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Mechanism of Deoxyadenosine-Induced Proc. NatL Acad. Sci. USA Vol. 79, pp. 2673-2677, April 1982 Medical Sciences Mechanism of deoxyadenosine-induced catabolism of adenine ribonucleotides in adenosine deaminase-inhibited human T lymphoblastoid cells (combined immunodeficiency disease/deoxycoformycin/lymphocytic leukemia/adenylate deaminase/5'-nucleotidase) ALDO S. BAGNARA* AND MICHAEL S. HERSHFIELDt Departments of Medicine and Biochemistry, Division of Rheumatic and Genetic Diseases, Duke University Medical Center, Durham, North Carolina 27710 Communicated by James B. Wyngaarden, January 13, 1982 ABSTRACT Loss of ATP accompanying accumulation of Ado (13, 14) and dAdo (15, 16) have been shown to inhibit and dATP has recently been reported to occur in the erythrocytes and irreversibly inactivate S-adenosyl-L-homocysteine hydrolase lymphoblasts of patients with T lymphocytic leukemia during (AdoHcyase; EC 3.3.1.1), which can lead to accumulation of S- treatment with deoxycoformycin, an inhibitor of adenosine de- adenosyl-L-homocysteine (AdoHcy), a potent inhibitor oftrans- aminase (adenosine aminohydrolase, EC 3.5.4.4) that causes the methylation reactions (17). accumulation of deoxyadenosine. We have studied the mecha- It is not clear that all consequences of ADA deficiency are nisms responsible for adenine ribonucleotide depletion in cultured caused by inhibition of ribonucleotide reductase or AdoHcy human CEM T lymphoblastoid cells treated with deoxycoformycin accumulation. The size of the dATP pool in dividing cells is and deoxyadenosine. Accumulation of dATP was accompanied by than that ofthe ATP pool and is even smaller depletion of total soluble adenine ribonucleotides without change usually 1% or less -* IMP in nondividing cells (18). Whereas ATP is involved in many cel- in the adenylate energy charge, by the route ATP-> AMP lular processes in various cellular compartments, utilization of - inosine -- hypoxanthine; conversion of IMP to AMP and de novo purine synthesis were inhibited in these cells. ATP degra- dATP is restricted largely to processes related to DNA repli- dation did not occur in a mutant of CEM that was incapable of cation and repair in the nucleus. dATP, when present in much phosphorylating deoxyadenosine, or in a B cell line with very lim- higher than normal concentrations, might act as an inhibitor or ited ability to accumulate dATP. We found that dATP and ATP otherwise affect some enzymes or metabolic processes that have were both able to stimulate markedly the deamination ofAMP by evolved with specificity for ATP. There have recently been re- lymphoblast AMP deaminase; dAMP was a poor substrate for this ports that marked depletion of ATP accompanies dATP accu- enzyme (Km = 2.4 mM, vs. 0.4 mM for AMP). Similarly, dATP mulation in the erythrocytes (19) and leukemic T cells (20) of as well as ATP caused marked activation of IMP dephosphoryla- patients undergoing treatment with dCF. These findings sug- tion by a lymphoblast cytoplasmic nucleotidase. Inhibition of in- gest an as yet unexplained effect of dATP on ATP metabolism tracellular AMP deaminase with coformycin prevented degrada- that could contribute to profound lymphopenia when ADA is tion ofadenine ribonucleotides without affecting dATP accumula- inhibited or deficient. We have investigated the effects ofdAdo tion. We propose that ATP-dependent phosphorylation of deoxy- on dCF-treated cultured human lymphoid cell lines, and here adenosine generates ADP and AMP. Simultaneously, dATP ac- we report that dATP accumulation activates the catabolism of cumulation stimulates deamination of AMP, but not dAMP, and adenine ribonucleotides, causing marked ATP depletion. This the dephosphorylation of IMP to inosine. Coupling of AMP deg- effect appears to be due, in part, to stimulation by dATP ofAMP radation to ATP utilization in deoxyadenosine phosphorylation deamination and IMP dephosphorylation. maintains the adenylate energy charge despite net depletion of cellular ATP. MATERIALS AND METHODS Heritable deficiency ofadenosine deaminase (ADA; adenosine Materials. [8-"'C]Ado, [8-14C]adenine, and [2-3H]dAdo aminohydrolase, EC 3.5.4.4) causes a form ofsevere combined were obtained from Moravek Biochemicals (Brea, CA); [8- immunodeficiency disease (1, 2). In addition, a potent inhibitor 14C]AMP and [8-14C]IMP were from Amersham. Polyethyl- of ADA, 2'-deoxycoformycin (dCF), has recently been shown eneimine (PEI)-cellulose thin-layer plates were from Merck. to be effective in the treatment ofsome lymphoid malignancies dCF (Pentostatin) was a gift from Warner Lambert-Parke Davis (3-6). In both genetic and dCF-induced ADA deficiency, lym- (Detroit, MI). Other materials were purchased as described in phopenia is thought to result from toxic effects of adenosine the references cited. (Ado) and 2'-deoxyadenosine (dAdo) and ofthe metabolites that Cell Culture. The hypoxanthine phosphoribosyltransferase accumulate when their deamination is prevented. (EC 2.4.2.8)-deficient (HPRT-) derivatives of the human ma- Greatly increased concentrations of dATP have been found lignant T lymphoblastoid cell line CCRF-CEM (cell line AG1) in the erythrocytes (7, 8), lymphocytes, and bone marrow (9) (21), and of the human splenic B lymphoblastoid cell line WI- ofchildren with the genetic immunodeficiency disease and also L2 (cell line AGR9, clone 35-1) (22), have been described. Cells in the erythrocytes and lymphoblasts of patients undergoing treatment swith dCF (4-6). dATP accumulation has long been Abbreviations: ADA, adenosine deaminase; Ado, adenosine; dAdo, 2'- known to block DNA synthesis in cultured cells (10), anieffect deoxyadenosine; AdoHcy, S-adenosyl-L-homocysteine; AdoHcyase, generally attributed to inhibition by dATP of ribonucleoside- AdoHcy hydrolase; dCF, 2'-deoxycoformycin; AXP, adenine ribonu- cleotides; dAXP, adenine deoxyribonucleotides; PEI, polyethyleneim- diphosphate reductase (EC 1.17.4.1) (11, 12). More recently, ine; HPRT, hypoxanthine phosphoribosyltransferase. * Permanent address: School ofBiochemistry, University ofNew South The publication costs ofthis article were defrayed in part by page charge Wales, Kensington, NSW, Australia. payment. This article must therefore be hereby marked "advertise- tTo whom reprint requests should be addressed: Box 3049, Duke ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. University Medical Center, Durham, NC 27710. 2673 Downloaded by guest on October 2, 2021 2674 Medical Sciences: Bagnara and Hershfield Proc. Natl. Acad. Sci. USA 79 (1982) were cultured in RPMI 1640 medium (GIBCO) supplemented stored at 40C. The partially purified enzyme was free of 5'-nu- with 10% horse serum, nonessential amino acids, and 1 mM cleotidase and ADA activities. pyruvate under an atmosphere of 5% CO2 in air; cells were stud- Enzyme Assays. AdoHcyase (15) and ADA (25) were assayed ied in their logarithmic phase ofgrowth (4-8 x 105 cells per ml). as described. 5'-Nucleotidase was assayed by measuring the Cultures were deemed free of mycoplasma contamination by rate. of formation of [l4G]inosine from [8-'4C]IMP in a 50-,ul the inability of cell-free extracts to synthesize [14C]Ado- from assay consisting of 50 mM Tris HCl at pH 6.5, 10 mM MgCl2, [I4C]adenine and ribose 1-phosphate or to convert [3H]dAdo bovine serum albumin at 1 mg/ml, 20 p.1 of enz'me, and 0.5 to [3H]adenine (each in the presence of 5 p.M dCF to inhibit mM [8-'4C]IMP (5 mCi/mmol; 1 Ci = 3.7 x 101 becquerels). ADA); or by a cytochemical stain procedure (23). With each After 30 min of incubation at 370C, 10-pgl aliquots were with- method mycoplasma-contaminated cultures were used as pos- drawn and spotted with nonradioactive inosine marker onto itive controls. PET-cellulose thin layers. The [14C]IMP was then separated Measurement of Intracellular Adenine Ribo- and- Deoxy- from [I4C]inosine by development in methanol/water, 1:1 (vol/ ribonucleotides. Total adenine ribo- and deoxyribonucleotides vol), and.the inosine area was identified under ultraviolet light, in HC104 extracts of cells were measured by HPLC after de- and its radioactivity was measured. AMP deaminase was as- phosphorylation of these nucleotides to Ado and dAdo, respec- sayed either spectrophotometrically (during the purification tively, by a modification (21) of a previously described proce- procedure) (26) or by following the conversion of [8-14C]AMP dure (16). to [14C]IMP. The standard isotopic assay contained, in a volume Labeling of Purines with ['4C]Adenine. Samples of whole of 25-50 p.1, 100 mM Hepes at pH 7.0, 150 mM KCl, 1 mM culture (100 pAl) were withdrawn at appropriate times after ad- ATP, 2mM MgCl2, 1 mM [8-14C]AMP (5 mCi/mmol), and 5-10 dition of [8-'4C]adenine, and they were acidified with 25 p.1 of .ul ofenzyme. Samples (5 ,ul) were withdrawn overatime course 4 M HC104 at 00C. After centrifugation (Beckman Microfuge, (usually 0-30 min) and spotted with IMP marker onto PEI-cel- 30 s, 40C), 100 ,ul ofsupernatant was neutralized with a mixture lulose thin layers. The [14C]IMP was then separated from of KOH and KHCO3 (16, 21). One aliquot of the neutralized [14C]AMP by development in 0.15 M sodium formate (pH 3.0), cell extract was used to determine the specific activity of the identified; and cut out, and its radioactivity was measured. adenine nucleotide pool: the nucleotides were first dephos- phorylated by incubation with venom phosphodiesterase and RESULTS alkaline phosphatase in the presence of an ADA inhibitor (16, Accumulation ofAdenine Deoxyribonucleotides and Catab- 21), then the radiochemical concentration ofthe Ado generated olism of Adenine Ribonucleotides in T Lymphoblasts. The ac- was determined by HPLC (16, 21) and liquid scintillation count- cumulation of total adenine deoxyribonucleotides (dAXP) dur- ing. A second aliquot ofthe original neutralized cell extract was ing incubation of T lymphoblasts with various concentrations subjected to thin-layer chromatography on PEI-cellulose (see of exogenous dAdo in the presence of 5 ,uM dCF was studied below) to measure the radioactivity incorporated into ATP; over a 20-hr period (Fig. 1A). At 2 p.M dAdo the total dAXP ADP, and AMP.
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