Effects of Deoxyadenosine Triphosphate

[CANCER RESEARCH 40. 3555-3558. October 1980] 0008-5472/80/0040-OOOOS02.00 Effects of Deoxyadenosine Triphosphate and 9-/?-D-Arabinofuranosyl- adenine S'-Triphosphate on Human Ribonucleotide ReducÃasefrom Molt-4F Cells and the Concept of "Self-Potentiation"1

Chi-Hsiung Chang2 and Yung-Chi Cheng3

Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill. North Carolina 27514

ABSTRACT conceivable that the intracellular activity of this enzyme is influenced or even regulated by the balance of the steady-state Deoxyadenosine triphosphate (dATP) acted as a noncom level of various nucleotides. That is, changes in pool sizes of petitive inhibitor with respect to the specific nucleoside tri nucleotides could influence the reduction of different ribonu- phosphate activator for the reduction of all four common ribo- cleoside diphosphates and result in changes in deoxyribonu- nucleoside diphosphates catalyzed by the reductase derived cleotide levels. Among the nucleotides examined, ATP acted from human Molt-4F (T-type lymphoblast) cells. The inhibition either as an activator for pyrimidine ribonucleoside diphos constant of dATP for different ribonucleotide reduction reac phate reduction or as an accessory activator for purine ribo tions was different, indicating that the binding of the nucleoside nucleoside diphosphate reduction. dATP is the only nucleotide triphosphate activator or substrate could modify the binding which inhibits reduction of all 4 natural ribonucleotides. The affinity of dATP to the enzyme. dATP also acted as a noncom Ki's (obtained by the replots of intercepts and slopes versus petitive inhibitor with respect to cytidine diphosphate (CDP) for inhibitors) were reported previously (4), but the detailed exper reductase-catalyzed CDP reduction. 9-/8-D-Arabinofuranosyl- adenine S'-triphosphate acted as a competitive inhibitor with imental results were not reported; these are included in this paper. It should be noted that the regulation of the enzyme respect to either adenosine triphosphate or guanosine triphos derived from Molt-4F cells is similar to that of the enzymes from phate for CDP or for adenosine diphosphate reduction, respec various sources such as Escherichia coli (9, 10) or other tively. The inhibition constant was 15 /Â¿MforCDP reduction and mammalian cells (12-14). However, they do differ in their 4 Â¡IMfor adenosine diphosphate reduction. 1-/8-o-Arabinofu- ranosyladenine S'-triphosphate could not substitute for aden kinetic properties. Since 9-/?-D-arabinofuranosyladenine exerts cytotoxic ef osine triphosphate or guanosine triphosphate as the activator fects after its conversion to a nucleotide, the effect of ara-ATP4 for CDP or adenosine diphosphate reduction, respectively. The effects of 9-ÃŸ-D-arabinofuranosylcytosine 5'-triphosphate and on this enzyme is important for a better understanding of its action. The effects of ara-ATP and ara-CTP on a crude prepa 5-iodo-2'-deoxyuridine 5'-triphosphate on ribonucleotide re ductase were also included for comparison. The "self-potentia- ration of rat tumor ribonucleotide reductase were examined by Moore and Cohen (11 ). It was concluded that ara-ATP inhibited tion" mechanism of the action of 9-ÃŸ-D-arabinofuranosyladenine and 5-iodo-2'-deoxyuridine is discussed. the reduction of all 4 common diphosphate ribonucleotides to about the same extent; inhibition appeared to be similar to that produced by dATP at a lower concentration. ara-CTP was a INTRODUCTION weak inhibitor of this enzyme activity (11 ). Since the purity and source of the enzyme that we studied are different, the effects Ribonucleotide reductase is one of the key enzymes respon of ara-ATP on the partially purified ribonucleotide reductase sible for the synthesis of deoxyribonucleotides which are re from human Molt-4F cells were examined in detail, and the quired for DMA synthesis. Therefore, this enzyme could be effects of ara-CTP and IdUTP on this enzyme are included for considered a target for anticancer agents. The activity of this comparison. enzyme in cells could also influence the cytotoxic effects of certain antitumor agents such as 1-/?-D-arabmofuranosylcyto- sine, 1-/?-D-arabinofuranosyladenine, 5-iodo-2'-deoxyuridine, MATERIALS AND METHODS and 5-fluoro-2'-deoxyuridine. Thus, it is of importance to un Materials. Nucleoside di- and triphosphates (sodium salts), derstand the kinetic behavior of this enzyme. DTT, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid We have partially purified ribonucleotide reductase from a were purchased from Sigma Chemical Company, St. Louis, human T-type lymphoblast tumor line (Molt-4F) (2). The kinetic Mo. ara-ATP, IdUTP, and ara-CTP were purchased from P-L behavior of this enzyme in utilizing ADP, GDP, CDP, and UDP Biochemicals, Inc., Milwaukee, Wis. Ammonium salts of all '"C- as substrates indicated that the enzyme can be regulated by labeled nucleotides were supplied by Amersham/Searle Corp., natural nucleoside triphosphates or diphosphates (3, 4). It is Arlington Heights, III. Dowex-1 CI was obtained from Bio-Rad Laboratories, Richmond, Calif. Materials required for cell cul ' This work was supported by USPHS Project Grant CA-27449 from the tures were purchased from Grand Island Biological Co., Grand National Cancer Institute. NIH. This is Paper 5 in a series on human ribonucleotide reductase from Molt-4F cells. Island, N. Y. All other chemicals were of reagent grade. 2 Present address: Biochemistry Department. Southern Research Institute. 2000 Ninth Avenue South, Birmingham, Ala. 35205. 3 An American Leukemia Society Scholar. To whom requests for reprint should " The abbreviations used are: ara-ATP. 9-/i-D-arabinofuranosyladenine 5'- be addressed. triphosphate; ara-CTP. 9-/i-D-arabinofuranosylcytosine 5'-triphosphate; IdUTP. Received February 4. 1980; accepted July 9. 1980. 5-iodo-2'-deoxyuridine S'-triphosphate; DTT. dithiothreitol.

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Preparation of the Enzyme Components. The methods for (A) culturing Molt-4F cells and the purification of Components A \fu 50 ntercept and B of this ribonucleotide redactase have been described (x-x) 30 previously (2). The final preparations of Components A and B are not homogeneous as judged by electrophoretic techniques. However, they were purified to such an extent that nucleotide phosphatase and nucleoside diphosphate kinases, which would interfere with kinetic studies of the enzyme, were not present in the purified Components A and B. All of the studies reported here were performed using the reconstituted enzyme from highly purified components. 05 LO I5 2.0 Enzyme Assays. CDP reducÃasewas assayed by the method ATPJ , mMJ of Steeper and Steuart (15) with the use of Dowex 1-borate ion-exchange chromatography. Under "standard conditions," l/v I Slope Intercept 5O the assay mixture contained, in a final volume of 0.2 ml, (x-x) [14C]CDP (0.2 /iCi; 0.15 ITIM),DTT (3 mvi), MgCI2 (6 rriM), ATP

(5 rriM), and a specific amount of the enzyme. ADP reductase dATP.uM -50 -30 -IO IO 30 50 activity was determined by the method of Cory et al. (6). Under 50 dATP, uM standard conditions, the assay mixture contained, in a final volume of 0.2 ml, [14C]ADP (0.4 jnCi; 0.3 rriM), DTT (3 ITIM), MgCI2 (6 ITIM), dGTP (5 HIM), and a specific amount of the 20 40 60 enzyme. UDP and GDP reductase activities were determined CDPJ,mM-1 by the method reported previously (3). Under standard condi tions, the assay mixture contained, in a final volume of 0.2 ml, [14C]UDP (0.2 /iCi; 0.15 rriM) or [14C]GDP (0.2 Â¿iCi;0.15 mw), Chart 1. Effects of dATP on the Lineweaver-Burk plots of the reciprocal of DTT (3 rriM), MgCI2 (6 rriM), ATP (5 ITIM)for UDP reduction or activator concentration (A) or substrate concentration (B) with respect to the dTTP (5 rriM) for GDP reduction, and a specific amount of the reciprocal of reaction velocity (nmol of dCDP produced in 1 hr). Standard incubation conditions were used except for addition of activator (A) or substrate enzyme. An enzyme sample heated for 2 min in a boiling water (8) at the indicated concentration. Each assay contained 14 /xg of purified bath prior to the addition of the labeled substrate served as the Component A and 12 fig of purified Component B of ribonucleotide reductase reaction blank. The incubation was at 37Â° for 60 min; the obtained from Molt-4F cells. reaction was linear with respect to time and enzyme concen tration during the incubation period. Protein Determination. Protein concentrations were deter mined by the method of Bradford (1 ). Bovine albumin was used as the standard. -IO -6 -2 246 8

RESULTS dATP, Inhibition by dATP of the Enzyme Activity for CDP, ADP, UDP, and GDP Reduction. In the preceding report (4), the inhibition constants of dATP on various ribonucleotide reduc tions were summarized without describing the kinetic data. 0.4 I.2 2.0 Detailed kinetic data are described here in Charts 1 and 2 for dGTP-l, mM-l purposes of comparison with the effects of ara-ATP. dATP behaves as a noncompetitive inhibitor of CDP reduction with respect to substrate (CDP) or activator (ATP) according to the definition of Cleland (5). The double reciprocal plot of initial velocity versus substrate or activator is shown in Chart 1. The -D -6-2 24 68 values of KÂ¡intercept and K, slope for dATP with respect to ATP for CDP reduction were calculated based on the replot dATP,/iM and gave the same value of 40 JUM(Chart 1/4). The values of Ki intercept and KÂ¡slope for dATP with respect to CDP for CDP

reduction were 60 and 21 fiM, respectively (Chart IB). -I.2 -0.4 Q4 I.2 2.0 Since both GTP and dGTP act equally well as the activator GTP-1, mM-1 for ADP reduction, the effect of ATP was examined with respect to GTP or dGTP on the reduction of ADP. These results are shown in Chart 2. dATP inhibited ADP reduction noncompeti- Chart 2. Effect of dATP on the Lineweaver-Burk plots of the reciprocal of activator concentration with respect to the reciprocal of reaction velocity (nmol tively with respect to both activators. The KÂ¡of dATP with of dADP produced in 1 hr). Standard incubation conditions were used except for respect to dGTP (Chart 2A) was the same as the value of dATP addition of activator at the indicated concentration. Each assay contained 14 fig of purified Component A and 12 jig of purified Component B of ribonucleotide with respect to GTP (Chart 2ÃŸ).The values of K, intercept and reductase obtained from Molt-4F cells. A, dGTP as activator for ADP reduction; Ki slope were 2 and 6 /IM, respectively. B, GTP as activator for ADP reduction.

3556 CANCER RESEARCH VOL. 40

The effects of dATP with respect to ATP for UDP reduction results are shown in Table 1. ara-CTP was a less potent and with respect to dTTP for GDP reduction also were exam inhibitor than was ara-ATP or IdUTP when the comparison was ined. The Ki was 55 JIMfor UDP reduction and 1.5 PPMfor GDP made under the same conditions. ara-CTP inhibition of UDP reduction; the same values were obtained from K( slope and KÂ¡ reduction was more pronounced than was inhibition of CDP intercept. reduction (data not shown). The Effects of ara-ATP on the Reduction of CDP and ADP. The type of inhibition of ara-ATP with respect to activator for DISCUSSION the reduction of CDP and ADP is shown in Chart 3. ara-ATP inhibited the reduction of CDP (Chart 3A) and ADP (Chart 36) Detailed experimental results on the effects of dATP on the competitively with respect to their nucleoside triphosphate reduction of the 4 natural ribonucleoside diphosphates cata activators. The KÂ¡ofara-ATP is 15 Â¡IMwith respect to ATP for lyzed by Molt-4F ribonucleotide reducÃase are presented in CDP reduction and 3.8 Â¡IMwith respect to ATP for ADP reduc this paper. dATP behaved as a noncompetitive inhibitor for the tion. We observed that GTP could decrease the apparent Ka of reduction of all 4 ribonucleotides, but the KÂ¡'sfor each ribo ATP for CDP reduction (4), and we posed the question of nucleotide reduction are different. ADP reduction was more whether the inhibition pattern or inhibition constant of ara-ATP susceptible to inhibition by dATP than was reduction of the might be changed when GTP and ATP are both present in the other 3 ribonucleotides. The different KÂ¡'sfor various ribonu CDP reduction. There was, however, no effect of ATP on ara- cleotide reductions indicated that the binding sites of dATP on ATP inhibition of CDP reduction in the presence of GTP, and ribonucleotide reducÃase are not rigid and thai Ihe affinily of the KÃ•of ara-ATP with respect to ATP in the presence of 0.5 dATP for the enzyme could be modified by other nucleolides. HIM GTP for CDP reduction was 15 UM, the same as that for Since dATP could lurn off its own synthesis more efficiently, ara-ATP in the absence of GTP (data not shown). ara-ATP this could prevenl Ihe conlinous synthesis of dATP. Otherwise, could not substitute for ATP in stimulating the reduction of CDP the accumulalion of inlracellular dATP could evenlually lurn off or UDP. Ihe synlhesis of dCTP and dTTP. Such deplelion of dTTP or The Effects of ara-ATP, ara-CTP, and IdUTP on UDP Re dUTP could affecl the synthesis of dGTP since Ihey are re duction Catalyzed by Molt-4F Enzyme. Previously, we ob quired for the reduction of GDP to dGDP. Thus, Ihe inlracellular served that the inhibition of UDP reduction was more pro concentration of dATP could be a critical faclor in regulating nounced than that of ADP, GDP, and CDP reduction by dCTP the synlhesis of DNA. The cyloloxicily of deoxyadenosine may and dTTP under our assay condition (4). Thus, we examined be relaled lo Ihe accumulation of dATP to a level which inhibils the effects of ara-CTP and IdUTP on UDP reduction catalyzed Ihe formation of olher deoxynucleolides. by this enzyme and included ara-ATP for comparison. The ara-ATP, an analog of dATP or ATP, was found to compete with ATP by inhibiting CDP reduction with the KÂ¡of 15 /Â¿M, which is lower than the Ka of ATP. In addition, Â¡Iinhibiled the reduction of ADP competitively with GTP (data not shown) or dGTP (Chart 3) with a KÃŒof3.5 [iM. Since dATP acted as a noncompetitive inhibitor for bolh ADP and CDP reduclion wilh respecl lo Iheir nucleoside Iriphosphate activator and since ara-ATP binds mutually exclusively with ATP for the reduction of CDP and mutually exclusively wilh GTP or dGTP for Ihe reduclion of ADP, the notion that ara-ATP and dATP exert similar inhibition of this enzyme, as suggested by Moore and Cohen (11 ) for Ihe ral tumor enzyme, does not apply in our -2.0 -1.4 -.8 .2 .6 I.O 14 1.8 22 ATP-I, mM-l system. Furthermore, ara-ATP inhibition on the reduclion of various ribonucleolides lo Ihe same extent as observed by these invesligalors does noi agree with our observations. ara- ATP was clearly demonstraled lo have differenl inhibitory po tency toward various ribonucleolide reduclions calalyzed by Moll-4F enzyme. Our resulls do agree with their reporl lhat the inhibitory potency of ara-CTP was weaker than that of ara-ATP,

Table 1 Effects of ara-CTP. IdUTP. and ara-ATP on UDP reduction catalyzed by Molt-4F ribonucleotide reducÃase The concentrations of ATP and UDP used in the assays were 5 and 0.15 rnw, respectively. The amount of enzyme used was 14 /ig of purified Component A and 12 (ig of purified Component B. Additivesara-CTPara-CTPara-CTPIdUTPara-ATPara-ATPConcentration(mw)0.5140.50.10.4Activity (%)10063471745829

Chart 3. Effects of ara-ATP on the Lineweaver-Burk plots of the reciprocal of activator concentration with respect to the reciprocal of reaction velocity (nmol of deoxyribonucleotides produced in 1 hr). Standard incubation conditions were used except for addition of activator at indicated concentration. Each assay contained 14 pg of purified Component A and 12 /jg of purified Component B of ribonucleotide reducÃase obtained from Molt-4F cells. A, reduction of CDP; B, reduction of ADP.

OCTOBER 1980 3557

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1980 American Association for Cancer Research. C-H. Chang and Y-C. Cheng at least in the case of UDP reduction. We observed that IdUTP 2. Chang, C.-H., and Cheng, Y.-C. Demonstration of two components and also could inhibit the reduction of UDP more effectively than association of adenosine diphosphate-cytidine diphosphate reducÃase from did ara-ATP when they were compared under the same con cultured human lymphoblast cells (Molt-4F). Cancer Res., 39. 436-442, 1979. ditions. The nature of the inhibition exerted by ara-CTP and 3. Chang, C.-H., and Cheng, Y.-C. Substrate specificity of human ribonucleo IdUTP is still under investigation. tide reductase from Molt-4F cells. Cancer Res., 39. 5081-5086, 1979. 4. Chang, C.-H., and Cheng, Y.-C. Effects of nucleoside triphosphates on The observation that ara-ATP inhibited the formation of dADP human ribonucleotide reductase from Molt-4F cells. Cancer Res., 39:5087- and that IdUTP inhibited the formation of dCDP or dUDP 5092, 1979. suggested that these nucleoside analogs may enlarge their 5. Cleland, W. W. Determining the chemical mechanism of enzyme-catalyzed cytotoxic effects by the mechanisms of "self-potentiation." reactions by kinetic studies. Adv. Enzymol., 45: 273-387, 1977. 6. Cory, J. G., Russell, F. A., and Mansell, M. M. A convenient assay for ADP That is, decreased formation of deoxyadenylate or deoxypyr- reductase activity using Dowex-1-borate columns. Anal. Biochem., 55. 449- imidine nucleotides through their inhibitory effects on ribonu- 456, 1973. 7. Derse, D.. and Cheng, Y.-C. Comparative studies of the kinetic properties of cleotide reducÃase would result in less competition of dATP herpes simplex induced and HeLa BU DNA polymerases. Fed. Proc., 39: with ara-ATP or of dTTP with IdUTP at the target site (e.g., DNA 1956, 1980. 8. Dicioccio, R., and Srivastava, B. I. S. Kinetics of inhibition of deoxynucleo- polymerase) (7, 8). This would in turn potentiate their action at tide-polymerizing activities from normal and leukemic human cells by 9-/J- the DNA level. In other words, ara-ATP or IdUTP could poten D-arabinofuranosyladenine S'-triphosphate and 1-/i-o-arabinofuranosylcy- tiate their action at the DNA level. In other words, ara-ATP or tosine 5'-triphosphate. Eur. J. Biochem., 79: 411-418, 1977. 9. Larsson, A., and Reichard, P. Enzymatic synthesis of deoxyribonucleotides. IdUTP could potentiate their own action by inhibiting the for IX. Allosteric effects in the reduction of pyrimidine ribonucleotides by the mation of their competing dATP or dTTP. It should be pointed ribonucleoside diphosphate reductase system of Escherichia coli. J. Biol. out that this self-potentiation may be mediated not only through Chem., 241: 2533-2539, 1966. 10. Larsson, A., and Reichard, P. Enzymatic synthesis of deoxyribonucleotides. their actions on ribonucleotide reducÃasebut also through Iheir X. Reduction of purine ribonucleotides; allosteric behavior and substrate aclion on olher deoxynucleotide-metabolizing enzymes, such specificity of the enzyme system from Escherichia coli B. J. Biol. Chem., 247. 2540-2549, 1966. as dCMP deaminase or TMP synlhelase. 11. Moore, E. C., and Cohen, S. S. Effects of arabinonucleotides on ribonucleo tide reduction by an enzyme from rat tumor. J. Biol. Chem., 242. 2116- 2118, 1967. ACKNOWLEDGMENTS 12. Moore, E. C., and Hulbert, R. B. Regulation of mammalian deoxyribonucle- otide biosynthesis by nucleotides as activators and inhibitors. J. Biol. Chem., We wish to thank Linda Roberts and Susan Grill for their excellent technical 241:4802-4809, 1966. assistance. 13. Murphee. P., Cannellakis, Z. N., and Beali, P. T. Regulation by nucleotides of the activity of partially purified ribonucleotide reductase from rat embryos. Cancer Res., 28. 860-863, 1968. REFERENCES 14. Reichard, P., Cannellakis, Z. N., and Cannellakis, E. S. Studies on a possible regulatory mechanism for the biosynthesis of deoxyribonucleic acids. J. 1. Bradford, M. M. A rapid and sensitive method for the quantitation of micro- Biol. Chem., 236: 2514-2519, 1961. gram quantities of protein utilizing the principle of protein-dye binding. Anal. 15. Steeper, J. R., and Steuart, C. D. A rapid assay for CDP reductase activity Biochem., 72. 248-254, 1976. in mammalian cell extracts. Anal. Biochem., 34: 123-130, 1970.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1980 American Association for Cancer Research. Effects of Deoxyadenosine Triphosphate and 9-β -d-Arabinofuranosyladenine 5′-Triphosphate on Human Ribonucleotide Reductase from Molt-4F Cells and the Concept of ''Self-Potentiation''

Chi-Hsiung Chang and Yung-Chi Cheng

Cancer Res 1980;40:3555-3558.

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