Grem J L, Mulcahy R T, Miller E M, Allegra C J, Fischer P H
Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison 53792.
Biochem Pharmacol. 1989 Jan 1;38(1):51-9. doi: 10.1016/0006-2952(89)90148-2.
We have reported previously that dipyridamole increases the toxicity of 5-fluorouracil and alters fluorouracil metabolism in HCT 116 cells, producing a selective increase in fluorodeoxyuridine monophosphate (FdUMP) levels by blocking the efflux of fluorodeoxyuridine. Dipyridamole also blocks deoxyuridine efflux and prolongs the intracellular half-life of deoxyuridine monophosphate (dUMP). The significance of the effect of dipyridamole on FdUMP and dUMP levels was explored further. In cell growth experiments, 1-50 microM deoxyuridine enhanced the cytotoxicity of 5 microM fluorouracil in a dose-dependent manner, and greater than or equal to 10 microM deoxyuridine increased the augmentation of fluorouracil toxicity produced by 0.5 microM dipyridamole. The effect of deoxyuridine on [6-3H]fluorouracil metabolism was studied. After 4 hr, 25 microM deoxyuridine increased the amount of [3H]FdUMP formed 2- to 4-fold relative to that of fluorouracil +/- dipyridamole alone. The mechanism by which deoxyuridine increased FdUMP was examined by measuring the distribution of [2'-3H]deoxyuridine metabolites following exposure of 25 microM deoxyuridine +/- 5 microM fluorouracil. Tritium appeared in the FdUMP peak at 4 and 24 hr in cells exposed to fluorouracil and deoxyuridine, indicating that [3H]deoxyribose was transferred to fluorouracil. A large buildup of [3H]dUMP was seen in cells exposed to fluorouracil plus deoxyuridine for 4 and 24 hr compared to exposure to [3H]deoxyuridine alone, suggesting that dUMP may also inhibit catabolism of FdUMP. Since the increased FdUMP levels produced by dipyridamole did not appear to correlate with further depletion of thymidine triphosphate pools, the incorporation of [3H]fluorouracil metabolites into nucleic acids was monitored by cesium sulfate density centrifugation. Fluorouracil-RNA increased as a function of time (1, 2 and 13 pmol/10(6) cells after 4, 8 and 24 hr), but fluorouracil-DNA was detected only after 24 hr (0.5 pmol/10(6) cells). Dipyridamole however, did not appear to alter the pattern of incorporation of fluorouracil into either RNA or DNA. Perturbations of endogenous dUMP levels by fluorouracil and dipyridamole were then studied. In cells exposed to fluorouracil alone, dUMP pools were unchanged from control at 2 hr, but they had increased 9-fold by 4 hr (3362 pmol/10(6) cells). Simultaneous exposure to fluorouracil and dipyridamole resulted in a 1.5-fold (566 pmol/10(6) cells) and 13.6-fold (5049 pmol/10(6) cells) increase over control dUMP levels after 2 and 4 hr respectively. The dUMP pools continued to enlarge through 24 hr. The effect of fluorouracil on DNA fragility was examined.(ABSTRACT TRUNCATED AT 400 WORDS)
我们之前报道过，双嘧达莫会增加5-氟尿嘧啶的毒性，并改变HCT 116细胞中氟尿嘧啶的代谢，通过阻断氟脱氧尿苷的外排使氟脱氧尿苷一磷酸（FdUMP）水平选择性升高。双嘧达莫还会阻断脱氧尿苷外排，并延长脱氧尿苷一磷酸（dUMP）的细胞内半衰期。我们进一步探究了双嘧达莫对FdUMP和dUMP水平影响的意义。在细胞生长实验中，1 - 50 microM的脱氧尿苷以剂量依赖的方式增强了5 microM氟尿嘧啶的细胞毒性，且大于或等于10 microM的脱氧尿苷增强了0.5 microM双嘧达莫所产生的氟尿嘧啶毒性增强作用。研究了脱氧尿苷对[6-3H]氟尿嘧啶代谢的影响。4小时后，相对于单独使用氟尿嘧啶和/或双嘧达莫，25 microM的脱氧尿苷使形成的[3H]FdUMP量增加了2至4倍。通过测量25 microM脱氧尿苷和/或5 microM氟尿嘧啶处理后[2'-3H]脱氧尿苷代谢物的分布，研究了脱氧尿苷增加FdUMP的机制。在暴露于氟尿嘧啶和脱氧尿苷的细胞中，4小时和24小时时氚出现在FdUMP峰中，表明[3H]脱氧核糖转移到了氟尿嘧啶上。与单独暴露于[3H]脱氧尿苷相比，暴露于氟尿嘧啶加脱氧尿苷4小时和24小时的细胞中出现了大量[3H]dUMP积累，这表明dUMP可能也抑制了FdUMP的分解代谢。由于双嘧达莫所产生的FdUMP水平升高似乎与三磷酸胸苷池的进一步消耗无关，因此通过硫酸铯密度离心监测了[3H]氟尿嘧啶代谢物掺入核酸的情况。氟尿嘧啶-RNA随时间增加（4、8和24小时后分别为1、2和13 pmol/10(6)细胞），但仅在24小时后检测到氟尿嘧啶-DNA（0.5 pmol/10(6)细胞）。然而，双嘧达莫似乎并未改变氟尿嘧啶掺入RNA或DNA的模式。然后研究了氟尿嘧啶和双嘧达莫对内源性dUMP水平的干扰。在仅暴露于氟尿嘧啶的细胞中，2小时时dUMP池与对照相比无变化，但4小时时增加了9倍（3362 pmol/10(6)细胞）。同时暴露于氟尿嘧啶和双嘧达莫分别在2小时和4小时后使dUMP水平比对照增加了1.5倍（566 pmol/10(6)细胞）和13.6倍（5049 pmol/10(6)细胞）。dUMP池在24小时内持续增大。研究了氟尿嘧啶对DNA脆性的影响。（摘要截断于400字）
