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. The specific activity measurement allowed the accumulated over 5-hr was 100 nmol per 109 cells, which is calculation ofthe concentrations ofthese individual nucleotides. 4-5 times the normal dAT.P content ofthese cells. At dAdo con- Purine ribonucleotides present in HC104 extracts of cultures centrations of 100 p.M andd00 ,uM, 1,000-3,700 nmol ofdATP were resolved on PEI-cellulose thin layers after the nucleosides per 109cells accumulated over 3-5 hr, values approaching the and bases had been washed to the top of the sheet by three or normal four successive developments in methanol/water, 1:1 (vol/ cellular concentration of ATP. After 20 hr in medium vol). The nucleotides were then separated essentially as de- containing 100 p.M or 500 p.M dAdo, the concentration ofdAXP scribed (24). The nucleosides and bases were separated-on sim- 4 A-F- ilar thin layers by a single development in 1-butanol/glacial acetic acid/water, 5:3:2 (vol/vol). Preparation and Partial Purification of Cell Extracts for = 3 U) Enzyme Assays. For isolation of cytoplasmic 5'-nucleotidase 80 a) c; (EC 3.1.3.5) a pellet of lymphoblasts (=109 cells) was resus- 0 0) 0 pended at 0°C in 2.0 ml of 25 mM Tris HCl, pH 7.4/15 mM Toa) 0-2 L- KCVl1 mM dithiothreitol/1 mM EDTA/0.25 M sucrose, and a the cells were gently disrupted in a Dounce homogenizer. The 75 -5 extract was centrifuged (20,000 x g, 10 min, 40C), and the re- E E sulting supernatant was then recentrifuged (100,000 x g, 60 CL min, 4°C). The supernatant was either used directly as a source of enzyme or was fractionated by gel filtration chromatography 0 0 on a 2.5 x 90 cm column ofUltrogel AcA 34 (LKB) (15, 16). For 0 1 2 3 4 5 20 0 1 2 3 4 5 20 isolation of AMP deaminase (EC 3.5.4.6) a pellet of lympho- blasts (=10'cells) was resuspended at 0°C in 1.5 ml ofTris HCl, Time, hr mM Nonidet P40, and the cellskwere pH 7.4/1 EDTA/0.5% FIG. 1. (A) Accumulation of dAXPfrom exogenous dAdo inhuman lysed by three freeze-thaw cycles. After centrifugation (5 min, T lymphoblasts. CEM AGI cells (4-6 x 105 per ml) were incubated in 12,000 x g, 4°C) the supernatant was loaded onto a 1.5 X 90 growth medium containing 5 pM dCF for 20 min before dAdo was cm column of Ultrogel AcA 34. The column was eluted with 25 added. Samples of culture (10 ml) were removed at the indicated times mM Tris HCl, pH 7.4/50 mM KCVl1 mM dithiothreitol. Frac- and the intracellular dAXP was quantified. Concentrations of dAdo tions containing AMP deaminase activity were pooled and added were 2 pMM(e), 20 pM (A), 10) p.M(d), and 500 pM (o). Analysis on a 1.5 X 10 cm column of DE52 DEAE-cellulose of several extracts by anion-exchange HPLC before the nucleotides loaded were dephosphorylated showed that >90% of the dAXP accumulated (Whatman) equilibrated with the same buffer and then eluted was present as dATP. (B) Degradation of adenine ribonucleotides in with a linear gradient from 50 mM to 500 mM KC1 in the same humanTlymphoblasts (cell lineAG1) duringdAXPaccumulation. The buffer. AMP deaminase emerged at -200 mM KC1. Active frac- data shown were generated in the same experiment shown in A, and tions were pooled, concentrated by ultrafiltration to 'z2 ml, and the symbols are as described for A. Downloaded by guest on October 2, 2021 Medical Sciences: Bagnara and Hershfield Proc. NatL Acad. Sci. USA 79 (1982) 2675

in these cells was substantially diminished, indicating that ofAXP, and Ado would be competing with 500 AM dAdo, also dAXP synthesized during the first few hours of incubation was a substrate for Ado kinase (16, 21); dAdo is also a potent inac- later degraded. tivator ofAdoHcyase (15, 16). The experiment shows that AXP During the period ofincubation with dAdo we found a dAdo catabolism proceeds via deamination ofAMP to IMP, followed concentration-dependent and time-dependent depletion ofad- by dephosphorylation of IMP to inosine and phosphorolysis to enine ribonucleotides (AXP) (Fig. 1B). In cells incubated with hypoxanthine. The inosine and hypoxanthine produced from 100 or 500 ,uM dAdo there was essentially complete loss ofAXP catabolism of AXP could not have arisen from Ado (generated after 20 hr (Fig. 1B). This net loss ofAXP from the lymphoblasts via dephosphorylation of AMP) because our experiments were presumably involved dephosphorylation to produce mem- performed in the presence of 5 ,M dCF, which quantitatively brane-permeant nucleosides or bases, which would be lost dur- inhibits ADA. An additional observation of interest is that AXP ing the centrifugation and washing ofcells before extraction with was degraded under conditions in which the adenylate energy HC104. In order to determine the metabolic route and end charge-i. e., ([ATP] + 1/2[ADP])/([ATP] + [ADP] + products of AXP catabolism, we therefore performed similar [AMP])-remained essentially constant, with values in excess experiments with cells in which the adenine ribonucleotides of 0.92 (Fig. 2A). were labeled by preincubation with [I4C]-adenine, and we ac- In previous studies (21) we have shown that CEM T cells counted for labeled material in culture medium as well as in accumulate dAXP from dAdo at 40-100 times the rate of the B cells. cell line WI-L2, a difference that is characteristic ofother T and Labeling ofPurines with [8-"C]Adenine and dAdo-Induced B lymphoid cell lines (27-30). The experiments in Table 1 show Catabolism of ["C]-Labeled AXP. After 2 hr of incubation of that 500 AM dAdo did not stimulate AXP degradation in the WI-L2 or CEM cells with 2.5 or 5 AM [14C]adenine, a steady AGR9 derivative of WI-L2 or in an adenosine kinase- and de- state was reached with respect to the amount of label in the oxycytidine kinase-deficient derivative of CEM that is essen- soluble intracellular AXP pool; this steady state was maintained tially incapable of dAdo phosphorylation (21). Thus, AXP deg- for about 3 hr, until the exogenous ['4C]adenine was totally radation is not mediated by dAdo itself but by nucleotides utilized (results not shown). Then the amount of label in AXP derived from dAdo. fell slowly, with =60% of the steady-state level of label in the Activation of Soluble 5'-Nucleotidase and AMP Deaminase culture still in the AXP pool after a further 3 hr of incubation. by dATP and ATP. Because dAdo-induced catabolism of AXP Using the above conditions, we have followed the metabolic occurred via deamination of AMP and dephosphorylation of fate of the [14C]adenine-labeled adenine ribonucleotides after IMP, we examined the effects ofATP and dATP on the activities the addition of 500 ,uM dAdo in T and B lymphoblasts. This of lymphoblast AMP deaminase and 5'-nucleotidase. At con- concentration ofdAdo was chosen because it caused substantial centrations above 1 mM both triphosphates markedly stimu- catabolism ofAXP to occur during the period ofoptimal labeling lated the dephosphorylation of 0.5 mM IMP by a 100,000 X g of the AXP pool after complete utilization of free [14C]adenine supernatant prepared from CEM cells (Table 2). Upon frac- by the culture. The studies were performed with HPRT- cells tionation of such an extract by gel filtration chromatography, to minimize any reutilization of hypoxanthine. In the T cells, stimulation by dATP of a high molecular weight (>200,000) 5'- [I4C]ATP was converted primarily to inosine and hypoxanthine nucleotidase activity could be demonstrated (not shown). This (Fig. 2), while less than 5% of AXP degraded was converted to activity was highly labile upon storage at 4°C and has not been Ado (Fig. 2B). Although this might be a slight underestimate further characterized, other than to show that it is also activated of the amount of Ado produced, to the extent that some Ado by arabino-ATP and is weakly inhibited by orthophosphate. might have been converted to AMP or to AdoHcy, we believe AMP deaminase activity partially purified from CEM cells that reutilization would be minimal: phosphorylation by Ado to remove ADA and 5'-nucleotidase activities was similarly kinase could not be significant in view of the net degradation stimulated by both ATP and dATP, especially when they were present as their respective Mg2+ complexes (Table 3). Activa- tion was maximal between 1 and 2 mM with either ATP or dATP, or with mixtures of these nucleotides (not shown). Thus, I1.00 8 A B activation ofAMP deaminase would be expected to be maximal 0.90 .C in cells that were at various stages of ATP depletion and dATP 0.85 accumulation, the condition that we observed in intact cells. In other studies not shown, we determined that partially

E 0 \ ; | E Table 1. Comparison of AXP catabolism in T cells and B cells AXP catabolized, ~0.60 gmol/3 hr per x x 109 cells E 0.4 E 500 ,uM No 0.2 Cell line dAdo dAdo

0 0E T cells 0 1 2 3 0 1 2 3 AG1 (HPRT-) 2.13 0.09 Time, hr RC3b (HPRT-, AK-, dCK-) 0.29 0.08 B cells FIG. 2. Catabolism of AXP induced by 500 AM dAdo in human T AGR9, clone 35-1 (HPRT-) 0.50 0.37* lymphoblasts (cell line AG1). Cells (7 x 105 per ml) were labeled for 3 hr with 2.5 AM [8-"C]-adenine (43.9 mCi/mmol) in the presence of Conditions were as described forFig. 2 exceptthatresults were quan- 5 dCF. dAdo was then added and samples were taken for estimation tified by calculations based on the specific activity of the AXP pool. of 14C label in nucleotides (A) and nucleosides and bases (B). (A) *, AK- and dCK-, deficient in Ado kinase and deoxycytidine kinase. ATP; o, ADP; o, AMP; x, IMP; A, adenylate energy charge. (B) e, Hy- * The relatively high basal rate of purine breakdown and excretion poxanthine; o, inosine; o, Ado. observed with this cell line is consistent with previous studies (31). Downloaded by guest on October 2, 2021 2676 Medical Sciences: Bagnara and Hershfield Proc. Natl. Acad. Sci. USA 79 (1982)

Table 2. Activation of T lymphoblast cytoplasmic 5'-nucleotidase Table 4. Effect of dAdo on de novo purine synthesis by ATP and dATP De novo synthesis cpm in 5'-Nucleotidase activity dAdo, cpm/60 min % of guanine/cpm Assay conditions relative to control AM3I per 106 cells control in adenine Control 1.00 0 37,030 100 0.72 +ATP 2 33,260 90 0.80 0.25 mM 1.09 10 24,960 67 1.08 0.75 mM 1.22 50 16,120 44 1.58 2.0 mM 6.57 200 8,190 22 3.03 5.0 mM 9.51* +dATP Duplicate aliquots of CEM AG1 cells (1.28 x 106 cells in 2 ml of 0.25 mM 1.01 growth medium) were incubated for 3 hr at 370C in the presence of 5 0.75 mM 1.11 1MdCF andthe indicated concentration of dAdo. Then 20 Al of sodium 2.0 mM 3.18 [14C]formate (Amersham, 0.18 mM, 59 mCi/mmol) was added, and after 60 min at370C the cells wereharvestedand thelabel incorporated 5.0 mM 9.30* into total cellular purines and into the guanine and adenine derived 5'-Nucleotidase activity was measured in the 100,000 x g super- from these nucleotides was measured as described (31, 33). natant of an extract of T lymphoblasts (cell line AG1). In the control assay using 0.5 mM [8-14C]IMP without added nucleoside triphos- phates, the activity was 0.65 ,umol of IMP dephosphorylated per hr per in particular on the ability to convert IMP to AMP. We have 109 cells. evaluated these possibilities in CEM cells, using a technique * Reaction was essentially complete in <30 min. that simultaneously measures both the overall rate of labeling of de novo synthesized purines with [14C]formate and the rel- ative rates ofconversion ofnewly synthesized IMP to adenylates purified AMP deaminase from CEM cells could deaminate vs. guanylates (31, 33). After a 3-hr exposure to 5 ,uM dCF and dAMP as well as AMP. In the presence of either ATP or dATP various concentrations ofdAdo, we observed clearcut inhibition at 1 mM the apparent Km for AMP was 0.4 mM, and that for of the overall rate of de novo synthesis that was due to a con- was velocities for AMP and dAMP dAMP 2.4 mM. The maximal siderably greater inhibition of the IMP -- AXP branch than of were no evidence of the deamination of identical. We found the IMP -- GXP branch of the pathway (Table 4). In this ex- dAMP by either intact CEM or WI-L2 cells under conditions periment we found no evidence of contamination of the dAdo in which deamination ofAMP was active-i.e., we observed no used with adenine, or of the formation of adenine from dAdo formation of [3H]deoxyinosine or [3H]hypoxanthine when (as could occur in mycoplasma-contaminated cultures), ruling these cells were incubated with 5 AM dCF and 100 ,M out the possibility that the observed effects were due to con- [3H]dAdo for up to 5 hr. version of adenine to AMP, and not to dAdo (although AMP To verify the critical role ofAMP deaminase in the catabolism synthesis from adenine would be unlikely in the face ofnet loss of AXP, we examined the effects of inhibiting this enzyme by ofAXP). Further examination of the basis for the observed in- preincubating cells with the AMP deaminase inhibitor cofor- hibition is beyond the scope of this report. mycin (32) (obtained from the National Cancer Institute). In a single experiment, incubation of CEM AG1 cells with 5 ,uM dCF and 500 ,uM dAdo for 3 hr resulted in the degradation of DISCUSSION 1.1 Amol of AXP per 109 cells; in the presence of 10 ,uM co- We have shown that in the ADA-inhibited CEM human T lym- formycin AXP degradation was 0.6 ,umol per 109 cells; with 100 phoblastoid cell line phosphorylation of dAdo to dAXP is ac- ,uM coformycin AXP degradation was completely prevented. companied by catabolism of adenine ribonucleotides. This ca- In all cases the AXP pool was maintained in a highly phos- tabolism proceeds via deamination of AMP to IMP, and phorylated state (adenylate energy charge >0.93), and the dephosphorylation of IMP to inosine, followed by phosphorol- amount of dAXP accumulated was the same. ysis to hypoxanthine. This is also the major pathway for AXP Effects of AXP Accumulation on de novo Purine Synthesis breakdown observed in mammalian cells treated with 2-deoxy- and on Formation of AMP from Newly Synthesized IMP. The glucose (34) or fructose (35), starved for glucose and oxygen, or -) -- occurrence of net loss of AXP via the route AMP IMP treated with inhibitors of oxidative phosphorylation (36). Ca- inosine suggests that dAXP accumulation could be associated tabolism via the alternative pathway of direct dephosphoryla- with inhibitory effects on de novo synthesis of adenylates, and tion of AMP to adenosine does occur to a lesser extent in some cell lines (36, 37), but apparently only when there is a significant decrease in the adenylate energy charge. For example, in stud- Table 3. Activation of AMP deaminase by ATP and dATP ies with the WI-L2 human B cell line (36) adenosine formation Assay Nucleotide, mM MgC12, Relative was observed only when this charge fell to <0.6 from its value no. ATP dATP mM activity of :0.9 under physiologic conditions. There have been reports that dCF (32, 38), and particularly 1 - - - 1.0 its 5'-phosphorylated derivative (39) can inhibit AMP deami- 2 - - 1.0 1.0 nase from several sources, and that intact mouse L1210 cells can 3 1.0 - - 35.2

4 1.0 - 1.0 67.0 convert dCF to its 5'-phosphorylated derivative to a small ex-

5 - 1.0 - 16.9 tent (40). However, we doubt that at the concentrations ofdCF 6 - 1.0 1.0 58.8 used in our studies the drug caused significant inhibition ofthe intracellular enzyme: we observed that AXP degradation pro- The standard isotopic assay mixture was modified to contain 0.5 mM ceeded via AMP deamination, and we found little evidence of [14C]AMP (5 mCi/mmol) and nucleotides and Mg2e as indicated. A AMP dephosphorylation. Similarly, Henderson et al. (38) found relative activity of 1.0 represents 0.62 nmol of AMP deaminated per min per ml of enzyme. AMP deaminase was partially purified by Ul- that 1 kLM dCF had negligible effect on AMP deaminase activity trogel AcA 34 and DE52 chromatography. in intact L1578Y mouse lymphoma cells. If the presumption is Downloaded by guest on October 2, 2021 Medical Sciences: Bagnara and Hershfield Proc. Natl. Acad. Sci. USA 79 (1982) 2677 AT P 4. Koller, C. A., Mitchell, B. S., Grever, M. R., Mejias, E., Mal- dAdo speis, L. & Metz, E. N. (1979) Cancer Treat. Rep. 63, 1949-1952. 5. Mitchell, B. S., Koller, C. A. & Heyn, R. (1980) Blood 56, dATP 556-559. 6. Grever, M. R., Siaw, M. F. E., Jacob, W. F., Neidhart, J. A., Miser, J. S., Coleman, M. S., Hutton, J. J. & Balcerzak, S. P. AMP (1981) Blood 57, 406-417. 7. Cohen, A., Hirschhorn, R., Horowitz, S. D., Rubinstein, A., Polmar, S. H., Hong, R. & Martin, D. W., Jr. (1978) Proc. Nati Acad. Sci. USA 75, 472-476. 8. Coleman, M. S., Donofrio, J., Hutton, J. J., Hahn, L., Daoud, IMP A., Lampkin, B. & Dyminski, J. (1978) J. Biol. Chem. 253, 1619-1626. 9. Donofrio, J., Coleman, M. S., Hutton, J. J., Daoud, A., Lamp- nosine kin, B. & Dyminski, J. (1978)J. Clin. Invest. 62, 884-887. Biosynthesis , 10. Klenow, H. (1962) Biochim. Biophys. Acta 61, 885-896. de novo Hypoxanthine 11. Moore, E. C. & Hurlbert, R. B. (1966) J. Biol. Chem. 241, 4802-4809. 0- Inhibition demonstrated 12. Reichard, P., Canellakis, Z. N. & Canellakis, E. S. (1961)1. Biol Chem. 236, 2514-2519. E>> Activation by dATP 13. Kredich, N. M. & Martin, D. W., Jr. (1977) Cell 12, 931-938. 14. Kredich, N. M. & Hershfield, M. S. (1979) Proc. Nati Acad. Sci. USA 76,2450-2454. FIG. 3. Schema outlining the effects of dAdo phosphorylation that 15. Hershfield, M. S. (1979)J. Biol Chem. 254, 22-25. promote the catabolism of adenine ribonucleotides. 16. Hershfield, M. S. & Kredich, N. M. (1980) Proc. Natl Acad. Sci. USA 77, 4292-4296. 17. Borchardt, T. (1977) in The Biochemistry of Adenosylmethionine, correct that phosphorylation of dCF is catalyzed by the same eds. Salvatore, F., Borek, E., Zappia, V., Williams-Ashman, H. kinases that phosphorylate dAdo (40), it seems unlikely that G. & Schlenk, F. (Columbia Univ. Press, New York), pp. in our 151-171. much phosphorylation ofdCF would have occurred stud- 18. Munch-Peterson, B., Tyrsted, G. & Dupont, B. (1973) Exp. Cell ies of dAdo-induced AXP catabolism. Res. 79, 249-256. Based on the studies we have reported, we propose the fol- 19. Siaw, M. F. E., Mitchell, B. S., Koller, C. A., Coleman, M. S. lowing explanation for dAdo-induced AXP catabolism (Fig. 3): & Hutton, J. J. (1980) Proc. Natl Acad. Sci. USA 77, 6157-6161. ATP-dependent phosphorylation of dAdo continuously gener- 20. Yu, A. L., Bakay, B., Kung, F. H. & Nyhan, W. L. (1981) Cancer ates ADP and AMP. At the same time accumulation of dATP Res. 41, 2677-2682. or maintain the state of activation of, AMP 21. Hershfield, M. S., Fetter, J., Small, W. C., Bagnara, A. S., Wil- would stimulate, liams, S. R., Ullman, B., Martin, D. W., Jr., Wasson, D. B. & deaminase, channeling the AMP formed into IMP; dAMP in Carson, D. A. (1982)J. Biol Chem. 257, in press. cells is not an effective substrate for this enzyme. dATP also 22. Lever, J. E., Nuki, G. & Seegmiller, J. E. (1974) Proc. Natl activates a cytoplasmic 5'-nucleotidase, promoting dephos- Acad. Sci. USA 71, 2679-2683. phorylation ofthe IMP generated from AMP. At the same time 23. Chen, T. R. (1977) Exp. Cell Res. 104, 255-262. the ability to convert IMP to AMP is inhibited, and the de novo 24. Crabtree, G. W. & Henderson, J. F. (1971) Cancer Res. 31, purines is The inosine derived from IMP 985-991. synthesis of blocked. 25. Hershfield, M. S., Snyder, F. F. & Seegmiller, J. E. (1977) Sci- and the hypoxanthine derived from inosine diffuse out of the ence 197, 1284-1287. cell. This series of events has the overall effect of coupling the 26. Yun, S.-L. & Suelter, C. H. (1978)J. Biol Chem. 253, 404-408. degradation ofAMP with the utilization of ATP (in the primary 27. Reynolds, E. C., Harris, A. W. & Finch, L. R. (1979) Biochim. phosphorylation of dAdo) and thus maintains a high adenylate Biophys. Acta 561, 110-123. energy charge in the face of net AXP pool depletion. 28. Mitchell, B. S., Mejias, E., Daddona, P. E. & Kelley, W. N. The mechanism proposed here for dAdo-induced depletion (1978) Proc. Natl Acad. Sci. USA 75, 5011-5014. 29. Carson, D. A., Kaye, J. & Seegmiller, J. E. (1978) J. Immunol of AXP in dCF-treated cultured T lymphoblasts might well be 121, 1726-1731. responsible for the AXP depletion that has been observed in 30. Carson, D. A., Kaye, J., Matsumoto, S. S., Seegmiller, J. E. & vivo in the erythrocytes and lymphoblasts of dCF-treated pa- Thompson, L. (1979) Proc. NatW Acad. Sci. USA 76, 2430-2433. tients with leukemia (19, 20). It is less clear that this mechanism 31. Hershfield, M. S. & Seegmiller, J. E. (1977) J. Biol. Chem. 252, operates to cause lymphopenia in genetic ADA deficiency, be- 6002-6010. cause is already present at diagnosis and ATP de- 32. Agarwal, R. P. & Parks, R. E., Jr. (1977) Biochem. Pharmacol. 26, lymphopenia 663-666. pletion has not been striking in the erythrocytes ofaffected chil- 33. Hershfield, M. S. & Seegmiller, J. E. (1976)J. Biol. Chem. 251, dren (7-9). 7348-7354. 34. Lomax, C. A. & Henderson, J. F. (1973) Cancer Res. 33, We acknowledge the expert technical assistance of Joan E. Fetter and 2825-2829. W. Curtis Small. 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