PULMONARY DISTRIBUTION OF THIOPURINE NUCLEOTIDES IN LUNG-TRANSPLANT PATIENTS

R.Boulieu. A.Lenoir. J.F.Mornex

Azathioprine, a thiopurine compound that exhibits immunosuppressive activity is currently used with cyclosporine and corticosteroid in combined immunosuppmssive protocols in organ transplantation (1). Azathioprine undergoes a complex metabolic pathway and its pharmacodynamic activity is considered to depend on the formation of intracellutar thiopurine nucleotides (2). Despite the extensive use of azathioprine in lung transplantation, data on diffusion of thiopurine nucleotides into the lung are not available. Therefore, we investigated lung distribution of thiopurine nucleotides of 6-thioguanine (6TGN), 6-mercaptopudne (6MPN) and 6-thioxanthine (6TXN) in eigth lung-transplant patients under Abstracts azathioprine therapy. Thiopurine metabolites were determined in cellular fraction of bronchoalveolar fluid (BALl=) and in red blood cells from patients receiving 2.5 mg/kg daily of oral azathioprine. Nucleotides of 6TG, 6MP and 6TX were analysed by high performance liquid chromatography. Sample treatment consisted of PCA of deproteinisation of cell suspension and hydrolysis of nucleotides to their free bases by heating of the PCA extract (3). In cellular fraction of BALF, 6TGN were recovered in three patients at concentrations ranging from 827 to 1667 pmol/108 cells. In three other patients, the thiopurine nucleotide found was 6MPN at concentrations ranging from 240 to 330 pmol/108 cells and in two patients, no Oral Presentations thiopurine nucleotides were detectable. In red blood cells, 6TGN was recovered in all patients in the concentration range of 9 to 121 pmol/lO 8 cells. In five patients, 6MPN was present at concentrations from 2 to 138 pmol/I08 cells.

These preliminary results show that thiopurine nucleotides of 6TG and 6MP that are considered to be the active metabolites can diffuse into the lung at significant levels. Further investigations are needed to define the relationship between thiopurine nucleotide levels recovered in lung and the occurence of pulmonary rejection.

I R.L. Simmonds el al,Transplant. Proc.,18 (1986) 76. 2 G.B. Elion, G.H .Hitchings, In O. Eichler et al (eds), Handbook of Experimental Pharmacology, NY, t975, 404. 3 R. Bodlieu et al, J. Chromatogr., in press.

Laboratoire de Pharmacie Clinique, Institat des Sciences Pharmaceutiques el Biologlques, Lyon, France - Service Pharmaceutique el Service de Bronchopneumologie, H6pital Cardiologique, Lyon, France.

MECHANISTIC STUDY ON THE ANTIVIRAL AND CYTOSTATIC EFFECTS OF ADENINE NUCLEOTIDE ~ETABOLISM IN PRIMARY 5-THIEN-2-YI~ AND 5-FURAN-2-YL-SUBSTITLYFED PYRIMIDINE NUCLEO- RAT NEURONAL CULTURES SIDE ANALOGUES. 5. Brosh, E. Zoref-Shani, O- Sperlin~, C. Bohman~', J. Balzarini'}" P .Wigerinck A. Van Aerschot#, P. Herdewiin+, E- Danzi~er, Y. Sidi, ~rkk~req- F The metabolic fate of prelabeled adenine nucleotides (AdRN) A novel group of pyrimidine nucleoside analogues, containing a furan-2-yl- or a thien-2-. was studied in immature (~ d old) and mature (8-14 d old) yl- moiety at C-5 of the uracil or cytosine ring have been synthesized (1). The primary rat neuronal cultures. The label in AdRN decreased compounds markedly inhibit herpes simplex virus type 1 (HSV-1) repllcatinn in cell with time, appearing in acid insoluble derivatives and in culture. The halogenated 5-thian-2-yl-substituted 2'-deoxyuridine (dUrd) and I-g-D- hypoxan%hine. The combined addition, for 4 h, of ribofuranosyluracil (araU) derivatives show a 50%-effective concentration (EC50) 2'-deoxycoformycin and of S'-amino-S'-deoxyadenosine ranging between 0.10 and 0.19 I~M. These values are only 1.5- to 3-fdld higher than resulted in both cultures in a decrease of about 6M in the recorded fur (E)-5-(2-bromovinyl)-dUrd (BVDU), an antiherpetic compound that has label in AdRN, associated with a similar increase in the proved efficacious in the treatment of HSV-1 infections in humans. The affinity of the label in adenine and adenosine. Addition of alanosine test compounds for HSV-1 thymidine (dThd) kinase (TK) was evaluated. HSV-1 TK resulted in both cultures in a 2-3 fold increase in the was purified approximately 50,000-fold from marine mammary carcinoma (FM3A) ceils label in hypoxanthine and inosine, associated with a deficient in cytosol thymidine kinase and transfected with the HSV-I TK gene (FM3A significant decrease of the labellng in AdRN. Enhanced TK'/HSV-1 TK+). 5-Furan-2-yl-dUrd, 5-thien-2-yl-dUrd, and 5-(5-bromothlen-2-yl)- degradation of AdRN was accomplished by incubation for 2 h dUrd emerged as the most potent inhibitors of thymidine phosphorylation by HSV-1 with iodoacetate and antimycin. Under such conditions, the TK. Their 50%-inhibltory concentrations (IC50) ranged from 2.4 to 3.5 p.M. The immature cells lost 58 ~ and the mature cells 74 ~ of the compound concentrations required to inhibit thymidine phosphorylatinn by cytosot label from AdRN, most Of it to bypoxanthine and inosine, but also to adenosine. Coaddition of 2'-deoxycoformycin, tbymidine kinase were at least 55- to more than 200-fold higher. However, we found no decreased markedly the label in bypoxanthine and inosine, correlation between the inhibitory effect of the test compounds on HSV-1 replication and increased the labeling of adenosine and adenine. In and their inhibitory effect on HSV-1 TK catalyzed phosphorylation of dThd. These data both cultures, the enhanced degradation was associated with suggest that the presumed target for the antiviral action of the compounds, that is HSV- a complete halt of transfer of label from AdRN to the acid 1 DNA polymerase, may have a different structure-activity relationship (SAR) for these insoluble fraction. The results demonstrate that in the compounds than HSV-I TK. The effect of the compounds on cell proliferation, cultured neurons, AdRN are metabolised to acid insoluble incorporation of DNA_ precursors into trichloroacetic acid-insdlublc material, and derivatives, or degraded to hypoxanthine. Under tritium release from [5-3H]dCyd in wad-type FM3A and FM3A TK'/HSV-1 TK § ceils physiological conditions AdRN are degraded through both was also investigated. FM3A TK'/HSV-1 TK + ceil growth was strongly inhibited in the dephosphorylation and deamination of AMP. Enhanced presence of either 5-furan-2-yt-dUrd, 5-thien-2-yl-dUrd or 5-thien-2-yl-dCyd (the IC50 degradation of AMP proceeded mainly through concentrations varying from 1.4 to 4.6 I~M), whereas growth of the corresponding wild- dephosphorylation in immature cultures, and equelly via type FM3A cells was not inhibited at concentrations up+ to 500 ~M. The cytostatic both pathways in the mature cells. Under physiological action of these compounds for FM3A TK-/HSV-1 TK cells could be ascribed to conditions, the flux of label from AdRN to the acid preferential phosphorylation by the virus-encoded TK to their monophosphate form, insoluble derivatives is more intense in the immature followed by inhibition of cellular thymidylate symhase. In this respect, the test neurons. The adenosine-AM~ cycle operates in the compounds behaved similarly to BVDU (2,3). However, in contrast with BVDU that cultured neurons. Both arms of the adenine nucleo%ide cycle had previously been shown to be an effective substrate for dThd pbosphorylase (4), we (IMP to AMP and AMP to IMP) are operating in the cultured found that 5-furan-2-yl-, 5-thien-2-yl-dUrd and 5-thien-2-yl-dCyd showed poor, if any neurons, but, the intensity of this cycle has not yet been hydrolysis by dThd phosphorytase. clarified and it is not known yet if this cycle has any 1. P .Wigerinck et al. J. Med. Chem. 34 (199t) 2383 slgnificant role in this tissue, other than synthesis and 2. J. Balzarini et al. Mol. Pharmacol. 32 (1987) 410 degradation of AMP. 3. J. Balzarini and E. De Clercq. Methods Find. Exp. CAin. Pharmacol. 11 (1989) 379 4. C. Desgranges etal. Biochem. Pharmacol. 32 (1983) 3583 Dep%s. of Clin. Bfochemistry and of Medicine D, Beilinson Hed. Center, Petah Tikva and Dept. Of Chemical Pathology, +Laboratory of Experimental Chemotherapy, and %Laboratory of Pharmaceutical Sackler School of Medicine, Tel Aviv University, Israel. Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, B- 3000 Leuven, Belgium

l'harma~/ I I bdd L ~ Sc wm r Vo,o..~ ,s N.. ,~9~ F7 DETECTION OF POINT MUTATIONS AT THE HUMAN HPRT-LOCUS USING THE RAPID DIHYDROPYRIMIDINE DEHYDROGENASE ACTIVITY IN HUMAN SINGLE STRAND CONFORMATION POLYMORPHISM (S$CP) ANALYSLS PERIPHERAL BLOOD MONONUCLEAR CELLS AND LIVER: POPULATION CHARACTERISTICS, NEWLY IDENTIFIED DEFICIENT Renate Buroemeister. Elke Rotzer, Wolf Gutensohn PATIENTS, AND CLINICAL IMPLICATION IN 5-FLUOROURACTL CHEMOTHERAPY Complete deficiency of HPRT causes the Lesch-Nyhan syndrome (LNS) which is characterized by hyperuricemia, mental retardation, choreoathetosis, and compulsive R.B. Diasio, Z. Lu,. and R. Zhan~ self4"nutilation. Partial deficiency of HPRT leads to a severe form of gout and nephtolithiosis. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in catabolism of 5-fluorouracil (FUra), one of the most widely used anticancer drugs. Here we describe three mutations in three nonrelated families previously studied by Previous studies from our laboratory 1,2 demonstrated the clinical importance of direct sequencing of PCR products of the human HPRT gene. DPD in cancer patients particularly in those with DPD deficiency who experience severe FUra toxicity (including death) following FUra treatment 3,4. We now suggest * Patient IJ shows an insertion of one G in exon 3 of the HPRT gene. He represents a case that measurement of DPD activity may be useful in routine screening of cancer of the classical Lesch-Nyhon syndrome. There is no residual activity of HPRT in his patients prior to FUra treatment and describe the following serial studies. First, erythrooyte lysate. His mother is heterozygous for the disease whilst his sister shows no mutations, development of a sensitive, accurate, and precise DPD assay and a storage method * Patient GS has a mutation in exon 7 (CGA -> TGA, Are --> Stop). He shows a partial to stabilize DPD activity, permitting large scale DPD screening in cancer patients. HPRT-deficlency, a residual activity of 1,8% of normal is found in his erythrooyte Iysate. Second, demonstration of a normal distribution (Gaussian distribution) of human His mother shows no rt,,utation, so it is assumed that this is a case of a new mutation. DPD activity from peripheral blood mononuclear cel!s (PBM-DPD) in a population * Patient KM has a point mutation in exon 2 (ATr --:, ITT, lle --> Phe) and he shows a study, l~aselines (mean -.+ SD) for PBM-DPD with fresh and frozen samples were partial HPRT-deticiency. His residual activity of HPRT is 2.7% of normal, His mother is 0.425 +- 0.124 and 0.189 *-. 0.064 nmol/min/mg protein, respectively. The 95% and heterozygous. 99% distribution ranges for both fresh and frozen samples were also determined, Patients GS and KM represent clinical borderline cases, including not only the symptoms providing criteria for detection of DPD deficient patients. Third, identification of 9 of gout, but also some of the neurological symptoms of LNS lacking the the typical new patients with profound or partial DPD deficiency. Fourth, determination of a autoaggressive behavior. baseline for human liver DPD activity, which was shown to be 0.360 -,-0.182 For the rapid arid simple detection of point mutations we used the single strand nmol/min/mg protein (frozen samples). Fifth, preliminary evaluation of liver DPD conformation polyrnorphlsm (SSCP) analysis. from deficient patients. Low liver DPD activity in 2 deficient patients correlated with Single stranded DNA molecules ore capable of forming unique secondary structures low PBM-DPD activity. Using a polyclonal antibody demonstrated decreased DPD whose nature is dependent upon their base sequences, protein in the liver cytosol from DPD deficient patients compared to normal The type and degree of secondary structure formation in turn determines subjects. These results may be useful in improving the effectiveness and/or lessening electrophoretic mobility under nondenaturing conditions. Mutations within a given DNA the toxicity of FUra chemotherapy. (Grant NCI CA 40530). segment alter this mobility and the "mobility shift" may be detected on high resolution native PolyacryJamid gels. 1 G.D. Heggie, et al. Cancer ges. 47 (I9871 2203. 2 B.E. Harris, et al. Cancer Res. 50 (19901 197. We demonstrate that SSCP analyf~s can reliably and reproducibly identitiy the mutations we had found previously in three exons of the human HPRT gene. 3 R.B. Diasio, et al. J. Clin. Invest. 81 (19881 47. All mutations detected by direct DNA sequencing were also detecled by SSCP in all 4 B.E. Harris, et al. Cancer 68 (19911 409. probands tested. Individuals homozygous and heterozygous for the mutations in this X s Z. Lu, et al. J. Biol. Chem. 267 (1992) 17102. chromosomal disease are olearly distinguishable. The simpl~ity of this technique together with its speed and reliability commend it for Departments of Pharmacology and Medicine, Division of Clinical Pharmacology, routine use in molecular diagnostic medicine. Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. Institut fGr Anthropologie und Humongenetik, Goethestr. 31, W-8000 MOnchen 2, FRG

THE iSOLATED RAT SUPERIOR CERVICAL GANGLION (SCG) AS A MODEL FENOFIBRATE: TISSUE FOR THE STUDY OF NEURONAL PYRIMIDINE RECEPTORS. A LIPID LOWERING DRUG WITH URICOSURIC PROPERTIES

G.P.Connollv & P.J. Harrison. U. Gresscr,

In the cardiovascular system there is some evidence in favour of the presence of Fenofibrate, a fibric acid derivate, is used for the treatment of hyperlipoproteinemias. extracellular receptors that are activated specifically by uridine 5'-triphosphate (UTP) In addition to its lipid lowering properties feaofibratr shows a uricosoric effect. In rather than adenosine 5'-triphosphate (ATP) i.e., pyrimidinoceptors, however it is healthy volunteers feoofibrate lead to a 2-fold, benzbromarone t9 a 4-fold increase of unknown if these receptors also exist within the nervous system. We bare uric acid clearanceI. In hyperuricemic outpatients the decrease in plasma uric acid investigated this possibility using methods described before 1. over a period of one year was 30 % of the initial value2. Other authors found a de- crease of plasma uric acid of 20 %3, 4 In comparison hczafibrate and placebo had no SCG were placed in a chamber and perfused with physiological salt solution+(PSS) uricosuric effect3. Most of these studies were performed to measure the lipid lowering at 25+ I~ 7.4 equilibrated with 5% CO,V95%O2 containing 2raM K and effect of fenofibrate and therefore give no detailled informations on extent and course 0. I mM Ca24. The d.c. potential between the ganglion and the postganghomc nerve of time of the uricosuric effect of fenofibrate. Aim of our study was to get detailled was recorded across a greased barrier, Adenosine and pyrimidines were added to the informations on these points in comparison to a well-known uricosaric drug. perfu~ate for 2 rain and other drugs for 1 rain. Responses in the presence of atropine We compared the uricosurie and lipid lowering effects of a single daily dose of feno- (2aM) or suramin (300,aM) were determined after I5 or 30 rain preincubation. fibratc (200 mg, micronized, Knoll AG) with benzbromaxone (100 rag, ratiopharm) in On the same ganglia UTP was more potent thatl a/bMeATP in producing a cross-over study with 7 healthy volunteers on a low purine dict. Our results confirm concentration -dependent depolarisatioos. Depolarisations caused by aBMeATP were the observations of a distinct uricosoric effect of fenofibrate. In 7 healthy volunteers a abolished by suramin, whereas depolarisation caused by UTP were potentiated, and single dose of 200 mg fenofibrate lead to an average additional renal uric acid excre- hyperpolarisations to adenosine, depolarisatioas to muscarine, DMPP or K + tion of 204 mg Coenzbromarone: 376 rag) within 14 hours after drug ingestion. So remained unaltered (Table 11. In the presence of suramirr, atropine abolished fenofibrate lead to 54 % of the additional renal uric acid excretion in comparison to depolarisations caused by muscarine (100nM, 276 _+ 33~V cf 6 _ 7~tV, n=4, benzbromarone in the same volunteers. If we include that the normal dally dose of P<0.01) but not those caused by 100~M UTP (263 -+ 67,aV cf200 4- 41/xV, n=4). beazbromarone would be 80 rag, the relative uricosurie effect of feoofihrate is more impressive. Effect of suramin on thexesoonse (uV, me,.a~t +/- s.e.mean (4-9 SCGI. Between hyperurieemia and hypertriglyceridemia there is a significant correlation .= P < 0_01.*~* P < 0.01. Paired t-test/to denoladsing and h yoemolarisin~_aeooists. (p<0,001) 5. Therefore many patients need a uric acid lowering and a lipid lowering A~anist ~/bMeATP UTP Adenosine Muscarine DMPP K + treatment. Fenofibrat preferentially is a lipid lowering drug, but with its clear uricos- Tceatment 100ttM 100~M I00#M 100nM 10uM 3mM uric properties it could be a good possibility in many patients with both metabolic dis- CONTROL 76+I1 216_+38 -1405:35 231_+32 1214-27 136+_33 eases. The uricosaric effect of fenofibrate is large enough to normalize uric acid in most patients with hyperlipidemia and hyperuricemia. SURAMIN -5_10"** 2834-44"*-161• 268+29 139_+35 1144-28 With its combined lipid lowering and uricosuric properties fenofibrate will help to shed light on the pathophysiology of the correlation between hyperlipidemias and hy- The inability of atropine to alter depoiarisation to UTP indicates that UTP did not peruricemia. depolarise ganglia by releasing acetylcholine to activate muscarinic receptors or We give detailled informations on the influence of fenofibrate and benzbromarone on activate muscarinic receptors directly. The selective antagonism of the response to plasma uric acid, renal uric acid excretion, triglycerides, HDL- and LDL-cholesterol. uBMeATP and not UTP by suramin is consistent with the presence of both P2- purinoceptors and receptors for UTP. Our results provide evidence for the presence 1. J.P. Desager et at., J Clin Pharmacol 10: 560-564, 1980 of pyrimidinoceptors in the nervous system and suggest that the rat SCG may be a 2. C. Harvengt et at., Artery 7 (I): 73-82, 1980 useful model system for studying these receptors. 3. M.D. Bestow et at., Metabolism 37: 217-220, 1988 4. J. SteinmetZ et at., Clinica Chimica Acta 112: 43-53. 1981 1. G.P. Connolly & T.W. Stone. J. Auton. Pharmacol., 13 (1993) 213-224. 5. B.S. Gathof et at., Adv Exp MeAl Bioi 309A: 231-234, 1991

Department of Physiology, University College London, Gower Street, London. U.K. Medizin. Poliklinik, Universit~t Miinchen, Pettenkoferstr. 8a, 80336 Mfinchen, FRG

Vo~um~ 15 Nr. a I~93 THE ACTIVE SITE OF HUMAN ECTO-5'-NUCLEOTIDASE. MAGNETIC RESONANCE IMAGING ENLARGES THE DIAGNOSTIC DELINEATION OF ESSENTIAL AMINOACID RESIDUES BY SITE POSSIBILITIES IN HYPERURICEMIA AND GOUT DIRECTED MUTAGENESIS AND AN ATTEMPT TO DISSECT CATALYSIS FROM OTHER FUNCTIONS OF THE MOLECULE. I. Kamilli I, U. Gresser 1, R. Rauch 1 , D. Hahn 2 W. Gutensohn, R.Resta, L.F.Thompson In patients with chronic gout uric acid deposition appears in bones near to joints, in Recent research has yielded a somewhat clearer picture of the synovial membrane, in bursae, tendon sheats and other bradytrophical tissues. the biological function(s) of ecto-5'-nucleotidase (5'-NT). As an enzyme it may contribute to the removal of potenti- Whereas bone tophi and superficial tophi are well accessible, tophi in non super- ally toxlc purine metabolites in the neighborhood of dying ficial tissues, e.g. heart valves, eyes, spine and peripheral nerves, can not be iden- cells or regulate critical ligand concentrations for puri- tified and we find them only surgically or by autopsy. The purpose of this study nerglc receptors. In addition 5'-NT was shown to act as an was to investigate the valuation of scintigraphy and magnetic resonance imaging accessory molecule in T-lymphocyte activation and here it is less clear, whether the catalytic property of the mole- (MR/) in comparison to clinics and conventional radiological procedures for de- cule is required or rather its particular type of membrane monstration of uric acid deposition. anchorage via glycan-phosphatidyl-inositol. We attempted to dissect such possible functions. Starting from 5'-NT cDNA Twentyfive patients with chronic gout followed in the Medizinische Poliklinik par- and guided by indirect evidence from chemical modification ticipated in this study. In every patient (n=25) radiographs of the feet, big toe, and sequence comparisons the codons for three conserved hl- stidlne residues were changed to alanine codons. Wild type ankle joints, knees, hands, wrists and elbows (joint region=350) and an early (5 cDNA and mutant constructs were expressed in the 5'-NT-ne- min) and late (2 hours) static scintigraphy (99mTcPP) were obtained. In 9 patients, satire T-lymphoblastoid cell line Jurkat. Surface expres- 7 feet, 6 hands and one elbow a MRI was done. Coronal, axial and sagittal T1 and sion of 5'-NT in the transfectants was monitored by T2 weighted MR images with and without Gd-DTPA were obtained. immunofluorescence using monoclonal antibodies (mAbs) and was seen in the wild type as well as the mutants. Enzyme We compared the findings in MRI, clinical evaluation, x-ray and scintigraphy in activity on the other hand was high in the wild type trans- every joint region. In MRI in 14 (100%) joint regions soft-tissue tophi were found, fectants, but was completely abolished in the 3 different whereas in clinical evaluation and x-ray only in 8 joint regions tophi were registra- His to Ala mutants. Using the same mAbs it could be clearly ted and in scintigraphy only in 11 joint regions higher concentrations of activity shown that mutant 5'-NT devoid of enzymatic activity could still function in signal transduction during stimulation of were found. In MRI tophi appear in TI weighted images as hypotense inhomoge- the Jurkat transfectants and thus this property could be nous structures and in T2 weighted images as hyperintense structures. With Gd- separated from catalysis. The roles of the 3 different His DTPA tophi appear in T1 weighted images as structures enhancing the contrast residues in 5'-NT enzymatic activity-have still to be defi- medium; in larger tophi there is only a peripheral enhancement and a central zone ned more precisely. They might be involved: i) in the cata- lytic reaction proper as acceptor of phosphate in a short- with a tow signal intensity. In contrast to all the other diagnostic possibilities only lived intermediate ii) as an essential constituent of a in MR/bone marrow edema are obtained as a sign of inflammation. nucleotide binding site and iii) in zinc coordlnation. MRI is the only method to mark off the complete extension of tophi. Therefore Illstitute of Anthropology and Human Genetics, University of Munich, Goethestr. 31, D 8000 Munich 2, Fed. ReD.Germany. MR/enlarges the diagnostic possibilities in hyperuricemia and gout, especially the Oklahoma Medical Research Foundation, 825 N.E. 13th Street, diagnosis of the extend of tophi in non superficial tissues, e.g. eyes, spine, skull- Oklahoma City, OK 73104, USA. base and perineural tissues such as the carpal tunnel.

I Medizinische Poliklinik der LMU-Mftnchen, Pettenkoferstr. $a, 80366 Mfinchen, Germany 2Radiologische Polikliaik der LMU-Mfinchen, Pettenkoferstr. 8a, 80366 Mtnchem Germany

AICARIBOSIDE INHIBITS GLYCOLYSIS IN HEPATOMA CELLS HIGH DOSE 6-MERCAPTOPURINE INFUSIONS IN CHILDREN WITH LYMPHOMA

F. Javaux. MF. Vincent. G. Van den Bemho CW Keuzenkams-Jansen RA De Abrea JPM B6kkerink MAH vd Heiiden

AICAriboside, the nucleoside corresponding to AICAR (ZMP), an intermediate The mode of action of 6-mercaptopurine (6MP) in vitro is based on incorporation into of de nove purine synthesis, inhibits glycolysis in isolated rat hepatocytes (t). DNA and RNA as thioguanine nuclcotides and inhibition of the purine de hove synthesis Since many tumour cells depend on a high glycotytic rate (2) to provide the by methylthioinosinic monophosphate (MetlMP). We studied the metabolic routes of 6MP energy required for proliferation, the effect of AICAriboside was investigated in in 6 lymphoma patients receiving 24 hours infusions of 6MP. the rat hepatoma cell line FTO-2B. Pharmacokinetics, intracellular biochemistry and pharmacology were studied in plasma, erythrocytes and mononaclear cells. Blood samples were collected at 0, 4, 20, 24, 28 and In intact FTO-2B. cells, addition of 250 l.tM AICAriboside resulted in the 48 hours after start of the 6MP infusion (100 rag/m: in 0.5 hr followed by 1200 mg/m2 in accumulation of 5 mM ZMP. Production of lactate with 10 mM glucose was 23.5 hr). 6MP, its metabolites, ribonucleotides, nuclcosidas and bases were measured by inhibited by 65% within 2 h. Half-maximal inhibition was reached with 50-100 HPLC. .: p.M AICAdboside. Iodotubercidin, an adenosine kinase inhibitor, suppressed In plasma steady state levels of 6MP were reached soon after administration of 6MP in a the effect, implying that phosphorylation of AICAriboside by this enzyme was concentration range of (~50 FM. Methylmercaptopurine (MeMP) appeared at t=4 (0.3-3 required. Further investigations revealed that AICAriboside displayed two p.M) and the concentration decreased rapidly, like 6MP, after the end of the infusion. major effects: Methylmercaptopurineriboside (MeMPR) appeared at t=20 and remained at a steady state (i) It decreased the release of 3H20 from [2-3H]glucose, synchronously with level (0-0.4 /~M) from t=24 till t=48. Two patients had a tumor lysis syndrome before the inhibition of the production of lactate. This ,indicates inhibition of glucose start of the 6MP treatment. They demonstrated extremely high 6MP levels (35-60 ~.M) and transport or of an early step of glucose metabolism: phosphorylation into longer 6MP half-life times. Relatively high MeMP levels (2-3 #M) and no MeMPR could glucose-6-P (by hexokinase in FTO-2B cells), or isomerisation of glucose-6-P be detected in these patients. Only these two patients had measurable amounts of thioxan- into fructose-6-P. thine at extremely high levels (10-40/~M). (ii) It decreased the concentration of fructose-2,6-P 2, the main stimulator of MetlMP in erythrocytes was detected at t=4 and reached, like MeMPR, a steady state phosphofructokinase (PFK)-I. This effect, which required at least 4 h of level (15-2000 pmol/10m cells) at t=24. MetlMP in mononuclear cells (2-20 pmol/l(P ceils) incubation, did not result from inactivation of PFK-2, but most probably from a was detected from t=20. The patients with a tumor lysis syndrome reached lower MatlMP decrease of fructose-6-P, due to inhibition of an early step of glucose levels as compared to the other children. Intraeellular thioguanine nuclcotides could not be metabolism. Fructose-l,6-P 2 decreased, most likely owing to the diminished detected in any of the patients, probably because these metabolites are rapidly incorparated stimulation of PFK-I. into DNA. These data show large differences in 6MP metabolism in lymphoma patients. Patients In cell-free extracts of FTO-2B cells, phosphoglucose isomerase was 70% without tumor lysis are probably able to incorporate 6MP into DNA and RNA and their inhibited by 5 mM ZMP. Implication of this effect in the inhibition of glycolysis blood cells contain MetlMP for more than 24 hours. In plasma of these patients no was, however, ruled out because residual activity remained 100-fold higher thioxanthine and less MeMP was detected. So, the anabolic route of 6MP in the cells of than that of hexokinase. Although no direct inhibition of hexokinase activity by these patients is high. In contrast, in patients with a tumor lysis syndrome the catabolic ZMP could be evidenced as yet, it is tentatively concluded that A$CAriboside, route of 6MP is predominant: high levels of thioxanthine and relatively large amounts of by a still to be defined mechanism, primarily inhibits this enzyme. At longer MeMP. time intervals, the inhibitory effect of AICAriboside on glycolysis is reinforced Our hypothesis for the predominant catabolic route of 6MP in tumor lysis patients is based by the subsequent decrease of fructose-2,6-P2. on an excess of free nuclcosides and bases, derived from the lysis of tumor cells. The purine nuclcosides and bases may then compete with 6MP for the enzyme hypoxanthine- 1 M.F. Vincent, F. Bontemps, G. Van den Berghe, Biochem. J. 281 (1992) 267 guanine phosphoribosyltransferase. This hypothesis is supported by the finding of high 20. Warburg, K. Posener, E. Negetein, Biochem. Z. 152 (1924) 309 plasma levels of hypoxanthine (6 and 85 #M) and xanthine (25 and 350/~M) in these two patients. Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology, UCL 7539, Center for Pediatric Oncology, University Hospital, P.O.B. 9101, Avenue Hippocrate 75, B-1200 Brussels, Belgium. 6500 HB Nijmegen, The Netherlands. This project is supported by the DutchCancer Society (NUKC-92-79).

Volume 15 N[ 4 IQo~ FC~ HISTOCHEMICALAND CYTOCHEMICALDEMONSTRARqON OF THE MOLECULAR DEFECT IN TWO MUTANT FORMS OF HPRT DIHYDROOROTATE DEHYDROGENASE AND OXIDASE A.M. Marinaki. E.H. Harley.

M. L6mer. C. Becker. E. We~erle HPRTcaDe Town is a partially defective form of HPRT in which the enzyme demonstrates the unusual property of having acquired substrate inhibition by its purine base substrates I. Following PCR amplification of reverse In the present study the enzyme dihydroorotate:ubiquinone o~dore- transcribed /RRNA, products were directly sequenced and ductase (DHO DH] was investigated in tissue samples of rat and man, showed a A502G transition resulting in a change in Asp 135 and in a mouse mamma carcinoma cell llne. to Asn. The latter is located in the putative PRPP Since the rnitochondrial DHO DH is connected to the functional respira- binding domain and is conserved in all phosphoribosyl- tory chain 1, the enzyme activity could beevaluated tn s/tu by channel - transferases. This mutation requires re-interpretation of ling the redox equivalents to artificial electron acceptors after cyanide- HPRT active site kinetics. blocking of the chain at the stage of cytochrome oxidase. HPRTRo_dboschne from a Lesch-Nyhan.. patient shows virtually The principle of the techniques used for the detection of the dehydroge- no actlvlty. PCR ampliflcatlon of cDNA yielded a smaller nase activity was to employ a colourless, soluble tctrazolium salt which product than normal due to a 47 base pair deletion is reduced to a deeply coloured, insoluble formazan product. This could corresponding to exon 7. be detected by light microscopy, and, in a first approach, by flow cyto- metry if a fluorescent formazan was produced. The specificity of the 1 L.M. Steyn and E.H. Harley. J. Biol. Chem., 259 (1984) dihydroorotate-dependent formazan production was confn'med using 338. enzyme-inhibiting drugs of high potency. The activity-pattern of DHO Dept of Chemical Pathology, University of Cape Town, DH was comparable to that of succinate dehydrogenase, a marker of in- Observatory 7925, South Africa. ner mitochondrial membrane. High activities were found in tissues with known transport/excretlon/regeneration/proliferatlve activities. The additional oxidase activity of the DHO DH, which was measured with the purified enzyme 2, was demonstrated/n s/tu by employing the cerium/diaminobenzldine method for trapping the hydrogen peroxlde resulting from direct reaction with oxygen. An unusual high o~ddase activity could be detected in the cardiac muscle by light microscopy. This was verified by ultrastructural studies which localized a cerium precipitate of high electron density at the inner mitochondrial membrane.

I M. LSffler, Cell Prolif. 25 (1992), 169. 2 G, Lakaschus and M. L6fller. Biochem. Pharmacol. 43 (1992}. 1025. We thank C. Schalk. Institute of Molecular and Tumor Biology, University of Marburg for FACS analyses, and S. Angerm1~dler, Institute of Anatomy, Uni- versity of Heidelberg for electron microscopy. Financial support by the Sander-Stlftung and the Kulemann-Stiftung is grate- fully acknowledged-.

Institut for Physiologische Chemie, Klinikum der Philipps-Universitftt Marburg, Karl-von-Frisch-Str., D-35011 Marburg, Germany.

ON THE ROLE OF THE IONIC COMPOSITION OF THE CULTURE FAMILIAL JUVENILE HYPERURICAEMIC NEPHROPATHY IS ALSO A MEDIUM FOR THE ROLE OF CELLULAR UPTAKE OF PHOSPHATE DISEASE OF CHILDHOOD. AND FOR THE RATE OF PHOSPHORYLATION OF ADDED PURINE RIBOSIDE TO THE CORRESPONDING TRIPHOSPHATE M.B. McBride. H.A. Simmonds, F. Moro, J.S. Cameron, C.S. Oqg, D.G. Williams, S. Riqden and G. Haycock.

IV[. Marcussen, El Overgaard-Hausen and H. Klenow Familial juvenile hyperuricaemic nephropathy (FJHN) is a dominant condition with high penetranee which affects a Under a number of conditions large amounts of the triphosphates of added group of young men and women who develop early progressive adenosine or analogs of adenosine may accumulate in Ehrlich ascites renal dysfunction. FJHN has often been considered 'familial nephritis' until an isolated attack of gout tumor cells. Almost all of the phosphate present in these triphosphates (rare in renal failure) has highlighted the underlying stem from the extracellular orthophosphate (P.~ and their rate of disorder. Gout and hypertension are inconsistent accumulation may, therefore, in part, depend on the rate of uptake of Pi. features. The genetic basis remains unknown. In media with high Na-concentration like Ringer HEPES buffer and at low pH (e.g. 6.5) the intraceUular concentration of Pi (about 10 raM) increases FJHH has, hitherto, been considered rare. Detailed by a factor of only about 2 with increasing extracelhilar Pi (up to 45 re_M). studies of 88 cases from 14 kindreds have been reported from 7 countries, excluding the UK, 36 of these cases have Neverthaless the presence of purina riboside (nebular/he) may cause not been children. We have now studied 90 individuals from 34 only a drastic decrease in intraceUular Pi but also a rapid uptake of kindreds in which the index case presented with gout and extracellular Pi resulting in accumulation of large amounts of purina found that the hallmark of the disease is hyperuricaemia riboside trlphosphate. The linear increase in purine riboslde triphosphate associated with a low clearance of uric acid relative to may in about 2 h result in a total content of low ~nolecular phosphate creatinine (FEur), disproportionate to age, sex and degree compounds that is about 10-fold that of cells not treated with purine of renal failure. On this criteria we identified 65 affected individuals, 25 of whom were reputedly healthy. riboside. It thus appears that the intracellular phosphorylatiou of purine The study included 27 children (one a female seen with riboside facilitates a greatly increased net uptake of Pv In media-buffers gout at 9 years). A reduced FEur has been found in 18 of with a high K-concentration increasing concentration of extraceUular Pi the children, including 5 with as yet normal renal gives rise to an unexpected high rate of accumulation of intracellular Pi. function, suggesting that renal urate hypoexcretion Under such conditions, or at high pH values (e.g. 7,9), the total Pi uptake precedes a fall in the GFR. was almost unaffected by the presence of purine ribosids even though Our findings indicate that FJHN is also a disease of considerable amounts of it were converted to the triphosphate. childhood. They underline the importance of detailed family studies in all kindred members. This is essential Dept. of Medical Biochemistry and Genetics, Laboratory of Medical since, in our experience; a) allopurinol appears to have Biochemistry B, The Panum Institute, Blegdamsvej 3, DK-2200 ameliorated the progression of the renal damage in Copenhagen N, Denmark. patients treated for up to 25 years; b) a successful prognosis appears to depend on the renal function at diagnosis and the absence of hypertension, or its adequate control.

Purine Research Laboratory, Renal and Paediatric Nephrology Units, UMDS Guy's Hospital, London, UK.

~] 0 l'h,lr.m,y H~,tld ~ ,q,wmc INCREASED RENAL VASCULAR RESISTANCE IN FAMILIAL ADENYLOSUCCINATE LYASE DEFICIENCY: A CASE REPORT NEPROPATHY ASSOCIATED WITH HYPERURICEMIA OR GOUT. C. Salerno, A. Lomonte, O. Giardini, C. Crifo'

ME Miranda. JG Puin FA Mateos. JO V~zouez. Adenylosuccinate lyase deficiency is a recently described inborn error of purine metabolism discovered in Benelux countries [i] and diagnosed hitherto in 12 We determined the renal plasma flow (RPF), renal vascular children, belonging to 4 nationalities (Belgian, Dutch, resistance (RVR) and filtration fraction (FF) in 11 subjects Moroccan and Turkish) [2]. Affected children are normal at belonging to 3 families with familial nephropathy associated birth, but psychomotor retardation and autistic features often become evident in the first two years. with hyperuricemia or gout (McKusik, 162000). Ten patients We have found a new case of adenylosuccinate lyase showed hyperuricemia, 5 had gout, 8 decreased glomerular deficiency in the course of a systematic screening of filtration rate (GFR) and 5 arterial hypertension. All the 11 urines of more than 100 patients with severe psychomotor patients showed a decreased RPF (267+110 mL/min/1.73 m 2 retardation belonging to the Italian Association of Parents of Autistic Subjects. HPCL detection of [reference, 709+30]), increased RVR (28.0_+16 mmHg/mL/l.73 m2 succinyladenosine (S-Ado) and succinylaminoimidazole [reference, 7.6_+ 0.6]) and normal FF(22.5_+3.1% [reference, carboxamide riboside (SAICA-R) was the technique of choice 20.7+1.2]). There was a significant correlation between RPF and for the screening. The affected girl (now 9 years old) is the second 24 hour urinary urate excretion (r=0.74, P<0.01). A decreased RPF daughter of unrelated Italian parents. A few months after and increased RVR was present in one patient wih normal GFR, birth, frequent crying attacks, motor restlessness and blood pressure and serum uric acid levels. Another patient showed hypertonicity were noticed. Eye contact was difficult, hyperuricemia, decreased RPF and increased RVR as the only while reaction to auditory stimuli was exaggerated. Psichomotor development was delayed (she was standing manifestations of the disease. After 4 years of follow-up his GFR alone at 15 months and walking after 2 years; up to now, decreased by 30% despite an adequate contro~ of hyperuricemia. she speaks unintelligibly in short sentences with an In summary, patients with familial nephropathy associated with incorrect structure and seems to understand very little of what is said to them). At age of 5 years, generalized hyperuricemia or gout show a decreased RPF and increased RVR tonic-clonic convulsions were noted for the first time. even in the absence of other manifestations of the disease. The There is an intermittent external strabism with crisis of observed correlation between RPF and uric acid excretion convergence spasm. Striking bouts of extreme agitation, suggests that altered renal hemodynamics may account for the especially involving the arms and legs, are common. Growth and head circumference are normal. urate underexcretion, hyperuricemia and the entire clinical The patient excretes approximately 130 nmol S-Ado / picture of this familial syndrome. min in the urine, while [S-Ado] in plasma is about 3 uM. The [S-Ado] to [SAICA-R] ratio is about 1.2. Serum urate concentration is 2-4 mg/dl. Intravenous injection of Dpt of Internal Medicine and Clinical Pharmacology. La Paz 200 mg / kg fructose causes a decrease of serum urate University Hospital. Paseo de la Castellana, 261. 28046 Madrid, concentration of about 1 mg/dl and a slight increase of Spain. serum [Mg]. Erythrocyte adenylosuccinate lyase appears to be unstable with time.

i. J. Jaeken & G. Van den Berghe, Lancet 2 (1984) 1058. 2. F. Van den Bergh, PhD Thesis, univ. Louvain, 1992, 45.

Dept. of Human Bio~athology, Dept. of Biochemical Sciences and Inst. of Paedriatrics, University of Roma La Sapienza, 00161 Roma, Italy

2',2'-DIFLUORO-DEOXYCYTIDINE INCORPORATES INTO RNA AND DNA DEPRESSED PURINE CATABOLITE PRODUCTION AND INHIBITS THEIR SYNTHESIS. IN THE POSTISCHEMIC HEAR3~ A 31p-NMR ASSESSMENT OF CYTOSOLIC METABOLITES. Smolenski, M.H. Yacoub. A.M-L. Seymour, V.W.T. Ruiz van Haperen, G. Veerman, J.B. Vermorken, G.J. Peters. Brief period of ischemia causes increase in adenosine and total purine catabolite release from the heart. However, if ischemia of similar duration 2',2'-difluoro-deoxycytidine (Gemcitabine, dFdC) is a deoxycytidine analogue is repeated, this increase in purine catabolite release is markedly reduced. with established anti-tumour activity in the clinic, dFdC is assumed to exert its The mechanism of this phenomenon is unclear. In this study an isovolum- antitumour effect through incorporation into DNA. Since the RNA-directed effects ic Langendorff perfused rat heart model was used. Hearts were subjected to (A) 1 min ischemia at 40 rain of perfusion, 10 rain ischemia at 50 rain were not clearly defined, we studied the incorporation of dl:dC into DNA and of perfusion and 1 rain ischemia at 85 rain of perfusion or to (B) 3x I RNA and its effect on nucteic acid synthesis. min ischemia at 40 rain, 60 rain, and 85 min of perfusion. ~ntracellular We determined incorporation of dFdC into nucleic acids of A2780, a human concentrations of ATP, phosphocreatine (PCr) and intracellular inorganic ovarian carcinoma cell line, Colon 26-10, a mudne colon carcinoma cell line and phosphate (P) were assessed using 3~p nuclear magnetic resonance in CCRF-CEM, a human leukemic cell line. For this purpose we used two spectroscopy (NMR). Purine catabolite concentration in the coronary different methods; an acid precipitation assay and a CsCI density gradient, after effluent was determined by HPLC. Mechanical function of the heart (left exposing the cells for 4 or 24 h to 0.1 or 1 I~M dFdC. Synthesis of DNA was ventricular developed pressure - LVDP) was determined using a balloon determined by [2-14C]-thymidine (14C-TdR) incorporation, RNA synthesis by [5- inserted into the left ventricle. 3H]-uridine (3H-UR) incorporation. Both in (A) and (B) P. increased two fold during 1 min ischemia at 40 min of perfumon. PCr decreased concomitantly by 30% but no change n ATP Incorporation of dFdC into DNA was time and concentration dependent. A2780, was seen. Prior 1 min ischemia at 85 rain of perfusion, ATP concentra- the most sensitive to dFdC, incorporated most dFdC (3-'7 fold compared to C26- tion was 65%, Pi was 60% and PCr 180% of the initial values in group 10). We also observed incorporation into RNA, which was only concentration (A) whilst these concentrations remained unchanged relative to starting dependent, within the time period used. The solid tumour cell lines incorporated levels in group (B). During 1 rain ischemia at 85 rain, concentrations of P. much more dFdC into RNA (6-14 fold) than the leukemic cell line, especially at and PCr reached the same levels as during the first ischemic incident 1 I~M dFdC. dFdC incorporation into RNA was confirmed by separation of RNA both in group (A) and (B). Relative change in comparison to values and DNA using CsCI gradient centrifugation. The same pattern was observed ,~nmediately before was thus much more pronounced in group (A). Purina catabotite release increased by 0.5/JM immediately after 1 rain ischemia as w~th the acid precipitaion assay, while contamination of RNA by DNA was at 40 min, but only by 0.05 pM at 85 min in group (A). These increases less than 0.4%. As to the synthesis of the nucleic acids, in all three cell lines in purine release remained unchanged in group (B) during 1 rain ischemia dFdC inhibited DNA synthesis completely, at 1 I~M after 24 h exposure. RNA at 40, 60 and 85 rain. Contractile function (LVDP) recovered to value synthesis could be inhibited completely only in CCRF-CEM ceils, after 24 h before 10 rain ischemia in group (A) and was maintained constant in exposure to 1 p.M of dFdC. In the two solid tumeur cell lines RNA synthesis was group (B). also inhibited, however to a maximum of 60%. In summary, despite ten fold reduction in purine catabolite release, myo- In conclusion, dFdC interferes with both DNA and RNA synthesis. The latter cardial concentrations of P. and PCr during 1 rain ischemia before and effect has not been recognized before and it has to be determined wether it is after 10 rain ischemia wer~ similar and lower was only ATP concentra- related to either anti-tumour activity or toxicity. tion during the second 1 rain ischemic event. However, PCr level was higher and P. concentration lower prior to second 1 rain ischemic inci- dent. Conse~luently, decreased nucleotide catabolite production in our experimental model seems to be related to reduction of nucleotide pool Dept. of Oncology, Free University Hospital, P.O. Box 7057, 1007 MB size and/or increased phosphorylation status before ischemia, but in- Amsterdam, The Netherlands. volvement of other factors still can not be excluded. M.R.S. Group, Cardiothoracic Surgery, National Heart and Lung Institute at Harefield Hospital, Harefield, Middx UB9 6JH, U.K.

Volume 15 Nr 4 199] MUTATIONAL LOSS OF DEOXYCYTIDINE KINASE (dCk) DETERMINATION OF PHOSPHORIBOSYLPYROPHOSPHATE ACTIVITY AS A CAUSE OF ACQUIRED 1-S-D- SYNTHETASE ACTIVITY. A NEW SIMPLIFIED METHOD. ARABINOFURANOSYLCYTOSINE- (AraC) AND 5 -AZA-2'- R.J. Torres. F.A. Mateos. J.G. Pui~, DEOXYCYTIDINE- (AzadC) RESISTANCE IN ACUTE MYELOID Phosphorlbosytpyrophosphate synthetase (PRPPs) superactivity is an inherited LEUKEMIA (AML). inborn error of metabolism which causes gout with over-excretion of uric acid associated, in some instances, with neurodevelopmental impairment. Screening for A.P.A+ Steqmann. M.W. Honders, R. Willemze. J.E. defects in PRPPs activity is complicated by the variety of kinetic alterations detected Landeqent. among affected families, being the most common the catalytic and the regulatory defeCts. Currently employed methods for measurement of PRPPs activity are Acquired drug resistance to the dC-analogues AraC cumbersome and expensive, requiring an auxiliary enzyme reaction, a radioactive and AzadC in the treatment of AML, one of the major nucleobase, and multiple incubations. The aim of the present study is to describe a obstacles for longterm beneficial therapy, is often simplified, single step, nonisotopic method for determination of PRPPs in associated with deficiency of the pyrimidine hemolysate. nucleoside kinase dCk. Recently, in human T-cell Charcoal-treated hemolysate is incubated in a pH 7.4 reaction mixture lines this lack of enzymatic activity was shown to containing: 50 mM TfisC1H, 5 mM MgC12, 1 mM EDTA, 0.4 mM DTT, 0.5 mM be associated with mutations in the dCk-gene, ATP, 0.35 mM Ribose 5-phosphate, 32 mM NaH2PO4 and 0.25 mM PI-P5 leading to severely reduced levels of the enzyme. We flladenosin 5' pentaphosphate (Ap5A, an inhibitor of the adanylate kinase). The used cell lines derived from an in vivo rat model reaction is stopped by addition of 0.1 M EDTA, and the samples are centrifuged in for AML to study mutational loss of dCk activity and Amicon cones. The resulting filtrate is injected into an HPLC ,aBondapak C18 the mechanisms underlying these mutations. One cell column and eluted with 0.2 M KH2PO4, pH 6, at 1.3 ml/min. Absorbance is line (RCL/0) is sensitive to the drugs and has a measured at 254 nm, and PRPPs activity is expressed as nmol of AMP generated/h/ normal dCk activity, a second (RCL/A) displays AraC- mg hemoglobin. and AzadC-resistance that was induced ex vivo in the Adenylate kinase activity is fully inhibited by Ap5A, allowing the accurate animal model; it shows no dCk-activity for AraC and determination of AMP. The method is sensitive and reproducible, being PRPPs AzadC, as well as a severely impaired enzyme activity linear with the amount of added hemolysate and with time of incubation, and activity for the metabolization of dC. We exposed mean and variance values (91.9 _+ 17.8 nmollh/mg) compare closely with those RCL/O cells to gradually increasing concentrations reported using other, more complicated, assay procedures (1). There is a significant of either AraC or AzadC and thus generated two correlation between the PRPPs activity measured by this one step assay and the independant AraC- and AzadC-resistant cell lines PRPPs activity determined by a modification of the two step assay employing (RCL/0-A and RCL/D resp.). Using RT-PCR-techniques adenine phosphoribosyltransferase as coupled enzyme (r=0.993, p<0.001). The with oligonucleotide primers chosen from the human hyperbolic curve relating Pi concentration to initial reaction velocity is shifted to dCk coding region , all AraC-resistant cell lines sigmoidal by addition of 0.02 mM GDP whioh inhibite PRPPs activities only at Pi were shown to express dCk-specific sequences with concentrations < 2 raM. This suggezt that this method should provide sensitive and aberant restriction sites as compared to amplicons accurate screening for regulatory as well as catalytic defects underlying PRPPs generated from the (normal) dCk-mRNA coding region of RCL/0. Southern blot analysis of RCL/A and RCL/0- superactivity. A genomic DNA revealed a genomic rearrangement of We conclude that the new method presented has important adventages over the dCk gene. In the AzadC-resistant RCL/D no the currently employed methods, and may provide a valuable diagnostic tool for a aberrant PCR-amplicons or Southern hybridization better understanding of the metabolic basis of uric acid overproduction and gout. patterns were detected. SSCP analysis however suggests a point mutation in the RCL/D dCk-gene. The I. Losman, et al. J Lab Clin Med 1984; 103: 932. data suggest distinct mutational events, underlying AraC- and AzadC-resistance respectively, in this Clinical Biochemistry and Internal Medicine Sections. La Paz University Hospital. model. Paseo de la Castellana n" 261. 280,16 Madrid. Spain.

THE IMPORTANCE OF THE METHYLATION ROUTE FOR 6- I~GULATION OF ~ BIFUNCTIONAL CYTOSOLIC 5'-NUCLEOTIDASFJNUCLEOSIDE MERCAPTOPURINE CYTOTOXICITY IN MOLT F4 HUMAN PHOSPHOTRANSFERASE LYMPHOBLASTS. *M,G. Tozzi. R Pes], M Turriani,S. Allegrini. M. CamicL P.L Ioa~ E,H, Stet+ R.A. O~ Abreu. G.M. Vo~els-MenLi0k. L.HJ. Lamboov. J.P+M. B~kkerink. J.M.F. Tri)bels. Cytosolic 5'-anclentidase, an ubiquitous enzyme which preferentially hydrolyzes 6- 6-Mercaptopurine (6MP) is used in the maintenance treatment of children with hydroxypufine nucleoside monophosphate% is regulated by energy charge, 2,3- acute lymphoblastic leukemia. Extensive intranellular metabolism of 6MP is diphosphogiycerate and phosphate (1-3). The enzyme appears to be present at higher required for its oytotoxicity. First, 6MP is converted into thin-IMP (tIMP) by the concentration in cells and organs with high nucleic acid turnover (4). Cytosolic 5'- enzyme hypoxanthine guanine phosphoribosyltransferase, tIMP is a branch-point nuclcotidase behaves as a bifunctional enzyme since it catalyzes the transfer of phosphate in 6MP metabolism. The first route of tIMP metabolism is conversion into from a nucleoslde monophosphate to a nucleoside aceeptor, probably forming an thioguanine nocleotides by enzymes of the purine interconversion pathway. In this enzyme-phosphate intermediate, thus operating a mononucleotide int~annversion (5). In route IMP dehydrogenase (IMPDH) is the rate-limiting enzyme. Thioguanine the absence of a suitable nuchioside, the enzyme-phosphate complex is hydroiytically nuclenddes are incorporate~l into DNA of the cells, inducing delayed cytotoxicity. cleaved. The understanding of the regulation of the two activities of this enzyme is In general it is accepted that this is the main caus~ for 6MP cytotoxicity. The depending on the possibility to measure the rate of formation of the three reaction second route for tIMP metabolism is methylation into methyl-thio-IMP (Mo- products: phosphate, nuclenside, and mononucleotide, in the same assay condition. dMP). This reaction is catalyzed by the S-adenosylmethionine (SAM) dependent Cytosolic 5' nucleotidase, has been purified to electrophoretic homogeneity from calf enzyme thiopurine methyltransfera.se in a conversion by which S-adenosyl-L- thymus. Its characteristics resulted to be identical to those of the enzyme purified from homocysteine is formed. Me-tIMP is a strong inhibitor of the purine de novo human colon carcinoma (2).We developed analytical methods utilizing HPLC and synthesis. Inhibition of this pathway leads to a depletion of porine nucleotides, and Capillary Electrophoresis to follow both enzyme activities as a fimction of pH and energy induces inhibition of DNA synthesis. This metabolic route may also contribute to charge variation, as well as nudeoside acceptor and nucleotide donor concentrations. cellular cytotoxieity. Our preliminary results indicate that the two activities show different optimum pH. In this study the importance of the methylation route for 6MP cytotoxicity was Furthermore both activities are increased in the presence of high energy charge, hut they studied in Molt F4 human malignant lymphoblasts. Cells were treated with 6MP display different ratios. In fact, 5'-nucleotidase is the main activity at low energy charge combined with mycophenolic acid, an inhibitor of IMPDH, and therefore an and slightly acidic pit, while phosphotransferase is favoured at high energy charge and inhibitor of formation of thioguanine nucleotides. As a result of addition of 0.5 pH around the neutrality. The study of the regulation of the enzyme is of great #M MPA to treatment with 2 .uM 6MP, less thio-GMP and more Me-tIMP were importance for the understanding both of its physiological role, and of its role in the formed. Under these conditions cytotoxicity was increased as compared to 6MP "activation" or "inactivation" of purine prodmgs. alone. As a result of addition of AICAR, an intermediate of the purine de novo synthesis to the treatment with 6MP and MPA, cytotoxicity resembled that of 1. Zimmermann H. Biochem. J. 385 (1993) 344. MPA alone. We therefore concluded that methylation is important for 6MP 2. Tozzi M.G. et al. Arch. Biochem. Biophys. 291 (1991) 212. cytotoxicity. 3. Itoh l~ et al. Biochem. J. 235 0956) 847. Furthermore, we determined the effects of 6MP on the SAM and the SAH 4. Itoh R. and Yamada K. Int. J. Biochem. 23 (199l). concentradons in Molt F4 cells. Treatment with 6MP decreased SAM, and 5. Worku Y. and Newby A.C. Biochem. J. 205 (1982) 503. increased SAH COncentration as compared to untreated ceils. SAM is an important methyl-donor in eukariotic cells, and is involved in methylation of DNA, RNA, Dipartimento di Fisinlogia e Biochimica, Universlt~i di Pisa, via S. Maria 55, 56100 Pisa proteins and phospholipids. So, any effect on the SAM/SAH ratio may exert Italy effects on these methylation reactions. Therefore, imbalance of the SAM/SAH *Istituto di Chimica Biologica, Universitl di Sassari, via Muroni 22/A, 07100 Sassari, ratio may he an other important mechanism by which 6MP induces deregulation of Italy cellular processes, which may ultimately result in cellular cytotoxicity.

Center for Pediatric Oncology SE Netherlands, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. This project is supported by the Dutch Cancer Society (NUKC 89-13)

F]2 +"+',,.,..,r Jl;,,l,l +~: +'q+,mr EIq'HANCED EXPRESSION OF THE CTP-SYNTHETASE GENE INTERFERON MODULATION OF 5-FLUOROURACIL IN COLON TUMOR CELL AFTER DEPLETION OF ITS END PRODUCT CTP LINES AT DIFFERENT FOLATE LEVELS

A.A. van den Berg, J.R. Meinsma, H. van Lenthe, C.L van der Wilt, P. Noordhuis, K. Smid, H.M. Pinedo, G.J. Peters A.H. van Gennip and A.B.P. van Kuilenburg. The relatively low response rate of standard 5-fluorouracil (FU) therapy ~n The activity of the enzyme CTP-synthetase in human leuke- advanced oolorsctal cancer can be increased by biochemical modulation at mic cells is increased in a transformation associated different levels. Leucovorin (LV) modulates by increasing the reduced folate manner, compared to that observed in normal human blood pools, which might be limiting for maximal inhibition of thymidylate synthase cells. Therefore, inhibitors of CTP-synthetase have been ('iS), mediated by the FU metabolite FdUMP. The mechanism by which proposed to be good candidate anti-leukemic drugs. The relationship between the expression of the CTP-synthetase interferons (IFN-a and IFN-7) modulate FU activity are not completely elucidated, gene and the size of the CTP pool in human cells is not but IFN-~ may increase FdUMP pools in the cell, thereby increasing FdUMP known. However, this relationship might be very important bindng to TS and TS inhibition, while IFN-7 has shown an effect at the level of in the light of the sensitivity of cells for inhibitors TS expression. of CTP-synthetase. Therefore, we studied this relation- We used two colon cancer cell lines (C26-10, murine and WiDr, human) to study ship in HL-60 human promyelocytic cell-line cells. the modulating effects of LV and IFNs. In these cell lines the antiproliferative effects of FU and FUdR, a FU derivative which is 'a more potent inhibitor of TS, HL-60 cells were incubated with 25 MM of 3'-deazauridine could not be modulated with LV (10 ~.M). The folates in the culture medium (2.3 (DAU), with i00 ~M of cytidine (Cyd) or 1.25 % DMSO. I~M) might be sufficient to achieve maximal TS inhibition. Sublines of the colon Initially, this led to a progressive depletion of the CTP celt lines (C26-10/F and WiDr/F) were adapted to physiological levels of folates pool (DAU), an increased CTP pool (Cyd) or an unchanged and maintained at 0.25 nM folinic acid. Sensitivity for FU and FUdR remained CTP pool (DMSO). RNA was purified from all HL-60 cells. unchanged but addition of LV (10 #M) increased FUdR mediated growth The expression of the CTP-synthetase gene at the level of inhibition 2-fold in C26-10/F. Activity of TS was increased in C26-10/F (1.3x) and its mRNA was determined after hybridisation with radio- WiDr/F (2.3x) compared to the parental lines. labeled DNA probes of the CTP-synthetsae gene. During Neither in the parental C26-10 nor in the subline C26-10/F IFN-~z or IFN-7 (100 incubation with DAU, the amount of CTP-synthetase mRNA U/ml) of human origine could enhance FU antitumor effect. IFN-7 was an active was increased in CTP depleted cells after 2 to 24 hours. Beyond 24 hours, the mRNA encoding the CTP-synthetase was modulator in WiDr and WiDr/F and a 2-fold increase of FU activity was observed. decreased and finally became undetectable. Also, in the In contrast to WiDr, WiDr/F also showed sensitivity to IFN-~ modulation (about presence of DMSO, the mRNA for CTP-synthatse diappeared 2-fold increase of FU activity), while IFN-r and IFN-y in combination more than within 48 hours. Because the HL-60 cells differentiated additively enhanced FU activity. into functional neutrophilic cells, both after treatment Addition of thymidine (TdR) in these growth inhibition tests reversed the with DAU and treatment with DMSO, it might be that the antiproliferative activity of FU and FUdR alone and that of combinations of these down-regulation of the CTP-synthatse gene is directly drugs with LV. The antiproliferative effect of high dose single IFN-~ or IFN-7 related to the maturation process. In cells incubated could not be reversed by TdR. with Cyd, the changes that Occurred in the amounts of TS activity measured in intact cells by incorporation of 3H-deoxyuridine into DNA CTP-synthetase mRNA are being studied now. So far, we was inhibited by FU in all cell lines. IFNs did not influence the extent of 3H- conclude that in HL-60 cells the expression of the CTP- deoxyuridine incorporation, but enhanced TdR incorporation. synthetase gene, at the level of the mRNA transcript, is These experiments underline the importance of folate levels in the modulation up-regulated after depletion of the intracellular CTP of FU activity. At physiological folate levels LV was useful as a modulator that pools.(Supported by the Dutch Federation for Pediatric acted at the level of TS inhibition, since TdR reversed the growth inhibitory Oncology Research, Grant SKK 89-01). effects. Although IFNs were more active at physiological folate levels, the inhibition of TS was not directly modulated by IFNs. Academic Me4ical Center, Deps. Pediatrisc Oncology and Clinical Chemistry, F-0-105, Meibergdreef 9/ 1105 AZ, Amsterdam, The Netherlands. Free University Hospital, dept. Oncology, P.O. BOX 7057, 1007 MB Amsterdam, The Netherlands

ALTERNATIVE SPLICING EVENTS IN THE 5'-END OF THE HUMAN COMPARISON OF DIHYDROPYRIMIDINE DEHYDROGENASE ACTIVITIES AMPD2 PRIMARY TRANSCRIPT GENERATE N-TERMINAL BETWEEN LEUKOCYTES AND FIBROBLASTS IN A FAMILY OF A PA- VARIANTS OF ISOFORM L. TIENT WITH THYMINE-URACILURIA F. Van den Berah. R.L Sabina A.H. van GENNIP, H. van LENTHE, N.G.G.M. ABELING, H.D. BAKKER AND A.B.P. van KUILENBURG Higher eukaryotes express multiple isoforms of AMP deaminase (EC 3.5.4.6.). In humans, at least three variants termed M(uscle), L(iver) and Thymine-uraciluria can be the result of a deficiency E(rythrocytes) have been characterized. Previous molecular studies have of one of the enzymes: dihydropyrimidine dehydrogenase reported three genes: AMPD1 (1), AMPD2 (2) and AMPD3 (3) that (DHPD, EC 1.3.1.2) or dihydropyrimidinase (DHP, EC produce transcripts encoding tsoforms M, L and E respectively. 3.5.2.2). DHPD activity can be measured in lymphocytes, Documented alternative splicing events in the 5'-ends of the AMPD1 and monocytes, fibroblasts and liver. In contrast, DHP ac- AMPD3 primary transcripts are predicted to generate isoform M and E tivlty is not expressed in leukocytes or fibroblasts and variants, respectively, differing at or near their N-terminii. therefore has to be measured in liver. As DHPD deficiency is a recently discovered disorder, it is important to Multiple human tissue Northern blot analysis (2) has demonstrated an investigate whether the defect is equally expressed in the tissues mentioned. We had the opportunity to study approximately 4 kb AMPD2.transcript in non-muscle tissues. These leukocytes and fibroblasts in the family of a patient results indicate a lack of several hundred basepairs from the 5'-end of with the defect. existing cDNA sequence. Additionally, a second smaller and more Patient BRB, a boy aged 2 yr, was investigated for abundant AMPD2 transcript appears to be expressed predominantly, if metabolic abnormalities because of psychomotor not exclusively, in the brain. retardation and ocular abnormalities. He appeared to have Our aims were to identify the extreme 5'-end of the AMPD2 gene, and to thymine-uraciluria. It was decided to investigate also investigate suspected alternative splicing events in ffs primary transcript. his parents and 2 brothers, although healthy, for this abnormality. For this purpose 24 h urine samples, blood A human cerebellum kgtl0 cDNA library was used to isolate additional and skin biopsies were collected from the patient and his AMPD2 clones using, as a probe the previously described 5' human T- family. In the patient thymine-uraciluria was confirmed, cell lymphoblast clone: Hut6A (2). Sixteen positive plaques were isolated while the pyrimidine excretion profiles of the family and the recombinant inserts from twelve were subcloned and sequenced. members were normal. Nucleotide sequence alignments between these and existing human The activity of DHPD was measured in lymnhocytes and AMPD2 cDNA clones has demonstrated three divergent extreme 5'-ends, cultured fibroblasts after incubation with 2-~C thymine which contribute at least 500 bp to the known sequence. Accordingly, a by HPLC analysis and on-line radioactivity measurement of human AMPD2 genomic clone was used to identify three exons which the reaction products. DHPD activities were undetectably contain the new 5'-end sequences. Subsequent RNase protection low in the lymphocytes and fibroblasts from the patient. analyses were used to demonstrate both cassette and mutually exclusive In one brother they were normal, while the other brother alternative splicing events involving the three identified exons. At the and the parents showed intermediate values. The results predicted protein level, our results indicate at least two different extreme from the measurements in both tissues led to identical N-terminii. These data, therefore, infer an additional level of complexity to conclusions, giving support to the assumption that leuko- isoform L expression in human tissues and cells. cytes as well as fibroblasts are appropriate for confir- mation of the diagnosis on the enzyme level. 1. R.L. Sabina, et al. J. Biol. Chem., 265 (1990) 9423. 2. M.T. 8ausch-Jurken, et aL J. Biol. Chem., 267 (1992) 22407. Academic Medical Center (AMC), Divisions of Pediatrics 3. D.K. Mahnke-Zizelman, R.L. Sabina. J. Biol. Chem., 267 (1992) and Cllnical Chemistry, Meibefgdreef 9, 1105 AZ Amster- 20866. dam, The Netherlands Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee Wi 53226, USA.

I~h,lrm,lcy l~'~,r/,I C" q, Jc..' ...... ~ .... ~ FI 3 INHIBITION OF CTP SYNTHETASE INDUCES DIFFERENTIATION OF HL-60 PURINE NU~DE METABOLISM IN HGPRT DEFICIENT RAT CELLS AND DOWN-REGULATION OF THE C-MYC ONCOGENE. NEUROMA CELL LINE A.B.P. Van Kuilenburs, A.A. Van den Bers. J.R. Meinsma. R.J. Slin-oerland and A.H. V~n Gennip. E__~.Zoref-Shani, Y_:_.BromberH, S__~.Brosh~ Y__~.Sidi, O_.~.SperlinK CTP synthetase and IMP dehydrogenase are the two 'key-enzymes' for the de novo biosynthesis of CTP and GTP, respectively. Inhibition of IMP dehydrogenase depletes the inlxacellulur guanine nucleotide pools and induces granulocytic differentiation An HGPRT-deficient rat neuroma cell line (BI03 4C), was of HL-60 ceils. Furthermore, the induction of HL-60 differentiation to granulocytes is utilized as a model tissue in search for the biochemical basis associated with specific changes in the protooncogene expression (1,2). These findings of the neurological manifestations of the Lesch-Nyhan have suggested that guanine ribonucleotides play an important role in the regulation of the syndrome (LNS). differentiation of leukemie-myeloid cells. So far, the role of CTP synthetase and cytosine The cells exhibited an almost complete absence of uptake of nucleotides in the process of differentiation and regulation of oncogene expression in HL-60 cells has hardly been studied. Therefore, we studied the pattern of c-myc guanine and of hypoxanthine into intact cell nucleotides (0.92 expression when HL-60 cells were induced to differentiate with either DMSO or with an % and 0.69 % of normal, respectively); a significant increase inhibitor of CTP synthetase. in the availability of PRPP; a 3 to 4 fold acceleration of the When HL-60 ceils were incubated with 25 I.tM 3'-deazauridine a rapid depletion of rate of nucleotide synthesis de novo; a normal excretion of the CTP ribonucleotides was observed within 2 h which persisted at least until 24 h. The xanthine, but 15 fold increase in the excretion of decline in the CTP levels reflects the inhibition of CTP synthetase by deazaUTP which is hypoxanthine into the culture media; a normal cellular purina a competitive inhibitor of the enzyme. However, a small increase in the UTP pool was nucleotide content (including the absence of Z-eucleotides), observed which can be explained by the fact that uridine and deazauridine are both but slightly enhanced turnover of adenine nucleotides and an metabolised by the same uridine/cytidine kinase. The progressive increase in the levels of elevated [rl'Pcontent. ATP and GTP until 24 h, is most probably due to stimulation of the purine de novo pathway by PRPP which accumulates after inhibition of the pyrimidine de novo pathway. The results suggest that under physiological conditions The mRNA levels of the c-myc oncogene was followed up to 72 h of exposure of guanine salvage does not occur in the normal neurons but that HL-60 ceils to either DMSO or 3'-deazauridine. Treatment of HL-60 ceils with DMSO hypoxanthine salvage is of great importance in the homeostasis resulted in a very rapid decline of the c-myc oncogene transcript and no c-myc mRNA of the adenine nucleotide pool. However, in the HGPRT- could be detected after 2 h. Despite the very rapid loss of c-myc mRNA, the HL-60 cells deficient neurons the lack of nucleotide salvage synthesis continued to progress through one cell cycle. Therefore, there appeared to be no direct from hypoxanthine is adequately compensated by the enhanced de link between the c.myc gene expression and the onset of proliferation arrest when HL-60 novo nucleotide synthesis. The results did not furnish cells were induced to differentiate with DMSO. evidence in support of the possibility that GTP or ATP The inhibition of CTP synthetase by deazaUTP resulted in a progressive decline of the c-myc transcript to undetectable levels within 48 h. Since CTP synthetase has an depletion, or Z-nucleotide accumulation occur in HGPRT- important role in providing the necessary precursors for RNA and DNA biosynthesis, the deficient neurons, but point to the possibility that elevated observed decrease in proliferation rate might be due to the depletion of CTP nucleotides. hypoxanthine concentration in the brain ~nay have an The proportion of HL-60 cells capable of generating an oxidative burst, a phenomenon etiological role in the pathogenesis of iNS. characteristic of mature granulocytic cells, increased to approximately 50% after 5 days. Our results demonstrate that the inhibition of CTP synthetase is associated with the down-regulation of the c-rayc oncogene which is a prerequisite in the process of Dept. of Chemical Pathology, Sackler School of Medicine, Tel terminal differentiation of HL-60 cells. Aviv University, Tel Aviv and Depts. of Clinical Biochemistry 1) S.M. Khurbanda et al. Cancer Res., 48 (1988) 5965. and of Medeicine D, Beilinson Medical Center, Petah-Tikva, 2) L.S. Mitchell et al. Differentiation, 49 (1992) 119. Israel.

Academic Medical Centre, Divs. EKZIkinder AMC and Clinical Chemistry, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

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