Mechanism of Deoxycytidine Rescue of Thymidine Toxicity in Human T-Leukemic Lymphocytes

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[CANCER RESEARCH 40, 1718-1 721, May 1980] 0008-5472/80/0040-OOOOS02.00 Mechanism of Deoxycytidine Rescue of Thymidine Toxicity in Human T-Leukemic Lymphocytes

Richard M. Fox,1 Edith H. Tripp, and Martin H. N. Tattersall

Ludwig Institute for Cancer Research, University of Sydney. Sydney, New South Wales. 2006 Australia

ABSTRACT sustain an excessive accumulation of dTTP at low concentra tions of exogenous dThd (2, 4, 17). Cultured malignant human lymphocytes are highly sensitive to growth inhibition by thymidine (50% inhibitory dose = 10~5 In this paper, we report a study of dCyd reversal of dThd growth inhibition in a cultured human leukemic T-cell line highly M). Growth inhibition reflects sustained elevation of the deoxy- sensitive to dThd. We have correlated changes in intracellular thymidine 5'-triphosphate pool associated with secondary el dNTP during growth reversal and demonstrate that elevated evation of the deoxyguanosine S'-lriphosphale pool and reduc dTTP pools appear to inhibit cell growth via an allosteric effect tion in the deoxycytidine S'-triphosphate (dCTP) pool. Deoxy on ribonucleotide reducÃase rather than having a direct effect cytidine was capable of partially reversing thymidine growth on the DNA replicating process. inhibition at a concentration of 0.5 /Â¿M,and growth recovery was virtually complete at 8 fiM. The dCTP pool remained MATERIALS AND METHODS depressed until growth inhibition reversal by deoxycytidine was complete, and at a higher concentration of deoxycytidine the Cell Lines and Culture. The cell lines studied were: (a) dCTP rose above control levels, but the deoxythymidine 5'- CCRF-CEM, derived from a patienl wilh acule lymphoblaslic triphosphate and deoxyguanosine S'-triphosphate pools re leukemia and characterized as a T-cell type (7); and (b) LAZ- mained elevated. 007, an Epstein-Barr virally Iransformed lymphocyle wilh B- These results support the view that thymidine growth inhibi cell surface markers. These lines were generously provided by tion induces a critical deficiency of dCTP via allosteric inhibition Dr. H. Lazarus (Sidney Farber Cancer Inslilule, Boslon, Mass.). of ribonucleotide reducÃase rather than inhibiting DMA repli The cells, in suspension cullure, were grown in Roswell Park cation directly by elevated deoxythymidine S'-triphosphate or deoxyguanosine 5'-triphosphate pools. Memorial Inslilule Tissue Cullure Medium 1640 supplemenled wilh 10% felal calf serum. The lines had similar doubling times (= 24 hr). INTRODUCTION Isotopes and Chemicals. 3H-Labeled dNTP (dTTP, dCTP, dATP, and dGTP) were purchased from The Radiochemical dThd, at mw levels, will inhibit growth of most cultured Centre, Amersham, England. Unlabeled dTTP, dCTP, dATP, mammalian cells. This toxic effect is believed to reflect a and dGTP were purchased from Sigma Chemical Co., St. Louis, sustained increase in the dTTP pool, which allosterically affects Mo, and from P. L. Biochemicals, Milwaukee, Wis. Micrococcus ribonucleotide reducÃase. This results in increased levels of luteus DNA polymerase and tÃ©mplalesfor the DNA polymerase dGTP and reduction in the level of dCTP. The inhibition of cell assay for dNTP, poly(deoxyadenylate-deoxythymidylate) and growth by dThd2 is reversed by dCyd which increases the poly(deoxyinosinate-deoxycylidylate) were purchased from dCTP pool, suggesting that dCTP concentration is critical in Miles Laboralories, Elkhart, Ind. Unlabeled dCyd and dThd controlling DMA synthesis (1, 6, 8, 9). were obtained from Calbiochem, La Jolla, Calif. An alternative suggestion has been that dNTP pools may Deoxynucleoside Inhibition of Cell Growth. These were react with a regulatory protein for DMA polymerase, modulating performed as described previously by us (4). DNA replication. Studies using this regulatory protein isolated DNA Polymerase Assay for dNTP. This was performed as from calf thymus have shown that, in the presence of this described previously by us except lhal cells were exlracted protein, the activity of DNA polymerase a is inhibited by dGTP with 60% ethanol ralher lhan with 60% methanol (1 3). This in a concentration-dependent manner. It was proposed that assay is a modificalion of the melhod described by Soller and physiologically or pharmacologically induced increases in Handschumacher (11). Resulls are Ihe means of duplicale dNTP pools could inhibit DNA synthesis by this mechanism (5, assays. 12, 15). Recently, it has been observed that the growth of cultured RESULTS human leukemic lymphoblasts of T- and null-cell origin is Deoxynucleoside Sensitivity of the T-Cell Line. We have inhibited by concentrations of dThd some 2 orders of magni previously reported Ihis cell line to be highly sensitive lo dThd tude below rriM levels. The mechanisms of this increased sen sitivity appear to reflect a reduced catabolism of deoxynucleo- (50% inhibilory dose, 30 /IM) (4), and Ihese cells were also sides and/or deoxynucleotides, which allows these cells to found lo be sensilive lo deoxyguanosine wilh lesser degrees of sensilivily to deoxyadenosine and dCyd. The T-cell is more sensitive lo Ihese nucleosides than is the Epstein-Barr virus- ' To whom requests for reprints should be addressed. transformed B-lymphocyle line (Table 1). 2 The abbreviations used are: dThd. thymidine; dCyd, deoxycytidine; dNTP. deoxynucleotide triphosphates. Influence of dThd on dNTP Pools of the Sensitive T-Cell Received October 26, 1979; accepted February 1, 1980. Line. The dThd-sensilive T-cell line (CEM) and the dThd-re-

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1980 American Association for Cancer Research. dCyd Rescue of dThd Toxicity sistant B-cell line (LAZ-007) were incubated with dThd (60 Table 3 Influence of dCyd incubation on T (ALL)3 CCRF-CEM cell dNTP pools /Ã•M),and at 1 and 48 hr the dNTP pools were assayed. The sensitive T-cell line shows a prompt elevation in the dTTP pool Cells were grown in suspension culture in Roswell Park Memorial Institute Culture Medium 1640 supplemented with 10% fetal calf serum and maintained at 1 hr which is sustained at 48 hr. This is accompanied by a in log growth. Cells were diluted with fresh medium to 2 x lOVml and grown for marked although lesser rise in the dGTP pool at 1 hr and 24 hr, and dCyd was added (10 /IM and 0.1 mM). At 48 hr, cells were harvested sustained until at least 48 hr, while the dATP level falls at 1 hr, by centrifugation and extracted with ethanol and the dNTP pools were measured by the DNA polymerase assay (11, 13). rising to just above control levels at 48 hr. By contrast, the levelslOjiMdCydof control dCTP pool is depressed at 1 hr and remains depressed at 48 hr (Table 2). 0.170 dCyd66 The changes in dNTP levels in the resistant B-cell line (LAZ- dATP 007) are less marked. A slight rise in the dTTP pool occurred dCTP 302 284 dGTP 285 180 at 1 hr, but the level returned to normal at 48 hr. dTTP% 265mw 243 Rescue of dThd Growth Inhibition by dCyd. dCyd alone has ALL, acute lymphoblastic leukemia. no effect on the growth of the dThd-sensitive cell line GEM at concentrations of <0.1 mM (Table 1). The effect of simultane Table 4 Influence of dCyd reversal of dThd growth inhibition of T (ALL)" CCRF-CEM cell ous addition of dCyd (3 fiM to 0.1 mM) on dThd (60 JIM) growth dNTP pools inhibition was examined. This concentration of dThd sup Cells were grown in suspension culture in Roswell Park Memorial Institute pressed growth to 32% of control, but 3 U.MdCyd reversed this Culture Medium 1640 supplemented with 10% fetal calf serum and maintained inhibition to 82% of control growth, and higher doses (>10 in log growth. Cells were diluted with fresh medium to 2 x 105 /ml and grown for 24 hr, and dThd plus or minus dCyd was added at the concentrations indicated. JUMdCyd) completely restored growth. At 48 hr after the addition of deoxynucleosides, the cells were harvested by Influence of dCyd Reversal of dThd Growth Inhibition on centrifugation and extracted with ethanol. and the dNTP pools were measured dNTP Pools. dCyd alone at growth noninhibitory doses (10 by the DNA polymerase assay (11, 13). /Â¿Mto0.1 mw) lead to 2- to 3-fold elevation of the dCTP, dTTP, valuesExperimentdThd % of control and dGTP pools and reduction of the dATP to 66 to 70% of control levels (Table 3). dThd alone caused a marked elevation /IM)dThdalone (60 of the dTTP and dGTP pool, little change in the dATP pool, and at:I.(60 /IM) + dCyd reduction in the dCTP pool to 40% of control value (Table 4). 3i/MII. 10flMIII. The lowest level of dCyd used in this experiment (3 /Â¿M)partially 30^MIV. 0.1 mwdATP98103534959dCTP4024346248355dGTP825712450687442dTTP15201250523869923 Table 1 ALL, acute lymphoblastic leukemia. Deoxynucleoside sensitivity of cultured lymphocytes Cells were grown in suspension culture in Roswell Park Memorial Institute Culture Medium 1640 supplemented with 10% fetal calf serum and maintained reversed dThd growth inhibition without significantly altering in log growth. Cells were diluted with fresh medium to 2 x 10Vml and grown for dNTP levels, and in particular the dCTP pool remained low. 24 hr. deoxynucleosides were added (0.1 to 5 /iM). and at 120 hr cell counts Higher concentrations of dCyd completely reversed growth were made. The percentage of inhibition of growth was then calculated, and the 50% inhibitory dose was calculated. inhibition and led to elevation of dCTP above control, but only 50% inhibitory dose (M) slightly lowered the highly elevated dTTP and dGTP pools. Despite rescue of growth, the dATP pool fell to =50% of control, a finding similar to that seen after incubation with dCyd lineT Cell nosine20 sine(mM)0.3>3dCyd (mM)0.3>3 (ALL)3CCRF-CEMB alone (Table 3). /IM2 fiM2 The finding that dCyd (3 /IM) could partially reverse dThd (Epstein-Barr virus- mMDeoxygua-mwDeoxyadeno- growth inhibition, without altering dNTP pools, in particular transformed) LAZ-007dThd30 without elevating the dCTP pool was surprising. This phenom ALL, acute lymphoblastic leukemia. enon was therefore examined in more detail. Cells were incu bated with dThd (60 U.M),and a concentration range of dCyd Table 2 that was capable of giving minimal to complete reversal of dThd Influence of dThd incubation on dNTP pool levels in cultured lymphocytes toxicity was used (0.5 to 16 JUMdCyd). The dTTP and dCTP Cells were grown in suspension culture in Roswell Park Memorial Institute Culture Medium 1640 supplemented with 10% fetal calf serum and maintained pools were assayed at 1 and 48 hr (Chart 1). At 0.5 Â¡IMdCyd, in log growth. Cells were diluted with fresh medium to 2 x 10Vml and grown for the dThd-inhibited growth was reversed from 26% of control 24 hr, and dThd was added (60 JIM). At 1 and 48 hr after the addition of dThd, growth rate to 37% with progressive reversal to 94% at 16 ^M the cells were harvested by centrifugation and extracted with ethanol, and the dNTP pools were measured by the DNA polymerase assay (11, 13). dCyd. T(ALL)a CCRF-CEM B (Epstein-Barr virus-trans In the cells treated with dThd without dCyd rescue, the dCTP formed) LAZ-007 pool had fallen to 51 % at 1 hr and 44% at 48 hr. At 1 hr, all doses of dCyd used (0.5 to 16 Â¡IM)elevated the dCTP pool to ofcontrolsAfter ofcontrolsAfter normal or supranormal levels (116 to 168% of control). How (pmol/10ecells)25.514.32053.6%48hr11963450545Control(pmol/106cells)4.54.62.816.7%1hr7287120286After48hr928262131ever, at 48 hr, the dCTP pool did not reach control levels until 1hr7061207417After 8 fiM dCyd was used, despite the fact that lower concentrations dATPdCTPdGTPdTTPControllevels of dCyd were partially reversing growth inhibition. At >8 /AM dCyd, and dCTP rose =2-fold of control levels. The dTTP pool in the cells treated with dThd alone was All, acute lymphoblastic leukemia. markedly elevated at 1 (760%) and 48 (1069%) hr. In cells

MAY 1980 1719

cell growth and cause sustained elevation of the dTTP pool associated with secondary elevation of the dGTP pool and reduction in the dCTP pool. Little change is seen in the dATP pool. These findings are similar to those reported for most o . mammalian cell lines (1, 6, 8, 9) requiring rriM levels of dThd for growth inhibition. The increased sensitivity of the malignant lymphocyte lines is believed to reflect deficiencies in deoxynucleoside or deoxynucleotide catabolism allowing sustained dTTP elevation at low concentrations of dThd (2,4,17). These observations suggest that the mechanism of dThd inhibition in the sensitive leukemia lines and relatively resistant mammalian cell lines, i.e., dNTP pool imbalance via feedback inhibition of ribonucleotide reducÃase, is identical.

0.5 1.0 2.0 16.0 The changes in dNTP pools following incubation with dThd d Cyd (um) appear to be explained by the known in vitro allosteric prop erties of ribonucleotide reducÃase. It is believed that the in Chart 1. Cells were grown in suspension culture in Roswell Park Memorial Institute Culture Medium 1640 supplemented with 10% fetal calf serum and creased cellular content of dTTP following exposure to dThd maintained in log growth. Cells were diluted with fresh medium to 2 x 10Vml inhibits the reduction of CDP to dCDP, thereby reducing the and grown for 24 hr, and dThd plus or minus dCyd was added at the concentra tions indicated. At 48 hr after the addition of deoxynucleosides cell growth was dCTP pool. dTTP stimulates the reduction of GDP to dGDP determined by counting, the cells were harvested by centrifugation and extracted thereby increasing the dGTP pool (1 ). with ethanol, and the dCTP pool was measured by the DMA polymerase assay An alternate mechanism for dThd-induced growth inhibition (11,13).Â«, growth (percentage of control); O, dCTP pool (percentage of control). has been proposed by Grindey ef al. They have studied the changes in dNTP pools in CCRF-CEM cells following exposure to inhibitory concentrations of dThd used after rescue with dCyd (5). At a concentration of 40 JUMdThd, which inhibited growth by 73%, the dTTP pool was elevated to 8 times the control levels, and the dGTP and dATP pools were also in creased 3- to 5-fold. The dCTP pool was reduced by 42%. On prevention of growth inhibition by dCyd, the dCTP pool in creased to control value while the dTTP and dGTP pools fell to 3 to 4 times the control levels. Grindey ef al. consider the fall in dCTP concentration follow ing exposure to dThd to be too small to account for growth inhibition. They postulate that the increase in the other dNTP pools may be partially responsible for the growth inhibition (5). A potential mechanism for this has been cited, invoking a regulatory protein for DMA polymerase a which has been 0.5 1.0 2.0 4.0 8.0 16.0 dCyd(uM) isolated recently from calf thymus. In the presence of this protein, activity of DNA polymerase a is inhibited by dGTP in a Chart 2. Cells were grown in suspension culture in Roswell Park Memorial concentration-dependent manner (12). They postulate that Institute Culture Medium 1640 supplemented with 10% fetal calf serum and maintained in log growth. Cells were diluted with fresh medium to 2 x 105/ml physiologically or pharmacologically induced increases in and grown for 24 hr, and dThd plus or minus dCyd was added at the concentra dNTP pools would be capable of inhibiting DNA synthesis by tions indicated. At 48 hr after addition of deoxynucleosides, cell growth was determined by counting, the cells were harvested by centrifugation and extracted this mechanism. with ethanol, and dTTP pool was measured by the DMA polymerase assay (11, In studying dCyd reversal of dThd growth inhibition of the 13). â€¢¿,growth(percentage of control); O, dTTP pool (percentage of control). lowest dose of dCyd, we initially used (3 /ÃŒM)partlyreversed dThd growth inhibition without elevating the dCTP pool. This exposed to dThd and dCyd, the dTTP pools were slightly lower paradoxical finding was further examined, and lower doses of at 1 hr from 560 to 384%); but at 48 hr they were higher than dCyd were used in rescue experiments. It was found that 0.5 in the dThd-alone-treated cells at low doses of dCyd (2483 to /IM dCyd would partially reverse the inhibition of 60 /Â¿MdThd, 3448%); but at higher doses of dCyd, the dTTP levels were and this reversal was virtually complete (=95% of control similar to those in cells not rescued with dCyd (Chart 2). growth) at 8 to 16 /AMdCyd. The dCTP pool remained low until dCyd alone (0.5 to 16 U.M)had little effect on the dTTP pool growth inhibition was reversed entirely, and at this point the (apart from a slight reduction at 1 hr). dCTP pool exceeded control levels by 2-fold, but the dTTP and dGTP pools remained elevated severalfold above control val DISCUSSION ues. The failure of the dCTP pool to elevate during exposure to Cultured malignant human lymphocytes of T- and null-cell increasing concentrations of dCyd (which progressively re origin appear to be unusually sensitive to deoxynucleosides, versed the growth inhibition) suggests that as exogenous dCyd particularly dThd and deoxyguanosine. In these cells, dThd is phosphorylated to dCTP it is immediately consumed in the concentrations, which are 1 to 2 orders of magnitude lower DNA-replicative process. Only when growth inhibition is re than those required to inhibit most mammalian cell lines, inhibit versed totally does the dCTP pool normalize and actually

1720 CANCER RESEARCH VOL. 40

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1980 American Association for Cancer Research. dCyd Rescue of dThd Toxicity overshoot; i.e., the dCTP provided by salvage kinase from malignant human T cell lines. Proc. Nati. Acad. Sei. U. S. A., 76. 2430- 2433, 1979. dCyd exceeds that normally produced by the reduction of CDP. 3. Fox. R. M.. Mynderse, J. F.. and Goulian. M. Incorporation of deoxynucle- These observations support the view that dThd growth inhi otides into DNA by diethylaminoethyldextran-treated lymphocytes. Biochem bition reflects a critical deficiency of dCTP for DMA replication, istry, 16: 4470-4477. 1977. 4. Fox, R. M.. Piddington. S. K.. Tripp. E. H., Dudman. N. P.. and Tattersall, M. despite the fact that the measured dCTP pool is only reduced H. N. Thymidine sensitivity of cultured leukemic lymphocytes. Lancet, 2. by =50%. One possible explanation is that this residual dCTP 391-393, 1979. pool is unavailable to the replicative DNA polymerase. Although 5. Grindey, G. B.. Wang. M. C., and Kinahan, J. J. Thymidine induced pertu- bations in ribonucleoside and deoxyribonucleoside triphosphate pools in little is known about compartmentalization of dNTP, it appears human leukemic CCRF-CEM cells. Mol. Pharmacol., 16: 601-606. 1979. potentially important. Skoog and Bjursell (10) have shown 6. Lowe, J. K., and Grindley, G. B. Inhibition of growth rate and deoxynucleoside triphosphate concentrations in cultured leukemic L1210 cells. Mol. unequal distribution of dNTP between the cytoplasm and nu Pharmacol., 12: 177-184, 1976. cleus and that this distribution of dNTP changes during the cell 7. Minowada. J. Markers of human leukemia-lymphoma cell lines reflect hae cycle. Furthermore, studies of dNTP incorporation into DNA of matopoietic cell differentiation. In: B. Serrou and C. Rosenfeld, (eds.), Human Lymphocyte Differentiation: Its Application To Cancer. INSERM isolated cell nuclei have demonstrated a Km of isolated DNA Symposium No. 8. pp. 337-344. Amsterdam: North Holland Publishing Co.. polymerase 10- to 100-fold that of enzyme in the nucleus, 1978. suggesting that the enzyme in the cell is in the form of a 8. Reichard, P. From deoxynucleotides to DNA synthesis. Fed. Proc.. 37. 9- 14, 1978. complex of high affinity (16). Alternatively, DNA replication 9. Reynolds, E. C.. Harris, A. W.. and Finch, L. R. Deoxyribonucleoside might require a critical concentration of dCTP which might triphosphate pools and differential thymidine sensitivities of cultured mouse serve a regulatory function. lymphoma and myeloma cells Biochim. Biophys. Acta, 560. 110-123, 1979. Clearly, growth reversal by dCyd occurs with only minimal 10. Skoog, L., and Bjursell. F Nuclear and cytoplasmic pools of deoxyribonu reduction of the markedly elevated dTTP and dGTP pools. This cleoside triphosphates in Chinese hamster ovary cells. J. Biol. Chem., 249 6434-6438. 1974. suggests that excesses of dNTP are not directly inhibitory to 11. Solter, A. W., and Handschumacher, R. E. A rapid quantitative determination DNA replication. Similarly, studies of lysed cell or permeable of deoxyribonucleoside triphosphates based on the enzymatic synthesis of cell systems incubated with exogenous dNTP demonstrate DNA. Biochim. Biophys. Acta, / 74: 585-590, 1969 12. Steinberg, J. A., Often, M., and Grindey, G. B. Isolation of a DNA polymerase saturation of incorporation of labeled dNTP into DNA but not Â«-associated regulatory protein from calf thymus. Cancer Res.. 39. 4330- inhibition at excess dNTP (3, 14). 4335, 1979. 13. Tattersall, M. H. N., Lavoie. A..Ganeshaguru, K., Tripp, E. H.. and Hoffbrand. A. V. Deoxyribonucleoside triphosphates in human cells: changes in disease ACKNOWLEDGMENTS and following exposure to drugs. Eur. J. Clin. Invest., 5 191-202. 1975 14. Tseng, B. Y., and Goulian, M. DNA synthesis in human lymphocytes: inter mediates in DNA synthesis, in vitro and in vivo. J. Mol. Biol.. 99: 317-337. We thank S. Knott and Dr. I. Taylor for their expert maintenance of these cell 1975. lines and A. Rattenbury for her patient typing of the manuscript. 15. UICC Workshop on Human Tumour Sampling for Biochemical Pharmacolog ical Studies of Target Determinants of Drug Action (report prepared by E. Mihich, D. J. R. Laurence, D. M. Laurence and S. Eckhardt). UICC Tech. REFERENCES Rep. Ser., 43: 12. 1979. 16. Winnacker, E. L.. Magnusson, G , and Reichard. P. Replication of polyoma 1. Bjursell, G., and Reichard, P. Effects of thymidine on deoxyribonucleotide DNA in isolated nuclei I. Characterization of the system from mouse fibroblast triphosphate pools and deoxyribonucleic acid synthesis in Chinese hamster 3Y6 cells. J. Mol. Biol., 72. 523-537, 1972. ovary cells. J. Biol. Chem., 248. 3904-3909. 1973. 17. Wortmann, R. L.. Mitchell, B. S., Edwards. N. L.. and Fox. I. H. Biochemical 2. Carson, D. A., Kay. J.. Matsumoto, S., Seegmiller, J. E., and Thompson. L. basis for differential deoxyadenosine toxicity to T and B lymphoblasts: role Biochemical basis for the enhanced toxicity of deoxyribonucleosides toward for 5' nucleotidase. Proc. Nati. Acad. Sei U. S. A.. 76. 2434-2437. 1979.

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Richard M. Fox, Edith H. Tripp and Martin H. N. Tattersall

Cancer Res 1980;40:1718-1721.

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