Purinogenic Immunodeficiency Diseases: DIFFERENTIAL EFFECTS OF DEOXYADENOSINE AND DEOXYGUANOSINE ON DNA SYNTHESIS IN HUMAN T LYMPHOBLASTS James M. Wilson, … , Peter E. Daddona, William N. Kelley J Clin Invest. 1979;64(5):1475-1484. https://doi.org/10.1172/JCI109606. Deoxyadenosine and deoxyguanosine are toxic to human lymphoid cells in culture and have been implicated in the pathogenesis of the immunodeficiency states associated with adenosine deaminase and purine nucleoside phosphorylase deficiency, respectively. We have studied the relative incorporation of several labeled nucleosides into DNA and into nucleotide pools to further elucidate the mechanism of deoxyribonucleoside toxicity. In the presence of an inhibitor of adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA], 5 μM], deoxyadenosine (1-50 μM) progressively decreased the incorporation of thymidine, uridine, and deoxyuridine into DNA, but did not affect uridine incorporation into RNA. This decrease in DNA synthesis was associated with increasing dATP and decreasing dCTP pools. Likewise, incubation of cells with deoxyguanosine caused an elevation of dGTP, depletion of dCTP, and inhibition of DNA synthesis. To test the hypothesis that dATP and dGTP accumulation inhibit DNA synthesis by inhibiting the enzyme ribonucleotide reductase, simultaneous rates of incorporation of [3H]uridine and [14C]thymidine into DNA were measured in the presence of deoxyadenosine plus EHNA or deoxyguanosine, and in the presence of hydroxyurea, a known inhibitor of ribonucleotide reductase. Hydroxyurea (100 μM) and deoxyguanosine (10 μM) decreased the incorporation of [3H]uridine but not of [14C]thymidine into DNA; both compounds also substantially increased 3[ H]cytidine incorporation into the ribonucleotide pool while reducing incorporation into the deoxyribonucleotide pool. In contrast, deoxyadenosine plus EHNA did not show this […] Find the latest version: https://jci.me/109606/pdf Purinogenic Immunodeficiency Diseases DIFFERENTIAL EFFECTS OF DEOXYADENOSINE AND DEOXYGUANOSINE ON DNA SYNTHESIS IN HUMAN T LYMPHOBLASTS JAMES M. WILSON, BEVERLY S. MITCHELL, PETER E. DADDONA, and WILLiAM N. KELLEY, Departments of Internal Medicine and Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 A B S T RA C T Deoxyadenosine and deoxyguanosine reducing incorporation into the deoxyribonucleotide are toxic to human lymphoid cells in culture and have pool. In contrast, deoxyadenosine plus EHNA did not been implicated in the pathogenesis of the immuno- show this differential inhibition of [3H]uridine incor- deficiency states associated with adenosine deaminase poration into DNA, and the alteration in [3H]cytidine and purine nucleoside phosphorylase deficiency, re- incorporation into nucleotide pools was less impressive. spectively. We have studied the relative incorporation These data show an association between accumu- of several labeled nucleosides into DNA and into nu- lation of dATP or dGTP and a primary inhibition of cleotide pools to further elucidate the mechanism of DNA synthesis, and they provide support for ribonu- deoxyribonucleoside toxicity. In the presence of an cleotide reductase inhibition as the mechanism re- inhibitor of adenosine deaminase [erythro-9-(2-hydroxy- sponsible for deoxyguanosine toxicity. Deoxyadeno- 3-nonyl)adenine [EHNA], 5 ,uM], deoxyadenosine sine toxicity, however, appears to result from another, (1-50 ,utM) progressively decreased the incorporation or perhaps a combination of, molecular event(s). of thymidine, uridine, and deoxyuridine into DNA, but did not affect uridine incorporation into RNA. This INTRODUCTION decrease in DNA synthesis was associated with increasing dATP and decreasing dCTP pools. Likewise, The association of at least two inborn errors of purine incubation of cells with deoxyguanosine caused an metabolism with immunodeficiency diseases has made elevation of dGTP, depletion of dCTP, and inhibition possible the investigation of these diseases at the of DNA synthesis. molecular level. Adenosine deaminase (ADA)' defi- To test the hypothesis that dATP and dGTP accumu- ciency results in severe combined immunodeficiency lation inhibit DNA synthesis by inhibiting the enzyme disease with impairment of T- and B-lymphocyte ribonucleotide reductase, simultaneous rates of in- function (1). Purine nucleoside phosphorylase (PNP) corporation of [3H]uridine and [14C]thymidine into deficiency has been associated with T-lymphocyte DNA were measured in the presence of deoxyaden- dysfunction in nine patients (2-9). The mechanism(s) osine plus EHNA or deoxyguanosine, and in the whereby these enzyme deficiency states affect lympho- presence of hydroxyurea, a known inhibitor of ribo- cyte development and/or function has not been fully nucleotide reductase. Hydroxyurea (100 ,tM) and elucidated. However, the recent findings of markedly deoxyguanosine (10 ,M) decreased the incorporation elevated dATP levels in ADA-deficient erythrocytes of [3H]uridine but not of [14C]thymidine into DNA; both (10, 11) and of dGTP levels in PNP-deficient erythro- compounds also substantially increased [3H]cytidine cytes (12) in conjunction with high-circulating or urinary incorporation into the ribonucleotide pool while levels of the corresponding deoxyribonucleoside in these patients (3, 13) provide support for the hypothesis that deoxyribonucleosides play an important role in the J. M. Wilson is a National Science Foundation Predoctoral Fellow. Dr. Mitchell is the recipient of National Institutes of Health Clinical Investigator Award 1 K08 AM 00442. ' Abbreviations used in this paper: ADA, adenosine Received for publication 17 January 1979 and in revised deaminase; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine; form 23 July 1979. PCA, perchloric acid; PNP, purine nucleoside phosphorylase. J. Clin. Invest. (© The American Society for Clinical Investigation, Inc. 0021-9738/79111/1475/10 $1.00 1475 Volume 64 November 1979 1475-1484 pathogenesis of the immune dysfunction in these two deoxyribonucleosides into DNA, into RNA, and into diseases and suggest that deoxyribonucleosides or their ribonucleotide pools to assess the effects of deoxya- metabolites may be toxic to lymphoid cells. denosine and deoxyguanosine on ribonucleotide re- The purine deoxyribonucleosides have indeed ductase activity. Whereas our results provide more been shown to have lymphocytotoxic effects in vitro. direct evidence for the inhibition of ribonucleotide Deoxyadenosine in the presence of an inhibitor ofADA reductase in deoxyguanosine toxicity, they also suggest inhibits [3H]thymidine incorporation into DNA by that additional factors may play a role in the toxicity peripheral blood lymphocytes (14) and is toxic in micro- of deoxyadenosine. molar concentrations to a variety of mouse (15, 16) and human (17, 18) lymphoid cell lines. Deoxyguanosine METHODS in the absence of a PNP inhibitor is likewise extremely toxic to cells in culture (18-20). In addition, Radioisotopes and chemicals. [3H]Cytidine (28.0 Ci/ lymphoid mmol), [3H]uridine (24.2 Ci/mmol), [3H]deoxyuridine (21.9 the rather striking selectivity of both deoxyadenosine Ci/mmol), [3H]dTTP (18.3 Ci/mmol), and [3H]dCTP (22.3 and deoxyguanosine toxicity for human lymphoblasts Ci/mmol) were obtained from New England Nuclear (Boston, of T-cell origin, as opposed to B-cell origin (18, 21), has Mass.), and [U-_4C]thymidine (515 mCi/mmol) and [U-'4C]- provided a potential explanation for the prominent deoxycytidine (462 mCi/mmol) were obtained from Amersham Corp (Arlington Heights, Ill.). All unlabeled ribonucleosides T-cell dysfunction in patients with ADA and PNP defi- and deoxyribonucleosides, Micrococcus lysodeikticus DNA ciency, respectively. polymerase, calf thymus DNA, and Dowex 50 x 4 200-400 The cytotoxic effects of deoxyadenosine and deoxy- (Dow Chemical Co., Midland, Mich.) were purchased from guanosine on cultured cells correlate well with an Sigma Chemical Co. (St. Louis, Mo.). Affi-Gel 601 was ob- the corresponding deoxyribonucleoside tained from Bio-Rad Laboratories (Richmond, Calif.). Erythro- accumulation of 9-(2-hydroxy-3-nonyl)adenine (EHNA) was provided by triphosphate and are also associated with a decrease in Burroughs-Wellcome Co., Research Triangle Park, N. C. the intracellular dCTP pool (15, 16, 19, 20). These ob- Cell cultures. The Molt-4 T-lymphoblast line was ob- servations, in conjunction with substantial reversal of tained from Hem Research, Inc., Rockville, md., and was toxicity in several culture systems with exogenous originally derived from a patient with acute lymphoblastic leukemia. Cultures were maintained in exponential growth in deoxycytidine (16-20), have led many investigators RPMI-1640 medium plus 10% horse serum. to suggest inhibition of the enzyme ribonucleotide Deoxyribonucleotide pool measurements. Cells were reductase by dATP or dGTP as the mechanism under- incubated at a concentration of 106/ml in RPMI plus 10% heat- lying cellular toxicity (10, 16, 17, 20). Ribonucleotide inactivated horse serum at 37°C for 1 h with or without ad- catalyzing the reduction of ditives. Levels of dATP, dGTP, and dTTP were determined in reductase is responsible for 1 x 106 cells; dCTP levels were determined in 1 x 107 cells. At all ribonucleoside diphosphates, and, thus, this enzyme the end of the incubation period, cold RPMI medium (5 vol) appears to be important in the control of DNA syn- was added. The cells were sedimented at 450 g for 5 min at 4°C thesis. It is subject to strong negative allosteric