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Characterization of Novel High Affinity Purine Base Long Term Expression of Human Adenosine Deaminase in Transport Systems in 549 Cells

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Characterization of Novel High Affinity Purine Base Long Term Expression of Human Adenosine Deaminase in Transport Systems in 549 Cells CHARACTERIZATION OF NOVEL HIGH AFFINITY PURINE BASE LONG TERM EXPRESSION OF HUMAN ADENOSINE DEAMINASE IN TRANSPORT SYSTEMS IN 549 CELLS. MURINE HEMATOPOIETIC CELLS. JW Belmont GR MacCre- 1 1 Joanne Beck and Buddy Ul lman, 14 gor. KA Moore. FA Fletcher. D Hawkins and CT Caskey. The Oregon Health Sciences University, Institute for Molecular Genetics and Howard Hughes Department of Biochemistry, Portland, Oregon, U.S.A. Medical Institute, Baylor College of medicine, Hous- Mutant cell lines have been isolated from wild type 549 ton, Texas 77030 USA. cells that possess a novel high affinity purine base transport Twenty-two defective retrovirus vectors carrying human adeno- system that is not found in parental cells or any other sine (ADA) coding sequences were tested for efficient transduc- mammalian cell line. These cells are capable of transporting tion of human ADA enzyme activity. These vectors were designed purine bases at a rate 10 to 30-fold greater than wild type to test functional aspects of construction including the use of cells. The Km value of this novel nucleobase transporter for internal transcriptional units, the importance of the Moloney purine bases is exceptionally low, approximately 10-100 sequences 563-1038 (gag+), and alterations in the 3' LTR. micromolar. The high affinity transporter does not recognize Promoters from the human ADA and cFos genes, herpes virus nucleosides or pyrimidine nucleobases and can be distinguished thymidine kinase, and cytomegalovirus immediate early region from other previously described nucleobase transporter in gene were tested as alternatives to the viral LTR promoter. mammalian cells on the basis of its high affinity for ligands Several vectors all containing the gag+ region allow high titer and its sensitivity to inhibition by DPA and NBtPR. That the virus production. These vectors were then tested in bone marrow expression of this high affinity purine base transport system transplant experiments. Two vectors, one utilizing the Moloney is independent of the NBMPR- and DPA-sensitive nucleoside LTR promoter and one with the HSVTK promoter, gave expression of transport system has been demonstrated by the insertion of the ADA in CN-C. CN-S and in the blood of reconstituted animals. high affinity nucleobase transport mutation into nucleoside Infection efficiency ranged from 15-85% in CN-S by Southern transport-deficient 549 cells. Two classes of high affinity analysis. Expression efficiency was also variable. All animals purine base transport mutants were generated that can be had human ADA in blood 2-4 weeks post-transplant but at 10.12 differentiated on the basis of their substrate specificities, weeks levels of the enzyme declined. Four oE 14 long term affinities for purine bases, and sensitivities to inhibitors of recipients however, demonstrated expression for up to 5 months. nucleoside transport. Tba data suggest that the expression of PCR analysis on nonexpressors indicated a loss of cell with the the high affinity transport probably requires the unmask~ngor proviral DNA suggesting that the initial expression resulted alteration of a specific genetic locus that is silent or from infection of more mature progenitors. Immuno-staining is different in wild type cells. being used to characterize the lineages and distribution oE ADA producing cells in the transplant recipients. pRPP AND PURINE NUCLEOTIDE METABOLISM IN HUMAN STIMULATION BY GLYCERITE-2.3-BISPHOSPHATE : A COEMON LYMPHOBLASTS WITH BOTH PRPP SYNTHETASE SUPERACTIVITY p PROPE~TYOF CYTOSOLIC PURINE 5'-NUCLEOTIDASES IN VAR- 12 AND HGPRT DEFICEENCY. M.A. Becker, M. Kim, K. 13 IOUS TISSUES. F. Bontemps, M.F Vincent, F. Van den Husain. University of Chicago, Chicago, Illinois, Bergh, C. van Waeg & G. Van den Berghe. International USA. Institute of Cellular and Molecular Pathology, Labor- atory of Physiological Chemistry, Brussels, Belgium. PRPP and purine nucleotide synthesis and culture growth in Human erythrocytes contain a cytosolic purine 5'-nucleotidase response to medium additions were compared in cloned human B (5'-Nase) (Bontemps et al., Adv. Exp. Med. Biol. 195B:283-90, lymphoblast lines selected for resistance to growth inhibition 1986; Biochem. J. 250:687-96, 1988) which, similarly to other by 6-thioguanine (TG) and in the respective normal and PS- cytosolic 5'-Nases, is more active on IMP and GMP than on AMP, superactive parental cell lines. TG-resistant cell lines were is stimulated by .\TP and, in addition, by glycerate-2,3-bisphos- severely deficient in HGPRT (<0.2% normal activity). Cells with phate (2.3-DPC), the main phosphate ester in human erythrocytes. both HGPRT deficiency and PS superactivity retained the kinetic Both stimulators increase activity to the same extent at satura- defect in PS of the parental cells but had PRPP concentration ting concentrations, but 2.3-DPG is more potent tl~an ATP at 20-fold greater than normal cells and 6- and 5-fold greater, subsaturating concentrations. To investigate if 2.3-DPG was able respectively, than the parental PS-superactive cells and cells to stimulate cytosolic 5'-Nase in other tissues, studies were defective in HGPRT alone. Nevertheless, PRPP generation in performed on high-speed supernatants, freed from small molecules HGPRT-deficient lines was minimally increased (<8%) compared by dialysis or by filtration on G-25. 5'-Nase activity was with parental cell lines and purine synthesis de novo in the measured at pH 7.2 by release of labelled nucleosides and bases cell line with both enzyme defects was comparable to that in the from 0.2 mM [814c]-1~p. At 5 mM, 2,3-DPG was found to PS-superactive parental line. stimulate the activity of cytosolic purine 5'-Nase to the same Regardless of the status of PS activity, all HGPRT-deficient extent as 5 mM ATP, in liver, cardiac and skeletal muscle, cell lines showed growth patterns typical of HGPRT deficiency in brain, spleen and erythrocytes from rat, and in human polymorph- HAT medium and in medium with 8-azaguanine. However, growth of onuclear leukocytes, mixed peripheral blood lymphocytes and HGPRT-deficient cell lines, including the line with PS platelets. Depending on the tissue, maximal stimulation varied superactivity, was more sensitive than that of parental lines to between 2- and 14-fold. 2.3-DPG was little hydrolyzed in the inhibition of purine synthesis de novo by 6-methylthioinosine various preparations and was without effect on the membranous (MMPR), especially when adeninmprovided, most likely 5'-Nase. 2.3-DPC is thus a common stimulator of cytosolic purine reflecting compromised guanine nucleotide synthesis in HGPRT- 5'-Nase of various tissues and can be used to iddntify and deficient cells when purine synthesis de novo is blocked and reveal this enzymic activity in crude preparations. adenine is the sole source of purine for salvage. INHERITED SUPERACTIVITY OF PRPP SYNTHETASE (PS): MECHANISM OF ATP CATABOLISM INDUCED BY DEOXYADENO- - ASSOCIATION OF PURINE OVERPRODUCTION AND SENSORI- SINE AND OTHER NUCLEOSIDES IN ADENOSINE DEAEIINASE-IN- NEURAL DEAFNESS. M.A. Becker, J.G. hig, F.A. HIBITEO HUMAN ERYTHROCYTES. Franqoise Bontemps & A1' u 16 Mateos, M.L. Jimenez, M. Kim, H.A. Simmonds. Univ. Georges Van den Berghe, International Institute of of Chicago, Chicago, IL, USA; Cuidad Sanitaria La Cellular and Molecular Pathology, Laboratory of Paz, Madrid, Spain; Guy's Hospital, London, England. Physiological Chemistry, Brussels, Belgium. In fibroblasts from an 8 year-old male (VRG) with tophaceous In adenosine deaminase (ADA) deficiency and during treatment gout, uric acid overproduction, and sensorineural deafness, and with deoxycoformycin (dCF), depletion of ATP accompanies nccumu- from his gouty mother, superactivity of PS due to purine nucleo- lation of dATP in erythrocytes and lymphocytes (Bagnara & tide inhibitor-resistance was found. Compared with normal Hershfield, PNAS, 79: 2673-77, 1982 and refs therein). Stimula- cells, VRG fibroblasts showed increases in the following: PRPP tion of AMP deaminase and cytosolic 5'-nucleotidrse by dATP was concentration (3-fold) and generation (2-fold); purine synthesis proposed to account for this. However, both enzymes are not more de novo (2.5-fold); purine base salvage (2.6-fold); ATP and GTP stimulated by dATP than by ATP. In human erythrocytes in which concentrations (1.4-fold); and excretion of newly synthesized ADA was inhibited with 1 uM dCF, a 3- to 30-fold stimulation of purines (5-fold). Derangements in PS and in PRPP and purine adenine nucleotide (AN) catabolism was recorded upon addition of synthesis were attenuated in cells from the mother, suggesting dAdo, but also of other substrates of adenosine kinase (AK) such that VRG is a hemizygote for an X chromosome-linked defect and as Ado, Ara-A, tuberdicin and 6-ECLPR. Concomitantly, there was a his mother is a heterozygote. Comparison was made of affected reproducible increase, from 10 uM up to 100 uEI, of AMP and of males in 8 families with PS superactivity. VRG was intermediate IMP, indicating an increased activity of AMP deaminase. The in range of expression of PS superactivity and in severity of effects of Ado were suppressed upon prior inhibition of AK by the enzyme defect as measured by the degree of aberration of 10 VPI iodotubercidin (ITu). Strikingly, they were also suppres- PRPP and purine nucleotide synthesis in fibroblasts. Metabolic sed if ITu was added 2 h after ado, although dATP was maintai- abnormalities were more severe in VRG than in most patients (in ned under this condition. ITu also abolished ATP catabolism whom expression is limited to early adult-onset gout with uric induced by the other nucleosides. With all nucleosides, UIP acid overproduction), but less severe than in 2 patients with increased during their phosphorylation and decreased after inhi- more complex defects in PS associated with uric acid over- bition of AK. Studies with erythrocytic ILYP deaminase, at low production and neurodevelopmental abnormalities, including substrate dnd physiological concentrations of effectors, showed deafness in hemizygous male children and heterozygous women.
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