunit of the enzyme. In the present study we investigated the antineoplastic effects of Amidox alone and in combination with Arabinosylcytosine (Ara-C) in HL-60 human promyelocytic leukemia cells. In growth inhibition experiments with logarithmically growing HL-60 cells Amidox yielded an IC50 of 30µM. The colony formation of HL-60 cells was inhibited at an IC50 of 20µM as determined by a soft agar assay. We also investigated in a possible reduction of intracellular dNTPs after incubation with Amidox as could be expected by inhibition of RR. Exposure of the cells to 75 and 100µM Amidox for 24 hours was shown to significantly decrease intracellular dCTP, dGTP and dTTP pools, whereas dATP concentration increased, as determined by HPLC. Combination of an RR inhibitor like Amidox with Ara-C could lead to synergistic antineoplastic effects by various mechnisms.The promotion of phosphorylation of Ara-C to Ara-CTP, the active compound, by deoxycytidinekinase, which underlies feedback inhibition by dCTP, by decreased dCTP levels could be one of them. In our experiments the combination of Amidox with Ara-C yielded additive cytotoxic effects in growth inhibition assays and additive inhibition of colony formation in soft agar assays. We could show that - after preincubating the cells with 75 and 100µM Amidox for 24 hours and subsequent exposure to Ara-C for 4 hours - intracellular Ara-CTP levels increased by 576% and 1143%, respectively. In conclusion, Amidox was shown to have potent antineoplastic effects in HL-60 human promyelocytic leukemia cells in concentrations that can be achieved in vivo. It increases the formation of Ara-CTP but yields additive effects with Ara-C concerning growth inhibition and colony formation. Nevertheless the compound might be further investigated for the treatment of leukemia.

169 P78

Mechanism of action of trifluorothymidine (TFT) in combination with a thymidine phosphorylase inhibitor (TPI)

O.H. Temmink1, O.H. Temmink1, M. De Bruin1, E.M. Comijn1, M. Fukushima2, G.J. Peters1 1VU University Medical Center, AMSTERDAM, The Netherlands 2Taiho Pharmaceuticals, HANNO-SHI, Japan

Trifluorothymidine (TFT) is a thymidine analog which is activated by thymidine kinase (TK) to TFT-MP which may inhibit thymidylate synthase (TS), involved in thymidine nucleotide synthesis. TFT-TP can be incorporated into DNA, possibly resulting in DNA damage and cell death. TFT can be broken down to trifluoromethyluracil (TFMU) by thymidine phosphorylase (TP), which can be inhibited by TP inhibitor (TPI) to enhance the bioavailability of TFT in vivo (the combination TAS-102). We studied the effect of TPI on TFT sensitivity, in situ TS inhibition, TFT metabolism and its incorporation into DNA. 5-Fluorouracil (5-FU) resistant H630 cell lines were cross-resistant to TFT (72 h exposure), in contrast to antifolate resistant WiDr cells, although all resistant variants had increased TS levels. However, at short term exposure (4 h) the 5FU- resistant H630 cells and antifolate resistant WiDr cells were both cross-resistant to TFT. TPI did not enhance TFT sensitivity in these and Colo320TP1 (TP transfected) cells. The concentration of TFT to get 50% inhibition of TS in both intact H630 and WiDr cross-resistant cells was 40- to 300-fold higher compared to the parental cells. In situ TS inhibition was very rapid in FM3A/0 mammary cancer cells (>90% after 4h), but also recovered rapidly (back to 75% after 20 h removal of TFT) when compared to 5FU. The addition of TPI to the medium did not increase TS inhibition by TFT. In the colon cancer cell lines WiDr, H630, Colo320 (TP deficient) and the TP transfected Colo320TP1 TFT incorporation into DNA was about 1.09, 0.77, 1.46 and 0.96 pmol/hr/106 cells, resp., and the accumulation of TFT phosphate derivatives was 1.39, 1.37, 2.73 and 1.29, resp. TPI increased the accumulation of TFT phosphate derivatives in Colo320TP1 cells to 2.42 pmol/hr/106 cells (p<0.05). In the presence of TPI TFMU formation was completely inhibited in all cell lines. The formation of TFMU by Colo320TP1 cells was also inhibited by almost 50% at equimolar thymidine concentrations. It can be concluded that cancer cells take up and activate TFT very rapidly, possibly due to favourable enzyme properties. TPI increased TFT phosphorylation only at very high TP levels.

170 P79

The effect of different fluoropyrimidines with or without a thymidine phosphorylase inhibitor (TPI) on the expression of platelet derived endothelial cell growth factor / thymidine phosphorylase (PD- ECGF/TP)

K. Smid1, M. De Bruin1, T. Van Capel1, K. Hoekman1, M. Fukushima2, H.M. Pinedo1, G.J. Peters1 1VU University Medical Center, AMSTERDAM, The Netherlands 2Taiho Pharmaceuticals, HANNO-SHI, Japan

TP is an enzyme catalyzing the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1- phosphate. TP is similar to PD-ECGF, which plays a role in angiogenesis and has been shown to be upregulated in several solid tumors. Fluoropyrimidines such as 5-fluorouracil (5FU), 5′-deoxy-5- fluorouridine (5′DFUR) and trifluorothymidine (TFT), are substrates for PD-ECGF/TP. These are either activated (5FU and 5′DFUR) or inactivated (TFT) by TP. We therefore studied whether treatment with 5′DFUR and TFT with or without a specific TP inhibitor (TPI) would affect PD-ECGF/TP expression and function. Three human colon cancer cell lines, WiDR, HT29 and Lovo were treated for 72 h with 5′DFUR or TFT at their IC50 concentration with or without 10 µM of TPI or with TPI alone. PD-ECGF/TP expression was analyzed at the mRNA level with competitive template reverse transcriptase polymerase chain reaction (CT-RT-PCR), the protein expression was measured using western blotting and the activity with 14C- thymidine as a substrate. In Lovo cells TFT plus TPI treatment increased TP protein expression 1.7-fold, but there was no profound effect of other treatments, TP-mRNA levels in cells treated with 5′DFUR and TFT showed a 5- and 1.4-fold increase, respectively. In WiDR cells 5′DFUR plus TPI treatment reduced TP protein expression 2-fold; TFT and TFT plus TPI increased TP protein 2- and 3-fold, respectively. TPI and 5′DFUR treatment reduced TP-mRNA 1.6- and 5-fold, respectively, which was only accompanied by a lower TP protein expression in 5′DFUR treated cells. In HT29 cells TP protein and TP-mRNA expression of the 5′DFUR and 5′DFUR plus TPI treated cells were lower, 3.6- and 2.75-fold, respectively. In all cell lines 5′DFUR and TFT treatment did not affect TP activity, however, treatment with TPI (alone or in combination) completely abolished TP activity in all cell-free extracts. In conclusion: different effects on TP were observed at equitoxic drug concentrations, indicating that substrate and endproduct regulation of TP is drug and cell line dependent, and may affect the effects of drugs.

171 P80

Combination studies of trifluorothymidine (TFT) with antifolates and the DNA synthesis inhibitors irinotecan, oxaliplatin and gemcitabine

O.H. Temmink1, M.F.M. Hoogeland1, M. Fukushima2 G.J. Peters1 1VU University Medical Center, AMSTERDAM, The Netherlands 2Taiho Pharmaceuticals, HANNO-SHI, Japan

Trifluorothymidine (TFT) is a fluorinated pyrimidine analog which can be activated by thymidine kinase (TK) to TFT-MP which may inhibit thymidylate synthase (TS), the only de novo enzyme involved in thymidine nucleotide synthesis (dTTP). TFT-TP can also be incorporated into DNA, possibly resulting in DNA damage and cell death. TS converts deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) and its inhibition leads to dTTP depletion in the cell causing dUTP to be misincorporated into DNA.The antifolates AG337, ZD1694 and GW1843 are direct TS inhibitors. We studied the effect of antifolates, irinotecan (as the topoisomerase inhibitor SN-38), oxaliplatin and gemcitabine on TFT sensitivity by using cytotoxicity assays. A panel of 5 colon cancer cell lines were used for the experiments. Multiple drug effect analysis was performed using specialized computer software based on the Chou and Talalay method (Calcusyn, Biosoft 1996). Effects were expressed as mutually nonexclusive case combination index (CI) in which a CI<1 indicates synergism and CI>1 antagonism. Three experimental procedures were used to test the interaction of the drugs: either one of the drugs was kept at a constant concentration (~IC25) or two drugs were added in a 1:1 IC50 equimolar ratio. The combination of TFT with irinotecan showed additive to antagonistic effects in most cell lines (0.9

172 P81

Combination studies of 5-fluorouracil with UCN-01 or staurosporine

J. Sigmond, E.M. Comijn, G.J. Peters VU University Medical Center, AMSTERDAM, The Netherlands

Cytotoxic effects of 5-fluorouracil (5-FU) have been reported to be RNA related or mediated by and inhibition of thymidylate synthase (TS). The latter effects are cell cycle dependent, and possibly also related to p53 expression. Therefore we evaluated the interaction between 5-FU and UCN-01, an inhibitor of cell cycle dependent kinase 2 and protein kinase C (PKC) and another PKC inhibitor, staurosporine. Growth-inhibitory effects of 5-FU combinations were evaluated by multidrug effect analysis and were related with the role of induction of apoptosis or cell cycle arrest (with FACS analysis). Variants of the colon cancer cell line Lovo with wildtype (Lovo B2), mutant p53 (Lovo 175x2) and functionally inactive p53 (Lovo Li) were used.Lovo B2 and Lovo Li cells showed similar IC50 values for 5-FU, staurosporine and UCN-01, while Lovo 175x2 cells were relatively more resistant. Combination indices for the 5-FU combinations showed synergism in Lovo B2 and Lovo 175x2 while in Lovo Li moderate antagonism was observed. To evaluate effects on cell cycle distribution, IC100 concentrations were used. In all Lovo variants, an S phase arrest was induced after exposure to 5-FU (50 µM). Exposure for 24 hr to staurosporine (0.05 µM) or UCN-01 (0.5 µM) induced accumulation of cells in the G2/M phase or G0/G1, respectively, shifting to an S-phase arrest after 48 hr. Simultaneous exposure to 5-FU combinations showed an average cell cycle distribution of both drugs when used alone. In sequential drug administration, the cell cycle distribution was determined by the first drug. More induction of apoptosis by IC100 of UCN-01 was found in Lovo 175x2 compared to other Lovo variants, which required no functional p53. Additive effects in induction of apoptosis were observed in both simultaneous and sequential 5-FU combinations. Based on induction of apoptosis 5-FU addition prior to the PKC inhibitors seemed preferable. When staurosporine was used as the second drug, induction of apoptosis in Lovo B2 and Lovo 175 was 2-fold higher than the sum of both drugs alone. These mechanistic studies offer a good basis for evaluation of development of combination therapies with 5-FU.

173 P82

5’-nucleotidases levels measured in peripheral blood cells from patients with chronic and acute leukemia

M. Razmara1, S. Eriksson2, F. Albertioni1 1Karolinska Institute, STOCKHOLM, Sweden 2Swedish Univ. of Agricultural Sciences, UPPSALA, Sweden

Treatment of leukemia has been improved significantly during the last three decades, mainly based on the introduction of new nucleoside analogs like cladribine (CdA, 2-chloro-2'-deoxyadenosine) and fludarabine. These nucleosides must be activated by enzymes such as deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Key catabolic enzymes responsible for degradation of nucleotides are 5´-nucleotidases. Five major 5´-nucleotidases have been described, 5´-ectonuclotidase (membrane-bound), cytosolic 5´-nucleotidase I (cN-I), cytosolic 5´-nucleotidase II (cN-II) and both cytosolic and mitochondria 5´(3´)-deoxyribonucleotidases (dNT-1, dNT-2). We have investigated the activity levels of cN-I and cN-II two key enzymes in the dephosphorylation of nucleoside analogs used in chemotherapy. Enzyme assays were performed in cell extracts from 32 patients with acute myeloid leukemia (AML) and chronic lymphoblastic leukemia (CLL) by using a selective HPLC assay. We found that the activity of cN-I (using adenosine monophosphate as substrate) was similar in AML and in CLL. The activity of cN-II (using inosine monophosphate as substrate), was much higher than cN-I both in AML and in CLL cell extracts. We also investigate the degradation of CdAMP in same cell extracts. The activity of CdAMP degradation was significantly correlated to the activity of cN-I (R= 0.60). The activity of cN-II did not show a correlation to CdAMP degradation. These results suggest that CdAMP was most probably dephosphorylated by cN-I. Whether high levels of cN-I independently identify a poor prognosis group of patients remain to be investigated

174 P83

Cyclic guanosine monophosphate role in human carcinoma pathogenesis

G. Spoto1, M. Di Nicola1, F. Santoleri1, S. Soscia1, M. Fioroni2, C. Rubini2, A. Piatelli1 1University of 'G. D'Annunzio', CHIETI, Italy 2University of Ancona, ANCONA, Italy

Cyclic guanosine monophosphate (cGMP) is an essential second messenger for different cellular signals and is hydrolyzed to guanosine 5’-monophoshate by phosphodiesterases (PDEs), whose activity is regulated by different inputs originated from some signaling-systems. The cGMP is important in inhibiting PDE3, stimulating PDE2 and activation of kinase protein. The aim of this study was a correlation between cyclic guanosine monophosphate and human oral squamous cell carcinoma progression. Although the cyclic nucleotide role in carcinoma growth and invasion has not been clearly defined, its function as second messenger of many signaling molecules suggest that it may be a key factor in neoplastic progression. We know that in cancerouses progression there is not only excessive cell proliferation, but also a cell death decrease, which we know is regulated by cGMP. The cGMP differences between healthy gingival tissues used as controls, Gingival Squamous Cell Carcinoma, (OSCC ), with (N+) and without (N-) lymph node metastases were evaluated. The difference between control (0.32 ± 0.10 µM) and N- (0.53 ± 0.24 µM) and N+ (0.56 ± 0.24 µM) values showed an important increase of cGMP contemporaneously to an increase of neoplastic tissue trasformation; this may suggest a probable cGMP involvement in this proliferation mechanism. Moreover, our study showed an higher PDE activity (5.74 ± 1.40 µM) values in N+ than in control samples (5.59 ± 1.15 µM). In another study, two colon adenocarcinoma cell lines at different stages of differentiation were analysed. In cell lines with a better differentiation degree an high cGMP concentration (0.0976 µM) was present. cGMP and enzyme activity, simultaneously confirm that this second messenger is really influenced by carcinoma progression. These data showed an increase of intracellular cGMP concentration in cancer specimens; the organism reacts increasing PDE activity, to try to stabilize cGMP on basal levels. Therefore, these results were characterized from an elevated standard deviation, that explain both an altered hydrolysis PDE mediated and an altered guanilate cyclase activity. In conclusion we belive that elevated cGMP intracellular concentration in human cancer may be interpreted as a positive cell because it is knows that cGMP (PDE3A inhibitor) is important in neoplastic proliferation inhibition.

175 P84

Cyclic nucleotides and neuroblastoma differentiation

A. Giacomello, E. Messina, F. Lupi, L. Barile University of Rome 'La Sapienza', ROME, Italy

An elevation of the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma cells. The rise of cGMP levels during 1-10 µM retinoic acid and 50 nM mycophenolic acid treatment suggests the possible involvement of this cyclic nucleotide in morphological neuroblastoma differentiation. In line with this hypothesis LAN-5 and SHEP human neuroblastoma cells elaborated a network of neuritic processes during a 6 days treatment with 0.1 to 1 mM 8-p-CPT-cGMP or dbcGMP, with a neurite outgrowth similar to that obtained using 1mM dbcAMP. In neuroblastoma cyclic nucleotides hydrolysis is achieved by members of at least five phosphodiesterases (PDEs) families (PDE1 to PDE5). The effect of the specific inhibitors of PDE1 (MMPX), PDE3 (Ciclostamide, Quazinone, Milrinone, Zardaverine), PDE4 (Rolipram, Etazolate, Zardaverine), PDE5 (Zaprinast, MY-5445, Dipyridamole) on neuroblastoma growth and differentiation have been studied. LAN5 and SHEP human neuroblastoma cells were treated for 3 to 6 days with each specific inhibitor at concentrations ranging from 0 to 10 x IC50 µM. Spectrophotometric quantification of cell proliferation and viability (cell proliferation reagent WST-1) showed a decreased cell proliferation only in response to Zaprinast and MY-5445. After 6 days of incubation of both cell lines in the presence of these two PDE5 inhibitors an increase in neurite outgrowth was observed. Preliminary experiments showed that at least the inhibitory effect of zaprinast on cell growth was more marked than the inhibition of PDE5 activity and that at the 10 x IC50 drug concentrations of specific PDE inhibitors intracellular cGMP levels obtained with Zaprinast or MY-5445 were comparable to those obtained with dipyridamole, a PDE5 inhibitor without significant effects on neuroblastoma growth and differentiation. These results suggest that Zaprinast and MY-5445 could act by interfering with other molecular events than direct cGMP-PDE inhibition. In conclusion we have demonstrated an involvement of cGMP in neuroblastoma differentiationa and a significant antiproliferative and differentiating action of Zaprinast and MY-5445. These observations suggest that these PDE5 inhibitors could have a potential role in the management of neuroblastoma patients.

176 P85a

Differentiation-inducing Activity of Purine and Pyrimidine Derivatives on Myelogenous Leukemia Cells: The most suitable analog depends on the leukemia type

Y. Honma Saitama Cancer Center Research Institute, SAITAMA, Japan

The patient outcomes following the treatment of acute myelogenous leukemia (AML) have improved with progress in intensive chemotherapy. However, the therapy is limited by the unresponsiveness of certain leukemias and severe adverse effects. The current results of treatment for AML clearly call for improvement. Differentiation therapy has been successful as a treatment for acute promyelocytic leukemia (APL), a subgroup of AML, but it dose not clinically apply to other hematopoietic malignancies. The further development of differentiation therapy for leukemia requires new and potent inducers of differentiation. Many purine and pyrimidine derivatives can induce the differentiation of human and murine leukemia cells. The sensitivity to the derivatives varies for each leukemia cell line. Therefore, the differentiation- inducing effect of various purine and pyrimidine analogs on several human myelogenous leukemia cells was examined and reviewed the association of differentiation-inducing activity of purine and pyrimidine derivatives with leukemia cell types. Monocytoid leukemia cells were more sensitive to deoxyadenosine analogs than were erythroid or myeloid leukemia cells. APL cells were less sensitive to purine and pyrimidine analogs than other AML cells at inhibiting cell growth and inducing differentiation. 1-β-D-Arabinofuranosylcytosine (araC) is effective to induce differentiation of many AML cells, but not of APL cells. However, a cytidine deaminase-resistant analog of araC (DMDC) induced differentiation of APL cells, with dose-response curves similar to those of other AML cells. Among naturally occurring nucleobases, adenine effectively enhanced the differentiation of myeloid leukemia HL-60 cells, while other nucleobases, adenosine or deoxyadenosine alone did not. Since N6-modification potentiated the ability of adenine analogs to induce differentiation, we examined the effects of various N6-modified derivatives on differentiation. All of the derivatives except zeatin were very potent at inducing differentiation, and isopentenyladenine had the greatest effect. Novel synthetic analogs of purines and pyrimidines were also examined to induce differentiation of AML cells, and several compounds might be useful for differentiation therapy of AML. However, the antileukemic analog that is the most suitable for the treatment of AML may depend on the leukemia subtype, since the sensitivity of leukemia cells to these analogs varied greatly among leukemia subtypes.

177 P85b

6-mercaptopurine (6-MP) metabolism in man and its fingerprints in erythrocytes (RBC)

S. Gutsche, A. Kotalczyk, H. Iven Medical University of Luebeck, LUEBECK, Germany

Introduction: The metabolism of 6-MP in nucleated cells is well known. Catabolism by xanthinoxidase leads to thiouric acid, anabolism via HGPRT and purine interconversion to cytotoxic thioguanine-nucleotides (TGN). Thiopurine S-methyltransferase (TPMT) – an enzyme with a genetic polymorphism – methylates 6- MP and most of the metabolites on various steps of the 6-MP pathway. While the metabolism to 6-TGN can only take place in nucleated cells, 6-TGN is determined in RBC which are also used to phenotype TPMT activity. This raises the question whether the results obtained in RBC are always represen-tative for other tissues? Materials and methods: We phenotyped TPMT-activity and measured 6-MP metabolites in RBC of more than 250 paediatric ALL patients treated according to the German BFM-2000 protocol. For each patient up to 13 samples were collected covering the whole treatment period of 2 years. Measurements were performed using an enzymatic assay for the TPMT activity and HPLC for the metabolites 6-TGN, 6-methyl-MP (6- MMP), and 6-methyl-TG (6-MTG). Results: After oral administration of 6-MP, 6-MMP accumulates in RBC and follows any change in dosing rapidly while 6-TGN (and 6-MTG) accumulate more slowly. In the individual patient steady state 6-MMP concentrations and 6-TGN +6-MTG concentrations correlate to the 6-MP dose. However, while the regression line of 6-TGN+6-MTG versus 6-MP dose goes through the origin, 6-MMP hits the abscissa approximately at a 6-MP dose of 20mg/m². In the majority of patients TPMT activity was low at diagnosis increasing dramatically during the next 20 weeks of treatment. There was no correlation between TPMT activity and the dependent metabolites or leukocyte count. Conclusions: Erythrocytes are a mirror of the 6-MP metabolism. They reflect 1) the patients compliance, 2) a saturable first pass metabolism probably useful for a better individualisation of therapy, 3) the effective formation of cytotoxic 6-TGN´s as a basis for drug monitoring and 4) a certain increase of the TPMT activity, dependent on transfusion of erythrocytes, enzyme induction and/or a renewal of the erythrocyte pool. Especially TPMT activity in RBC´s and its predictive value for the therapeutic efficacy should be a point of further discussion. Department of Pharmacology, *Center for Pediatrics , Medical University of Luebeck, Ratzeburger Allee 160. D-23538 Luebeck, FRG . These studies were supported by the Deutsche José Carreras Leukaemie Stiftung.

178 P86a

ATP-site directed potential inhibitors of nucleoside triphosphatases (NTPase)/helicases and polymerases of hepatitis C and other selected Flaviviridae viruses

T. Kulikowski1, M. Bretner1, S. Schalinski2, M. Lang2, H. Schmitz2, P. Borowski2, S. Behrens3 1Polish Academy of Science, WARSAW, Poland 2Bernhard Nocht Institute for Tropic. Med, HAMBURG, Germany 3Justus Liebig University, GIESSEN, Germany

5`O-(4-fluorosulfonylbenzoyl)-esters of ribavirin (FSBR), adenosine (FSBA), guanosine (FSBG) and inosine (FSBI) were obtained by acylation of the 5’-OH of adenosine, guanosine, inosine, and ribavirin with 4- fluorosulphonylbenzoyl chloride (FSBCl) in HMPA. The above derivatives were tested as inhibitors of NTPase/helicase and polymerase activities of selected Flaviviridae viruses, such as the hepatitis C virus (HCV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and dengue virus (DENV). When the helicase activity of viral NTPase/helicases was tested under standard conditions, only moderate inhibition was obtained (IC ≥ 120µM) and in the case of FSBG even an activation was seen. The preincubation of the enzymes with the FSB-derivatives induced or amplified inhibitory effect, approximately 10– to 100–fold. Comparative studies revealed that the extent of inhibition of the unwinding activity is enzyme specific. When the polymerase activity of HCV was investigated as a function of increasing concentrations of the FSB-derivatives, all the compounds inhibited the enzyme with the following order of efficiency: FSBA

179 P86b

Pre-treatment TPMT phenotyping/genotyping : an excellent guide for initial thiopurine drug dosing

E.A. Shobowale-Bakre1, A. Ansari1, M. Arenas1, A.M. Marinaki1, L.D. Fairbanks1, J.A. Duley2, J.D. Sanderson1 1Guy's Hospital, LONDON, United Kingdom 2University of Queensland, QUEENSLAND, Australia

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme. Deficiency of the enzyme is predictive of thiopurine drugs (i.e. azathioprine, mercaptopurine and thioguanine) intolerance. In Caucasian and African populations, 10-14% are carriers while 0.3% of the individuals are totally TPMT deficient. Two-thirds of these carriers have a high risk of drug induced side-effects on a full dose and therapy is typically withdrawn due to bone marrow suppression. In some clinics, these patients are either switched onto more toxic drugs or given surgical intervention. To test the hypothesis that carriers should tolerate half the normal dose, we identified 28 TPMT carriers who had pre-therapy TPMT phenotyping done in our laboratory mainly from gastroenterology and dermatology clinics. Two patients had autoimmune hepatitis, 6 skin and or oral ulceration and 3 eczema. Each patient was initially given azathioprine (aza) at a dose of 0.5 mg/kg which was then increased to 1mg/kg as required for steroid-sparing according to departmental guidelines. Clinical notes were reviewed after one year of treatment for adverse effects and clinical response (defined as withdrawal of steroids: complete, partial or non-response). TPMT phenotype results were confirmed in all patients by genotyping for the three most common TPMT allelic variants: TPMT *3A (G460A-Ala154Thr and A719G-Tyr240Cys + G460A-Ala154Thr), TPMT*3C (A719G-Tyr240Cys) and TPMT*2 (G238C-Ala80Pro). Six of the 28 patients experienced side-effects requiring drug withdrawal (2 pancreatitis, 4 nausea/malaise). The 4 patients that experienced nausea were switched to low dose 6-mercaptopurine and no further side effects were observed over the treatment period. 24 of the 26 carriers who remained on drug therapy responded favourably. In conclusion, TPMT heterozygotes showed an excellent clinical response (86%) to thiopurine therapy at reduced thiopurine dose. TPMT testing is thus a useful tool for guiding thiopurine drug dosing, thereby avoiding more toxic drug therapy and complicated surgical intervention.

180 P87

A time saving and rapid HPLC-method for the determination of 6-thioguanine and 6-thioguanine (-nucleotides)

A. Kotalczyk, S. Gutsche, H. Iven Medical University of Luebeck, LUEBECK, Germany

Introduction: After oral administration of 6-mercaptopurine (6-MP) or its prodrug azathiopurine (AZA), 6- MP enters the nucleotide pool as thioinosine-monophosphate and is further metabolised to thioguanine- nucleotides. On the other hand 6-thioguanine (6-TG) is directly metabolised to thioguanine- nucleotides. The latter may be incorporated into DNA and RNA and is considered responsible for therapeutic outcome and/or toxicity. We therefore monitor 6-TG concentrations routinely in RBC of children treated for ALL and in patients treated with AZA because of autoimmune diseases using the method of Erdmann et al. (Biomed Chromatogr 4: 47-51;1990) with slight modifications. This method takes advantage of the fact, that 6-TG can be transformed into a highly fluorescent derivative by oxidation with potassium permanganate. This pre column derivatisation requires multiple steps and is tedious and time consuming. Therefore we developed and improved an HPLC- method for 6-TG (- and its mono-, di-, and trinucleotides) using H202 and UV- irradiation (Beam Boost) for post column oxidation of 6-TG. Methods: We used a Waters Nova Pack C-18, 39x 150 mm column. The eluent consisted of 0,02m phosphate buffer pH 7.9, acetonitrile (85/15; v/v) containing 700mg/ l tetra-n-butylammonium-hydrogen sulphate. Flow rate was 0.5 ml/min with no recirculation of eluent. Post column: 0.3% H202 at a flow rate of 10 µl/min was added to the eluent before it entered the photochemical reaction-unit (UV:254 nm, coil-length 5 m). Fluorescence was monitored at Ex 315nm, Em 390nm. The sensitivity of the system for an aqueous 6- TG solution is 10 pg absolute. The system is very sensitive to any change of the pH of the eluent. Pre-treatment: RBC were haemolysed in 5 volumes of aqua dest. and kept at 95° C for 15 min. After centrifugation the clear supernatant was used directly for analysis. Results: A modification of column temperature did not improve the chromatograms. The nucleotides decomposed when the column temperature was more then 40° C. We used this system to study the time and concentration-dependent uptake of 6-TG into erythrocytes in vitro. In accordance with the result of Kong et al. (Biochem Pharmacol 24: 807-813;1975) we found a rapid uptake and metabolism of 6-TG to 6- thioguanosine- mono-, di-, and predominantly -tri-phosphate. Uptake and metabolism was almost complete after 90 min of incubation. The metabolism was near saturation at a concentration of 3-5 µg 6-TG/100 µl erythrocytes. Beyond this concentration, in addition to the 6-TG nucleotides the free base also accumulated in the erythrocytes. In contrast to 6-TG, 6-MP was only detected as thioinosinic acid (6-MP- monophosphate).Within 24 hours only 1% of the 6-TG in RBC was metabolised to 6-methyl-TG by thiopurine S-methyltransferase (TPMT). This also held true for 6-MP. Department of Pharmacology, *Center for Pediatrics, Medical University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, FRG. These studies were supported by the Deutsche José Carreras Leukaemie Stiftung.

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Real time RT-PCR methodology for quantification of TPMT gene expression

M. Lindqvist, S. Almer, C. Peterson, P. Söderkvist University of Linköping, LINKÖPING, Sweden

Thiopurine methyltransferase (TPMT) catalyses the methylation of thiopurines such as mercaptopurine, thioguanine and azathioprine, which are used to treat acute lymphoblastic leukemia, autoimmune disorders and inflammatory bowel diseases. Levels of TPMT enzymatic activity are controlled by a genetic polymorphism, an important factor responsible for individual differences in thiopurine toxicity and therapeutic efficacy. Ninety percentages of Caucasians are homozygous for two active alleles, 10 % are heterozygous for one inactive allele and 0.3 % are homozygous for two inactive alleles. The active TPMT gene is located on chromosome band 6p22.3 in humans and more than 10 variant TPMT alleles have been described. A TPMT processed pseudogene, with a DNA sequence 96 % identical to that of the open reading frame of TPMT cDNA is located on chromosome band 18q21.1. The aim of this study was to design and develop a real time PCR method, using Taqman technology, for quantification of TPMT mRNA in leukocytes and to use this method for correlation studies between gene expression and enzyme activity. The PCR product was sequenced and thereby confirmed to be the TPMT cDNA. The housekeeping gene Cyclophilin (CYC) was used as internal control and standard curves were created both for TPMT and CYC using RNA from a MOLT-4 cell line. Quantitative analysis of TPMT mRNA was performed in comparison to CYC mRNA. TPMT enzyme activity was determined in lysates from red blood cells (RBC) using a radioactive assay with tritiated S-adenosyl methionine as a methyl donor. The TPMT mRNA expression was measured in leukocytes from 39 individuals. In the group presumed to be homozygous for two active alleles (TPMT value 9.40-19.6 U/ml RBC, n=29), the correlation coefficient between the mRNA expression and the enzyme activity was statistical significant (p<0.001). No significant correlation was found in the group of individuals with TPMT activity lower than 9 U/ml pRBC (TPMT values 0.3-8.50, n=10). The real time PCR method will be a valuable tool in future studies of TPMT enzyme activity regulation. Hopefully, this will contribute to better prediction of the effects of thiopurines in individual patients.

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Quantitative detection of deoxycytidine kinase by real time light cycler PCR in comparison with conventional competitive template RT-PCR

J. Sigmond, J.R. Kroep, W. Loves, G.J. Peters Dept. Medical Oncology, VU University Medical Center, AMSTERDAM, The Netherlands

Deoxycytidine kinase (dCK) is essential for the phosphorylation of several deoxyribonucleoside analogues that are widely used as chemotherapeutic agents. Deoxyribonucleoside analogues such as cytosine arabinoside (Ara-C) or gemcitabine (2’2’-difluorodeoxycytidine) are used in leukemias and solid tumors, respectively. For gemcitabine it has been shown that pretreatment dCK levels can predict the response to gemcitabine in vivo. For evaluation of dCK mRNA expression, conventional Competitive Template Reverse transcriptase Polymerase Chain Reaction assays were performed (CT-RT-PCR). However, real time PCR enables more rapid and sensitive detection of mRNA expression using either SYBR Green or fluorescent probes using a Light Cycler (LC). We developed and optimized a sensitive real time PCR assay for dCK with SYBR green detection. For this purpose we used cDNA from human xenografts previously used to establish a relation between dCK activity and gemcitabine sensitivity (Kroep et al, Mol. Canc. Ther. 1:371-6, 2002). A significant correlation between LC-PCR and CT-RT-PCR (Pearson: r=0.956; p<0.0001) was found. In addition, we observed a good correlation (Pearson: r=0.972; p=0.003) between LC-PCR and enzyme activity. Furthermore, gemcitabine sensitivity was correlated (Pearson: r=0.695; p=0.048) with dCK mRNA measured with LC-PCR. The assay was applicable to determine dCK mRNA levels in needle biopsy specimens of various human tumors. Our data demonstrate that Light cycler is a rapid and reliable method for the detection and quantitation of dCK mRNA levels which can be used in the clinic to predict chemotherapeutic agent sensitivity.

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Immunocytochemical detection of deoxycytidine kinase

I. Hubeek1, G.J. Peters1, A.J.F. Broekhuizen1, I. Talianidis2, E.R. Van Wering3, U. Creutzig4, G. Giaccone1, P. Van der Valk1, G.J.L. Kaspers1 1VU University Medical Center, AMSTERDAM, The Netherlands 2Institute of Molecular Biology, HERAKLION, Greece 3DCOG, THE HAGUE, The Netherlands 4AML-BFM Study Group, MÜNSTER, Germany

Deoxycytidine kinase (dCK) is responsible for the activation of several clinically important anticancer agents. These nucleoside analogs are widely used for the treatment of malignancies and are dependent on phosphorylation by dCK. To determine the expression level of dCK protein in several different malignancies we have set up an immunocytochemical method, using a rabbit-anti human dCK antibody and applied this method in 97 samples. Immunocytochemical staining was performed on cryopreserved cytospins of 31 childhood acute myeloid leukemia (AML), 29 childhood acute lymphoblastic leukemia (ALL), 10 retinoblastoma and 12 pediatric brain tumor samples. Furthermore, the leukemic cell lines CCRF-CEM, U937 and HL60 were used as positive controls. Negative controls were performed by omitting the first antibody. dCK protein expression was determined by scoring the intensity of the staining: negative (0), low (1+), intermediate (2+), high (3+), or very high (4+), by two independent investigators. In the leukemic cell lines U937 and CCRF-CEM and in the different tumor types a predominantly cytoplasmic staining was observed. In 4 out of 31 AML samples and in 2 out of 29 ALL samples we also observed staining in the nucleus. The AML and retinoblastoma samples showed an intermediate to (very) high expression of dCK. The expression level of dCK in ALL samples ranged from low to (very) high. In contrast, the majority of brain tumor samples expressed low to intermediate levels, with 2 samples expressing no dCK. In the NCSLC tissue samples the expression of dCK ranged from low to very high. All NCSLC samples expressed the dCK protein. The interobserver variability between two observers showed agreement in 72/82 (88%) cases (kappa = 0.83). On Western blotting the antibody detected dCK appropriately at 30 kD, without additional bands. In conclusion, immunocytochemistry is an effective method to determine the expression level of dCK in patient samples. In the vast majority of samples dCK was located exclusively in the cytoplasm. It was possible to discriminate between patient samples with a low, intermediate and (very) high expression level of dCK. There were clear differences in dCK expression observed between tumor types.

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Determination of the deoxycytidine kinase activity in cell homogenates with a non-radiochemical assay using reversed-phase HPLC. Identification of a novel metabolite of 2-Chlorodeoxyadenosine

J. Bierau1, A.B.P. Van Kuilenburg1, R. Leen1, A.H. Van Gennip2, H. Caron1 1Academic Medical Center, AMSTERDAM, The Netherlands 2Academic Hospital Maastricht, MAASTRICHT, The Netherlands

Deoxycytidine kinase (dCK) is a deoxynucleoside kinase with a broad substrate specificity. The natural substrates of dCK are dCyd, dAdo and dGuo [1]. However, dCK also phosphorylates therapeutically important deoxynucleoside analogues, such as 1-ß-D-arabinofuranosyl cytosine (cytarabine), 2',2'-difluorodeoxyctytidine (gemcitabine). and 2-Chlorodeoxyadenosine (cladribine, CdA). In fact, it is the rate-limiting enzyme in the activation of these cytotoxic nucleoside analogues [2]. To date, all procedures to measure dCK activity are based on the method described by Ives and Durham [3], and rely on thin-layer chromatography or weak ion- exchange paper chromatography to separate the radioactive substrate (CdA or dCyd) from the corresponding nucleoside-5'-monophospate. These procedures proved to be extremely laborious and time consuming. Another major disadvantage of these traditional analytical methods is that the formation of other metabolites, which might hamper accurate measurement of dCK activity, may not be detected. We have developed a non-radioactive procedure to measure the deoxycytidine kinase activity in crude cell free homogenates. 2-Chloro-deoxyadenosine (CdA) was used as the substrate and was separated from its 5'- monophosphate by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 40 minutes. The minimum amount of CdA that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3 hours. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 µg/ml in the assay. An amount of 8 x103 cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. Under the standard reaction conditions described in literature, CdA was not only converted to its 5'- monophosphate but also to 2-Chloroadenine and, surprisingly, also to 2-Chlorodeoxyinosine, in MOLT-3 cells. Therefore, our results demonstrate that CdA is also a substrate for adenosine deaminase. In order to disqualify artefacts, the cell lines were tested and found to be mycoplasma negative.

References

1. Arner ES and Eriksson S, Pharmacol.Ther. 67: 155-186, 1995. 2. Liliemark JO and Plunkett W, Cancer Res. 46: 1079-1083, 1986. 3. Ives DH and Durham JP, J.Biol.Chem. 245: 2285-2294, 1970.

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Application of liquid chromatography - mass spectrometry for identification and quantitation of metabolic alterations in chronic renal failure

E.M. Slominska1, R.T. Smolenski2, M. Lipinski1, H. Dziewit1, N. Leaver2, B. Rutkowski1, J. Swierczynski1, R.T. Smolenski3 1Medical University of Gdansk, GDANSK, Poland 2Imperial College, LONDON, United Kingdom 3University of Gdansk, GDANSK, Poland

Metabolic abnormalities are the primary consequence of chronic renal failure (CRF) but its full spectrum has not been fully elucidated. Because of the limitations of standard HPLC with UV detection for analysis complex samples such as in CRF we aimed to develop liquid chromatographic/mass spectrometric (LC/MS) procedure for analysis of alterations of blood concentration of nucleotide metabolites in CRF. An MS compatible reversed-phase chromatographic system has been developed for analysis and identification of nucleosides and bases in plasma samples. Despite common view that only crude chromatographic separation is necessary while applying mass detection, we found that adequate chromatographic separation is essential to achieve sufficient sensitivity and recovery and to simplify the identification of unknown metabolites. In addition, optimization of conditions of ion source and mobile phase was critical for sensitivity due to profound effect on ionization efficiency. Full mass range analysis allowed detection of metabolites in micromolar concentration. However, several fold improvement of the sensitivity was possible in selective ion monitoring mode or even 1000x sensitivity improvement while operating in MS2 (fragmentation) mode. Analysis of CRF plasma samples in addition to well established alterations demonstrated less well characterized changes such as accumulation of adenine, nicotinamide and its metabolites: N-methyl-2-pyridone-5-carboxamide (2PY) and N-methyl-4- pyridone-5-carboxamide (4PY). Adenine concentration increased from 71±32nM in healthy subjects to 632±124nM in CRF. 2PY concentration increased from 0.36±0.10µM in healthy subjects to 19.3±6.4µM in adult patients with CRF while in children with similar degree of CRF 2PY reached the concentration 7.5±2.3 µM; 4PY was 2.10 µM±0.86 and nicotinamide was 0.29 µM±0.07. Adenosine blood concentration was not was not different from controls in children or adults with CRF. In addition to the effect of age on 2PY accumulation which was smaller in children with similar degree of CRF both plasma adenine and nicotinamide metabolite concentrations correlated with renal function impairment, transiently decreased following hemodialysis and permanently decreased following successful renal transplantation. Conclusion: LC/MS has unprecedented advantages for studies of metabolic alterations in CRF. It allows identification of new changes as well as rapid development of analytical techniques for its detailed analysis.

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Detection of prothrombin and osteopontin in a renal stone found in a hyperuricemic patient using 2D-PAGE and LCMS analysis

K. Kaneko Teikyo University School of Medicine, TOKYO, Japan

Objectives: The liquid chromatography/mass spectrometry (LCMS) technique has recently been applied on analysis of various proteins. A renal stone found in a hyperuricemic patient was analyzed with the two- dimensional polyacrylamide gel electrophoresis (2D-PAGE) and LCMS. Methods: A renal stone (size: 7-9 mm) from a patient with hyperuriceamia was examined. After the infrared spectrophotometry (IR) analysis, the stone was extracted with EDTA and guanidine hydrochloride following dialysis, concentration, and was applied to 2D-PAGE. Several proteins were isolated, treated with trypsin digestion, and analyzed with a nano-LC/mass spectrometer (LCMS). The LCMS was equipped with a nanospray ionization and an ion trapping detection system. A protein search from the obtained MS/MS spectra was undertaken, and the containing proteins determined. Results: IR analysis showed the renal stone was found to be mainly composed of calcium oxalate. In 2D- PAGE, several proteins were detected in the acid rich area (pI 3-4). After LCMS analysis, osteopontin and prothrombin were determined to be contained as acidic proteins. These proteins are both related to calcium binding. It is suggested that Ca-related proteins play an important role in the growing process of the calcium oxalate stones. Conclusion: The analytical technique using 2D-PAGE and LCMS was so sensitive that it was possible to detect small quantities of proteins even in a renal stone. In order to investigate the cause of pathological stone recurrence, analysis of each stone in detail is thought to be helpful.

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A non-radioactive sensitive assay to measure 5-fluorouracil incorporation into DNA of solid tumors

P. Noordhuis, U. Holwerda, J.A.M. Van Laar, C.L. Van der Wilt, G.J. Peters VU University Medical Center, AMSTERDAM, The Netherlands

The mechanism of action of 5-fluorouracil (5FU) has been associated with inhibition of thymidylate synthase and incorporation of 5FU into RNA and DNA. In cell line models this incorporation is usually measured with radioactive 5FU, but this is not applicable in patients. Previously we described a method for measurement of 5FU incorporation into RNA, based on a sensitive 5FU assay using gas-chromatography coupled to mass-spectrometry (GC-MS). This assay was adapted for measurement of 5FU into DNA. For that purpose we isolated DNA from cells and tissues using standard methods and degraded DNA enzymatically with DNAse, nuclease, alkaline phosphatase and purified thymidine phosphorylase, to 5FU, which after derivatization was measured with GC-MS. The assays were validated using the colon cancer cell line WiDr. In parallel experiments it was found that the incorporation as measured with radiolabelled 5FU and non-labelled 5FU (with GC-MS), were similar. The murine Colon 26-B tumor was used as an in vivo model. Incorporation into DNA after treatment with maximal tolerated doses of 5FU or fluorodeoxyuridine (FUdR) was maximal at 2 hours and was 15.4 and 71.0 fmol/µg DNA respectively. The level of incorporation remained high for about 3 days after which a decrease was observed to ± 2 fmol/µg DNA after 10 days both for 5-FU and FUdR. In contrast to the RNA incorporation, DNA incorporation did not correlate with the intra-tumoral 5-FU concentration, neither for 5FU alone or FUdR. This is possibly because FUdR can also be incorporated into DNA directly in addition to the pathway via 5FU. Consequently FUdR treatment as compared to 5FU resulted in a higher incorporation into DNA. In conclusion, a sensitive non- radioactive assay for 5FU incorporation into DNA was developed. This method is also applicable for human tissues.

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