[CANCER RESEARCH 43, 5145-5150, November 1983]
Enhancement of the Incorporation of 5-Fluorodeoxyuridylate into DMA of HL-60 Cells by Metabolic Modulations1
Masao Tanaka,2 Kiyoji Kimura, and Shonen Yoshida
Nagoya National Hospital, Department of Medicine, 4-1-1, Naka-ku, Nagoya, 460 [M. T., K. K.¡,and Department of Biochemistry, Institute for Developmental Research, Aichi Prefecture Colony, Kasugai, Aichi, 480-03 [S. Y.], Japan
ABSTRACT into DNA in vivo. We also detected a significant amount of incorporation of The exposure of HL-60 human promyelocytic leukemia cells FdUMP into DNA of human promyelocytic leukemia cells (HL-60) to 0.5 MM5-fluoro-2'-[3H]deoxyuridine (FdUrd) for 16 hr resulted incubated with [3H]FdUrd. More importantly, it was found that in the incorporation of 5.14 ±0.31 (S.D.) x 10~7 mol FdUrd into the incorporation was enhanced by the pretreatment of the cells DMA per mol of DMA nucleotide, which corresponds to 0.146 ± with either dThd or MTX. 0.082 pmol FdUrd per 107 cells. Pretreatment with 50 MMdeoxy- thymidine for 24 hr led to a 2.7-fold increase in the incorporation MATERIALS AND METHODS of this analogue into newly synthesized DNA during the ensuing 16-hr exposure to 0.5 MM [3H]FdUrd. Pretreatment with 0.5 MM Cell Culture. Human promyelocytic leukemia cells (HL-60), transferred methotrexate for 3 hr also increased the [3H]FdUrd incorporation twice weekly, were maintained as suspension culture in a 5% CO2 into newly synthesized DNA approximately 5-fold. The coexist atmosphere at 37° in Roswell Park Memorial Institute Medium 1640 ence of deoxythymidine or methotrexate with [3H]FdUrd, how (Grand Island Biological Co., Grand Island, N. Y.) supplemented with ever, led to decreased incorporation of FdUrd into DNA. 10% heat-inactivated fetal calf serum (Grand Island Biological Co.), 100 More than 50% of the radioactivity in DNA separated by units of streptomycin per ml, and 100 ^g of penicillin per ml. In all experiments to be described, 75-sq cm tissue culture flasks (Corning Cs2S04 equilibrium density gradient centrifugation was proven to be fluorodeoxyuridylate by means of its binding to Lactoba- Glass Works, Corning, N. Y.) were inoculated with 30 ml of cell suspen cillus case/' deoxythymidine monophosphate synthetase. sion at 105 cells/ml. Chemicals. dThd was obtained from P-L Biochemicals Inc., Milwau kee, Wis. MTX was purchased from Lederle Laboratories, Pearl River, INTRODUCTION N. Y. FUra and FdUrd were supplied from Mitsui Pharmaceuticals Inc., Tokyo, Japan. 6-[3H]FdUrd (specific activity, 20 Ci/mmol) was obtained FUra3 and FdUrd are well-known clinical anticancer agents. from Moravek Biochemicals, City of Industry, Calif. 6-[3H]FUra (specific Their active metabolite is generally thought to be FdUMP, which activity, 3.83 Ci/mmol) was obtained from Amersham International Ltd. is a potent inhibitor of thymidylate synthetase (3,7). On the other (Amersham, United Kingdom). FdUMP was purchased from Sigma Chem hand, FUra and FdUrd are converted to 5-fluorouridine mono- ical Co., St Louis, Mo. Thymidylate synthetase, prepared from MTX- phosphate and subsequently incorporated into RNA of mam resistant Lactobacillus case/ by the methods of Crusberg (4), was malian cells via its triphosphate form (12,18). This 5-fluorouridine obtained from New England Enzyme Center, Tufts University School of Medicine, Boston, Mass. The enzyme preparation formed 8.2 ^mol TMP monophosphate-containing RNA is also deleterious to cell per hr per mg protein at pH 7.0 and 30°.Snake venom phosphodiester- growth. ase was from Miles Laboratories, Elkhart, Ind. Bovine pancreatic DNase A third possible mechanism, incorporation into DNA, has not was from P-L Biochemicals. 5,10-Tetrahydrofolate was prepared nonen- been proven until recently, except in the case of a bacteriophage, zymatically by the method of Moran (14). which normally contains uracil in place of thymine in its DNA. Incorporation of [3H]FdUrd and [3H]FUra into Nucleic Acids. HL-60 Danenberg ef al. (5), using L1210 murine leukemia cells, dem cells in logarithmic growth phase were washed twice with 0.01 M Na2- onstrated that detectable amounts of FdUrd can be incorporated PO4 (pH 7.3) containing 0.15 M NaCI (phosphate-buffered saline) and were suspended at 1.5 x 106 cells/ml in Roswell Park Memorial Institute into DNA. The presence of FUra residues in DNA has been Medium 1640 supplemented with 10% fetal calf serum. The cells were demonstrated recently in other cell lines (9, 10). Further, the incubated with [3H]FdUrd (10 MCi/ml; 0.5 MM)or [3H]FUra (10 MCi/ml; 2.6 enhancement of FdUrd incorporation into DNA by concurrent ¿IM)fora period varying from 4 to 16 hr. To determine the relative rate incubation with dThd was demonstrated by Major ef al. (11) of synthesis of RNA and DNA, the cells were incubated simultaneously using MCF-7 human breast carcinoma cells. Herrick et al. (8) with H332PO4(10 i
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Relative rate of incorporation of fluorinated pyrimidine into the newly synthesized, since the ratio of [3H]FdUrd to the newly synthe synthesized DNA or RNA was estimated from the ratio of "H/32?. The sized DNA (3H/32P)in the DNA band was nearly constant through sedimentation patterns were reproducible through 2 or 3 independent exposure (Chart 1). After 16 hr of continuous exposure, 107 cells experiments, and the representative results are shown in the charts and incorporated 0.131 pmol of [3H]FdUMP into DNA, corresponding tables. to 5.14 x 10~7 mol [3H]FdUMP into 1 mol total DNA nucleotides Identification of FdUMP in the DNA Region. Hydrolysis of the DMA estimated from UV adsorption at 260 nm. As clearly shown in to nucleotides was carried out with bovine pancreatic DNase and snake venom phosphodiesterase according to the method of Singer (15). Chart 1, a larger amount of radioactivity was incorporated into FdUMP in hydrolysate was measured by the method of Danenberg ef a/. RNA. The incorporation was also time dependent. This incorpo (5). In brief, aliquots of the DNA hydrolysates were incubated with 37 ration may have resulted from catabolic conversion of FdUrd into nmol of dTMP synthetase, 0.1 M Tris-HCI, pH 7.5, 0.1 mw 5,10-methyl- FUra, followed by phosphorylation into FUTP, a substrate for enetetrahydrofolate, 20 HIM fi-mercaptoethanol in a final volume of 1.0 RNA synthesis. ml for 2 hr at 32°.After the incubation, 1 ml of a charcoal suspension Enhancement of [3H]FdUrd into DNA by dThd Pretreatment. [0.1 g of charcoal, 2.5 mg/ml of bovine serum albumin and high-molec As shown in Chart 2, the preincubation with 0.1 mw dThd for 3 ular-weight dextran (0.1 mg/ml) in 0.1 N HCI] was added. The mixture hr resulted in a 2-fold increase in the incorporation of FdUrd into was centrifuged for 20 min at 4400 x g, and 1.4 ml of the supernatant DNA. The enhancement of incorporation was also measured was counted for radioactivity in Aguasol (New England Nuclear). under various conditions, i.e., longer preincubation time and RESULTS various concentrations of dThd (Table 1). As listed in Table 1, the incorporation of [3H]FdUrd into DNA Incorporation of [3H]FdUrd into DNA. As shown in Chart 1, per cell increased 3-fold by the preincubation with 0.05 mw dThd a substantial amount of [3H]FdUrd was incorporated into DNA for 24 hr. The rate of incorporation of FdUrd into the newly by the exposure of logarithmically growing HL-60 cells to 0.5 MM synthesized DNA (3H/32P)also increased 2.7-fold by the pretreat [3H]FdUrd. The amount of incorporation increased linearly with ment of the cells with 0.05 mw dThd for 24 hr. The incorporation the incubation time. of radioactivity into RNA decreased only slightly by the pretreat The incorporation was proportional to the amount of DNA ment with 0.1 mM dThd (Chart 2B).
Ahr 8hr 16hr
30
1.8 ^ 6 1.6
0> 20 U "o O x E X o. E u o. o X
o o io 20 30 10 20 30 10 20 30 Fraction Number Chart 1. Incorporation of (3H)FdUrd and MP into nucleic acids of HL-60 cells. HL-60 cells in logarithmic growth phase (1.5 x 106/ml) were incubated with [3H]Fd(Jrd (10 >jCi/ml; 0.5 Õ/M)and H3MPO< (10 ¿iCi/ml)for 4, 8, and 16 hr. The total nucleic acids were purified and analyzed by cesium sulfate density gradient centrifugation ("Materials and Methods"). The radioactivity of [3H]FdUrd and ^P banded in the RNA region (between 1.69 and 1.65 g/ml) and DNA region (between 1.43 and 1.46 g/ml) of the gradient was determined. •,3Hradioactivity per 107 cells; O, MP radioactivity per 107 cells.
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for mammalian DNA polymerase «as well as DNA polymerase 0(17). Herrick ef al. (8), however, reported that MTX treatment had no detectable effect on the incorporation of FUra or FdUrd in MCF-7 human breast carcinoma cell DNA and that FUraexcision from DNA was enhanced by MTX treatment in the presence of excess dThd. The reason for the apparent discrepancy between their results and ours might be due to the different methods of MTX treatment (preincubation versus coexistence; Tables 2 and 3) or the difference in cell lines used as discussed above. By the treatment of HL-60 cells with 0.05 to 0.1 ITIMdThd,the too 200 incorporation of [3H]FdUrdinto the newly synthesized DNA was Hydrolysale Of ONA (pi) enhanced about 3-fold. When dThd is administered in large amount, the ensuing high levels of dTTP have been known to exert an end product feedback inhibition on a number of enzymes involved in the de novo and salvage pathways. This would alter deoxynucleotide pools as well as cell kinetics. The enhanced incorporation of FdUrd in DNA might be due to synchronizing 0. cells with thymidine block and increased total DNA synthesis £500 during the incubation period with [3H]FdUrd. In this study, the 32Pincorporation into DNA actually increased 1.7-fold over that in the control by the pretreatment of the cells with 0.3 HIMdThd (data not shown). However, the incorporation of [3H]FdUrd into DNA was enhanced to a higher extent, resulting in a 2.7-fold IO'2 W" enhancement of the rate of incorporation of this drug into newly dlIMP added imoi) synthesized DNA. At present, the metabolic change which in Chart 4. A, binding of [3H]FdUMP to dTMP synthetase from a hydrolysate of DNA from HL-60 cells incubated with 0.5 mt [3H]FdUrd for 16 hr at 37°. The duces this enhancement is not clear. Major et al. (11) observed binding experiment was carried out as described in "Materials and Methods." 8, the enhanced incorporation of FUra into DNA by concurrent inhibition of the binding to dTMP synthetase of radioactive materials from a incubation with dThd at concentrations much lower than those hydrolysate of DNA of HL-60 cells. FdUMP (•),dCMP(A), or dTMP (O) was added used here. to the reaction mixture (300 /J) containing DNA hydrolysate, dTMP synthetase, and 5,10-methylenetetrahydrofolate. The binding was measured as described in "Ma In conclusion, FdUrd can be incorporated into DNA, and the terials and Methods." incorporation was augmented up to 4-fold by the pretreatment of the cells with MTX and dThd. This magnitude of enhancement used. Our preliminary experiment indicates that the incorporation might not be dramatic, but the enhanced incorporation of FdUrd of FdUrd into DNA varies depending on the cell lines; CCRF- and FUra by the metabolic modulation may open up a new CEM (cultured human T-cell line) incorporates 10-fold as much viewpoint regarding the combination therapy containing these FdUrd into DNA as do HL-60 cells (data not shown). Kufe ef a/. drugs. (10) also detected FUra or FdUrd residues in L1210 murine leukemia cells and MCF-7 human breast carcinoma cells follow REFERENCES ing exposure to either FUra or FdUrd. They reported approxi 1. Aradalan, B., Buscalglia, M. D., and Sehen, P. S. Tumor, 5-fluorodeoxyuridine mately 0.8 pmol [3H]FdUrdresidues were incorporated into DNA concentration as a determinant of 5-fluorouracil response. Biochem. Pharma- of 107 MCF-7 cells, following 12 hr incubation with 0.1 ßM[3H]- col., 27. 2009-2013, 1978. 2. Caradonna, S. J., and Chen, Y. C. The role of deoxyuridine triphosphate FdUrd. This amount of FdUrd incorporation is in the same order nucleotidedehydrolase, uracil DNA glycosylase and DNA polymerase in the as that obtained in the present investigation. metabolism of FdUrd in human tumor cells. Mol. Pharmacol., 78: 513-520. By the treatment of the cells with 0.5 ^M MTX, the incorpora 1980. 3. Cohen, S. S., Flasks, J. D., Narner, H. D., Loesb, M. R., and Lichtenstein, J. tion of [3H]FdUrdinto the newly synthesized DNA was enhanced The mode of action of 5-fluorouracil and its derivatives. Proc. Nati. Acad. Sci. 5-fold. The effect of pretreatment with MTX, a potent inhibitor of U. S. A., 44: 1004-1012, 1958. 4. Crusberg, T. C., Leary, R. P., and Kisliuk, R. C. Properties of thymidylate dihydrofolate reductase, will be the depletion of the TTP pool synthetase from dichromethotrexate-resistant Lactobacillus case/. J. Biol. which competes with FdUTPfor DNA polymerase. On the other Chem., 245: 5292-5296, 1970. 5. Danenberg, P. V., Heidelberger, C., Mulkins, M. A., and Peterson, A. R. The hand, MTX has been reported to increase the level of dUTP up incorporation of 5-fluoro-2'-deoxyuridine into DNA of mammalian tumor cells. to 1000-fold over that in the control (6). FdUrd or FUra itself also Biochem. Biophys. Res. Commun., 702: 654-658, 1981. increases the intercellular dTMP pool and decreases the dTTP 6. Goulian, M., Bleile, B., and Tseng, B. Y. Methotrexate-induced misincorpora- pool due to the inhibition of thymidylate synthetase by FdUMP tion of uracil into DNA. Proc. Nati. Acad. Sei. U. S. A., 77: 1956-1960, 1980. 7. Heidelberger. C. Fluorinated pyrimidines. Prog. Nucleic Acid Res. Mol. Biol., 4: (1,13). It seems likely that the increase in dUMP pool would lead 1-50. 1965. to the increase in dUTP, which will ultimately overwhelm the 8. Herrick, D., Major, P. P., and Kufe, D. W. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1983 American Association for Cancer Research. Enhancement of the Incorporation of 5-Fluorodeoxyuridylate into DNA of HL-60 Cells by Metabolic Modulations
Masao Tanaka, Kiyoji Kimura and Shonen Yoshida
Cancer Res 1983;43:5145-5150.
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