On the Action of Fluorouracil on Leukemia Cells1

[CANCER RESEARCH 26 Part 1, 1611-1615,August 1966] On the Action of Fluorouracil on Leukemia Cells1

ALLAN R. GOLDBERG, JOHN H. MACHLEDT, JR., AND ARTHUR B. PARDEE Department of Biology, Princeton University, Princeton, New Jersey

Summary In the present study the lymphoid leukemia L1210 of the mouse and a FU-resistant line were investigated. The problem The uptake and metabolism of radioactive uracil, uridine, posed was to discover a site of FU inhibition in the sensitive phosphate, and 5-fluorouracil by the mouse L1210 leukemic cells. The results suggest that the "salvage" pathway of pyrimi leukocytes and a fluorouracil-resistant variant were investigated. dine synthesis (see Chart 1) is sensitive to FU, with a resulting The dual aims of the research were to locate a metabolic differ inhibition of nucleic acid synthesis in the sensitive cells. The re ence responsible for resistance, and to define the site of action of the inhibitor. The resistant cells possess a much less active "sal sistant cells do not depend on this pathway, and hence are not vage" pathway, from uracil to nucleic acids, owing to a weaker susceptible to the inhibitor. uridine phosphorylase activity. They depend on the de novo pathway for a supply of pyrimidine nucleotides. Also, the con Materials and Methods version of fluorouracil to phosphorylated derivatives and its Uracil-3H and uridine-3H were obtained from the New Eng incorporation into RNA is somewhat reduced. Fluorouracil is land Nuclear Corporation, 5-FU-3H from Schwarz BioResearch, postulated to be less effective against these cells because its main Inc., and Na2H32PO4from Volk Radiochemical Co. Nonradio- site of action is in the salvage pathway, on which the resistant active FU and FUR were gifts of Hoffman-LaRoche Corp. cells do not depend. Perhaps the sensitive cells are only moder Both L1210/V (sensitive to FU) and L1210/FU-XIII (re ately affected by fluorouracil because they can utilize the de novo sistant) cells were generously supplied by Dr. Doris Hutchison. pathway for growth, as can other tissues lacking an active sal They were maintained in ascitic form by serial transfers in 6- vage pathway. The relations of these results to other proposed 10-week-old BDFi mice weighing 20-25 gm (Jackson Memorial mechanisms of fluorouracil action and resistance are discussed. Laboratory)- To maintain FU resistance, the L1210/r cells were periodically exposed to FU (25 mg/kg), beginning 1 day after Introduction transplant. Fluoropyrimidines are able to inhibit the growth of both bac For in vivo incorporation studies, 32P was injected i.p. into terial and mammalian cells (1, 7, 14, 15, 21, 22). Several modes 7-day leukemic mice. Fluorinated pyrimidines and sodium of inhibition have been proposed, the principal ones being: (a) orotate were injected 30 min prior to phosphate. Leukemia formation of TMP2 is inhibited by FdUMP, thereby blocking cells were withdrawn after 2.5 hr, and fractionated as described DNA synthesis (5, 11); (6) abnormal RNA is produced by incor below. poration of FU in place of uracil, and this produces a toxic en For in vitro incorporation studies cells were centrifuged several zyme pattern (4, 16); (c) nucleotide synthesis is inhibited, result times in cold Krebs-Ringer-bicarbonate containing 60 mg/ml ing in a reduced rate of RNA or DNA synthesis, or both (24). heparin and 10 mg/ml glucose, until free of blood cells and as Considerable controversy exists as to which of these mechanisms citic fluid. The leukemia cells were then weighed in a tared cen is most important for inhibition in vivo. Extensive work has been trifuge tube, and were resuspended in cold Krebs-Ringer solution done with mutant cell lines that arise spontaneously after treat containing glucose. Fluorinated compounds, when present, were ment with FIT, owing to their importance in cancer chemother preincubated with the cells for 15 min at 37Â°C.The cells were apy. Several modes of resistance have been reported, but no then added to 50 ml Ehrlenmeyer flasks containing radioactive single basis takes precedence. These problems are treated fur compounds. They were subsequently gassed with 5% C02 in air ther in the discussion. and were equilibrated for a few minutes at 37Â°.Incubations were carried out for 45 min at 37Â°with slow shaking. The reac tion mixtures were transferred to test tubes immersed in ice 1This investigation was supported by Grants 5T1-GM-457 and water, were centrifuged, and the pellet was washed in cold Krebs- Al-04409 from the USPHS. 2The following abbreviations are used: FU, 5-fluorouraeil; Ringer to remove most of the extracellular radioactivity and FUR, 5-fluorouridine; FUDR, 5-fluorodeoxyuridine; FUMP, then was weighed. 5-fluorouridylic acid; FdUMP, 5-fluorodeoxyuridylic acid; UR, The cells were fractionated according to a modified Schmidt- uridine; UMP, uridylic acid; UDP, uridine diphosphate; UTP, Thannhauser procedure (23). Protein and nucleic acids were uridine triphosphate; TMP, thymidylic acid; L1210/S, L1210cells precipitated with cold 0.6 N perchloric acid. The precipitate was sensitive to FU (D. J. Hutchison's L1210/V strain); L1210/r, washed once with perchloric acid. The supernatant fractions L1210 cells resistant to FU (D. J. Hutchison's L1210/FU-XIII were pooled and neutralized with 4 N KOH. Nonlabeled carriers strain). (uracil, UR, UMP, UDP, UTP, or the corresponding fluorinated Received for publication November 19, 1965. compounds) were added. The pyrimidine derivatives were sepa-

AUGUST 1966 1611

dsportic a.->â€”Â»â€”>orotic a.- OMP TABLE 1 INFLUENCEOF FLUOROUHACILONPHOSPHATEINCORPORATION INTORNA OF L1210/S CELLS ANDL1210/R CELLS UMP RNA Each value represents the mean of triplicate determinations on pooled cells from 5 animals. Percentage changes from control uridine uridine kinase values are given in parentheses. Each animal received i.p. 1.0 phosphorylase gniole phosphate (1.5 X IO6cpm) in 0.1 ml 2.5 hr prior to sacrifice. uracil uridine + ribose-l-P Fluorouracil was also injected i.p. (25 mg/kg) in 0.1 ml isotonic CHART1. The "salvage" and de novo pathways of pyrimidine NaCl. biosynthesis. OMP, orotidine phosphate; UMP, uridine phos OF PHOSPHATEINTORXA phate; ATP, adenosine triphosphate. protein)L1210/S(m/imoles/mg EXPERIMENTi2GÃ•OUPControlFluorouracilControlFluorouracilUPTAKE

cells13.70.3 cells9.58.9 rated by descending paper chromatography in a solvent system of 100 ml isobutyric acid plus 60 ml 0.5 N NH4OH (3). Spots were located with an ultraviolet lamp, cut out, and counted with (-54)13.25.1 (-6)9.38.5 a Packard liquid scintillation counter. The perchloric acid precipitate was washed twice with 95% (-61)L1210/F (-9) ethanol and once with 1:1 ethanol-ether to remove lipids. RXA was extracted from the precipitate by hydrolysis in 1.0 N KOH for 16 to 20 hr at 37Â°,andthen was neutralized with perchloric acid. TABLE 2 The precipitate was washed once with perchloric acid. Superna- INFLUENCEOFOROTICACIDANDFLUOHOURACILONPHOSPHATE tants were combined and counted to determine the amount of INCORPORATIONINTORNA OF L1210/S CELLS radioactive precursor incorporated into RNA. Counts incor Each value represents the mean of triplicate determinations on porated into the residue, in part DNA, were also determined, pooled cells from 5 animals. Percentage changes from control but too few counts were found for conclusive results. values are given in parentheses. Each animal received i.p. 1.0 /imole phosphate (4 X 10scpm) in 0.1 ml 2.5 hr prior to sacrifice. Results Fluorouracil (25 mg/kg) and orotic acid (10 mg/kg) were injected i.p. in 0.1 ml isotonic NaCl. RNA synthesis in vivo was much more strongly inhibited by of phosphate FU in L1210/S than in L1210/r cells (Table 1). FU decreased AdditionNoneOrotic intoR.VA(m(

1612 CANCER RESEARCH VOL. 26

TABLE 3 TABLE 5 THE EFFECT OF FLUOROURACILONURACIL INCORPORATIONINTO UPTAKE OF FLUOROURACILINTO L1210/S AND L1210/K CELLS L1210/8 AND L1210/R CELLS The results were obtained from a typical experiment in which The results were obtained from a typical experiment in which cells were pooled from 3 animals. Percentage changes from L1210/S tells were pooled from 3 animals. Percentage changes from con values are given in parentheses. There were 109 mamÃ³les fluorouracil (2.7 X IO7cpm) in 2.0 ml total volume. trol values are given in parentheses. There were 175 nuimoles uracil (4.5 X 10' cpm) in 2.0 ml total volume. Fluorouracil was OFFLUOROUEACIL present at a concentration of 100 jug/ml. protein)L1210/S(wmoles/mg PRODUCTFluorouracil protein)L1210/SOF URACIL(wimoles/mg cells282 cells445 PRODUCTUracilUridine cells12901440 cells1770 (+63) Fluorouridine and fluorou- 8954L1210/r 56(-37)34 ridine phosphates" 1080(-5)82 FluorouracilControl (+11)595145 RNAUPTAKE (-37) and Â°Includes fluorouridine, 5-fluorouridylic acid, 5-fluorouridine uridine FluorouracilControl (-76)100 42(-49)40 diphosphate, and 5-fluorouridine triphosphate. phosphates"UNAGROUPControl

TABLE 6 FluorouracilUPTAKE 30 (-68)L1210/r 15 (-62) THE EFFECT OF FLUOROURIDINE ON URIDINE INCORPORATION INTO L1210/S AND L1210/R CELLS " Includes uridine, uridylic acid, uridine diphosphate, and The results were obtained from a typical experiment in which uridine triphosphate. cells were pooled from 3 animals. Percentage changes from control values are given in parentheses. There were 41 Ã±amÃ³lesuridine TABLE 4 (1.5 X 10s cpm) in 3.0 ml total volume. Fluorouridine was at a THE EFFECT OF FLUOROURACILON URIDINE INCORPORATION concentration of 100 ^g INTO L1210/S AND L1210/R CELLS protein)L1210/SOFURIDINEOi^moles/mg The results were obtained from a typical experiment in which PRODUCTUridine0Uridine cells were pooled from 3 animals. Percentage changes from con cells191 cells82 trol values are given in parentheses. There were 41 mamÃ³les uridine (1.5 X IO6cpm) in 3.0 ml total volume. Fluorouracil was at a concentration of 100 Mg/ml. FluorouridineControl 374(+95)1200 241)(+300)980

OF URIDIXK protein)L1210/S(wimoles/mg phos PRODUCTUridine"Uridinephosphates'RNAGROUPControlFluorouracilControlFluorouracilControlFluorouracilUPTAKEphates6RNAGROUPControlFluorouridineControl 490(-59)2730 328(-66)2730 cells11060 cells4947

FluorouiidineUPTAKE 628 (-77)L1210/r 547 (-80) (-45)43604220 (-2)35503550 " Uracil and other small acid-soluble molecules into which uridine may have been converted were detected in minor amounts. (-3)19101500 (0)19102050 b Includes uridylic acid, uridine diphosphate, and uridine triphosphate.

(-21)L1210/r (+7) phosphorylase. It is important to note that inhibition was nearly " Uracil and other small acid-soluble molecules into which the same with both types of cells; a resistance mechanism based uridine may have been converted were detected in minor amounts. on loss of sensitivity of uridine phosphorylase to FU seems to ' Includes uridylic acid, uridine diphosphate, and uridine be ruled out by these data. triphosphate. Similar experiments were performed using FUR as an inhibitor of the salvage pathway. FUR appeared to inhibit uridine kinase, obtained with uracil (Table 3). They can also be explained by a the second enzyme in the pathway, since a large pool of uridine lower uridine phosphorylase level, although the differences do accumulated when uracil and FUR were present together. But not seem to be so great. FUR did not inhibit conversion of uracil into phosphorylatecl Effects of FU on metabolism of uracil were next examined derivatives or incorporation into RNA. Perhaps the pool of uri (Table 3). The entry of uracil into the cells was little affected, dine was sufficiently high to overcome the inhibition of uridine indicating that the analog did not act to inhibit the transport kinase by FUR. step. However, the conversion of uracil to nucleotides, and its The inhibition of uridine utilization by FUR was investigated incoqjoration into nucleic acids was inhibited by about 50-70% (Table 6) and it was discovered that the intracellular pool of in both sensitive and resistant cells. Since FU had little effect on uridine was increased 2- to 3-fold by FUR. However, conversion the metabolism of uridine (Table 4), it appears to inhibit uridine to nucleotides and incorporation into RNA was decreased by

AUGUST 1966

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1966 American Association for Cancer Research. Allan R. Goldberg,John H. Machledt, Jr., and Arthur B. Pardee about 65% in both types of cells. Thus, again resistance cannot such as uridine phosphorylase, insensitive to FU was not prob be attributed to decreased affinity of the enzymes in the salvage able. Reichard et al. have reported a small effect of this sort in pathway for their substrates. their system (19). Similar experiments were performed with FUDR. No effects Three pieces of evidence suggest that inhibition by FU of the of this compound on formation of nucleotides or incorporation de novo pathway is not important in inhibiting growth. First, into RNA, of either uracil or uridine, in either sensitive or re orotic acid permitted vigorous RNA synthesis in the presence sistant cells was noted. These results do not of course exclude of FU, indicating that the part of the pathway from orotic acid possible inhibition of DXA synthesis by FUDR. to UMP was not inhibited. Second, Bresnick found minor feed back inhibitions of the pathway in hepatomas by FU and its derivatives FUR and FUMP (2). Third, there was ver}* little Discussion inhibition of nucleic acid synthesis in the resistant cells which The most striking difference noted in these experiments be seem to use mainly the de novo pathway; it cannot be blocked tween FU-sensitive and -resistant cells was the lower activity by FU. This pathway could not be blocked in the sensitive cells of the salvage pathway in the latter. This was due to the lower unless a 2nd mutation (aside from the 1 that reduced activity activity of uridine phosphoiylase, as measured in whole cells in of the salvage pathway) occurred when resistance developed. vitro. Reichard et al. (19, 20) earlier reported lower uridine phos- The principal present hypothesis is that FU inhibits DXA phoiylase and uridine kinase in resistant tumor cells, although synthesis by being converted to FdUMP, which inhibits thy- they later questioned the importance of these observations (20). midylate synthetase (8, 11). In the Ehrlich ascites cell, FUDR The most apparent effects of FF and FUR were inhibitions of inhibits DXA synthesis, but it is equally active in sensitive and the salvage pathway in both cell types. resistant cells (5, 6, 8). The hypothesis presented here is that FU and FUR inhibit Our experiments permitted an examination only of the action growth of the sensitive cells by reducing the rate of nucleic acid of FU on ribopyrimidine metabolism, and the consequences of synthesis, owing to inhibition of the salvage pathway which is this action on RNA synthesis. Inhibitions in the DNA pathway important in these cells. L1210/r cells are postulated to be re are not distinguished by existing experiments nor can they be sistant because they have a relatively inactive salvage pathway, by experiments on inhibition of incorporation of precursors (such and depend on the de novo pathway which is not inhibited by as formate) into DNA. An inhibition of DXA synthesis could FU. be caused by inhibition of the salvage pathway owing to a di This hypothesis of utilization of alternative pathways (17) by minished supply of both pyrimidine precursors, as well as by the sensitive and resistant cells is consistent with observations inhibition of thymidylate synthetase. Indeed, FU inhibited DXA made here and by others. Both FU and FUR inhibit tumor synthesis even when thymidine was present (15), a result which growth, and they are both active in inhibiting the salvage path would not be predicted if inhibition of TMP synthesis were way in sensitive cells. Lack of complete growth inhibition (9, solely involved. Bloch and Hutchison (1) have presented evi 10, 13, 14) can be attributed to the ability of these cells to draw dence that production of surplus pyrimidine intermediates can on the de novo pathway in the presence of the inhibitors. Cells overcome FUDR action in Streptococcusfaecalis. which have developed resistance to FU appeared also to have In vivo experiments would seem the most adequate for dis developed simultaneously considerable resistance to FUR (14; tinguishing these alternatives of in vivo action. The data are as D. J. Hutchison, personal communication) as would be expected follows. First, FUDR is a poor inhibitor of L1210 tumor growth if the salvage pathway became relatively unimportant, and yet relative to FU or FUR (14). The weak inhibitor}* effect of FUDR was the major site of inhibition by both compounds. The same can be explained by either unimportance of inhibition at this argument applies to the slight sensitivity of normal cells to FU point or possibly by poor conversion to FdUMP. Second, FUR since they principali}*use the de novo pathway (12, 18). is a poor inhibitor of resistant cells (14), a result which is con A further effect of decreased activity of the salvage pathway sistent with the hypothesis of involvement of the salvage path is that conversion of FU to other biologically active forms, prin way rather than of thymidylate synthetase. Thus, the hy cipally FUDR- and FU-containing RXA, is reduced. If FUDR pothesis of an inhibited salvage pathway would seem adequate is important in growth inhibition (see below), or if FU-contain to account for the inhibition of growth, and should be considered ing RNA is inhibitory, their influences would be less pronounced as a likely alternative to action of FdUMP upon thymidylate in the resistant cells. However, FUR, conversion of which should synthetase. not be different, is less inhibitor}* in L1210/r cells (14). The im portance of FU-RNA has been discounted by Heidelberger et References al. (8, 11) in the Ehrlich ascites cell. It should be stressed that 1. Bloch, A., and Hutchison, D. J. A Mechanism of Resistance only 1 FU-resistant cell line was investigated. Other mechanisms to Fluoropyrimidines. Cancer Res., 24: 433-39, 1904. of resistance could develop in other lines. 2. Bresnick, E. Feedback Inhibition of Aspartate Transcarbamy- lase in Liver and in Hepatoma. Ibid., 2%:1240-51,1962. The present data are not consistent with some other modes 3. Brockman, R. W., Davis, J. M., and Stutts, P. Metabolism of of inhibition. FU did not reduce the activity of the salvage path Uracil and 5-Fluoronracil by Drug Sensitive and Drug Re way by decreasing the entry of uracil into the L1210/S or L1210/r sistant Bacteria. Biochim. Biophys. Acta, 40: 22-32, 1959. cells. Reichard et al. (20) have previously concluded that inhi 4. Gros, F., Gilbert, W., Hiatt, H. H., Attardi, G., Spahr, P. F., bition of entry is not important in 4 tumor lines. The salvage and Watson, J. D. Molecular and Biological Characterization pathway was as inhibited by FU in the resistant as in the sen of Messenger RNA. Cold Spring Harbor Symp. Quant. Biol., sitive line; therefore, an alteration which makes some enzyme, 26: 111-26, 1961.

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5. HÃ¤ggmark,A. Studies on Resistance Against 5-Fluorouracil. Leukemia: Relative Responses of Antimetabolite Resistant II. Thymidylate Synthetase from Drug-Resistant Tumor Strains. Cancer Res. (suppl.), 22: 57-72, 1962. Lines. Cancer Res., 22: 568-72, 1962. 15. Lindner, A., Kutkam, T., Sankaranarayanan, K., Rucker, R., G. Hartmann, K-U., and Heidelberger, C. Studies on Fluorinated and Arrandondo, J. Inhibition of Ehrlich Ascites Tumor with Pyrimidines. XIII. Inhibition to Thymidylate Synthetase. 5-Fluorouracil and other Agents. Exptl. Cell Res. (suppl.), J. Biol. Chem., 236: 3006-13, 1901. 9: 485-508, 1963. 7. Heidelberger, C., Chaudhuri, N. K., Danueberg, P. B., 16. Naono, S., and Gros, F. Effets d'un Analogue de Base Nu Mooren, D., Griesbach, L., Duschinsky, R., Schnitzer, R. J., clÃ©iquesurla BiosynthÃ¨sede ProtÃ©inesBactÃ©riennes.Change Pleveri, E., and Scheiner, J. Fluorinated Pyrimidines, A New ments de la Composition Globale des ProtÃ©ines.Compt. Class of Tumor-Inhibitory Compounds. Nature, 179: 663-66, Rend., 250: 3527-29, 1960. 1957. 17. Potter, Y. R., and Heidelberger, C. Alternative Metabolie 8. Heidelberger, C., Ghobar, A., Baker, A. K., and Mukherjee, Pathways. Physiol. Rev., 30: 487-512, 1950. K. L. Studies on Fluorinated Pyrimidines. X. In Vivo Studies 18. Reichard, P., and SkÃ¶ld,O.Enzymes of Uracil Metabolism in on Tumor Resistance. Cancer Res., 20: 897-902, 1960. the Ehrlich Ascites Tumor and the Mammalian Liver. Bio- 9. Heidelberger, C., Griesbach, L., Montag, B. J., Mooren, D., chim. Biophys. Acta, 28: 376-385, 1958. Cruz, O., Schnitzer, R. J., and Grunberg, E. Studies on Fluori 19. Reichard, P., SkÃ¶ld,O., and Klein, G. Possible Enzymic nated Pyrimidines. II. Effects on Transplanted Tumors. Mechanism for the Development of Resistance Against 5- Ibid., 18: 305-17, 1958. Fluorouracil in Ascites Tumors. Nature, 183: 939-41, 1959. 10. Heidelberger, C.( Griesbach, L., Cruz, O., Schnitzer, R. J. 20. Reichard, P., SkÃ¶ld,O., Klein, G., Revesz, L., and Magnus- and Grunberg, E. Fluorinated Pyrimidines. VI. Effects of son, P.-H. Studies on Resistance Against 5-Fluorouracil. 1. 5-Fluorouridine and 5-Fluoro-2'-deoxyuridine on Transplanted Enzymes of the Uracil Pathway During Development of Re Tumors. Proc. Soc. Exptl. Biol. Med., 97: 470-75, 1958. sistance. Cancer Res., 22: 235-43, 1962. 11. Heidelberger, C., Kaldor, G., Mukherjee, K. L., and Danne- 21. Rich, M. A., Bolaffi, J. L., Knoll, J. E., Cheong, L., and Eidi- berg, P. B. Studies on Fluorinated Pyrimidines. XI. In Vitro noff, M. L. Growth Inhibition of a Human Tumor Cell Strain Studies on Tumor Resistance. Cancer Res., W: 903-9, 1960. by 5-Fluorouracil, 5-Fluorouridine, and 5-Fluoro-2'-deoxy- 12. Heidelberger, C., Leibman, K. C., Harbers, E., and Bhargava, uridineâ€”Reversal Studies. Ibid., 18: 730-35, 1958. P. M. The Comparative Utilization of Uracil-2-C" by Liver, 22. Scheiner, J. M., Kostelak, K., and Duschinsky, R. 5-Fluoro- Intestinal Mucosa, and Flexner-Jobling Carcinoma in the pyrimidines as Growth Inhibitors of Microorganisms. Federa Rat. Ibid., 17: 399-404, 1957. tion Proc., 16: 242, 1957. 13. Heidelberger, C., Sunthankar, A. V., Griesbach, L., and An 23. Schmidt, G., and Thannhauser, J. J. A Method for the Deter derson, S. Fluorinated Pyrimidines. XII. Effects of Simple mination of Deoxyribonucleic Acid, Hibonucleic Acid, and Nucleotides on Transplanted Tumors. Proc. Soc. Exptl. Phosphoproteins in Animal Tissues. J. Biol. Chem., 161:83-9, Biol. Med., 104: 127-29, 1960. 1945. 14. Hutchison, D. J., Robinson, D. L., Martin, D., Ittensohn, 24. SkÃ¶ld,O. Inhibition of Uracil-utilizing Enzymes by 5-Fluoro O. J., and Dillenberg, J. Effects of Selected Cancer Chemo- uracil and 5-Fluorouracil Nucleosides. Arkiv Kemi, 17: 51-7, therapeutic Drugs on the Survival Times of Mice with L1210 1960.

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Allan R. Goldberg, John H. Machledt, Jr. and Arthur B. Pardee

Cancer Res 1966;26:1611-1615.

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