Depression of Thymidylate Synthetase Activity in Response to Cytosine Arabinoside1

Home , Deoxycytidine

[CANCER RESEARCH 32, 1160-1169, June 1972] Depression of Thymidylate Synthetase Activity in Response To Cytosine Arabinoside1

DeWayne Roberts and Ellen V. Loehr St. Jude Children's Research Hospital, Memphis, Tennessee 38101 Â¡D.R., E. V. L.J and Department of Pharmacology, University of Tennessee Medical Units, Memphis, Tennessee 38101 [D. R.J

SUMMARY Cytosine arabinoside induces remission of acute granulocytic leukemia (11). After the administration of Depression of thymidylate synthetase activity by cytosine cytosine arabinoside to patients, changes in the enzyme arabinoside in CCRF-CEM cells was correlated with a blocking activity pattern of leukemic leukocytes occur (32). After drug of precursor incorporation into RNA and with cell lysis. With administration, a decrease in thymidylate synthetase activity is increasing concentrations of cytosine arabinoside in the observed as early as 1 hr and persists for 24 hr in some culture media, a proportional decrease of uridine patients. A concentration-dependent decrease in thymidylate incorporation into RNA and decrease in thymidylate synthetase activity also follows the addition of cytosine synthetase activity occurred and was accompanied by lysis of arabinoside to CCRF-CEM cultures (33). the cells. These effects continued to develop with drug Methotrexate elevates thymidylate synthetase activity in concentrations in excess of that required to block thymidine leukocytes from patients with acute leukemia (32), in rat liver incorporation into DNA. The addition of deoxycytidine at and transplantable tumors (27), and in cells of LI210 or concentrations that failed to reverse inhibition by cytosine CCRF-CEM cultures (32, 33). The simultaneous addition of arabinoside of thymidine incorporation into DNA reversed the cytosine arabinoside and methotrexate to CCRF-CEM cultures drug effects on uridine incorporation, thymidylate synthetase modulated the effect of each drug on thymidylate synthetase activity, and cell lysis. Inhibition of precursor incorporation activity (33). Intermediate levels of thymidylate synthetase into RNA was also observed with isotopically labeled glycine activity, between the level observed for each drug alone, and hypoxanthine. Cytidine could not be substituted for developed in cultures receiving both methotrexate and deoxycytidine to reverse drug effects on uridine incorporation cytosine arabinoside. The level of enzyme activity can be and thymidylate synthetase. Deoxycytidine blocked altered by changing the ratio of the 2 drugs. The intracellular accumulation of cytosine arabinoside, while methotrexate-induced elevation of thymidylate synthetase cytidine was ineffective in blocking the drug uptake. We activity in leukocytes correlates with recovery of the capacity conclude that depression of thymidylate synthetase activity by by these drug-intoxicated cells to incorporate deoxyuridine cytosine arabinoside is a secondary drug action requiring drug into DNA (32). By blocking the methotrexate-induced concentrations in excess of that required to inhibit thymidine elevation of thymidylate synthetase activity with a second incorporation into DNA and is attributed to an inhibition of drug, such as cytosine arabinoside, we considered that the RNA synthesis that results from the action of the drug. inhibition of DNA synthesis by methotrexate might be prolonged and the oncolytic action of the drug might be increased. INTRODUCTION The CCRF-CEM lymphoblast line provides an experimental system in which the response of thymidylate synthetase to Cytosine arabinoside, 1-0-D-arabinofuranosylcytosine, is an cytosine arabinoside and methotrexate resembles these analog of both deoxycytidine and cytidine. The functional drug-induced changes in patients' leukocytes (33). The present relationship to deoxycytidine has been emphasized by the studies examine the molecular basis for cytosine arabinoside inhibition of DNA synthesis (2, 6, 7, 9, 15, 17, 21-24, 32, 35) depression of thymidylate synthetase activity. and the inhibition of DNA polymerase (14, 16, 25, 29) incorporation into DNA (6, 7, 16, 29, 35, 38), and because its cytotoxicity and oncolytic activity can be reversed by deoxycytidine (4, 5, 12, 15, 31). Cytolysis by cytosine MATERIALS AND METHODS arabinoside is dependent on conversion to a nucleotide in a The CCRF-CEM cell line with lymphoblastic morphology reaction catalyzed by deoxycytidine kinase (6, 10, 19, 30. 36). The structural analogy with cytidine is expressed by incorpora originated from the blood specimen of a child with tion into RNA (6, 7, 35). lymphosarcoma that converted to acute lymphocytic leukemia (13). Cells were grown in suspension or spinner cultures as previously described (33). Drugs in aqueous solution were 1Supported by Research Grant CA-11148 and Clinical Center Grant added in 0.4-ml aliquots to 40 ml of Eagle's minimal essential CA-08480 from the National Cancer Institute, NIH, and by American medium plus 10% fetal calf serum at the time of inoculation Lebanese Syrian Associated Charities. with approximately 2 X IO7 cells, and, unless otherwise Received February 12, 1971 Â¡acceptedMarch 6, 1972.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1972 American Association for Cancer Research. Cytosine Arabinoside Depression of Enzyme Activity indicated, these suspension cultures were incubated for 20 hr o H2o at 37Â°.At the end of this time, aliquots of the cultures were â€¢¿OXMtiMCA A 0.03uMCA A O.luMCA removed for measurement of thymidine-methyl-3 H D I.O)lMCA incorporation into DNA; uridine-5-3H, glycine-l-14C, and e.ooor- hypoxanthine-8-3H incorporation into nucleic acids or RNA; 5,000 - and thymidylate synthetase activity. The thymidylate synthetase activity in homogenates of the cells was assayed as previously described (33). Thymidine incorporation into DNA was assayed with 2 fiM concentration, which saturated the rate-limiting reaction in these cells (33). Uridine incorporation into RNA was measured by the same general technique as thymidine incorporation into DNA, except that 1.25 juCi of uridine-5-3H, 26.1 Ci/mmole, were added in 25-julaliquots to 2-ml aliquots of the cultures. The trichloroacetic acid-insoluble radioactivity was collected on 0.45-jum MF Millipore cellulose ester filters and counted by liquid scintillation technique. This was a tracer level of uridine; and although incorporation was linear for more than 30 min, the rate-limiting step in its utilization was not saturated with Chart 1. The inhibition of thymidine incorporation into DNA by substrate. In control cultures, less than 10% of the CCRF-CEM cells after exposure to various concentrations of cytosine trichloroacetic acid-insoluble product remained insoluble after treatment with l N NaOH for 2 hr at 37Â°. arabinoside (CA). Hypoxanthine incorporation into nucleic acid was assayed with 1 MM hypoxanthine-8-3H, 0.42 ÃŸCi,by the same jeI Â»3hr INCUBATION technique as that used for uridine and thymidine. With -AINCUBATIONâ€” Oâ€”O 2O hr cytosine arabinoside, total intracellular pool and incorporation -^P5040302010i00.â€¢ Control-'\\-\\\\- hr after the cell suspension was washed with 0.9% NaCl solution on a Millipore filter and after an additional trichloroacetic acid wash was assayed as the total intracellular pool and the acid-insoluble pool, respectively, by liquid scintillation counting. The acid-soluble pool was calculated from the difference between these 2 measured pools. \' For the double labeling experiment, incorporation of glycine-l-I4C and hypoxanthine-8-3 H into RNA was assayed r01 *â€”¿""^T T by a different technique. The cells were collected by centrifugation at 500 Xg and washed with 0.9% NaCl solution O.IIOCYTOSINE 1 and cold 5% trichloroacetic acid. The isotope incorporated ARABINOSIDE (uM) into RNA was released into solution by suspension of the acid-insoluble precipitate in l N NaOH for 2 hr at 37Â°.The 1.0 B DNA and protein were reprecipitated by addition of an equivalent volume of l N HC1 to neutralize the NaOH at 0Â° 0.8 and then addition of trichloroacetic acid to give a final concentration of approximately 7%. After centrifugation at 500 X g for 5 min at 4Â°,aliquotsof the supernatant fluid were "o Â°'6 removed for liquid scintillation counting. Correction was made for ' 4C spilloverinto the 3H counts. 0.4

RESULTS 0.2

Time Course of Cytosine Arabinoside Inhibition of Thymidine Incorporation. Cytosine arabinosideinhibition of 001 0.1 K) thymidine incorporation into DNA by CCRF-CEMcells is time CYTOSINE ARABINOSIDE(;j.M) and concentration-dependent (Chart 1). The time coordinate Chart 2. The effect of various concentrations of cytosine arabinoside represents the period between addition of cytosine on CCRF-CEM cultures incubated for 3 or 20 hr in the presence of arabinoside, 0.01 to 10 /uM,to freshly inoculated cultures of drug. A, inhibition of thymidine incorporation into DNA; B, inhibition CCRF-CEM cells and the addition of labeled thymidine. For of cell division and the development of cell lysis.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1972 American Association for Cancer Research. De Wayne Roberts and Ellen V. Loehr the "zero" time point, approximately 15 min elapsed at room substrate as the control, respectively. With 0.1 to 10 nM temperature between addition of drug and initiation of the cytosine arabinoside, thymidine incorporation remained 30-min incubation with tritiated thymidine. Incomplete completely inhibited at 20 hr. inhibition of thymidine incorporation was observed with these Cytosine arabinoside, 0.01 to 0.04 Â¿/M,did not completely zero-time studies. A 3-hr lag occurred after transfer of the cells inhibit growth of the culture (Chart 2B). At higher drug before the control cultures began to increase their rate of concentration, 0.1 to 10 ;uM, partial lysis of the inoculum thymidine incorporation. An earlier and more complete occurred, and after 20 hr of exposure to 10 ^M cytosine inhibition of thymidine incorporation was observed with arabinoside the cultures contained only 20% of the cells added higher concentrations of cytosine arabinoside. With 0.01 //M in the inocula. Cell lysis became more pronounced as the drug cytosine arabinoside, the capacity to incorporate thymidine concentration was increased above the level required to inhibit into DNA was recovered; initiation of recovery was noted at 9 thymidine incorporation into DNA. The latter was completely hr for cultures exposed to 0.03 juM cytosine arabinoside. inhibited at 3 and 20 hr by 0.1 nU cytosine arabinoside. Cytosine Arabinoside Lysis of Cells and Inhibition of DNA Correlation between Cell Lysis, Depression of Uridine Synthesis. Recovery of the capacity to incorporate thymidine Incorporation, and Decrease in Thymidylate Synthetase into DNA and the effect on growth of cultures exposed to Activity after Cytosine Arabinoside. Chart 3, A, B, C, and D, cytosine arabinoside, 0.01 to 10/uM, were examined in greater shows the effects of various concentrations of cytosine detail at 3 and 20 hr after drug addition to freshly inoculated arabinoside on undine incorporation into RNA, thymidylate cultures (Chart 1A). After 20 hr with 0.01 or 0.02 MM synthetase activity, and the correlation between the effect of cytosine arabinoside, cultures incorporated thymidine at 69 the drug on these 2 parameters and cell lysis. As the and 46% of the rate observed for the control, although at 3 hr concentration of cytosine arabinoside was increased, uridine parallel cultures had incorporated 10 and 6%, as much incorporation into RNA was more effectively inhibited; 75%

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02 46 8 10 12 14 O 2 4 6 8 IO 12 14 THYMIDYLATE SYNTHETASE (p moles/hr/g PROTEIN) THYMIDYLATE SYNTHETASE ( ÃŸmolesTMP/hr/g protein) Chart 3. The effect of various concentrations of cytosine arabinoside (CA) on CCRF-CEM cultures grown for 20 hr in the presence of drug. A, depression of undine incorporation into RNA; B, depression of thymidylate synthetase activity; C, correlation between the depression of uridine incorporation and thymidylate synthetase activity; and D, the correlation between cell count and thymidylate synthetase activity.

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â€¢¿2.7000-, Chronological Sequence of Cytosine Arabinoside Effects. 1 oâ€”â€”Â«Control Inhibition of uridine incorporation into RNA was observed â€¢¿â€”â€¢CytosineArabinoside(IO>iM) Â§6000 within 2 hi after the addition of 10 /^M cytosine arabinoside to CCRF-CEM cultures (Chart 4/1). Lysis of cells, when assayed | 5000 - by decrease in cell number, required slightly longer for expression and was observed between 4 and 8 hr after drug Ã¨4000 H addition (Chart 45). Inhibition of uridine incorporation into RNA began to level off 4 hr after drug addition. Maximum cell I 3000 H lysis occurred between 4 and 8 hr after drug addition and _ 2000 - ^ Jxpt I dropped only slightly thereafter. Cellular debris was observed O _c in cultures as early as 2 hr after drug addition, although the g loco decrease in cell number occurred later. 's o Cytosine Arabinoside Inhibition of Precursor Incorporation into Nucleic Acid. Drug inhibition of precursor incorporation 468 IO 12 Hours into RNA was more dramatically shown with hypoxanthine than with uridine (Table 1). Cytosine arabinoside, 10 Â¿iM, inhibited 80% of the uridine incorporation and all incorporation of hypoxanthine into nucleic acids. Incubation for 2 hr at 37Â°in l N NaOH resulted in hydrolysis of 95% of Chronological Change in Cell Number of CCRF-CEM Cultures the trichloroacetic acid-insoluble uridine products in the o o Control control cultures and 82% of the hypoxanthine products. From â€¢¿-â€¢¿CytosineArabinoside I.O-i these data, the conclusion was drawn that 95 and 80% of the ExptI trichloroacetic acid-insoluble products of uridine and hypoxanthine, respectively, were incorporated into RNA by the control cultures. The trichloroacetic acid-insoluble products in the control cultures, which were not hydrolyzed by NaOH, presumably represent incorporation into DNA. As -â€¢ExptI expected, cytosine arabinoside completely inhibited the incorporation of uridine and hypoxanthine into DNA. The continued, although reduced, incorporation of pyrimidine nucleotide precursor into RNA is attributed to an exchange of o terminal cytidine nucleotides of tRNA. Resolution of cytoplasmic RNA by sucrose gradient showed that cytosine arabinoside more effectively inhibited uridine incorporation Chart 4. The time course of cytosine arabinoside effect on uridine into rRNA than that into the slower sedimenting fractions. incorporation and cell count. A, cultures of CCRF-CEM cells were Concentration Curve for Hypoxanthine Incorporation into preincubated at 37Â°with cytosine arabinoside, and at the indicated Nucleic Acid. Cytosine arabinoside inhibition of precursor time, aliquote were removed and incubated with labeled uridine; B. the cell count for cultures incubated with cytosine arabinoside. Expt, Table 1 experiment. The effect on RNA synthesis of 20 hr of exposure to cytosine arabinoside CCRF-CEM cultures were preincubated for 20 hr with 10 MM inhibition occurred at 10 juM concentration (Chart 3A). At 0.1 cytosine arabinoside and then incubated for 30 min with uridine-5-3H Ã•IMcytosine arabinoside, an elevation of uridine incorporation or hypoxanthine-8-3H. The incorporation into trichloroacetic into RNA occurred. This has been repeatedly observed. acid-insoluble product was assayed by collecting cells on a Millipore filter and washing them with 0.9% NaCI solution and trichloroacetic Thymidylate synthetase activity was more effectively acid. Alkali-stable and trichloroacetic acid-insoluble product was depressed at higher concentrations of cytosine arabinoside assayed by collecting cells and washing them with 0.9% NaCI solution, (Chart 3B). An elevation of thymidylate synthetase activity resuspending the cell pellet for 2 hr at 37Â°in l N NaOH, precipitating was not observed with 0.1 pM cytosine arabinoside, although with 5% trichloroacetic acid, and collecting cells on a Millipore filter for in other studies a slight stimulation was observed with lower liquid scintillation counting. concentrations of this drug (28). Uridine incorporation and Incorporation of precursor into RNA thymidylate synthetase activity increased together. As the (cpm/million cells) concentration of cytosine arabinoside was lowered from 10 to Uridine-5-3H Hypoxanthine-8-3H 1 /Â¿Mand then to between 1 and 0.4 /uM, the incorporation of Condition uridine was inhibited less than was thymidylate synthetase Trichloroacetic acid-insoluble product activity (Chart 3C). These changes in thymidylate synthetase Drug-treated 663 18 activity and uridine incorporation into RNA occurred at drug Control 3563 9689 concentrations in excess of that required completely to inhibit Residual product following alkaline hydrolysis Drug-treated O O thymidine incorporation into DNA but correlated with cell Control 165 1772 lysis by the drug (Chart 3D).

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1972 American Association for Cancer Research. De Wayne Roberts and Ellen V. Loehr incorporation into RNA was also observed when hypoxanthine rate. Pretreatment for 2 hr with cytosine arabinoside, 0.01 Â¿tM, was substituted for uridine. An extracellular concentration of reduced glycine incorporation to approximately 70% of the 6 Â¿/Mhypoxanthine saturated the cellular capacity for pretreatment rate where the rate remained almost constant for incorporation into the nucleic acids (Chart 5). The the remaining 3 hr. Preincubation with 0.1 and 10 Â¡Â¿Mcytosine rate-limiting reaction for incorporation has not been arabinoside reduced glycine incorporation to approximately identified. Preincubation of cultures with cytosine arabinoside one-fourth the pretreatment rate. for 2.5 hr prior to addition of the hypoxanthine reduced In this same study, the rate of hypoxanthine incorporation incorporation into the nucleic acids, and saturation of the by control cultures increased approximately 50% with 1 hr of rate-limiting reaction was observed with 2 pM hypoxanthine. preincubation before substrate addition and remained If the cytosine arabinoside inhibition of hypoxanthine approximately constant throughout the remainder of the incorporation were the result of a larger acid-soluble purine study. With 0.01, 0.1, and 10 /Â¿Mcytosine arabinoside, the nucleotide pool, then higher, rather than lower, concentrations hypoxanthine incorporation rate after preincubation for 5 hr of hypoxanthine should have been required to equilibrate was approximately 10% of the initial rate. Glycine and isotopically with the acid-soluble pool. hypoxanthine incorporation into RNA was inhibited by Development of Cytosine Arabinoside Inhibition of concentrations of cytosine arabinoside lower than those for Hypoxanthine and Glycine Incorporation into DNA. In a the inhibition of uridine. double labeling study with glycine-l-14C and hypoxanthine- Deoxycytidine Reversal of Cytosine Arabinoside Action. 8-3H, CCRF-CEM cultures were preincubated for 0 to 5 hr The simultaneous addition of deoxycytidine at concentrations with 0.01, 0.1, or 10/L/Mcytosine arabinoside prior to addition between 0.2 and lOpM did not reverse the inhibitory effect of of the isotopically labeled precursors (Chart 6, A and B). The 10 /nMcytosine arabinoside on thymidine incorporation (Chart rate of glycine incorporation initially increased in the control 1A). In control cultures, which lacked cytosine arabinoside, cultures and then decreased to slightly below pretreatment high concentrations of deoxycytidine lowered the incorporation of exogenous thymidine into DNA. Although in the assay the initial extracellular concentration of thymidine 5000 r was 2 AIM,there is no assurance that deoxycytidine inhibition of thymidine incorporation may not be the result of the Control conversion of deoxycytidine to either deoxyuridine or dUMP. Deoxyuridine would compete with thymidine for conversion to nucleoside monophosphate by thymidine kinase. dUMP, if normally rate limiting in the de novo synthesis of TMP, could O. I>J-M CA increase synthesis by the de novo pathway and dilute the IO>j.MCA exogenous precursor of DNA. !>>MCA Deoxycytidine, 0.2 to 10 Â¡M,produced very little effect on the growth rate of control cultures (Chart IB). As shown 0 24 6 8 10 earlier, a 20-hr exposure of the cultures to 10 Â£iMcytosine Hypoxanthine (>Â¿M) arabinoside resulted in cell lysis. The addition of deoxycytidine reversed the lytic action of 10 Â¿/Mcytosine Chart 5. Substrate curve for hypoxanthine incorporation into nucleic acid. CCRF-CEM cultures were preincubated for 2.5 hr at 37Â°with arabinoside, and the reversal was proportional to the cytosine arabinoside (CA) prior to a 1-hr incubation with concentration of deoxycytidine. As would be expected in the hypoxanthine-8-3 H. The cultures contained 0.61 million cells/ml. All absence of thymidine incorporation into DNA, no increase in values are corrected for adsorption or trapping of precursor by cell number was observed following the addition of unincubated cell cultures. deoxycytidine to cultures containing cytosine arabinoside.

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1164 CANCER RESEARCH VOL. 32

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Chart 7. The observed metabolic changes in CCRF-CEM cultures after growth for 20 hr in the presence of 10 MMcytosine arabinoside (C4) and various concentrations of deoxycytidine (CdR) were: A, the absence of a reversal of the drug inhibition of thymidine incorporation into DNA; B, reversal of the drug-induced cell lysis; C, reversal of drug-depressed uridine incorporation into RNA; D, reversal of drug-depressed thymidylate synthetase activity; and E, relationship between deoxycytidine reversal of cytosine arabinoside depression of uridine incorporation and thymidylate synthetase activity.

Deoxycytidine caused a slight inhibition of uridine The addition of deoxycytidine to the control cultures incorporation when the deoxycytidine concentration was 10 produced no effect on thymidylate synthetase activity (Chart Â¿/Mbut produced no effect at lower concentrations (Chart ID). As the concentration of deoxycytidine was increased in 1C). Deoxycytidine reversed cytosine arabinoside inhibition of the cultures to which cytosine arabinoside had been added, the uridine incorporation into RNA. The reversal was biphasic level of thymidylate synthetase activity increased. At 10 ^M with respect to deoxycytidine concentration. In the presence deoxycytidine, an elevation of thymidylate synthetase activity of cytosine arabinoside, a stimulation of the utilization of was observed in the presence of cytosine arabinoside. uridine was noted with the higher concentrations of Deoxycytidine reversed cytosine arabinoside inhibition of deoxycytidine. uridine incorporation and depression of thymidylate

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Chart 8. The inability of cytidine to reverse cytosine arabinoside inhibition of uridine incorporation. The CCRF-CEM cells were incubated for 20 hr in medium containing 10 MM cytosine arabinoside (CA) and various concentrations of cyti dine before the addition of uridine-5-3 H.

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synthetase activity (Chart IE). At between 0.02 and 2 juM Table 2 deoxycytidine, the reversal of cytosine arabinoside inhibition The effects of deoxycytidine and cytidine on cytosine arabinoside uptake of uridine incorporation and the reduction of thymidylate Cultures of CCRF-CEM cells were incubated for 2 hr at 37Â°with 10 synthetase activity occurred in parallel. However, at higher MM cytosine arabinoside-5-3 H (0.87 MCi)and 10 MMdeoxycytidine or concentrations of deoxycytidine, 4 and 10 Â¡M, which 10 MMcytidine. Uptake was terminated by pouring the culture onto a stimulated 1 or both parameters in the presence of cytosine Millipore filter and washing it with 0.9% NaCl solution before counting arabinoside, a change in the relationship between these 2 by liquid scintillation tp^hnique. Values were corrected for absorption parameters was observed. These effects by deoxycytidine on of radioactivity at Time zero. cytosine arabinoside-induced cell lysis, inhibition of uridine Condition Uptake (cpm/million cells) incorporation, and the depression of thymidylate synthetase activity occurred without reversal of the inhibition of Cytosine arabinoside 1580 thymidine incorporation into DNA. Cytosine arabinoside + deoxycytidine 190 Twenty hr elapsed between the addition of deoxycytidine Cytosine arabinoside + cytidine 1572 to the cultures and the collection or removal of cells for study. This period of time could have permitted a conversion of deoxycytidine to ribosides, ribotides, or other products that total radioactivity retained by cells that received only a 0.9% possibly could have reversed the effect of cytosine arabinoside NaCl solution wash. on RNA synthesis and in turn could have led to the responses The simultaneous addition of 10/nM deoxycytidine with 10 attributed to deoxycytidine. Cytidine and its nucleotides /uM cytosine arabinoside reduced the drug content retained by would presumably be among the products of these reactions. the washed cells to approximately 10% of the level observed Cytidine Effect on Cytosine Arabinoside Action. The with drug alone (Table 2). The simultaneous addition of 10 addition of 10 to 100 Â¡J.Mcytidine blocked uridine juM cytidine with cytosine arabinoside produced no effect on incorporation into RNA by control cultures (Chart 8). intracellular accumulation of radioactivity. A competition for Presumably, this occurred as the result of cytidine transport between cytosine arabinoside and both cytidine and deamination, although no information was obtained to deoxycytidine was observed with a cytosine arabinoside- support this assumption. With the combination of 10 ^M. resistant subline of LI 210 (20). Competition between cytidine cytosine arabinoside and cytidine, 10 to 100 nM, a more and cytosine arabinoside was not observed in the present effective inhibition of uridine incorporation was observed than study. The 10 nM concentration of drug and of nucleosides, with cytosine arabinoside alone. With lower cytidine which was lower than the levels used in the earlier study, or concentration, 0.4 to 4 p\l, uridine incorporation into RNA differences in the relative rates of uptake and phosphorylation was close to the rate observed for the control cultures. These may be the basis for the variation observed with the 2 cell cultures incorporated uridine at the same rate as cultures with types. only cytosine arabinoside. Cytidine produced no effect on the Cytosine arabinoside was not detected in the acid-soluble level of thymidylate synthetase activity in cells from control labeled pool, which was resolved into 3 components by cultures or from cytosine arabinoside-inhibited cultures. chromatography (39) with 6 volumes 0.5 N NH4OH plus 10 Uptake of Cytosine Arabinoside. Although drug uptake was volumes isobutyric acid on Whatman No. 1 paper. not saturated by 10 /LiMcytosine arabinoside-5-3H, uptake of Deoxycytidine severly inhibited the accumulation of the drug was linear for more than 3 hr. Radioactivity was radioactivity into these 3 components. The present evidence linearly distributed with time into 2 pools. The trichloroacetic suggests that deoxycytidine reversal of drug effects on acid-insoluble pool comprised slightly less than 20% of the CCRF-CEM cells resulted from a competition for

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1972 American Association for Cancer Research. Cytosine Arabinoside Depression of Enzyme Activity deoxycytidine kinase, which blocked nucleotide formation this effect was masked by an inhibition of exogenous uridine and drug activation. utilization. If an increase in de novo purine nucleotide synthesis by cytosine arabinoside diluted the exogenous hypoxanthine, DISCUSSION then an increased utilization of glycine should have been observed, rather than its inhibition. Increases in the size of the Inhibition of DNA synthesis by cytosine arabinoside has purine nucleotide pools should have been reflected by an been widely accepted as the cytotoxic basis for the action of increase in the hypoxanthine concentration required to the drug. Therefore, effect of the drug on thymidine saturate the rate-limiting step if this reaction were beyond the incorporation into DNA was the first parameter examined for pool. If the pool were beyond the rate-limiting step, then correlation with the depression of thymidylate synthetase similar saturation kinetics would have been expected. The activity. Thymidine incorporation was depressed observation that lower concentrations of hypoxanthine were approximately 90% after 20 hr of exposure to 0.06 jUM required to saturate the cytosine arabinoside depressed cytosine arabinoside and was completely inhibited by 0.1 Â¿(M incorporation is incompatible with these possibilities. drug. This is the range of drug concentration in which the cell The observation that depression of uridine incorporation, number dropped below the inoculum level. With further thymidylate synthetase activity, and lysis of cells by cytosine increase in the concentration of cytosine arabinoside, more arabinoside were prevented by deoxycytidine is compatible pronounced cell lysis occurred. Lysis of the CCRF-CEM cells with the hypothesis of a 2nd drug action (7, 38) although occurred earlier and at lower drug concentrations than has Graham and Whitmore (16) suggest that some of these been reported for L-cells (15). secondary drug effects should be attributed to inhibition of With higher concentrations of cytosine arabinoside, greater DNA polymerase. Deoxycytidine decreased the conversion of than 0.1 Â¿zM,thedepression of thymidylate synthetase activity cytosine arabinoside to nucleotides, as expected (19, 30, 34). became more pronounced as the drug concentration was However, the decrease in nucleotide formation was increased. In addition, an increase in cell lysis and a decrease in inadequate, under the present conditions, to reverse the precursor incorporation into RNA were observed. The inhibition of thymidine incorporation. These results are depression of thymidylate synthetase activity, uridine compatible with the observations that cytidine could not incorporation into RNA, and increase in cell lysis were block the effect of cytosine arabinoside on thymidylate correlated. These correlations developed at concentrations of synthetase activity or precursor incorporation into RNA. cytosine arabinoside that were in excess of that required to Deoxycytidine and cytosine arabinoside are converted to their block thymidine incorporation. respective nucleoside monophosphate form by deoxycytidine Inhibition of precursor incorporation into RNA developed kinase. Cytidine is not a substrate for this enzyme. more slowly than inhibition of thymidine incorporation. This The pharmacological action of cytosine arabinoside has might be expected from the requirement for higher drug been generally attributed to an inhibition of DNA synthesis. concentrations to inhibit the former and from the observation Dosage scheduling with repeated small dosage of drug at that drug uptake was linear for 3 hr with 10 /uM drug. spaced intervals (26, 37) or prolonged infusions have been Incorporation of isotope from the drug into an acid-insoluble used to maintain intracellular drug concentrations for product was also linear for 3 hr and presumably represents inhibition of all cells entering the S phase during the course of cytosine arabinoside or its deaminated product, uracil therapy (11). The very favorable therapeutic indices observed arabinoside (8), since mammalian cells are apparently unable for the rodent systems with these schedules have probably to cleave the glycoside linkage. been achieved to some degree because of certain known Inhibition of uridine incorporation into RNA was biological and biochemical variations between normal and incomplete after 20 hr of preincubation with 10 Â¡Mcytosine neoplastic cells. The presumptions are: (a) stem cell pools of arabinoside. The residual uridine incorporation was into normal, nondividing, or "resting" cells are insensitive to the low-molecular-weight RNA and may have represented terminal drug until "stimulated" to synthesize DNA, while the general exchange on tRNA. Hypoxanthine incorporation was inhibited neoplastic population may resume cell division soon after 95% by this drug concentration in 5 hr, while glycine completion of mitosis; and (6) at low doses of cytosine incorporation was inhibited approximately 75%. arabinoside, the greater drug activation by sensitive, neoplastic The correlation between the increased cell lysis and cells permits attainment of inhibitory intracellular drug levels decreased uridine incorporation with elevation of drug at lower extracellular drug concentrations (20, 21). The rapid concentration may have been the primary result of an altered blood clearance and inactivation of cytosine arabinoside (1,9) cell membrane, but no information is available to substantiate requires repeated drug administration at short intervals to this consideration. The possibility that depression of precursor maintain optimal extracellular drug concentrations and to incorporation resulted from increases in nucleotide pool size expose cells to drug as they enter the S phase. Two sites of or from greater de novo synthesis appears unlikely. The cytosine arabinoside action have been postulated (7, 38). The biphasic effect of cytosine arabinoside on uridine relationship of inhibition of DNA synthesis and incorporation incorporation is compatible with the suggestion that inhibition into DNA to these 2 sites of action has not been evaluated of de novo pyrimidine biosynthesis may have occurred at with respect to drug incorporation into RNA or drug lower drug concentrations, an occurrence which would explain interference with cytosine nucleotide function in intermediary the observation of increased uridine incorporation at these metabolism. The rapid lysis of cells, which is associated with drug concentrations. As the drug concentration was increased, higher concentrations of cytosine arabinoside, indicates the

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presence of a 2nd biochemical lesion in this lymphoblastic cell Distribution in Normal and Neoplastic Tissues and line of human origin. The only present correlation between the Interrelationships of Deoxycytidine and 1-0-D-Arabinofuranosylcy- cell culture studies and the response of leukemic leukocytes tosine Phosphorylation. Mol. Pharmacol., 5: 358-375, 1969. following cytosine arabinoside on a twice-weekly schedule to 11. Ellison, R. R., Holland, J. F., Weil, M., Jacquillat, C., Boiron, M., patients is the observation that cytosine arabinoside depressed Bernard, J., Sawitsky, A., Rosner, F., Gussoff, B., Silver, R. T., thymidylate synthetase activity in some patients and in Karanas, A., Cuttner, J., Spurr, C. L., Hayes, D. M., Blom, J., Leone, L. A., Haurani, F., Kyle, R., Hutchison, J. L., Forcier, R. J., cultures. and Moon, J. H. Arabinosyl Cytosine: A Useful Agent in the In conclusion, the addition of cytosine arabinoside to Treatment of Acute Leukemia in Adults. Blood, 32: 507-523, CCRF-CEM cultures altered a number of parameters in 1968. addition to DNA synthesis. The assumed 2nd locus of drug 12. Evans, J. S., and Menge, G. D. The Reversal of Cytosine action on this cell line is less sensitive than DNA synthesis to Arabinoside Activity in Vivo by Deoxycytidine. Biochem. drug inhibition. Whether this secondary effect of cytosine Pharmacol., 13: 989-994, 1964. arabinoside can be attributed to drug incorporation into DNA 13. Foley, G. E., Lazarus, H., Farber, S., Uzman, B. G., Boone, B. A., and termination of chain elongation (38) or to incorporation and McCarthy, R. E. Continuous Culture of Human Lymphoblasts into RNA (7) is not known. The effect of these higher from Peripheral Blood of a Child with Acute Leukemia. Cancer, 18: 522-529, 1965. concentrations of cytosine arabinoside results in a rapid cell 14. Furth, J. J., and Cohen, S. S. Inhibition of Mammalian DNA lysis and is accompanied by changes in the synthesis of RNA Polymerase by the 5'-Triphosphate of 1-0-D-Arabinofuranosylcyto- and level of thymidylate synthetase activity, as well as the sine and 5'-Triphosphate of 9-/3-D-Arabinofuranosyladenine. Cancer level of other enzymes (34). The present results are compatible Res., 28: 2061-2067, 1968. with the interpretation that high concentrations of cytosine 15. Graham, F. L., and Whitmore, G. F. The Effect of arabinoside interfere with RNA synthesis, an interference 1-(3-D-Arabinofuranosylcytosine on Growth, Viability, and DNA which in turn leads to a cessation of thymidylate synthetase Synthesis of Mouse L-cells. Cancer Res., 30: 2627-2635, 1970. synthesis. 16. Graham, F. L., and Whitmore, G. F. Studies in Mouse L-cells on the Incorporation of 1-0-D-ArabinosyIcytosine into DNA and on Inhibition of DNA Polymerase by 1-(3-D-Arabinofuranosylcytosine ACKNOWLEDGMENTS S'-Triphosphate. Cancer Res., 30: 2636-2644, 1970. 17. Karon, M., Henry, P., Weissman, S., and Meyer, C. The Effect of The authors gratefully acknowledge the assistance of Dr. Bruce Sells 1-0-D-Arabinofuranosylcytosine on Macromolecular Synthesis in and Miss Annett Lassiter with the sucrose gradient studies of cytosine KB Spinner Cultures. Cancer Res., 26: 166-171, 1966. arabinoside inhibition or uridine incorporation into RNA. 18. Karon, M., and Shirakawa, S. The Locus of Action of 1-0-D-Arabinofuranosylcytosine in the Cell Cycle. Cancer Res., 29: 687-696, 1969. REFERENCES 19. Kessel, D. Some Observations on the Phosphorylation of Cytosine 1. Borsa, J., Whitmore, G. F., Valeriote, F. A., Collins, D., and Bruce, Arabinoside. Mol. Pharmacol., 4: 402-410, 1968. W. R. Studies on the Persistence of Methotrexate, Cytosine 20. Kessel, D., Hall, T. C., and Rosenthal, D. Uptake and Arabinoside and Leucovorin in Serum of Mice. J. Nati. Cancer Phosphorylation of Cytosine Arabinoside by Normal and Leukemic Inst., 42: 235-242, 1969. Human Blood Cells in Vitro. Cancer Res., 29: 459-463, 1969. 2. Brewen, J. G., and Christie, N. T. Studies on the Induction of 21. Kessel, D., Hall, T. C., and Wodinsky, I. Transport and Chromosomal Aberrations in Human Leukocytes by Cytosine Phosphorylation as Factors in the Antitumor Action of Cytosine Arabinoside, Exptl. Cell Res., 46: 276-291, 1967. Arabinoside. Science, 756: 1240-1241, 1967. 22. Kessel, D., and Shurin, S. B. Transport of Two Non-metabolized 3. Camiener, G. W., and Smith, C. G. Studies of the Enzymatic Deamination of Cytosine Arabinoside. I. Enzyme Distribution and Nucleosides, Deoxycytidine and Cytosine Arabinoside, in a Species Specificity. Biochem. Pharmacol., 14: 1405-1416, 1965. Sub-line of the L1210 Murine Leukemia. Biochim. Biophys. Acta, 4. Chaube, S., Kreis, W., Uchida, K., and Murphy, M. L. The 163: 179-187,1968. 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I. 28. Maley, G. F., and Maley, F. Regulatory Properties and Subunit

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Structure of Chick Embryo Deoxycytidylate Deaminase. J. Biol. Synthetase Activity in CCRF-CEM Cells. Cancer Res., 31: Chem., 243: 4506-4512, 1968. 1181-1187, 1971. 29. Momparler, R. L. Effect of Cytosine Arabinoside 5'-Triphosphate 35. Salagi, S. Metabolism of 1-0-D-Arabinofuranosy Icytosine in L-cells. on Mammalian DNA Polymerase. Biochem. Biophys. Res. Cancer Res., 25: 1446-1453, 1965. Commun., 54/465-471, 1969. 36. Schrecker, A. W. Metabolism of 1-0-D-Arabinofuranosylcytosine in 30. Momparler, R. L., and Fischer, G. A. Mammalian Deoxynucleoside Leukemia LI210: Nucleoside and Nucleotide Kinases in Cell-free Kinases. I. Deoxycytidine Kinase: Purification, Properties, and Extracts. Cancer Res., 30: 632-641, 1970. Kinetic Studies with Cytosine Arabinoside. J. Biol. Chem., 243: 37. Skipper, H. E., Schabel, F. M., Jr., and Wilcox, W. S. Experimental 4298-4304, 1968. Evaluation of Potential Anticancer Agents. XXI. Scheduling of 31. Renis, H. E., and Johnson, H. G. Inhibition of Plaque Formation of Arabinosylcytosine to Take Advantage of its S-phase Specificity Vaccinia Virus by Cytosine Arabinoside Hydrochloride. Bacteriol. Against Leukemia Cells. Cancer Chemotherapy Rept., 51: Proc., 45: 140,1962. 125-165,1967. 32. Roberts, D., Hall, T. C, and Rosenthal, D. Coordinated Changes in 38. Waqar, M. A., Burgoyne, L. A., and Atkinson, M. R. Biochemical Patterns: The Effect of Cytosine Arabinoside and Deoxyribonucleic Acid Synthesis in Mammalian Nuclei: Methotrexate on Leukocytes from Patients with Acute Incorporation of Deoxyribonucleotides and Chain-Terminating Granulocytic Leukemia. Cancer Res., 29: 571-578, 1969. Nucleotide Analogues. Biochem. J., 121: 803-809, 1971. 33. Roberts, D., and Loehr, E. V. Methotrexate and Cytosine 39. Wyatt, G. R. Separation of Nucleic Acid Components by Arabinoside Modulation of Thymidylate Synthetase Activity in Chromatography on Filter Paper. In: E. Chargaff and J. N. CCRF-CEM Cells. Cancer Res., 31: 457-462, 1971. Davidson (eds.), The Nucleic Acids, Vol. 1, pp. 243-265. New 34. Roberts, D., and Loehr, E. V. Elevation of Thymidylate York: Academic Press, Inc., 1955.

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DeWayne Roberts and Ellen V. Loehr

Cancer Res 1972;32:1160-1169.

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