Cytotoxic and Biochemical Effects of Thymidine and 3-Deazauridine on Human Tumor Cells1

[CANCER RESEARCH 44, 2534-2539. June 1984]

Arnold Lockshin, John T. Mendoza, Beppino C. Giovanella,2 and John S. Stehlin, Jr.

St. Joseph Hospital Laboratory for Cancer Research, Houston, Texas 77002

ABSTRACT cells, thymidine-resistant human malignant B-cells have much lower ratios of these activities (6, 19). The cause of thymidine- Cytotoxicity and perturbations of the deoxyribonucleoside tri- induced inhibition of DNA synthesis in mammalian cells has been phosphate pools caused by thymidine were studied in thymidine- ascribed to depletion of the intracellular dCTP pool brought about sensitive and -resistant human tumor cells. Incubation with 1 mw by allosteric effects of dTTP on ribonucleotide reducÃase(2, 33, thymidine reduced cell viability by more than 90% in the three 36). Other investigators have concluded that the decrease in the sensitive cell lines (two melanomas and one adrenal carcinoma) dCTP pool is too small to account for growth inhibition of L1210 and reduced the growth rate without decreasing the viability of murine leukemia cells (14) and that high levels of dTTP (and/or resistant LO melanoma cells. Thymidine (1 HIM)greatly increased other dNTPs3) interact with a regulatory protein for DNA polym- the ratio of the deoxythymidine 5'-triphosphate to deoxycytidine 5'-triphosphate pools in the sensitive cells compared to LO cells erase (37). Yet another interpretation is that imbalance of the pyrimidine dNTP pools brought about by supranormal levels of and also caused larger relative increases in the pool sizes of deoxyguanosine 5'-triphosphate and deoxyadenosine 5'-tri- exogenous thymidine causes misincorporation of bases into DNA, which leads to mutagenicity and cytotoxicity (4). phosphate in the sensitive compared to the resistant cells. 3-Deazauridine inhibits the growth of microbial and murine 3-Deazauridine, known to inhibit synthesis of deoxycytidine 5'-triphosphate and cytidine 5'-triphosphate in other cell lines, tumor cells in culture and of transplantable mouse tumors in vivo (5, 39). In patients with acute leukemia, infusions of 3-deazauri potentiated the cytotoxicity of thymidine for thymidine-sensitive dine resulted in hematological improvement but no significant BE melanoma and LO cells. In LO cells, 3-deazauridine (50 UM) responses (41). 3-Deazauridine 5'-triphosphate, an intracellular decreased the intracellular pool of deoxycytidine 5'-triphosphate metabolite of 3-deazauridine, inhibits CTP synthetase from mu to the level obtained with 1 rnw thymidine. Lower concentrations rine tumor cells and from calf liver, thereby lowering intracellular of deoxycytidine as compared to cytidine were required to pro levels of CTP and indirectly those of dCTP (5, 26, 27). A second tect BE and LO cells against the cytotoxicity of thymidine plus ary effect of 3-deazauridine in these cells is inhibition by 3- 3-deazauridine. Deoxycytidine also was more effective than was deazauridine 5'-diphosphate of the reduction of CDP to dCDP cytidine in preventing loss of cell viability after exposure to mediated by ribonucleoside reducÃase (5). Cytidine and less thymidine or to 3-deazauridine individually. In these human mel effectively deoxycytidine or uridine counteract growth inhibition anoma cells, ribonucleotide reducÃase may be a major site of caused by 3-deazauridine in murine adenocarcinoma Ca755 and action of thymidine, of 3-deazauridine, and of both drugs in in L1210 cells, whereas Ihymidine or deoxyuridine do not have combination. this effect (5,26). 3-Deazauridine is not incorporated into nucleic These results indicate that in human tumor cells the cytotoxic acids (39). effect of thymidine correlates with greater perturbations of the The major known effects of thymidine and 3-deazauridine pyrimidine deoxyribonucleoside 5'-triphosphate pools and that metabolites on pyrimidine nucleotide biosynthesis are presenled thymidine and 3-deazauridine, which independently reduce the intracellular levels of deoxycytidine 5'-triphosphate, act synergis- in Chart 1. Other biochemical effecls include feedback inhibilion by dTTP of Ihymidine kinase (15) and dCMP deaminase (25) and tically against human tumor cells. inhibilion by 3-deazauridine and ils 5'-monophosphate nucleo- lide of cylidine deaminase and dCMP deaminase, respectively INTRODUCTION (10). We are exploring the potenlial Iherapeulic value of combining High concentrations of thymidine selectively kill certain human fhymidine wilh agenls lhal deplete the dCTP pool. Using human tumor cells In vitro (21), and continual infusions of thymidine tumor cell lines, we are interesled in determining (a) if differences cause regression of human tumor heterotransplants in nude in Ihe inlracellular dNTP pools are consislenl wilh sensilivily and (athymic) mice (22). Clinical evidence of antitumor effect has resislance to thymidine, and (b) if drugs which deplete dCTP been observed with T-cell leukemias, lymphomas, and metastatic pools enhance Ihe cyloloxicily of Ihymidine. We report here our melanomas when plasma levels of about 1 mw thymidine have studies wilh 4 human cell lines, 3 derived from melanomas and been maintained (3, 7, 19, 20). Thymidine must be converted to one from an adrenal carcinoma. A preliminary report of some of dTTP in order to cause cytotoxicity or cytostasis. The sensitivity Ihese findings has been published (24). of human malignant T-cells to thymidine has been attributed to the high levels of thymidine-phosphorylating activity relative to dephosphorylating activity in these cells. Compared to these T- MATERIALS AND METHODS

1 Supported by The Stehlin Foundation for Cancer Research and by the Sid Cells. The human melanoma cell lines CA, BE, and LO were obtained from this laboratory (13), and the SW-13 human adrenal tumor cells were Richardson Foundation, Dallas, TX. 2 To whom requests for reprints should be addressed. Received November 14, 1983; accepted March 14, 1984. 3The abbreviation used is: dNTP, deoxyribonucleoside 5'-triphosphate.

2534 CANCER RESEARCH VOL. 44

3 Deazaundine Thymidine polycytidylate copolymer was used for the dCTP assays. The polymeri zation reactions were conducted in 96-well culture plates, and aliquots (90 ÃÃl)ofeach reaction mixture were spotted on discs of Whatman GFA RNA ||X 1/\dCTPA1Ã•tDeoxycytidineglass fiber filters (pretreated with cold 5% trichloroacetic acid containing \*Â¿f 1% inorganic sodium pyrophosphate) placed on a manifold. After 10 min, ^v DAUOP each dish was washed 12 times with the cold trichloroacetic acid- /CTP \ICTP SynthetaseX T inorganic sodium pyrophosphate solution followed by 4 washes with MiT. 'JTP Â«}tCytidineI| 100% methanol and 3 with diethyl ether. For each cell line, dNTP DAUTP \\ determinations were performed on extracts from 3 separate experiments tRibonucleolÃde with at least 2 flasks/treatment. ReducÃasettt/dTTP/DNA RESULTS

Effect of Thymidine on Growth Rate and Cell Viability. At plating densities of 500,000 cells/75 sq cm flask, the logarithmic

Chart 1. Major effects of thymidine and 3-deazauridine metabolites on pyrimi- phase growth rates of all 4 cell lines were approximately the dme nudeotide biosynthesis, x, enzyme inhibition. See text for details. DAUDP, 3- same (doubling times ranging from 29 to 33 hr). Under these deazauridine 5'-diphosphate; DAUTP, 3-deazauridine 5'-trÃphosphate. conditions, 48-hr incubation of CA, BE, or SW-13 cells with 1 HIMthymidine prevented cell division and decreased cell viability from Dr. A. Leibovitz (23). Cells were maintained as monolayer cultures in Eagle's minima! essential medium containing penicillin (50 units/ml), by more than 90%. LO cells divided more slowly in the presence of thymidine (see below), but their viability was enhanced after streptomycin (50 ng/ml), and supplemental 2 mw glutamine, all from 24-hr incubation and was not impaired for up to 72 hr (Table 1). Grand Island Biological Co. (Grand Island, NY), and 10% heat-inactivated fetal calf serum from Reheis Chemical Co. (Phoenix, AZ). Cultures were Effect of Thymidine on Intracellular dNTP Levels. Chart 2 incubated at 37Â°in a humidified 5% CO2 atmosphere. Periodic tests for shows the changes in the dNTP pools caused by incubating Mycoplasma contamination were negative. these cell lines with 1 mwi thymidine. After 24 hr, the dTTP pools Chemicals. [mef/7y/-3H]dTTP (60 Ci/mmol), [5-3H]dCTP (30 Ci/mmol), of the sensitive lines increased 60-fold or more, whereas the [8-3H]dGTP, (18 Ci/mmol), and [8-3H]dATP (17 Ci/mmol) were purchased relative increase in the LO cells was less than half these values. â€¢%. from ICN Clinical Radioisotopes Div. (Irvine, CA). Lyophilized noncovalent double-stranded copolymers of deoxypolyadenylate-deoxypolythymidy- Table 1 late, deoxypolyinosinate-deoxypolycytidylate. and deoxypolyguanylate- Effect of 1 mu thymidine on viability of human tumor cells deoxypolycytidylate were obtained from P-L Biochemicals, Inc. (Milwau Cells (500.000) were plated in 75-sq cm culture flasks. After 24 hr, fresh medium kee, Wl), and Micrococcus /ÃºfeosDNA polymerase (EC 2.7.7.7) (specific with or without 1 mu thymidine was added. The cells were harvested at the times indicated and were replated for colony formation. activity, 469 units/mg protein) was purchased from Miles Laboratories control8)CA0.38 viability (fraction of (Elkhart, IN). Thymidine, 3-deazauridine, and other nonlabeled chemicals Thymidine treatment were from Sigma Chemical Co. (St. Louis, MO). (hr)24 Growth and Cytotoxicity Assays. Cells were seeded in 75-sq cm Â±0.06" Â±0.03 Â±0.07 Â±0.06 culture flasks and allowed to plate for 24 hr, at which time the medium 48 0.08 Â±0.04 0.05 Â±0.01SW-130.740.08 Â±0.02LO1.361.05 Â±0.10 was removed and replaced with fresh medium with or without drugs. At 72Cell 0.006 Â±0.003BE0.24 0.95 Â±0.04 indicated times, cells from one-half of the flasks were harvested with 8 Incubation without thymidine. trypsin to determine cell number with a Model ZBI Coulter Counter 6 Mean Â±S.E. (Coulter Electronics, Inc., Hialeah, FL). Aliquots from these flasks were replated as single-cell suspensions (2000 CA cells, 1500 BE cells, 4000 10.000-8.000 LO cells, or 2000 SW-13 cells) in 25-sq cm flasks to determine cell viability as measured by colony formation. Plating efficiencies were calculated as that fraction of cells seeded which formed colonies of 30 5,0004.000W 30T)S cells or more. For control (untreated) cells, these values for all 4 lines were similar (0.2 to 0.35). Some experiments with BE and LO cells were conducted by plating in 25-sq cm flasks for colony formation studies G!2.000uÂ£ 10OS,600 JdGTP*-iA_^^CA l}-p1"*dATP|â€¢: â€” only. The cells were allowed to attach overnight, and medium was added Â°Ã•Ãœ Â«14005 with or without drugs. After incubation equivalent to about 2 generation 1200-? times under these conditions for control cells (120 hr for BE cells and 72 1000-800400200^dTTP1ÃŒl1ifcNiiÃ•-.--1iPflJJ hr for LO cells), fresh medium was added, and colonies were allowed to 12001000200 form. Each experiment was performed at least 2 times. [i dNTP Assays. Flasks (75 sq cm) which had been seeded and incu T|iiSW-13 bated in parallel with those used for cell growth and viability assays were washed with phosphate-buffered saline [Na2HP04 (2.0 g/liter)-KH2PO4 11â€”pÂ£SW-13 (0.4 g/liter)-NaCI (8.0 g/liter)-KCI (0.2 g/liter)| and then quickly treated with 60% methanol for 20 min at 20Â°followed by cold 80% methanol to BE_ji.-â€¢."Â¿_^Ã„dCTPÃ±rEs^Ti^Ã±~^.ILO CA!Â«^1BEJWÃ LO5040 Chart 2. Effect of 1 mu thymidine on dNTP pools in human tumor cells. extract the Â¡ntracellulardNTPs (29, 38). Recovery of added dNTPs was Logarithmically growing cells (500.000/75-sq cm flask) were incubated with or 85 to 114%. In agreement with recent studies with human cells (12), we without thymidine. After 24- and 48-hr incubation, the cells in one-half of the flasks found no significant conversion of deoxynucleosides, or deoxynucleoside were harvested for determination of cell number. Cells in the other flasks were mono- or diphosphates to dNTPs, as has been observed when HeLa extracted with methanol for measurement of dNTP pools by the DNA polymerase assay. Note different scales on the ordinales. The dCTP levels in thymidine-treated cells were extracted with cold methanol (31). SW-13 cells were below the limits of detection (2 pmol/10* cells). D, no thymidine; The dNTPs were quantitated by a modification of the DNA polymerase E3,24-hr incubation with 1 HIMthymidine; SI, 48-hr incubation with 1 mw thymidine; assay described by Kinahan ef al. (17). The deoxypolyguanylate-deoxy- bars, S.E.

JUNE 1984 2535

The dCTP pools in untreated CA, BE, and SW-13 cells were in all 4 cell lines. These changes were also relatively greater in lower than those of untreated LO cells. After incubation with the thymidine-sensitive cells as compared to the resistant LO thymidine, the dCTP pools of the sensitive cells decreased by cells. 80% or more, whereas the decrease in LO cells was only 42%. Cytotoxicity of Thymidine plus 3-Deazauridine. Thymidine The pyrimidine dNTP pools did not change further after 48-hr plus 3-deazauridine caused greater than additive cytotoxicity to incubation. Thymidine caused the purine dNTP pools to increase the thymidine-sensitive BE melanoma cells (Chart 3). Thymidine (1 mM) reduced the growth rate of LO cells but not their viability. 1.4-,1.2 In combination with 3-deazauridine, the cytotoxicity was greater than additive (Chart 4). Similar results were obtained when the -g cells were plated in low number for colony formation assays only. Short-term (4-hr) incubation of 3-deazauridine prior to or LO~UÂ¡z1 simultaneously with thymidine also resulted in greater than ad ditive toxicity (Chart 5). The sequence of thymidine followed by .-â€¢ 3-deazauridine resulted in additive cytotoxicity only (data not OÂ«-â€¢z.pÂ¿ shown). Reversal of Cytotoxicity. Very low (0.04 to 1 /Â¿M)concentra 0.6-W5 tions of deoxycytidine counteracted the cytotoxicity of thymidine plus 3-deazauridine to BE cells; cytidine at these concentrations

0.4 was less effective (Chart 6). Much higher concentrations of IIIE0.2-f.m'â€¢ â€¢'râ€¢â€¢{â€¢â€¢W////////AÃcytidine iÃ®Â«SÃ• as compared to deoxycytidine were required to prevent cytotoxicity caused by thymidine plus 3-deazauridine to LO cells (Chart 7). Cytosine at concentrations of up to 1 HIMhad no effect aSÃThymidine aÃ± on the cytotoxicity of this drug combination. As shown in Table 2, lower concentrations of deoxycytidine as compared to cytidine 43 (//M) 0 40 15 4 40 15 also prevented cytotoxicity caused by thymidine or by 3-dea Deaiaund.ne I/,M| 0 10101010 zauridine alone to these cells. With one exception (thymidine at Chart 3. Effect of thymidine and 3-deazauridine on the viability of BE cells. Cells were plated and the following day incubated with the drugs indicated for 120 hr. a concentration of 16 mw), deoxycytidine at a concentration of Fresh medium was then added, and colonies were allowed to form. Bars, S.E. no greater than 5 /*M counteracted 95 to 100% of the cytotoxicity of thymidine and/or 3-deazauridine at the concentrations shown 6.0 -, in Charts 6 and 7 and Table 2. At the concentrations used in Control these experiments, neither deoxycytidine nor cytidine alone had any effect on the viability of BE or LO cells. Effects of 3-Deazauridine and Thymidine plus 3-Deazauri dine on dNTP Pools in LO Cells. 3-Deazauridine (50 /JM) de creased the intracellular dCTP pool by 63%, which was similar

. -: f?, nh

Ã•Ã•0.8 Thymidine plus U 3-Deazauridine ulE O Â§ 0.6 0.6 96 p 0.4

Viability of LO Cells 0.2 - Treatment After 48 Hr Incubation (Fraction of Control)

Thymidine 3-Deazauridine Control Thymidine 3 Dea/auridine Thymidine 3-Deazauridine 0.440.07Â±Â±Â±0.130.080.03 PluÂ» Followed By Thymidine plus 3-Deazauridine Thymidine 3-Deazauridine1.14 Chart 5. Effect of simultaneous or sequential administration of 3-deazauridine Chart 4. Effect of thymidine plus 3-deazaundine on the growth and viability of and thymidine on the viability of LO cells. Plated cells (80,000/25-sq cm flask) were LO cells. Plated cells (500,000/75-sq cm flask) were incubated with thymidine (1 incubated with 1 mM thymidine for 48 hr and/or 0.5 mm 3-deazauridine for 4 hr, HIM) and/or 3-deazauridine (50 ^M) and, at the times indicated, were harvested to where indicated. For thymidine plus 3-deazauridine, both drugs were added to the determine cell number and were replated for colony formation. â€¢,control; O, cells for 4 hr followed by thymidine alone for an additional 44 hr. After incubation, thymidine; A, 3-deazauridine; D, thymidine plus 3-deazauridine; bars, S.E. the cells were harvested and replated for colony formation Bars, S.E.

2536 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1984 American Association for Cancer Research. Thymidine and 3-Deazauridine to the decrease caused by 1 mw thymidine. The combination of zauridine had no effect on the dNTP assay as determined by thymidine plus 3-deazauridine did not lower further the dCTP incorporation of known amounts of dNTPs added to assay pool or increase the ratio of dTTP to dCTP (Table 3). 3-Dea- mixtures.

1.2-, DISCUSSION

Thymidine (1 mw) markedly altered the dNTP pools, especially the levels of the pyrimidine dNTPs, in the 3 thymidine-sensitive cell lines. After incubation with thymidine for 24 hr, the ratio of dTTP to dCTP changed from about 4 in untreated cells to 1900 o z in BE cells, to 3100 in CA 1 cells, and to an even higher value in SW-13 cells. In the thymidine-resistant LO melanoma cells, the lu increase in the dTTP pool was smaller; the dCTP pool decreased only to about the dCTP levels in untreated CA, BE, or SW-13 cells; and the ratio of dTTP to dCTP rose from 2.2 to only 130. These data suggest a correlation between cytotoxicity and the I magnitude of the changes in the dTTP pools and/or the dCTP ThymldmÂ«|pM| 80 80 80 80 80 80 80 200 200 200 200 200 200 200 pools. The purine dNTP pools, especially dGTP, also increased 3-DeazauridinÂ«(,.M| is 25 26 25 26 2B 25 BO so so 50 50 60 50 to a greater extent in the thymidine-sensitive cells, and it cannot Dnoiycytulim. (;.Ml O.O4 O.2O 1.0 0.040.20 1.0 be excluded that these changes contributed to the observed Cyttdine (..Ml 0.040.20 1.0 0.040.20 1.0 cytotoxicity. Longer incubations of sensitive cells with thymidine Chart 6. Effect of deoxycytidine or cytidine on the cytotoxicity of thymidine plus increased the cytotoxicity without further altering the dNTP pools 3-deazauridine for BE cells. Plated cells were incubated with the compounds indicated for colony formation assays. Control (untreated) flasks had 362 Â±9 indicating that events subsequent to DNA precursor pool imbal colonies. Bars, S.E. ance were the proximate cause of cell death. The question arose as to whether an agent which depletes the dCTP pool might potentiate the effect of thymidine on human tumor cells. For these experiments, we selected 3-deazauridine, which lowers the intracellular pool of dCTP (and CTP) and is not

Table 3 Effect of thymidine, 3-deazauridine, or thymidine plus 3-deazauridine on dNTP pools of LO melanoma cells Cells were plated as described in Table 1, and fresh medium with or without drugs was added 24 hr later. After 48-hr incubation, the cells of some flasks were trypsinized and harvested for determination of cell number with a Coulter Counter. Cells from other flasks were extracted with methanoi. and the dNTP pools were measured by the DNA polymerase assay. dNTPpools(pmoi/1o"cens)

Â¡S3 TreatmentControl ThymidmelmM; 2.0 2.0 2.0 3.Â« 3.8 3.Â« 3.Â«3.6 3.6 3.6 Â± 0.04" 3-DMuuridiiw (mM) 0.15 0.160.16 030 0.30030 0.300.30030030 Â±4.0 Â±0.8 Â±10 Thymidine (1 mM) 1870 Â±220 18 Â±0.2 122 Â±3.0 130 Â± 8.0 Dnoxycy1idine(,,Mi 0.20 1.0 1.0 4.0 3-Deazauridine(50MM)Thymidine0.3' 211 Â±212260 16Â±0.822 11Â±1.788 82 Â± Cylidine |,;M) 1.0 4.0 10 20 4.0 10 20 80 (1 mm) + 3- Â± 30dCTP43 Â±1.5dGTP10Â±4.0dATP63 Chart 7. Effect of deoxycytidine or cytidine on the cytotoxicity of thymidine plus 3-deazauridine for LO cells. Plated cells were incubated with the compounds (50MM)dTTP105deazauridine indicated for colony formation assays. Control (untreated) flasks had 1098 Â±71 Mean Â±S.E. colonies. Bars, S.E.

Table 2 Prevention by deoxycytidine or cytidine of cytotoxicity caused by thymidine or by 3-deazauridine BE cells or LO cells were plated and incubated with the mixtures indicated as described in "Materials and Methods."

Concentration of added Thymidine 3-Deazauridine nucteoskJe required to de crease cytotoxicity by 50%

with drug with drug atone*000.04 alone"0.06 CellsBE dine(MM)5 (MM)>100

BE 1.016.0 0.5 50 Â±0.036 BE 0.2 2 BE 40200Viability0.45 Â±0.040.33 0.05 0.5 LO Â±0.01 5 60 LO 4.0Viability 0.25 Â±0.04(MM)200 0.5 10 LOHIM4.0 Â±0.01Deoxycyti 0.5Cytidine 2 * Relative to controls, 362 Â±6 colonies for BE cells and 565 Â±15 colonies for LO cells. Â°MeanÂ±S.E.

JUNE 1984 2537

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1984 American Association for Cancer Research. A. Lockshin et al. incorporated into nucleic acids (5, 39). The combination of thy- for DMA synthesis and cell division may be depleted even though midine plus 3-deazauridine was synergistic against thymidine- the measured dCTP pool is reduced only by about 50% (11). sensitive BE cells and resistant LO cells. With LO cells, it was Postulation of such compartmentalization in LO cells would found that this drug combination was synergistic when 3-dea correspond to the hypothesis that greater perturbation of the zauridine was incubated prior to or simultaneously with thymi- dTTP:dCTP ratio contributes to greater cytotoxicity and that the dine. The lack of synergism when thymidine preceded 3-dea potentiation of thymidine is related to further depletion of a critical zauridine may have been due to the rapid normalization of dNTP dCTP pool in these cells. Further work, including enzyme studies, pools and resumption of DMA synthesis when exogenous thy will be required to elucidate more fully the mechanisms of action midine is removed from mammalian cells (2, 8). Removal of of thymidine plus 3-deazauridine and of each drug separately on thymidine but not of 3-deazauridine from LO cells was accom human melanoma cells. panied by rapid reversal of growth inhibition.4 Of potential practical importance is the effectiveness of very The cytotoxicity caused by thymidine plus 3-deazauridine was low concentrations of deoxycytidine in protecting cells from the prevented by lower concentrations of deoxycytidine as com toxicity of thymidine (42) as well as of 3-deazauridine and of pared to cytidine, suggesting that this drug combination depletes both drugs in combination. These results suggest that plasma a critical dCTP pool which can be repleted with exogenous levels of deoxycytidine may affect critically the responsiveness deoxycytidine. Deoxycytidine also was more effective than was to these drugs of tumors in animals or in patients. cytidine in protecting BE and LO cells against the toxicity of In these studies, we did not examine whether the combination thymidine or 3-deazauridine alone. Because of the known allo- of thymidine and 3-deazauridine selectively kills tumor cells rel steric inhibition of ribonucleotide reducÃaseby dTTP (33), it would ative to sensitive normal tissues or whether this drug combination be expected that lower concentrations of deoxycytidine as com acts synergistically against normal cells. Certain human and pared to cytidine would prevent thymidine toxicity (40). The murine tumor cells compared to nonmalignant cells of the same results with 3-deazauridine contrast, however, with earlier work type are considerably more sensitive to thymidine (21, 32). To with murine Ca755 adenocarcinoma and L1210 leukemia cells our knowledge, similar comparisons using 3-deazauridine are which showed that cytidine was much more effective than was not available. According to one study, however, 10 to 20 times deoxycytidine in protecting against 3-deazauridine (5,26). These higher concentrations of 3-deazauridine 5'-diphosphate were results led to the suggestion that CTP synthetase is the major produced in implanted L1210 cells as compared to the normal site of inhibition by metabolites of 3-deazauridine. Further sup mouse tissues (9). Increased anabolismi of 3-deazauridine in port for this conclusion was obtained from inhibition studies of tumor tissues would be expected to enhance tumor cell kill L1210 cell-free extracts and of purified CTP synthetase from calf relative to normal cells. If 3-deazauridine would potentiate the liver (26). For the BE and LO melanoma cells, a different principal cytotoxicity of thymidine without negating the greater sensitivity mechanism is indicated. of some tumor cells to this nucleoside, then this combination From the known pathways of pyrimidine nucleotide metabo could have potential therapeutic benefit. lism (Chart 1), cytidine can be a precursor for dCTP via ribonu cleotide reducÃase. Deoxycytidine, on the other hand, is not ACKNOWLEDGMENTS metabolized to CTP because the reduction of ribonucleoside diphosphates is irreversible enzymatically (28). Conversion of The authors wish to thank Dr. Peter V. Danenberg tor his critical reading of this manuscript, Carla Warneke for assistance in some of these experiments, Rosalva deoxycytidine to cytidine via cytosine is unlikely, as cytosine is Reyes-Hada for the art work, and Betty Harris and Gail Hirschhorn for expert not metabolized by mammalian cells (1,18), although it crosses preparation of this manuscript. the cell membrane (43). We found that cytosine had no effect on the cytotoxicity caused by thymidine plus 3-deazauridine. In BE REFERENCES and LO cells, there is no apparent requirement for supplemental 1. Bendich, A., Getter, H., and Brown, G. B. A synthesis of isotopie cytosine and CTP (from cytidine) even after considerable loss of cell viability a study of its metabolism in the rat. J. Biol. Chem., 777: 565-570,1949. due to thymidine and/or 3-deazauridine treatment, and the data 2. Bjursell, G., and Reichard, P. Effects of thymidine on deoxyribonucleoside strongly suggest that dCTP becomes critically deficient. Although triphosphate pools and deoxynbonucleic acid synthesis in Chinese hamster ovary cells. J. Biol. Chem., 248: 3904-3909,1973. 3-deazauridine metabolites may inhibit CTP synthetase in these 3. Blumenreich, M., Andreeff, M., Murphy, M. 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Arnold Lockshin, John T. Mendoza, Beppino C. Giovanella, et al.

Cancer Res 1984;44:2534-2539.

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