Inhibition of Mammalian DNA Polymerase by the 5'-Triphosphate of 1-$-D-Arabinofuranosylcytosine and the 5'-Triphosphate of 9../3'

[CANCER RESEARCH 28, 2061-2067, OctOber 19681

J. J. Furth2and SeymourS. Cohen Department of Pathok,gy and the Department of Therapeutic Research, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

SUMMARY DNA viruses (18, 20). At a more readily analyzable level, the compound has been found to inhibit the growth of cultured The 5'-tniphosphate of 1-,8-D-arabinofuranosylcytosine (ara CTP) was tested as a substrate or inhibitor of polynucleotide animal cells, as well as the multiplication of DNA viruses in synthesis using mammalian polymerases. The compound did such cells. These effects are obtained by a relatively specific not replace deoxycytidine triphosphate (dCTP) as substrate inhibition of DNA synthesis. These data have been summarized for DNA polymerase and did not replace cytidine triphosphate in a review of the properties of D-arabinosyl nucleosides (4). as substrate for RNA polymerase. The compound was found Several hypotheses have been suggested to account for the in to inhibit DNA synthesis catalyzed by DNA polymerase ; the hibitory action of the compound at the enzymatic level, and inhibition appeared to be competitive with dCTP. No inhibition the present status of these explanations will be considered in the Discussion. of RNA polymerase was observed. Further studies indicate that the inhibition of mammalian DNA polymerase by the York and LePage (22) indicated that ara-ATP inhibited the 5'-triphosphate of 9-$-D-arabinofuranosyladenine (ara-ATP) DNA polymerase of some ascites tumor cells. This report was is also competitive with the corresponding deoxytriphosphate. confirmed and extended by the present authors (8) . We then asked if ara-CTP did not similarly inhibit the DNA polymerase The data on the mode of action of 1-fl-D-arabinofuranosyl cytosine and of 9-,6-D-arabinofuranosyladenine have been sum of animal cells. This compound has now been found to inhibit the animal enzyme fairly effectively, possibly accounting in manized, and the present status of hypotheses on these mech anisms have been discussed. The data are consistent with the some measure for its inhibitory activity in vivo. view that ara-CTP and ara-ATP act in animal cells via their A preliminary report of this work has appeared (9). respective tniphosphates to inhibit the animal cell DNA poly MATERIALS AND METHODS merase, although ara-ATP, but not ara-CTP, may also act by The syntheses of ara-CTP and ara-ATP have been described inhibiting ribonucleotide reductase. previously (1, 8). INTRODUCTION The mammalian DNA polymerase used in these experiments, prepared as formerly described (1 1), was purified further by ara-C3 inhibits the development of tumors (7) and of some chromatography on phosphocellulose (21) . The eluate fractions containing DNA polymerase were combined and concentrated 1 This investigation was supported by USPHS Grants 7005 from the National Institute of Allergy and Infectious Disease and 10390 by ammonium sulfate precipitation. Enzyme was obtained from from the National Institute of General Medical Sciences. both bovine lymphosarcoma and calf thymus. Its properties 2 Research Career Development Awardee (GM-K3-12,888) of have also been reported in another communication ( 11) . While the USPHS. the rate of the reaction was proportional to time, an initial lag

3 The abbreviations used are : ara-A or arabinosyladenine, 9-fl-D- was invariably observed (Chart 1) . This lag could not be arabinofuranosyladenine ; ara-ADP, 9-fl-D-arabinofuranosyladenine overcome by incubating all components of the reaction (except 5'-diphosphate ; ara-ATP, 9-p-n-arabinofuranosyladenine-5'-tniphos enzyme) and adding the enzyme after 5 mm, or by incubating phate ; ara-C or arabinosylcytosine, 1-@-D-arabinofuranosylcytosine; all components except the labeled triphosphate and adding it ara-CTP, 1-p-n-arabinofuranosylcytosine-5'-triphosphate ; RNA after 5 mm. While this is probably the result of formation of an polymerase, nucleoside triphosphate :RNA nucleotidyltransferase, initiator region (2), we have not yet done the appropriate ex EC2.7.7.6 ; DNA polymerase, deoxynucleoside triphosphate : DNA periments to prove this hypothesis. In any event, the duration deoxynucleotidyltransferase, EC2.7.7.7 ; dATP, dCTP, dGTP, and of the lag does not appear to affect the interpretation of the dTTP, 5'-deoxynucleoside triphosphate derivatives of adenine, kinetic data reported beow. It should also be noted that a small cytosine, guanine, and thymine respectively ; ATP, CTP, GTP, and UTP, 5'-ribonucleoside triphosphate derivatives of adenine, amount of labeled substrate is incorporated in the absence of cytosine, guanine, and uracil respectively ; dTMP, deoxythymi dCTP. This value, which may represent terminal addition of a dine monophosphate ; CDP, cytidine diphosphate. few nucleotides to the DNA, has been subtracted in the analysis Received January 29, 1968; accepted June 26, 1968. of the inhibition by the arabinosyl nucleoside triphosphates.

OCTOBER 1968 2061

Chemical and Nuclear Corp. DNA was denatured by heating 1.0 at 100Â°C for 4 minutes, followed by rapid cooling in an ice bath.

0.S RESULTS 4 Studies with DNA Polymerase. ara-CTP does not appear 0 E to support DNA synthesis. As shown in Chart 1, in the corn

E plete system which contained 12.8 @.tusdCTP, 0.75 msmole of 0.6 14C-labeled thymidylic acid was incorporated into acid-insolu w ble material in 100 minutes. When ara-CTP was added in place I- 4 of dCTP, incorporation of 0.04 m@tmole of 14C-labeled thymi 0 0.4 dylic acid was the same as that observed when neither triphos 0. phate was added. 0 While ara-CTP does not support DNA synthesis, the analog I-) z inhibits the reaction with the normal substrate. In the experi 0.2 ment shown in Chart 1, ara-CTP was added either at 11 mm 0. after the reaction was initiated and dCTP had been added, or I- @0 at zero time with dCTP added 21 mm later. Incorporation of labeled substrate was inhibited to approximately the same 40 $0 120 extent in both cases. f (minutes) Increasing the concentration of ara-CTP relative to the dCTP concentration increased the inhibition. In the experi Chart 1. Effect of ara-CTP on DNA polymerase of calf thymus. ment shown in Chart 2, the concentration of ara-CTP was in The reaction mixture (0.50 ml) contained : 50 mus phosphate creased up to 2.5 @tMin the presence of 3.2 @usdCTP. In the buffer, pH 7.0 ; 4 mM MgCl2 ; 2 mus 2-mercaptoethanol; 40 jsM each of dATP and dGTP ; 48 @MdTTP-'@C (1.70 x 106 cpm/ @&mole);heated calf thymus DNA, 48 msmoles of deoxynucleotide; @ and 59 of the AS II fraction. ara-CTP (12.5 @tM)and/or dCTP (12.8 @M)were added to the times indicated. Incubation was at C 0 37Â°C.Aliquots were removed at the time indicated and placed C in water at O'C (0.30 ml) ; incorporation of labeled substrate into acid-insoluble material was determined as described previously 0 Id . S â€¢ â€¢ (8). @â€”Oâ€”@,dCTP added at 0 mm, no ara-CTP added; I-. @ 0â€”0â€”0 , dCTP added at 21 min, no ara-CTP added; 4 0 dCTP added at 0 min, ara-CTP added at 11 min; Vâ€”V----V, 0. dCTP added at 21 mm, ara-CTP added at 0 mm ; 0â€”0â€”0, no 0 U dCTP, no ara-CTP added ; â€¢, no dCTP, ara-CTP added at 0 z min. ara-CTP, 1-p-D-arabinofuranosylcytosine-5'-triphosphate ; dATP, 0. I- dCTP, dGTP, dTTP, deoxynucleoside-5'-triphosphate derivatives â€¢0

of adenine, cytosine, guanine, and thymine respectively ; dTMP, -I.e -0.5 0.5 1.0 5.5 tO 2@S thymidine-5'-monophosphate. ora-CTP (pM)

The method for obtaining the mammalian RNA polymerase Chart 2. The inhibition of calf thymus DNA polymerase as a used in these experiments has been published (10) ; further function of varying the ara-CTP concentration. The reaction mix purification was accomplished by chromatography on phospho ture (025 ml) contained 50 mus phosphate buffer pH 7.0, 4 mus cellulose. Hybridization experiments with this fraction confirm MgCl2, 2 mus 2-mercaptoethanol; 50 @tMeach of dATP and that the RNA product is a replica of the DNA used as template. dGTP ; 56 @M dTTP-'@C (1.33 x 106 cpm/@tmole) ; heated calf That is, RNA synthesized with calf thymus DNA as template thymus DNA, 12 m@tmoles of deoxynucleotide ; 32 MM dCTP; will hybridize with thymus DNA but not with T2 DNA. Con and varying concentrations of ara-CTP. The reaction was initiated versely, RNA synthesized with T2 DNA template hybridizes by the addition of enzyme (54 pig) and terminated after 60 min with T2 DNA but not with DNA of calf thymus. (W. Abrams at 37Â°C.Incorporation of labeled substrate was determined as and J. J. Furth, unpublished observations.) described previously (8). The interrupted lines intersecting the The routine assay of the polymerases was also as previously ordinate are the values (_i-@ obtained in Charts 3 and 5. reported (8) . In a few experiments, DNA polymerase was ara-CTP, 1-@-D-arabinofuranosylcytosine-5'-tniphosphate ; dATP, assayed by a modification of the filter disc method of Yoneda dCTP, dGTP, dTTP, deoxynucleoside-5'-triphosphate derivatives and Bollum (21) . dTTP-14C was obtained from Schwarz Bio of adenine, cytosine, guanine, and thymine ; dTMP, deoxythymi research. a-32P-labeled UTP was obtained from International dine-5'-monophosphate.

2062 CANCER RESEARCH VOL. 28

absence of inhibitor, 022 m@smoleof dTMP was incorporated; in the presence of ara-CTP, inhibition varied from 18% with 025 @tM ara-CTP to 54% with 2.5 @sM ara-CTP. When approxi mately equal concentrations of dCTP and analog were added, . the rate of incorporation of thyrnidlylate was inhibited 78% (Chart 1). In the experiment shown in Chart 3, the concentration of dCTP was varied from 1.9 @Mto 12.8 j@us, both in the presence 0.5 and absence of ara-CTP (1 .25 /LM). When the data was plotted 0 by the method of Lineweaver and Burk, straight lines could be C drawn intersecting on the ordinate, indicating that the inhibi E 0 tion was competitive. Id I- 0.5 Tests of Irreversible Inhibition by Preincubation. To ex 4 elude the possibility that the inhibitor is bound irreversibly 0 0. to the enzyme, an attempt was made to titrate the inhibitor 0 with enzyme. This was done by incubating enzyme and in U z hibitor for 5 mm before the addition of dCTP. As shown in Chart 4, no stoichiometry is observed relating inhibition to 0. I- enzyme concentration (ci. 15) . Furthermore, a double reciprocal @0 plot of data obtained with preincubation of enzyme and in hibitor results in straight lines also intersecting on the ordinate (Chart 5) . These experiments also indicate that the inhibitor ENZYME ADDED (jig) does not inactivate the DNA template. Chart 4. Attempted titration of DNA polymerase by ara-CTP. The reaction mixture (025 ml) contained : 50 mus phosphate buffer, pH 7.0; 4 mM MgCl2 ; 2 mus 2-mercaptoethanol; 40 j@us 0 each of dATP and dTTP ; 64 @tMdGTP_14C (1.15 x 10@cpm/ C @ @mole); 1.7 or 4.8 ara-CTP ; heated calf thymus DNA, 48 C m@tmoles of deoxynucleotide ; and varying amounts of enzyme. 0 Id dCTP (6.4 @LM)wasadded after 5 mm at 37Â°C.The reaction was 4 terminated 60 mm after the addition of dCTP. â€¢â€”.â€”â€¢,no @ 0 ara-CTP ; 0â€”0â€” 0 , 1.7 @Mara-CTP ; 42 ius ara 0. CTP. 0 U ara-CTP, 1-@-D-arabinofuranosylcytosine-5'-tr1phosphate ; C1ATP, z dCTP, dGTP, dTTP, deoxynucleoside-5'-triphosphate derivatives 0. of deoxyadenine, cytosine, guanine, and thymine ; dTMP, deoxy thymidine-5'-monophosphate. @0

Determination of [email protected] experiments reported in Charts 3 and 5 permit the calculation of the apparent Km of enzyme and dCTP. The affinity of enzyme and the ara-CTP can also be calculated from the data reported in Chart 2. Using the Chart 3. Inhibition of DNA polymerase of calf thymus by ara experiments reported in Charts 3 and 2, the Km for dCTP is CTP. The reaction mixture (025 ml) contained : 50 mus phosphate 4 @M,while the K@for ara-CTP is 0.72 @.tM.Use of the data re buffer pH 7.0; 4 mM MgCl2 ; 2 mus 2-mercaptoethanol; 40 @tus ported in Charts 5 and 2 results in a calculated Km for dCTP each of dATP and dGTP ; 36 @usdTTP-'@C (1.10 x 106 cpm/ of 2.5 ,.tM with a corresponding K4 of 1.1 gus. @tmole);heated calf thymus DNA, 24 m@tmolesof deoxynucleotide; These experiments (Charts 2, 3, and 5) were done with 125 @iMara-CTP; and varying concentrations of dCTP. The re 14CdTTP as the labeled deoxynucleoside triphosphate. To action was initiated by the addition of enzyme (152 @tgof the exclude the possibility that the results were due to the choice AS II fraction) and terminated after 60 mm at 37Â°C. Incorpora tion of labeled substrate into acid-insoluble material was deter of labeled substrate, the experiments were repeated using

@ mined as described previously (8). In plotting the data, the GTP-'4C (Chart 6) . Double reciprocal plots (@. vs -@) at two incorporation observed in the absence of dCTP (0.02 m,@mole) inhibitor concentrations resulted in points through which has been substracted. â€¢â€”â€¢â€”â€¢,no ara-CTP ; 0 â€”0â€”0 , +ara CTP. straight lines could be drawn intersecting on the ordinate, ara-CTP, 1-@-D-arabinofuranosylcytosine-5'-tniphosphate ; dATP, again indicating competitive inhibition. dCTP, dGTP, dTTP, deoxynucleoside-5'-triphosphate derivatives Experiments with ara-ATP. Since in our earlier experiments of adenine, cytosine, guanine, and thymidine ; dTMP, deoxythy with ara-ATP (8) some of the experiments suggested a form midine-5'-monophosphate. of inhibition not purely competitive, the inhibition by ara

OCTOBER 1968 2063

ATP was reexamined using the more highly purified enzyme. C ara-ATP inhibits, as shown previously, and the inhibition is 0 C competitive (Chart 7A) . Increasing the inhibitor concentration E at constant substrate levels results in decreased incorporation 0 of labeled deoxynucleotide (Chart 7B) . When the reciprocal LI I- 4 of the velocity of the reaction is plotted as a function of the 0 inhibitor concentration, the affinity constant of the inhibitor 3. can be calculated. This value comes out 1.3 @Mcompared to a 0 @ U Km dATP of 2.5 @tus(Chart 7C). z While these experiments were done with the DNA polymerase 0. obtained from calf thymus, ara-CTP was also found to inhibit I- the enzyme of bovine lymphosarcoma. For example, with 3.6, @0 7.2, and 14.4 @MdCTP, the presence of 62 @.tusara-CTP re sulted in inhibitions of 73%, 59%, and 33% respectively. Lack of Effect of ara-CTP on Mammalian RNA Polymerase. As previously observed in studies with ara-ATP, ara-CTP does dCTP (pM) not appear to support RNA synthesis with the mammalian enzyme (Table 1) . For example, in the complete system 91 Chart 5. Inhibition of DNA polymerase of calf thymus by ara jqzmoles of labeled UTP was incorporated into RNA (Line 2). CTP with preincubation of enzyme and inhibitor. The reaction When ara-CTP was substituted for CTP, there was no detect mixture and assay were essentially as described in the legend to able incorporation (Line 8). Chart 3, except that dCTP was added after the other components In contrast to the results obtained with DNA polymerase, had been preincubated for 5 mm at 37Â°C. â€¢â€”Sâ€”â€¢,no ann ara-CTP does not significantly affect the incorporation of UTP CTP; 0â€”0â€”0, +ara-CTP. ara-CTP, 1-p-n-arthinofuranosylcytosine-5'-triphosphate ; dCTP, into RNA in the presence of the normal substrate (Table 1, deoxycytosine-5'-triphosphate ; dTMP, deoxythymidine-5'-mono Lines 3â€”7). phosphate. DISCUSSION

These results with ara-CTP are consistent with the results previously reported for ara-ATP. ara-CTP does not support

0 the incorporation of labeled deoxytriphosphates into DNA but C does inhibit the incorporation of the proper substrate. In our C

0 LI 4 0 Table 1 0. Concentration (jiM)Uridylic 0 acid U z (jijimoles)CTPara-CTP00514091144.6941492981418.4941446.0841492.090092.0<5incorporated 0.

â€¢0

dCTP (pM)

Chart 6. Inhibition of calf thymus DNA polymerase by nra Effect of ara-CTP on RNA polymerase of calf thymus. nra CTP with dGTP-'@C as labeled substrate. The reaction mixture CTP, 1-fl-D-arabinofuranosylcytosine-5'-triphosphate ; ATP, CTP, (025 ml) contained : 50 mM phosphate buffer pH 7.0; 4 mus GTP, UTP, ribonucleoside-5'-triphosphate derivatives of adenine, MgCl2 ; 2 mus 2-mercaptoethanol; 40 @Meach of dATP and cytosine, guanine, and uracil respectively. The reaction mixture dTTP; 46 ,L@usdGTP'@C (1.55 x 106 cpm/1ttmole), 0.0, 0.9, or (0.50 ml) contained : 50 mus Tris-maleate buffer pH 8.1 ; 2 mus 3.5 @tMara-CTP ; heated calf thymus DNA, 48 m@tmoles of deoxy 2-mercaptoethanol; 160 @iMeach of ATP and GTP ; 94 @M nucleotide ; varying amounts of dCTP ; and 21.6 jig of enzyme. a-UTP-32P (3 :45 X 10@ cpm/j@mole) ; ara-CTP ; native calf The reaction was terminated after 60 mm at 37'C. â€¢â€”â€¢â€”â€¢,thymus DNA, 48.0 mj&moles of deoxynucleotide ; and enzyme @ no ara-CTP ; 0â€”0â€”0 , 0.9 AM ara-CTP ; 3.5 @tM (130 jig of the AS II fraction) . After incubation for 20 min at ara-CTP. 37Â°C,incorporation of labeled substrate into acid insoluble ma ara-CTP, 1-@-D-arabmofuranosylcytosine-5'-triphosphate ; dATP, terial was determined as described previously (8), except that 0.42 dCTP, dGTP, and dTTP, deoxynucleoside-5'-triphosphate deriva m@imole of ATP was added after the addition of 7% perchioric tives of adenine, cytosine, guanine, and thymidine ; dGMP, deoxy acid. Vessels in which incubation at 37Â°Cwas omitted served as guanine-5'-monophosphate. the control.

2064 CANCER RESEARCH VOL. 28

0 C 0 C 12 C 0 C

0 C 50 Id 0 I-. Id 4 4 S 0 -@ 0. 0 a. 0 U 0 z U z a. I- @0

as i.o 1.5 i.e 2.5 3.0

S 6 S 10 ara.ATP (pM)

dATP (pM) Charts 7A-C. Inhibition of calf thymus DNA polymerase by ara-ATP. The reaction mixture (0.5 ml) contained : 50 mus phos phate buffer, pH 7.0, 4 mM MgCl2, 2 m@ 2-mercaptoethanol; 50 @Meach of dCTP and dGTP; 56 @iMdTTP@14C (1.35 X 106 cpm/ @imole); and 54 @tgof enzyme. In the experiment reported in Charts 7B and 7C, the concentration of dATP was 2.3 @tus.Other wise the concentrations of dATP and ara-ATP were as noted. ass 0 Chart 7A. â€¢â€”Oâ€”â€¢,noara-ATP; oâ€”@---o, +1.7 @Mann I 2. ATP. I ara-ATP, 9-p-r,-arabinofuranosyladenine-5'-tniphosphate ; dATP, 0 dCTP, dGTP, dTTP, deoxynucleeside-5'-tniphosphate derivatives I-.Id 4 of adenine, cytosine, guanine, and thymidine ; dTMP, deoxythy midine-5'-monophosphate. a.0 0 Uz in animal cells for long intervals, permitting considerable en a. largement of the cells without division (4) . After prolonged I- exposure to these agents at inhibitory concentrations, the cells irreversibly lose the power to multiply. Inhibited cells are also observed to suffer chromosome breaks and other abnormalities ara.ATP (jiM) (17) . If deoxycytidine is added to cells with ara-C or shortly thereafter, the inhibition is not maintained, i.e., the inhibition is earlier studies the type of inhibition was not clearly resolved, specifically prevented in the first instance or is reversed in the one of the plots suggesting â€œmixedâ€•inhibition while the other second. Thorough reversal studies have not been reported for suggesting competitive kinetics. In the present experiments, no ara-A. The effects of this agent are not simply prevented or such discrepancy was noted ; competitive inhibition was always reversed by deoxyadenosine, the specific metabolite whose syn observed. While an element of uncertainty remains (due in large measure to inherent difficulties in the enzyme assay, the initial thesis or utilization might be imagined to be influenced by ara-A. lag, and the incorporation observed when one deoxytniphos phate is omitted) , the interpretation seems reasonably unam ara-A is lethal to certain strains of E. coli at concentrations biguous. On the other hand, D-arabinosyl compounds do not comparable to those at which it is inhibitory for animal cells, inhibit RNA synthesis in vivo, and neither mammalian nor the i.e., 1-2 x 10@'us. In such strains this agent inhibits DNA syn bacterial RNA polymerase are inhibited (1, 8 ; Table 1). thesis relatively specifically. On the other hand, ara-C has not It appears appropriate at this point to summarize the data been found to inhibit E. coli at concentrations comparable to bearing on various hypotheses that have been advanced con those at which it inhibits animal cells, i.e., 1-2 x 10Â°us. ann-C cerning the mode of action of D-arabinOsyl nucleosides at the has been used to inhibit DNA synthesis in E. coli at 4 x 10@ enzymatic level. M (13), but it is not clean that the mechanism of this inhibition ara-C and ara-A inhibit DNA synthesis in animal cells in is at all comparable to that seen in animal cells. With these tissue culture and are being used widely to produce such an biologic and biochemical data in mind, we can look to some of effect. The compounds do not affect RNA and protein synthesis the hypotheses on the sites of action of ara-C and nra-A.

OCTOBER 1968 2065

Hypothesis of the Inhibition of Ribonucleotide Reductase. triphosphates in the cell (3) . ara-CTP and ara-ATP are corn In animal cells and in E. coli, this enzyme effects the reaction: petitive inhibitors of dCTP and dATP respectively in DNA ribonucleoside thioredoxin deoxyribonucleoside synthesis by mammalian DNA polymerase.4 The K@for these diphosphate Mg+ + , allosteric effector diphosphate substances are reasonably low and suggest that these analogs The patterns of allosteric effectors and inhibitors are similar might be effective on this reaction within animal cells. The but slightly different for the reductase derived from tumor cells specificity of â€œreversalâ€•ofnra-C by deoxycytidine is in agree (16) or from E. coli. dATP is a powerful inhibitor with both ment with this mechanism. The complexity of reversibility of types of enzyme. inhibitions produced by nra-A may arise from the additional It has been proposed that a nucleotide derived from ara-C inhibition of nibonucleotide reductase produced by ara-ATP, inhibits the conversion of CDP to dCDP by this enzyme and an inhibition which results in multiple deficiencies of the deoxy that a product derived from deoxycytidine overcomes this site ribonucleotides. The latter type of inhibition has not been of the metabolic block. However, a tumor cell reductase is very demonstrable with ara-CTP. poorly inhibited in vitro by the diphosphate or triphosphate Neither ara-CTP nor ara-ATP inhibits the DNA polymerase of ara-C at relatively high levels, e.g., 50% inhibition was not of E. coli. This is consistent with the relative lack of inhibition reached at concentrations below 2 mus (16). On the other hand, produced by ann-C in E. coli, even in strains deficient in deoxy ara-ADP and ara-ATP, although less inhibitory than dATP, cytidine deaminase (unpublished data) . The inhibition of E. produced 50% inhibition at 0.07 mus in the conversion of CDP coli by ara-A can probably be explained by the inhibition of the to dCDP. It appears possible that ara-ATP does produce a bacterial ribonucleotide reductase by ara-ATP. This point has physiologically significant inhibition of the reductase, whereas not yet been tested experimentally, although dATP is known there is no evidence at present to indicate a comparable role to be a powerful inhibitor of the bacterial enzyme. for nucleotides of ara-C. It is not yet known for any system with certainty why a Hypothesis of the Incorporation of Aranucleotides in DNA. relatively specific inhibition of DNA synthesis is lethal in bac It has been reported by several workers that highly redioactive teria and eucaryotic cells and leads to chromosome breaks in ara-C is incorporated at low levels into cellular RNA and DNA the latter. Even@if the in vitro mechanisms of inhibition re (3, 19) . Such radioactive materials decompose to readily corded above are proven to operate within the cell, there will utilizable metabolites which can enter the nucleic acids. It is still be a serious gap in knowledge between the observed site obviously important for studies purporting to demonstrate in and mechanism of inhibition and the lethal consequences of corporation of an arabinonucleoside to be chemically rigorous, such arrest of DNA synthesis. presenting evidence that the nucleic acid in fact contains a bound arabinonucleotide. A reasonable degree of rigor, for ex ACKNOWLEDGMENTS ample, requires the isolation of a 3'-phosphate of the fed arab We are indebted to Miss Teresita Chichioco for excellent tech inonucleoside. Unfortunately, this type of evidence has neither nical assistance. been obtained nor have the data on incorporation so far ob tamed been reported in convincing detail (see Ref. 4). REFERENCES It is relevant that no incorporation of ara-ATP or ara-CTP 1. Cardeilhac, P. T., and Cohen, S. S. Some Metabolic Properties has been detected with the DNA polymerase or RNA poly of Nucleotides of l-ft-D-Arabinofuranosylcytosine. Cancer Res., merase of animal cells (8) or E. coli (1, 8) . Nor does E. coli @4:1595â€”1603,1964. polynucleotide phosphorylase incorporate ara-ADP (12) or 2. Cassani, G. R., and Bollum, F. J. Replication of Homopoly ara-CDP (1) into polynucleotides. deoxynucleotides. Federation Proc., @5:708, 1966. Nevertheless, if it is supposed that incorporation into DNA 3. Chu, M. Y., and Fisher, 0. A. Comparative Studies of Leu of ara-CTP or ara-ATP does occur by the action of DNA kemic Cells Sensitive and Resistant to Cytosine Arabinoside. polymerase, several possibilities can be envisaged: Biochem. Pharmacol., 14: 333â€”341,1965. (a) Thearanucleotidesarewithinthepolynucleotidechains. 4. Cohen, S. S. Introduction to the Biochemistry of D-Arabinosyl Nucleosides. Progr. Nucleic Acid Res. Mol. Biol., 5: 1â€”88, In this instance it might be asked why such incorporation would 1966. be lethal. In any case, such internal incorporation, if it does 5. Doering, A. M., Jansen, M., and Cohen, S. S. Polymer Syn exist, cannot be extensive. thesis in Killed Bacteria: Lethality of 2', 3'-Dideoxyadenosine. (b) The aranucleotides are terminal, blocking extension of J. Bacteriol., 9@: 565â€”574,1966. the polynucleotide chains. In such a case, the DNA of inhibited 6. Doering, A., Keller, J., and Cohen, S. S. Some Effects of D cells would be expected to be irreversibly blocked. In fact, after Arabinosyl Nucleosides on Polymer Synthesis in Mouse Fibro extended inhibition of bacteria by nra-A or of mouse fibroblasts blasts. Cancer Res., 26: 2444â€”2450,1966. by ara-A or ara-C, mere separation of the inhibitor from the 7. Evans, J. S., Musser, E. A., Mengel, G. D., Forsblad, K. R., cells permits a rapid incorporation of thymine or thymidine and Hunter, J. H. Antitumor Activity of 1-fl-D-Arabinofurano @ into DNA (5, 6) . Thus, cells inhibited with these agents do not sylcytosine Hydrochloride. Proc. Soc. Exptl. Biol. Med., 106: 350-353,1960. appear to contain irreversibly jammed polydeoxynucleotides. Hypothesis of the Inhibition of DNA Polymerase. This 4 Concurrent studies by Kimball and Wilson, using a crude ex hypothesis to explain the inhibitions produced by the ara tract of Ehrlich ascites tumor cells, also indicate that DNA poly nucleosides is most consistent with presently existing data. merase i.s inhibited by ara-CTP, and this inhibition is reversed by ara-C and possibly ara-A are converted to the corresponding deoxycytidine-5'-triphosphate (12).

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8. Furth, J. J., and Cohen, S. S. Inhibition of Mammalian DNA 15. Mathews, C. K., and Cohen, S. S. Inhibition of Phage-induced Polymerase by the 5'-Triphosphate of 9-p-n-Arabinofurano-. Thymidylate Synthetase by 5-Fluonodeoxyunidylate. J. Biol. syladenine. Cancer Res., @7:1528â€”1533,1967. Chem., P@38:367-370, 1963. 9. Furth, J. J., and Cohen, S. S. Effect of the 5'-Triphosphates 16. Moore, E. C., and Cohen, S. S. Effects of Arabinonucleotides on of 1-p-n-Arabinofuranosylcytosine and 9-fl-D-Arabinofurano Ribonucleotide Reduction by an Enzyme System from Rat syladenine in the Enzymatic Synthesis of Nucleic Acid in Tumor. J. Biol. Chem., 242: 2116â€”2118,1967. Mammalian Tissues. Proc. Am. Assoc. Cancer Res., 9: 23, 1968. 17. Nichols, W. W. In Vitro Chromosome Breakage Induced by 10. Furth, J. J., and Ho, P. The Enzymatic Synthesis of Ribo Arabinosyladenine in Human Leucocytes. Cancer Res., @4: nucleic Acid in Animal Tissue. I. The Deoxyribonucleic Acid 1502-1505, 1964. Directed Synthesis of Ribonucleic Acid as Catalyzed by an En 18. Renis, H. E., and Johnson, H. G. Inhibition of Plague Form zyme Obtained from Bovine Lymphosarcoma Tissue. J. Biol. ation of Vaccinia Virus by Cytosine Arabinoside Hydrochlo Chem., p40: 2602â€”2606,1965. ride. Bacteriol. Proc. (Am. Soc. Microbiology), p. 140, 1962. 11. Furth, LI., Rosenberg, M., and Ho, P.L. Comparison of the 19. Silagi, S. Metabolism of 1-fl-D-Arabinofuranosylcytosine in Requirements for Ribonucleic Acid Synthesis with the Re L Cells. Cancer Res., 25: 1446â€”1453,1965. quirements for Deoxyribonucleic Acid Synthesis in Animal 20. Underwood, G. E. Activity of 1-p-s-Arabinofuranosylcytosine Tissues. J. Cell Physiol., 69: 209â€”217,1967. Hydrochloride Against Herpes Simples Keratitis. Proc. Soc. 12. Kimball, A. P., and Wilson, M. J. Inhibition of DNA Poly Exptl. Biol. Med., 111: 660-664, 1962. merase by fl-n-Arabinosylcytosine and Reversal of Inhibition 21. Yoneda, M., and Bollum, F. J. Deoxynucleotide-Polymenizing by Deoxycytidine-5'-Triposphate. Proc. Soc. Exptl. Biol. Med., JPII: 429-432, 1968. Enzymes of Calf Thymus Gland. I. Large Scale Purification 13. Lark, C., and Lark, K. G. Evidence for Two Distinct Aspects of Terminal and Replicative Deoxynucleotidyl Transfenases. of the Mechanism Regulating Chromosome Replication in J. Biol. Chem., p40: 3385â€”3391,1965. Escherichia coli. J. Mol. Biol., 10: 120-136, 1964. 22. York, J. L., and LePage, G. A. A Proposed Mechanism for the 14. Lucas-Lenard, J. M., and Cohen, S. S. The Inhibition of Action of 9-p-s-Arabinofuranosyladenine as an Inhibitor of the Polynucleotide Phosphorylase by Certain Substrate Analogues. Growth of Some Ascites Cells. Can. J. Biochem. Physiol., Biophys. Acta 123: 471â€”477,1966. .44:19-28,1966.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1968 American Association for Cancer Research. Inhibition of Mammalian DNA Polymerase by the 5′ -Triphosphate of 1-β-d-Arabinofuranosylcytosine and the 5′ -Triphosphate of 9-β-d-Arabinofuranosyladenine

J. J. Furth and Seymour S. Cohen

Cancer Res 1968;28:2061-2067.

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