Paul H. Fischer

Augmentation of methotrexate cytotoxicity in human colon cancer cells achieved through inhibition of thymidine salvage by dipyridamole

In HCT 116 cells, a human colon cancer cell line, the levels of thymidine (0.6 microM) and hypoxanthine (9 microM) contributed to the tissue culture medium by the fetal bovine serum significantly reduced the growth inhibition and lethality produced by 0.1 microM methotrexate. Dipyridamole, an inhibitor of nucleoside transport, potentiated the growth inhibitory effects of methotrexate when the cells were grown in medium that was changed daily. However, when the medium was supplemented with dialyzed serum, methotrexate cytotoxicity was not increased by dipyridamole. Similarly, in cloning experiments, dipyridamole increased the cell killing produced by methotrexate. The potentiation of methotrexate toxicity produced by dipyridamole was mediated through inhibition of thymidine uptake. The uptake of 1 microM thymidine was inhibited 50% by 0.12 microM dipyridamole but neither hypoxanthine nor guanine uptake was decreased by dipyridamole (5 microM). As a result, the decrease in dTTP pools produced by methotrexate was augmented by dipyridamole. In contrast, dipyridamole did not influence the effect of methotrexate on ribonucleoside triphosphate pools. HCT 116 cells avidly salvaged low concentrations of thymidine, and methotrexate increased this capacity. Conversion of 0.11 microM thymidine to thymidine triphosphate was increased by 55%, from 16.6 to 25.7 pmoles/10(6) cells, following exposure to 1.0 microM methotrexate. Dipyridamole blocked this pool expansion. This study suggests that the salvage of physiological levels of thymidine may diminish the cytotoxic effects of methotrexate on human colon cancer cells. Inhibition of thymidine uptake by dipyridamole may be an effective strategy to increase the cytotoxicity of methotrexate.

Modulation of 5-iodo-2'-deoxyuridine metabolism and cytotoxicity in human bladder cancer cells by fluoropyrimidines.

Iododeoxyuridine (IdUrd) potentiated the lethal but not the growth inhibitory properties of fluorouracil (FUra) and fluorodeoxyuridine (FdUrd) in human bladder cancer cells (T24). The rate of incorporation of IdUrd into DNA was enhanced by both fluoropyrimidines, but to a significantly greater extent by FdUrd. Both inhibition of iododeoxyridylate dehalogenation and the depletion of thymidine triphosphate pools contributed to the increased incorporation rate. Inhibition of dehalogenation accounted for 67% of the observed stimulation in the case of FUra, but only 37% of the increase produced by FdUrd. The depletion of dTTP pools, both in the presence and absence of IdUrd, was greater after FdUrd than FUra exposure. The observed increase in the rate of incorporation of IdUrd appears to account for the enhanced toxicity seen with FdUrd, but other factors may be involved in the case of FUra. Since FUra and IdUrd appear to be mutually potentiating and do not share a dependence on thymidine kinase activity, this drug combination warrants further investigation.