Mechanism of Adenosine Triphosphate Catabolism Induced by Deoxyadenosine and by Nucleoside Analogues in Adenosine Deaminase-Inhibited Human Erythrocytes1

Home , Adenine, Deoxyadenosine, Nucleoside

(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

FranÃ§oiseBontemps2 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, Â»asinvestigated in normal human eryth utilizes ATP and generates ADP and AMP; (Â¿>)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 Â¿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 37Â°Cwith 2-3 (Â¡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 PÂ¡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 Â»\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, Â¿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 37Â°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 ÃŸCi/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 /Â¿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. The hemolysate, prepared from 1 Fig. 2 depicts the time course of the effect of various concen unit of fresh blood collected on acid-citrate-dextrose, was washed free trations of dAdo. Concurrently with the depletion of ATP, of hemoglobin and subjected to DEAE-Trisacryl chromatography in dose-dependent increases of ADP, AMP, and IMP were re 20 mM Tris-HCI buffer, pH 7.2, containing 1 mM dithiothreitol (buffer corded. Despite inhibition of ADA, no adenosine could be A), as described in Ref. 20. Fractions containing AMP-DA activity detected. Together with the increase in IMP, this indicates that eluted at about 100 mM NaCl from the DEAE-Trisacryl column. They the accumulation of inosine and/or hypoxanthine results from were pooled and 5-ml portions were applied to a 0.4- x 6-cm affinity chromatography column containing 0.1 ml of GTP-agarose, equili deamination rather than from dephosphorylation of AMP. Influence of Inhibition of the Phosphorylation of dAdo. Simi brated with buffer A. After a rinsing with 5 x 1 ml of buffer A, containing 0.5 M NaCl, AMP-DA was eluted by 10 mM 2,3-BPG. The larly to that of adenosine, the phosphorylation of dAdo can be eluate, collected in albumin (1 mg/ml) to stabilize the enzyme, was inhibited by the addition of ITu, a potent inhibitor of adenosine filtered on Sephadex G-25 (fine) to remove 2,3-BPG which is a strong kinase (21). When 10 ^M ITu was added at the beginning of inhibitor of AMP-DA (22). Specific activity, measured at 5 mM AMP. the incubation, together with dAdo (Fig. 3, Â¡eft),the accumu was above 200 nmol/min/mg of protein. The enzyme was free of lation of dATP as well as the depletion of ATP and the myokinase, 5'-nucleotidase, and adenosine kinase activities. elevations of AMP and IMP were prevented, while the produc Analytical Methods. The extraction of metabolites was performed as tion of inosine plus hypoxanthine was inhibited by more than described in Ref. 19. All extracts were neutralized within 30 min to 90%. These results accord with the observation that dAdo did avoid artifactual increases in AMP, which represents only 0.5-1% of not stimulate adenine nucleotide catabolism in an adenosine ATP under control conditions. ATP, dATP, ADP, AMP, IMP, and kinase- and deoxycytidine kinase-deficient cell line (14). Like the nucleotides produced from nucleoside analogues were separated by HPLC on a 12.5-cm Partisphere 5 SAX column by the method of wise, addition of ITu 90 min after dAdo, halfway through the incubation (Fig. 3, right), completely arrested the build-up of Hartwick and Brown (23). Detection was performed at 254 nm, except for the Rbv nucleotides which were analyzed at 205 nm. All the dATP. Strikingly, however, the addition of ITu also brought nucleotides formed from nucleoside analogues were clearly separated, about a complete arrest of the degradation of ATP. This was except those generated from N6-MeAdo, which slightly overlapped with accompanied by a return of the concentration of AMP to nearly the adenine nucleotides. Concentrations of the nucleotides were calcu its basal value, by a decrease in IMP and, after a 30-min latency, lated by comparison with nucleotide standards when available, or when not, as in the case of MMPR, by comparison with the absorbance of the nucleoside. The radioactivity in the adenine nucleotides and in adenosine, inosine, and hypoxanthine was determined by thin-layer chromatography as given previously (19). The concentration of the nucleosides and base was calculated from the specific radioactivity of the adenine nucleotides, which remained constant over the duration of the experiments. For determination of the nucleosides added to the erythrocyte sus pensions, these were briefly centrifuged, and the supernatant medium was deproteinized by heating for 1 min at 100Â°C.This extraction method avoided acid hydrolysis of dAdo to adenine. The concentrations of nucleosides were measured by reversed phase HPLC on a 3.9-mm x 30-cm Ci8-Â¿iBondapak column (Waters-Millipore, Bedford, MA), eluted at a flow rate of 1 ml/min with 10 mM NaH2PO4, pH 5.5, and a 0-15% gradient of methanol over 15 min. MMPR was eluted iso- cratically with 30% methanol. The methods used for the determination 0.2 0.5 0 0.2 0.5 of other metabolites have been given (19). [ dAdo ] (mM) Fig. 1. Effect of the concentration of dAdo on the synthesis of dATP and on the catabolism of ATP. Various concentrations of dAdo were added to erythro RESULTS cytes that had been preincubated successively with ['4C]adenine, in order to label their adenine nucleotides, and with dCF. in order to inhibit adenosine deaminase, Effects of Deoxyadenosine on the Concentrations of dATP and as described under "Materials and Methods.** Metabolites were measured after 3 ATP and on the Production of Purine Catabolites. Incubation of h of incubation. ATP and dATP were determined by HPLC. whereas inosine ADA-inhibited erythrocytes with dAdo resulted in a dose-de (INO) plus hypoxanthine (HX) were determined radiochemically and their con centrations were calculated from the specific radioactivity of the adenine nucleo pendent accumulation of dATP (Fig. 1, left); dAdo, even at the tides. Results shown are means of two experiments. 4984

can be phosphorylated intracellularly. As shown in Fig. 4, adenosine and a series of nucleoside analogues, added at 0.5 HIMconcentration, all stimulated, although to various extents, the catabolism of the adenine ribonucleotides into inosine and hypoxanthine. Maximal stimulation was obtained by Tu, fol lowed by, in order, N6-MeAdo, dAdo, MMPR, AICA riboside (not illustrated), Rbv, adenosine, and ara-A. Owing to the presence of dCF, it could be concluded that, as with dAdo, the inosine and hypoxanthine produced from the prelabeled ade nine nucleotides resulted from the deamination of AMP into IMP rather than from AMP dephosphorylation. In the presence of Tu and of adenosine, some dephosphorylation of AMP occurred, as evidenced by the appearance of labeled adenosine. To verify the similarity of the mechanism of action of the nucleoside analogues with that of dAdo, further experiments were performed with ITu. Results obtained with two of them, MMPR (Fig. 5, left), which is converted for 90% into a monophosphate, and Tu (Fig. 5, right), which is mainly converted into a triphosphate, are shown. Similarly to that induced by

12301 23 dAdo, the catabolism of the adenine nucleotides provoked by Time (h) after addition of dAdo these nucleosides was arrested by the subsequent addition of Fig. 2. Time course of the effect of various concentrations of dAdo on the ITu. This arrest was associated with an interruption of the concentration of ATP and of its catabolites. Erythrocytes were preincubated as synthesis of MMPR monophosphate, which reached the con in Fig. 1 and incubated thereafter in the absence or in the presence of dAdo at 0.1, 0.2, or 0.5 HIMconcentration. Results shown are means of two experiments. centration of about 1.2 /Â¿mol/mlof packed cells after 90 min, INO, inosine; HX, hypoxanthine. and of Tu triphosphate which reached about 0.8 ^mol/ml of packed cells after 60 min (not illustrated). As observed with dAdo, addition of ITu also provoked a decrease of AMP and IMP, as well as of the production of inosine and hypoxanthine. Similar results were obtained with adenosine, Rbv, N6-MeAdo, and AICA riboside (not illustrated). These experiments indicate that all the nucleosides provoke an increase in the activity of AMP-DA by a common mechanism, which is independent of the level of their phosphorylation. Influence of Supraphysiological Concentrations of 1',. Because Parks and Brown (24) had not observed a depletion of ATP after addition of various nucleosides to human erythrocytes incubated in 30 mivi PÂ¡,the influence of an elevation of the concentration of PÂ¡wasinvestigated. When PÂ¡inthe incubation medium was increased from its physiological level of 1.2, to 10 HIM.the effects on adenine nucleotide catabolism of adenosine, dAdo, ara-A, and MMPR, all at 0.5 HIMconcentration, were completely suppressed (results not shown). The effect of 0.5

01 2301 23 Time (h) after addition of 0.5 mM dAdo Fig. 3. Effect of ITu on the synthesis of dATP and on the catabolism of the adenine nucleotides induced by dAdo. Erythrocytes were preincubated as in Fig. 1. Incubations were performed in the presence of 0.5 mM dAdo alone (O, D) or together with 10 /AI ITu (â€¢,â€¢¿).whichwas added to the cell suspension, either initially, simultaneously with 0.5 mM dAdo (left), or 90 min later (right). Results shown are means of duplicates. INO, inosine; HX, hypoxanthine. by a marked decrease of the production of inosine plus hypo xanthine. These observations indicate that accumulation of dATP is not sufficient by itself to explain the catabolism of the 0123 adenine nucleotides. In addition, they suggest that the elevation Time (h) after addition of AMP may play a determining role in the induction of this Fig. 4. Effect of various nucleosides on the production of inosine (INO) plus catabolism. hypoxanthine (HX) from prelabeled adenine nucleotides. Erythrocytes were prein Effect of Nucleoside Analogues. The results depicted in Fig. cubated as in Fig. 1. Incubations were started with the addition of the nucleosides at 0.5 mM concentration. Inosine plus hypoxanthine were determined radio- 3 led to the hypothesis that the ability of dAdo to induce adenine chemically and their concentrations were calculated from the specific radioactivity nucleotide catabolism may be shared by other nucleosides that of the adenine nucleotides. Ado, adenosine; Co, control. 4985

1.0

0.8

So 0.6

to â€”¿ u o nÂ»_ 0.4 i ~ -ITu I 0.2 a.

0123 0123 Time (h) after addition of nucleosides Fig. 6. Influence of dAdo. Tu, and ITu on the concentration of intracellular PÃŒ.Erythrocyteswere preincubated as in Fig. 1. Incubations were started with the addition of 0.5 mM dAdo (left), or 0.5 mM Tu (righi). O, D, values measured in the absence of ITu; â€¢¿,â€¢¿valuesmeasured after the addition of 10 /IM ITu at the times indicated. Results are means of duplicates.

012301 2 Control 0.02-0.5mM dAdo Time (h) after addition of nucleosides O.SmM Ado Fig. 5. Effect of ITu on the catabolism of adenine nucleotides induced by O.SmM Ara-A MMPR and by Tu. Erythrocytes were preincubated as in Fig. 1. Incubations were O.SmM MMPR started with the addition of 0.5 ITIMMMPR (left) or 0.5 mivi Tu (right). O, D, O.SmM Tu values measured in the absence of ITu; â€¢¿,â€¢¿valuesmeasured after the addition of 10/IM ITu at the times indicated. Results are means of duplicates.//VO, inosine; HX, hypoxanthine.

IHMTu was strongly delayed, and at 0.1 HIMit was completely abolished. The accumulation of the nucleotide derivatives of the nucleosides mentioned was slightly increased in the presence of 10 IHMPÂ¡(not shown). Effects of dAdo and Other Nucleosides on the Concentration of the Effectors of AMP Deaminase in Erythrocytes. Human RBC AMP-DA is known to be stimulated by ATP and alkali metal cations and inhibited by PÂ¡and2,3-BPG (15, 16, 22, 25). 12 Both inhibitors were therefore measured in ADA-inhibited (nmol/ml of suspension) erythrocytes in order to assess if a decrease of their concentra Fig. 7. Correlation between the concentration of AMP and the rate of pro tion could explain the increased activity of AMP-DA induced duction of inosine (INO) plus hypoxanthine (HX) from prelabeled adenine by the addition of various nucleosides. The concentration of nucleotides. AMP concentrations are means of three measurements, performed 2,3-BPG, which was about 4-5 ^mol/ml of packed cells in after 30, 60, and 120 min of incubation, in the absence (O) or in the presence of nucleosides as indicated. Productions of inosine plus hypoxanthine were calcu control conditions, was not modified by the addition of the lated over the 120-min duration of the incubations. Results are from 6 separate nucleosides. However, the concentration of intracellular PÂ¡, experiments, r = 0.95; P < 0.001. Ado, adenosine. depending on the added nucleoside, decreased by 30 to 60% (results not shown). Nevertheless, when in experiments similar packed cells), there was almost no production of inosine and to those depicted in Figs. 3 and 5, the ATP degradation induced hypoxanthine. Beyond this value, a steep linear relationship by dAdo (Fig. 6, left) or by Tu (Fig. 6, right) was arrested by between the concentration of AMP and the rate of production the addition of ITu, intracellular PÂ¡reincreased only by 20%. of inosine plus hypoxanthine was observed; a 2-fold increase in The reason for the decrease in PÂ¡isnot clear. It does not seem AMP enhanced the production of these catabolites by a factor linked exclusively to the phosphorylation of the nucleosides of 10. The AMP threshold was not due to extracellular AMP, since it was also observed, although to a smaller extent, when since its concentration in the medium was below 0.4 nmol/ml, the synthesis of dATP was prevented by the addition of ITu at and the slight hemolysis recorded under control conditions was the beginning of the incubation (not illustrated). not increased by the addition of the various nucleosides. Correlation between the Concentration of AMP and the Rate Fig. 8 shows that the concentrations of AMP obtained with of Catabolism of the Adenine Ribonucleotides. With all nucleo the various nucleosides were closely related to their rates of sides studied, the concentration of AMP increased when their phosphorylation, with the exception of MMPR. This may be phosphorylation was allowed to proceed and adenine nucleotide linked to the fact that MMPR is converted to a monophosphate catabolism was induced, and AMP concentration decreased nucleoside, in contrast with the other nucleosides which are when both processes were switched off by ITu. The correlation metabolized to triphosphates. between the concentrations of AMP and the rates of production Kinetic Properties of Erythrocyte AMP Deaminase at Phys of inosine plus hypoxanthine, recorded under control condi iological Concentrations of AMP. The kinetic properties of tions and with various nucleosides, was therefore investigated. purified erythrocytic AMP-DA, including its stimulation by As shown in Fig. 7, below a threshold concentration of AMP ATP and K+, and inhibition by 2,3-BPG and PÂ¡,have been of 2 nmol/ml of suspension (corresponding to 10 nmol/ml of studied extensively (15,16,22,25). However, to our knowledge, 4986

Control â€¢¿0.02-0.5mM dAdo * O.SmM Ado â€¢¿O.SmMAra-A * O.SmM Tu = 30 60 T O.SmM MMPR E 3 E

a 20 40

Q- 2 - Q. < < 10 20

0 20 40 60 80 100 120 140 Rate ol nucleotide synthesis (nmol/h x ml of suspension) Fig. 8. Correlation between the rate of synthesis of nucleotide analogues and 10 0 2 5 10 the concentration of AMP. AMP concentrations are means of three measurements [ ATP ] or I dATP ] (mM) as in Fig. 7. Rates of synthesis of nucleotides from the added nucleosides were I PI ] (mM) calculated from the concentrations of the nucleotides measured at 30, 60. and Fig. 10. Influence of PÂ¡,ATP, and dATP on AMP deaminase activity. AMP- 120 min of incubation, r (excluding the MMPR data) = 0.96; P < 0.001. Ado, DA activity was measured at 50 Â¿IMAMP.The effect of P, (left) was measured in adenosine. the presence of 1 mM ATP and 3 mM 2,3-BPG; the effects of ATP (â€¢)anddATP (A) (right) were measured in the presence of 1 mM PÂ¡and3 mM 2,3-BPG.

0.5 mM dAdo or Tu, would thus increase the activity of AMP- 500 DA by no more than 20%. The influence of ATP and dATP on the activity of the enzyme in the presence of physiological 150 concentrations of its other effectors is shown in Fig. 10 (right). As described by others (14, 15), dATP and ATP were equally efficient as stimulators, the half-maximal effect being obtained at approximately 1 mM in the presence of 1 mM PÂ¡and 3 mM 2,3-BPG. Under the same conditions, ara-ATP was a 2-fold 100 less potent stimulator than ATP and dATP (not shown). The effect of other triphosphate nucleosides was not tested because they were not available. It was also verified that dAMP, which is not a substrate of AMP-DA (15, 16), was not a positive I Q. effector of the enzyme at low concentrations of AMP. Tu < 50 monophosphate and ara-AMP were also without effect on the activity of AMP-DA. 1mM ATP 3mM 2,3-BPG 1mM Pi DISCUSSION L Several mechanisms have been proposed to explain the loss 0.02 0.05 0.1 of ATP accompanying accumulation of dATP in erythrocytes AMP ] (mM) of leukemia patients treated with dCF and of children with Fig. 9. Influence of effectors on the substrate saturation curve of AMP ADA deficiency. Simmonds et al. (4) have claimed that eryth- deaminase. The activity of the enzyme was measured in the absence of additions (O) or in the presence of 1 mM ATP (â€¢)and of 1 mM ATP, 3 mM 2,3-BPG, and rocytic ATP is dependent, at least partly, on adenosine pro 1 mM I', (â€¢). duced by the transmethylation pathway and that this production could be impaired in ADA-deficient or -inhibited cells, because the kinetics of the enzyme had not been investigated at the of inactivation of S-adenosylhomocysteine hydrolase by dAdo micromolar concentrations of AMP which prevail in intact (Ref. 6, and references therein). This mechanism is, however, RBC. The influence of both the increase in AMP and the difficult to reconcile with the finding of increased concentra decrease in intracellular PÂ¡,recorded during the metabolism of tions of adenosine in the plasma and urine of ADA-deficient nucleosides, was therefore assessed. In the presence of 100 HIM patients (1,7). Recently, Snyder et al. (26) have proposed that KC1, but in the absence of other effectors AMP-DA displayed adenosine causes a substrate inhibition of adenosine kinase hyperbolic kinetics with a S0.5 for AMP of 0.4 HIM (Fig. 9). and/or a decreased phosphoribosylation of adenine. On the Addition of 1 mM ATP, the concentration of the stimulator in other hand, the study of Bagnara and Hershfield (14) in ADA- control erythrocytes, decreased the S0.s for AMP to 0.15 mM. inhibited lymphoblasts and our study in ADA-inhibited eryth Further addition of the inhibitors at their physiological concen rocytes show that dAdo increases the rate of degradation of trations, namely 1 mM for Pi and 3 mM for unbound 2,3-BPG, ATP and of total adenine nucleotides. The effect is already seen rendered the substrate saturation curve sigmoid and the enzyme at 20 MMdAdo, a concentration which is in the range of the nearly inactive up to 10 UM AMP. At 50 Â¿IMAMPand in the concentrations found in leukemia patients treated with dCF presence of 1 HIM ATP and 3 HIM 2,3-BPG, PÂ¡inhibited the (11, 12). Both studies also show that the stimulation of the enzyme activity by 40% at 1 HIM and by 95% at 10 HIM degradation of adenine nucleotides by dAdo results from an concentration (Fig. 10, left). A decrease in PÂ¡from 1 HIMto 0.5 increased activity of AMP-DA. Indeed, catabolism of AMP via mM, as recorded in ADA-inhibited erythrocytes incubated with dephosphorylation to adenosine can be ruled out because, de- 4987

spite inhibition of ADA, adenosine does not accumulate, and nucleosides, although we have observed a small (10-20%) stim inosine and hypoxanthine are produced. The accumulation of ulatory effect of their addition on the production of lÃ¡clate.5 inosine, recorded concomitantly with the high rates of build-up The rates of accumulation of nucleotides, shown in Fig. 8, of hypoxanthine, indicates that the activity of purine nucleoside although potentially also influenced by their concomitant deg phosphorylase becomes limiting under these conditions, most radation which may vary from one analogue to another, reflect likely owing to the low intracellular concentration of PÂ¡. approximately the affinities and substrate efficiencies of the We show in this work that the main factor responsible for respective nucleosides toward adenosine kinase from rabbit liver the increased activity of AMP-DA in erythrocytes incubated (29). Adenosine is, however, an exception since, although it is with dAdo is neither the accumulation of one of its stimulators, the best substrate of adenosine kinase, it induced only a limited dATP, nor a decrease of its inhibitors, PÂ¡and 2,3-BPG, but an elevation of adenine nucleotides, including AMP. This is most increase in AMP brought about by the utilization of ATP in likely due to the known inhibition of adenosine kinase by excess the phosphorylation of dAdo. That the elevation of AMP plays of substrate (26, 29). a major role in this catabolism is indicated by the observation The relationships between the rate of phosphorylation of that the arrest of the synthesis of dATP by ITu (Fig. 3) reverses nucleosides and the elevation of AMP and between the latter the elevation of AMP and stops the catabolism of ATP, despite elevation and the rate of AMP catabolism most likely explain the presence of 0.5 mM dATP in the cells. ITu, an inhibitor of why the depletion of ATP is usually smaller in erythrocytes of adenosine kinase, has no effect by itself on AMP-DA (19, 27). ADA-deficient children (3-6) than in patients treated with dCF The role of the elevation of AMP is validated by our results (9-11). Indeed, in the latter, higher concentrations of dAdo in obtained with other nucleosides, which are also substrates of the plasma have been recorded (11, 12). adenosine kinase (28, 29). Similarly to dAdo, the nucleoside Taken together, our results suggest that catabolism of the analogues induced an elevation of AMP and an acceleration of adenine nucleotides of human erythrocytes can be induced by the catabolism of the adenine ribonucleotides, which were both all nucleosides, and perhaps by other compounds, that are counteracted by the subsequent inhibition of adenosine kinase phosphorylated at a sufficient rate. The degradation is caused by ITu. by an elevation in erythrocytic AMP and proceeds by way of The relation between the intracellular concentration of AMP AMP-DA. This catabolism may be responsible for the anemia and the rate of production of purine catabolites (Fig. 7) shows recorded not only in ADA deficiency (6) and dCF treatment (9, that erythrocytes are very sensitive to an elevation of AMP 32) but also with other nucleoside analogues used in anticancer above a threshold concentration of 5-10 fiM. One hypothesis and in antiviral therapy (33). Whether a similar nucleoside- to explain this threshold, is that part of AMP, similarly to induced catabolism occurs in other tissues remains to be estab other erythrocytic metabolites (30), is bound to hemoglobin lished. Its existence may depend on various factors, among and thus not accessible to AMP-DA. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. Mechanism of Adenosine Triphosphate Catabolism Induced by Deoxyadenosine and by Nucleoside Analogues in Adenosine Deaminase-inhibited Human Erythrocytes

Françoise Bontemps and Georges Van den Berghe

Cancer Res 1989;49:4983-4989.

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