Studies on the mechanism of fluoropyrimidine cytotoxicity in l1210 cells: correlation with inhibition of thymidylate synthetase but not with incorporation into RNA.

The effects of 5-fluorouracil (FUra), 5-fluorouridine (FUrd), and 5-fluoro-2'-deoxyuridine (FdUrd) on L1210 cells were examined in an effort to determine whether the cytotoxicity of these fluoropyrimidines is more closely associated with incorporation of FUra residues into RNA or inhibition of thymidylate (dTMP) synthetase (5,10-methylenetetrahydrofolate: deoxyuridylate C-methyl-transferase, EC 2.1.2.45) by 5-fluoro-2'-deoxyuridylate (FdUMP). In different batches of cells exposed to equitoxic (LD50) doses of these drugs for 48 hr, the levels of free FdUMP, dUMP, and free dTMP synthetase were found to be very similar. However, the number of FUra residues incorporated into total cellular RNA were in the approximate ratio of 1:10:100 in cells treated with FdUrd, FUrd, and FUra, respectively. Although these results are consistent with a common DNA-directed mechanism of toxicity, thymidine (dThd), which should circumvent dTMP synthetase inhibition, did not rescue the cells from the effects of FUra. However, uridine (Urd), which should compete with FUra for incorporation into RNA, had no effect on the toxicity of FUra either. Urd at 10(-5) M did not decrease the amount of incorporation of 10(-7)M [(3)H]FUra into total RNA, but a limited fractionation of polysomal RNA showed about a 4-fold decrease of incorporation of FUra into mRNA in the presence of Urd. Urd and dThd did effectively decrease the cytotoxicity of FUrd and FdUrd, respectively. These observations suggest that cell rescue experiments may not be reliable indicators of the mechanism of cytotoxicity of antimetabolites with complex mechanisms of action.