[CANCER RESEARCH 38, 2357-2362, August 1978] 0008-5472/78/0038-OOOOS02.00 Cytotoxic and Metabolic Effects of Adenosine and Adenine on Human Lymphoblasts1 Floyd F. Snyder,2 Michael S. Hershfield,3 and J. Edwin Seegmiller Department of Medicine, University of California, San Diego, La Jolla, California 92093 ABSTRACT and further studies have shown that cytotoxic concentra tions of adenosine reduce pyrimidine ribonucleotides (12, The metabolic and growth inhibitory effects of adeno- 17, 21, 31) and NAD+ (17, 31) and cause an accumulation of sine toward the human lymphoblast line WI-L2 were po orotic acid (12, 31). Adenosine also increases cellular ade tentiated by the adenosine deaminase inhibitors erythro- nine nucleotides (12, 17, 31), including a transient increase 9-(2-hydroxy-3-nonyl) adenine (EHNA) and coformycin. in cAMP4 concentration (34, 35). In addition to studies in EHNA, 5 nu, or coformycin, 3.5 ¿iM,atconcentrations that cultured cells, increased adenine ribonucleotide pools have inhibited adenosine deaminase activity more than 90%, been found in erythrocytes (1, 25) and lymphocytes (28) of had little effect on cell growth or the metabolic parameters adenosine deaminase-deficient, immune-defective patients. studied. Adenosine, 50 /¿M,plusEHNA, 5 JUM,arrested cell The selective toxicity of adenosine to dividing lymphoid growth in both parent and adenosine kinase-deficient cells, including inhibition of both the response of human lymphoblasts, implicating the nucleoside as the mediator peripheral blood lymphocytes to mitogen and the growth of of the cytostatic effect. Adenosine, 50 /KM,in combination lymphoblastoid cell lines, is considered a possible basis for with the adenosine deaminase inhibitors reduced 14C02 the severe combined immunodeficiency disease associated generation from [1-14C]glucose by 38%, depleted 5-phos- with a hereditary absence of adenosine deaminase activity phoribosy 1-1-pyrophosphate by more than 90%, and re (11,24). duced pyrimidine ribonucleotide concentrations. Uridine, It has been suggested that the toxic effects of adenosine 10 or 100 ftM, reversed adenosine plus EHNA growth are caused by an increased cellular adenine nucleotide inhibition in WI-L2 but not in adenosine kinase mutants. pool, but some evidence has suggested a mechanism(s) of Adenine, 500 //M, which may be converted to the same toxicity that does not require conversion of adenosine or intracellular nucleotides as adenosine, reduced the adenine to nucleotides. Certain analogs of adenosine that growth rate by 50% in both parent and adenine phospho- cannot be phosphorylated are cytotoxic (19, 32), and we ribosyltransferase-deficient lymphoblasts. Although ade have found the toxicity of adenosine to persist in adenosine nine also depleted cells of 5-phosphoribosyl-l-pyrophos- kinase-less mutants of the WI-L2 human splenic lympho phate and reduced pyrimidine ribonucleotide by 50%, the blast line; mutants lacking adenine phosphoribosyltransfer- mechanisms of adenine and adenosine toxicity differ. In ase were as sensitive as was their parent line to growth contrast to the ability of uridine to reverse adenosine inhibition by adenine (16). We now report more detailed cytostasis, growth inhibition by adenine was not reversed studies on the biochemical effects of adenosine and ade by uridine, indicating that pyrimidine ribonucleotide de nine on these lymphoblast lines. In these studies we have pletion is not the primary mechanism of adenine toxicity. used 2 potent inhibitors of adenosine deaminase, coformy cin (26) and EHNA (27). We have compared the effects of INTRODUCTION adenosine and adenine since both purines can be con verted to the same intracellular nucleotides (Chart 1) and The mechanism(s) of cytoxicity for the naturally occurring might therefore be expected to have similar mechanisms of nuceoside, adenosine, and its related base adenine is not toxicity if their effects are related only to expanded adenine understood but remains important, with growing interest in nucleotide pools. the use of adenosine deaminase inhibitors in combination chemotherapy. Both adenosine (9, 10, 18, 30) and adenine (20) block mitogen-induced transformation of human lym MATERIALS AND METHODS phocytes. Adenine toxicity toward mouse L-cells was par Biochemicals. Radiochemicals were purchased from tially overcome by certain pyrimidines (2), and adenosine Amersham/Searle Corp., (Arlington Heights, III.): [8- toxicity was reversed by uridine in normal (12) but not in 14C]adenine, 59 mCi/mmol; [8-14C] adenosine, 59 mCi/ adenosine deaminase-deficient fibroblasts (4). Hilz and mmol; [8-14C]hypoxanthine, 59 mCi/mmol; [1-14C]glucose, Kaukel (17) first documented a decrease in intracellular 9.37 mCi/mmol; [6-14C]glucose, 5.0 mCi/mmol; and sodium DTP concentration in adenosine-inhibited HeLa cells (21), [14C]formate, 59 mCi/mmol. Adenine, adenosine, hypoxan- thine, uridine, and PP-ribose-P (sodium salt) were pur 1This work was supported by Grants AM-1362, AM-5646, and GM-17702 chased from P-L Biochemicals (Milwaukee, Wis.). EHNA from NIH and grants from the National Foundation and the Kroc Foundation. 2 Present address: Division of Pediatrics and Medical Biochemistry, Fac was provided by Wellcome Research Labs (Research Tri- ulty of Medicine, The University of Calgary, Calgary, Alberta T2N 1N4, Canada. To whom requests for reprints should be addressed. 3 Present address: Department of Medicine, Duke University Medical 4The abbreviations used are: cAMP, cyclic adenosine 3':5'-monophos- Center, Durham, N. C. 27710. phate; EHNA, eryr/ii-o-9-(2-hydroxy-3-nonyl) adenine; PP-ribose-P, 5-phos- Received December 13, 1977; accepted May 11,1978. phoribosyl-1 -pyrophosphate. AUGUST 1978 2357 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1978 American Association for Cancer Research. F. F. Snyder et al. ATP conditioned to more than 3 months of growth in medium II supplemented with 10% horse serum. This serum was ADP previously shown to deaminate less than 5 /¿molof 50 /XM II adenosine per 25 hr (30). The growth rate of WI-L2 lympho- blasts in this medium was inhibited 50% by approximately EHNA, Cofonnycln 200 /¿Madenosine, and 5000 /UM adenosine completely /If(ADENOSINE « INOSINE ' (AD arrested growth (Chart 2). The adenosine deaminase inhib DAP \ t ? TG A itors EHNA, 5 /iM, and coformycin, 3.5 /¿M(1 pig/ml), ADENINE HYPOXANTHINE inhibited lymphoblast adenosine deaminase activity more Chart 1. Purine interconversion showing sites of selected lymphoblast than 95% in either extracts or whole cells but had little mutation and enzyme inhibition. DAP, 2,6-diaminopurine resistant and effect on growth. WI-L2 grew at approximately 90% of the deficient in adenine phosphoribosyltransferase; MTV, 6-methylthioinosine resistant and deficient in adenosine kinase; TG . 6-thioguanme resistant and normal growth rate for 8 weeks in the presence of 5 /¿M deficient in hypoxanthine-guanine phosphoribosyltransferase; EHNA and EHNA with weekly subculturing and addition of EHNA. coformycin, inhibitors of adenosine deaminase; AMPS, adenylosuccinate. EHNA increased the sensitivity of WI-L2 lymphoblasts to growth inhibition by adenosine greater than 10-fold, such angle Park, N. C.), and coformycin was provided by Dr. H. that 100 ^M adenosine completely arrested growth in the Umezawa, Institute for Microbial Chemistry, Tokyo, Japan. presence of 5 /¿MEHNA (Chart 2). Complete growth inhibi Lymphoblasts. The human splenic lymphoblast line Wl- tion by 50 t¿Madenosine and 5 /¿MEHNA occurred after 24 L2 (23) was grown in suspension culture supplemented with hr of exposure or approximately 1 cell doubling (Chart 3/4) 2 nriM glutamine and 10% horse or fetal calf serum (Flow and was reversible for at least 72 hr of culture (Chart 30), Laboratories, Rockville, Md.) as previously described (16, demonstrating retention of cell viability despite growth 31). The isolation and characterization of clonal lympho arrest. The combination of 50 /J.M adenosine and 3.5 /¿M blast lines derived from WI-L2 deficient in adenosine kinase coformycin also arrested growth of WI-L2 lymphoblasts (EC 3.7.1.20) (MTI), both adenosine kinase and hypoxan after 24 hr. thine-guanine phosphoribosyltransferase (EC 2.4.2.8) (MTI- In studies with lymphoblast extracts, we found the appar TG), and adenine phosphoribosyltransferase (EC 2.4.2.7) ent Km's of adenosine kinase and adenosine deaminase for (DAP) were previously described (16). Cell counts were adenosine to be approximately 2 to 4 and 40 to 50 /¿M, measured by a Model ZB, Coulter counter. respectively. The maximal velocity of the deaminase was Metabolic Studies. Adenosine kinase, adenine phospho approximately 10-fold greater than that of the kinase. The ribosyltransferase, and hypoxanthine-guanine phosphori metabolism of extracellular adenosine by lymphoblasts ap bosyltransferase were assayed in lymphoblast extracts as peared to be governed by the characterisitics of these 2 previously described (16, 30). Adenosine metabolism was enzymes. Thus deamination was the principal route of studied in the intact lymphoblast as previously described adenosine metabolism for intact WI-L2 exposed to 80 /¿M (30). Intracellular PP-ribose-P concentrations (15, 16, 31) adenosine; the rates of deamination and phosphorylation and de novo purine synthesis (15) were measured as before. were 1035 and 74 pmol/106 cells/min, respectively. At a cAMP concentrations were measured according to the lower concentration of adenosine, 4 ¿¿Morapproximately method of Wastili