(CANCER RESEARCH 49, 4983-4989, September 15. 1989] Mechanism of Adenosine Triphosphate Catabolism Induced by Deoxyadenosine and by Nucleoside Analogues in Adenosine Deaminase-inhibited Human Erythrocytes1 FrançoiseBontemps2 and Georges Van den Berghe' Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology, ana University of Louvain, B-1200 Brussels, Belgium ABSTRACT explained this ATP depletion as follows: (a) dAdo, which accumulates when ADA is deficient or inhibited, is phospho- The mechanism of the depletion of ATP, recorded in the erythrocytes of adenosine deaminase-deficient children and of leukemia patients rylated to dAMP and subsequently to dATP, a process which treated with deoxycoformycin, »asinvestigated in normal human eryth utilizes ATP and generates ADP and AMP; (¿>)theaccumula rocytes treated with this inhibitor of adenosine deaminase. Deoxyadeno tion of dATP, which is greatly facilitated by the fact that dAMP sine, which accumulates in both clinical conditions, provoked a dose- is a poor substrate for AMP-DA (15, 16), stimulates both the dependent accumulation of dATP, depletion of ATP, and increases in the deamination of AMP to IMP and the dephosphorylation of production of inosine plus hypoxanthine. Concomitantly, there was an IMP to inosine (14, 17), thereby provoking adenine ribonucle- increase of AMP and IMP, but not of adenosine, indicating that catabo- otide catabolism. This mechanism is, however, difficult to rec lism proceeded by way of AMP deaminase. A series of nucleoside oncile with two observations: (a) dATP and ATP are equipotent analogues (9-/3-D-arabinofuranosyladenine, A*-methyladenosine, 6-meth- as stimulators of erythrocyte (15) and lymphoblast (14) AMP- ylmercaptopurine ribonucleoside, tubercidin, ribavirin, and ¿V-I-ribosyl- DA; (b) the sum of dATP and ATP in ADA-deficient (4, 5) or 5-aminoimidazole-4-carboxamide riboside) also stimulated adenine nu- in ADA-inhibited cells (9, 10, 13) is not or only slightly higher cleotide catabolism and increased AMP and IMP to various extents. The effects of deoxyadenosine and of the nucleoside analogues were prevented than that of ATP in control cells. These data and uncertainties by 5'-iodotubercidin, an inhibitor of adenosine kinase. Strikingly, they about the concentration of AMP in ADA-deficient or -inhibited were reversed if the inhibitor was added after the accumulation of cells prompted a reinvestigation of the mechanism of the deple nucleotide analogues and initiation of adenine nucleotide catabolism. tion of ATP induced by dAdo. This study was performed with Further analyses revealed linear relationships between the rate of phos- normal human erythrocytes in which ADA was inhibited by phorylation of deoxyadenosine and nucleoside analogues and the increase dCF. The effect of dAdo has been compared to that of other in AMP and between the elevation of the latter above a threshold purine nucleoside analogues which are also substrates of aden concentration of 10 UM and the rate of adenine nucleotide catabolism. osine kinase. Our results provide evidence that all these nucle Kinetic studies with purified erythrocytic AMP deaminase, at physiolog osides induce catabolism of the adenine nucleotides by a com ical concentrations of its effectors, showed that the enzyme is nearly mon mechanism, namely elevation of AMP. Part of this work inactive up to 10 UMAMP and increases in activity above this threshold. We conclude that the main mechanism whereby deoxyadenosine and has been presented at a symposium (18). nucleoside analogues stimulate catabolism of adenine nucleotides by way of AMP deaminase in erythrocytes is elevation of AMP, secondary to MATERIALS AND METHODS the phosphorylation of the nucleosides. Chemicals. [t/-uC]Adenine (270 Ci/mol) and [8-MC]AMP (55 Ci/ INTRODUCTION mol) were purchased from the Radiochemical Centre (Amersham, Buckinghamshire, England). ITu was from RBI (Natick, MA) and dCF Accumulation of dATP has been documented in erythrocytes was from Warner Lambert (Detroit, MI). Adenine nucleotides and (1-6), in lymphocytes and bone marrow cells (7), and in plate adenosine were from Boehringer (Mannheim, Germany). Other nucleo lets (8) of children with ADA4 deficiency. It is also found in tides, nucleosides, and GTP-agarose were from Sigma (St. Louis, MO). RBC (9-11) and lymphoblasts (12, 13) of leukemia patients The sources of all other chemicals have been given (19, 20). treated with the ADA inhibitor, dCF. In addition, dramatic Incubation of Erythrocytes. Fresh blood taken from a cubital vein of healthy human volunteers was collected on heparin. Isolation and depletions of ATP were recorded in the erythrocytes (9, 10) and washing of erythrocytes were performed in KRB, pH 7.4, containing 5 lymphoblasts (13) of patients treated with dCF, whereas smaller mM glucose and gassed with 95% O2-5% CO2 as described in Ref. 19. decreases were observed in the erythrocytes of some children The RBC were resuspended in the same medium as a 20% hematocrit with ADA deficiency (3-6). Bagnara and Hershfield (14), in and their adenine nucleotides were labeled by a 60-90-min prcincuba- vestigating ADA-inhibited human lymphoblastoid cells, have tion at 37°Cwith 2-3 (¡M[U-'4C|adenine. This was followed by two washes and resuspension as a 20% hematocrit in KRB with 5 mM Received 2/27/89; revised 6/1/89; accepted 6/14/89. The costs of publication of this article were defrayed in part by the payment glucose. Unless given otherwise, the concentration of P¡in the KRB of page charges. This article must therefore be hereby marked advertisement in buffer was 1.2 HIM.In the experiments in which this concentration was accordance with 18 U.S.C. Section 1734 solely to indicate this fact. increased to 10 mM. that of Ca-+ was reduced from 2.5 to 1.25 m\i. 1Supported by Grant 3.4539.87 of the Fund for Medical Scientific Research (Belgium) and by the Belgian State-Prime Minister's Office-Science Policy Pro Incubations were performed in carefully regassed and stoppered vials. In all experiments, suspensions were preincubated for 20 min with 1 gramming. ! To whom requests for reprints should be addressed, at Laboratory of Phys ^M dCF before addition of nucleosides, in order to allow tight binding iological Chemistry. UCL-ICP 75.39, Avenue Hippocrate, 75, B-1200 Brussels, of the inhibitor with ADA. We have shown previously that dCF has no Belgium. 1Director of Research of the Belgian National Fund for Scientific Research. effect by itself on erythrocytic adenine nucleotide catabolism (19). In 4The abbreviations used are: ADA. adenosine deaminase (EC 3.5.4.4); dAdo. some experiments, 10 »\iITu was used to inhibit adenosine kinase 2'-deoxyadenosine; AMP-DA, AMP deaminase (EC 3.5.4.6); KRB, Krebs-Ringer (21). bicarbonate; dCF, 2'-deoxycoformycin; ITu, 5'-iodotubercidin; 2,3-BPG, 2,3- Assessment of Inhibitor Requirements in Intact Erythrocytes. Prelim bisphosphoglycerate; ara-A, 9-fi-D-arabinofuranosyladenine; AICA riboside, ¿V-l- inary studies were performed in which the nucleotides and the catabo- ribosyl-5-aminoimidazole-4-carboxamide: N'-MeAdo, A*-methyladenosine; MMPR, 6-methylmercaptopurine ribonucleoside: Tu, lubercidin (7-deazaadeno- lites produced from the added nucleosides were measured by HPLC, as sine); Rbv, ribavirin (l-/i-n-ribofuranosyl-l.2,4-triazole-3-carboxamide); HPLC, described below. These showed that the addition of IMMdCF and 10 high-performance liquid chromatography. /aMITu inhibited by more than 90% the deamination of up to 0.5 mM 4983 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. MECHANISM OF NUCLEOSIDE-1NDUCED ATP CATABOL1SM adenosine, dAdo, and ara-A and prevented any detectable synthesis of lowest concentration, was not completely utilized over 3 h; nucleotides from the latter nucleosides and from Tu, MMPR, N6- dATP represented 80 to 90% of the total amount of deoxyri- MeAdo, Rbv, and AICA riboside. bonucleotides synthesized (not illustrated). Concomitantly, Assay and Partial Purification of AMP Deaminase. AMP-DA activity was measured by the production of [S-'TJIMP from |8-'4C]AMP. there was a decrease of ATP. At all concentrations of dAdo, Incubations were performed at 37°Cin a medium containing 50 HIM the sum of both triphosphates remained approximately equal A'-2-hydroxyethyl-piperazine-A'-2-ethanesulfonic acid buffer (pH 7.2), or became slightly higher than the initial concentration of ATP. 100 mM KCI, 5 mM MgCl2, [8-uC)AMP (0.05-0.1 ßCi/lest),and AMP The addition of dAdo also induced an accumulation of the at the concentrations indicated, in a total volume of 50 or 100 n\. At terminal purine catabolites, hypoxanthine and inosine (Fig. 1, appropriate time intervals, 10 M'of the incubation medium were spotted rifflit). the latter increasing only at high concentrations of dAdo on polyethyleneimine cellulose thin-layer chromatography plates on (see Fig. 2). The very small accumulation of purine catabolites, which carrier solutions (50 mimi) of AMP and IMP had been applied. recorded in the control condition, was about doubled upon After development in 1.4 M LiCl, activity was calculated from the addition of 20 /¿MdAdo, the lowest concentration used, and radioactivity appearing in IMP. In crude preparations, AMP-DA activ 30- to 50-fold increased in the presence of 0.5 mivi dAdo. The ity was measured at 5 mM AMP in the absence of Mg2* to prevent the dephosphorylation of AMP or IMP by S'-nucleotidase(s). Insignificant purine catabolites nearly completely accounted for the loss of ATP. All these results are qualitatively similar to those recorded amounts of inosine, hypoxanthine, and adenosine (less than 5% of by Bagnara and Hershfield (14) in ADA-inhibited lymphoblas- AMP or IMP) were produced during the assay under these conditions. AMP-DA was purified approximately 50,000-fold, with a yield of toid cells. 10-20%, by a two-step procedure.