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Biochemical Basis for Differential Deoxyadenosine Toxicity To Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2434-2437, May 1979 Medical Sciences Biochemical basis for differential deoxyadenosine toxicity to T and B lymphoblasts: Role for 5'-nucleotidase (deoxyadenosine kinase/deoxyadenylate kinase/immunodeficiency) ROBERT L. WORTMANN, BEVERLY S. MITCHELL, N. LAWRENCE EDWARDS, AND IRVING H. Fox Human Purine Research Center, Departments of Internal Medicine and Biological Chemistry, Clinical Research Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109 Communicated by James B. Wyngaarden, March 7, 1979 ABSTRACT Deoxyadenosine metabolism was investigated tained from Calbiochem. Erythro-9-[3-(2-hydroxynonyl)]- in cultured human cells to elucidate the biochemical basis for adenine (EHNA) was a gift from G. B. Elion of Burroughs the sensitivity of T lymphoblasts and the resistance of B lym- Wellcome (Research Triangle Park, NC). Horse serum was phoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20- to 45-fold greater capacity to synthesize deoxyadenosine nu- obtained from Flow Laboratories (Rockville, MD), and Eagle's cleotides than B lymphoblasts at deoxyadenosine concentrations minimal essential medium was purchased from GIBCO. From of 50-300 ,uM. During the synthesis of dATP, T lymphoblasts Amersham/Searle, [U-14C]deoxyadenosine (505 mCi/mmol), accumulate large quantities of dADP, whereas B lymphoblasts [8-14C]hypoxanthine (52.5 mCi/mmol), and [U-14C]deoxy- do not accumu ate dADP. Enzymes affecting deoxyadenosine adenosine monophosphate (574 mCi/mmol) were purchased; nucleotide synthesis were assayed in these cells. No substantial and, from ICN, [8-14C]adenosine monophosphate (34.4 mCi/ differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76) or mmol) was purchased (1 Ci = 3.7 X 1010 becquerels). All other deoxyadenylate kinase [ATP.(d)AMP phosphotransferase, EC reagents were of the highest quality commercially available. 2.7.4.111. The activity of 5'-nucleotidase (5'-ribonucleotide Cell Lines. The MOLT-4 (T cell lymphoblast) cell line was phosphohydrolase, EC 3.1.3.5) was increased 44-fold for AMP obtained from HEM Research (Rockville, MD). The cells were and 7-fold for dAMP in B lymphoblasts. A model for the regu- negative for surface immunoglobulins, contained 23% T-ro- lation of deoxyadenosine nucleotide synthesis by 5'-nucleotidase setting cells, and were originally derived from a patient with activity is proposed on the basis of the observations. acute lymphoblastic leukemia. The MGL-8 (B-cell lympho- Deoxyadenosine and adenosine concentrations are increased blasts) cell line was derived from a normal individual and was in individuals with adenosine deaminase deficiency and severe a gift from J. Epstein, Johns Hopkins University. These cells combined immunodeficiency disease (1, 2). The accumulation were positive for surface immunoglobulins (8-20%) and in- of deoxyadenosine leads to the increased concentrations of corporated [3H]leucine into protein that migrated as heavy and dATP and dADP in erythrocytes, peripheral blood lympho- light chains on a sodium dodecyl sulfate gel after immu- cytes, and bone marrow cells of these patients (2-4). The ele- noprecipitation. vated levels of deoxyadenosine nucleotides are believed to Deoxyadenosine Metabolism. T and B lymphoblasts were provide the biochemical basis for the immune dysfunction removed from flasks in which they were grown in continuous observed in the enzyme deficiency state. culture. Cells were washed twice in normal saline and 100 mM Although the precise mechanism for the immune dysfunction Tris-HCl, pH 7.4. Cells were suspended in Eagle's minimal is unclear, several studies in vitro have attempted to determine essential medium containing 10% dialyzed horse serum, 1.2 mM the underlying molecular pathology. The addition of deoxy- potassium phosphate at pH 7.4, 25 mM Tris-HCl at pH 7.4, and adenosine reduces the response of peripheral blood lymphocytes 0 or 5.0 ,uM EHNA. Cell counts were performed on the cell to mitogen stimulation when adenosine deaminase is inhibited suspensions. (1, 5). The combination of deoxyadenosine and adenosine Aliquots (50 ,l) of cell suspension (2.0-6.0 X 106 cells per ml) deaminase inhibition is also cytotoxic to T lymphoblasts but not were incubated at 37°C for 20-35 min, removed and placed B lymphoblasts (6, 7). Deoxyadenosine-mediated cytotoxicity on ice, and immediately mixed with substrate solution (50,ul) in T lymphoblasts is accompanied by increased concentrations that contained Eagle's minimal essential medium. The final of dATP. concentrations were as follows: 1.2 mM potassium phosphate Deoxyadenosine metabolism was investigated in cultured at pH 7.4, 20 ,uM adenine, 25 mM Tris-HCl at pH 7.4, 0 or 5.0 human cells to elucidate the biochemical basis for the sensitivity ,uM EHNA, and 20-400 jiM radiolabeled deoxyadenosine di- of T lymphoblasts and the resistance of B lymphoblasts to luted with nonisotopic deoxyadenosine. The mixture was in- deoxyadenosine toxicity. These studies have revealed a major cubated at 37°C. The reaction was stopped by heat inactivation difference in the capacity of T lymphoblasts to accumulate at 85°C for 2 min and cooled to 40C. After centrifugation at deoxyadenosine nucleotides compared to B lymphoblasts, 1500 X g for 10 min, 20 pA of the supernatant solution was ap- possibly because of a difference in activity of 5'-nucleotidase plied to two sheets of Whatman 3 MM chromatography paper previously spotted with 20 Al of 1 mg/ml nonradioactive dATP, (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5). dADP, dAMP, dIMP, adenine, deoxyinosine, deoxyadenosine, MATERIALS AND METHODS and hypoxanthine. Deoxynucleotides were separated by high-voltage electrophoresis in 50 mM sodium citrate at pH 3.9 Reagents. Deoxyadenosine, deoxyinosine, ATP, dATP, and deoxynucleosides and bases, in 50 mM sodium borate at pH dADP, dAMP, dIMP, Tris base, dithiothreitol, and EDTA were 8.9. Compounds were visualized with ultraviolet light and cut purchased from Sigma. Adenine and hypoxanthine were ob- out, and their radioactivities were measured in a liquid scin- tillation spectrometer system. The publication costs of this article were defrayed in part by page reaction mixture to block charge payment. This article must therefore be hereby marked "ad- Adenine was added to the hypo- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: EHNA, erythro-9-13-(2-hydroxynonyl)ladenine. 2434 Downloaded by guest on September 29, 2021 Medical Sciences: Wortmann et al. Proc. Natl. Acad. Sci. USA 76 (1979) 2435 xanthine phosphoribosyltransferase by depleting the intracel- labeled dADP and dATP formed from radiolabeled dAMP. lular phosphoribosyl pyrophosphate supply through its WUtili- Diluted cell extracts (25 ,gl) were mixed with a substrate solution zation in the adenine phosphoribosyltransferase reaction. To (75 ,l) providing final concentrations of 1.0 mM ATP, 5.0mM ensure that hypoxanthine formed from the degradation of la- magnesium chloride, 100 mM potassium chloride, 50 mM beled deoxyadenosine was not being salvaged by hypoxanthine TrisIHCI at pH 7.4, and 0.1 mM [U-14C]dAMP. The mixture phosphoribosyltransferase and contaminating the deoxynu- was incubated at 370C and the reaction was stopped by the cleotide pool, radiolabeled hypoxanthine was substituted for addition of 0.2 M EDTA and heating at 850C for 2 min. Product deoxyadenosine in the above system. Studies using 20 ,uM hy- formation was determined as described above with the ex- poxanthine demonstrated that less than 5% of the deoxynu- ception that high-voltage electrophoresis was performed in 50 cleotides could have originated from ribonucleotides. mM sodium citrate at pH 3.9. The reaction was linear with Viability of cells was checked at the end of each experiment protein concentration and time up to 45 min. Initial velocity in tubes not heated at 85°C by the nigrosin dye exclusion studies were performed with 12.5-100 MiM [U-14C]dAMP by method (8). Viability ranged from 94% to 98% in all experi- using similar methods. ments. Intact cell 5'-nucleotidase activity was quantitated by mea- Enzyme Assays. Membrane-depleted extracts of T and B suring the conversion of radioisotopic AMP or radioisotopik lymphoblasts were prepared for enzyme assays. Washed cells dAMP to radiolabeled nucleoside products as described by were frozen and thawed three times and centrifuged at 28,000 Edwards et al. (9). Lymphoblasts from continuous cell lines X g for 30 min at 4°C. The supernatant solutions were stored were harvested and suspended in Hanks' solution to 3.0-6.0 X at -70'C until use. Prior to assay the extracts were dialyzed in 106 cells per ml for assay. Aliquots (25 ,l) of cell suspension were normal saline with 1 mM Tris-HCI at pH 7.4 and 0.1 mM incubated in a total volume of 100 ,l with 40 mM Tris-HCI at EDTA for 6-14 hr. pH 7.5, 4.0 mM magnesium chloride, and 0.2 mM [8-14C]AMP Deoxyadenosine kinase activity (ATP:deoxyadenosine 5'- or [ U-14C]dAMP. The assay was linear with cell number and phosphotransferase, EC 2.7.1.76) was determined by the time up to 45 min. Initial velocity studies were performed with amount of radiolabeled deoxyadenosine nucleotides formed 5-50 ,M by using a similar method. from radiolabeled deoxyadenosine. Diluted cell extracts (50,ul) Concentrations. Proteins were measured by the method of were mixed with a substrate solution (50 ,l) to final concen- Lowry et al. (10) with bovine serum albumin as a standard. In trations of 5 mM ATP, 2 mM dithiothreitol, 40MuM EHNA, 200 the use of diluted radioisotopes, the concentration was deter- mM Tris-HCI at pH 7.4,20 mM magnesium chloride, and 0.5 mined by calculation from an absorbance measured at the ab- mM [U-14Cldeoxyadenosine. After 30-min incubation, the sorption maximum. reaction was stopped by heating at 85°C for 2 min.
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