Interaction of deoxyuridine with fluorouracil and dipyridamole in a human colon cancer cell line

Abstract

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 μM deoxyuridine enhanced the cytotoxicity of 5 μM fluorouracil in a dose-dependent manner, and ≥10 μM deoxyuridine increased the augmentation of fluorouracil toxicity produced by 0.5 μM dipyridamole. The effect of deoxyuridine on [6- 3H]fluorouracil metabolism was studied. After 4 hr, 25 μM 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 μM deoxyuridine ±5 μM 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 pmoI 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 4hr (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 4hr respectively. The dUMP pools continued to enlarge through 24 hr. The effect of fluorouracil on DNA fragility was examined. In cells prelabeled with [ 14C]thymidine, there was no evidence of single-strand breaks in high molecular weight DNA after 4 or 24 hr of exposure to fluorouracil alone or with dipyridamole as measured by alkaline elution. In contrast, fluorouracil produced alkaline labile sites in newly synthesized DNA. Alkaline labile sites were also produced by exposure to dipyridamole. Concomitant exposure to FUra with dipyridamole and/or deoxyuridine resulted in a striking increase in the alkaline labile sites in DNA. These results suggest that effects on deoxyuridine metabolism may be important components of the interaction between fluorouracil and dipyridamole.