Effects of leucovorin on idoxuridine cytotoxicity and DNA incorporation.
Leucovorin (LV) increased the growth inhibition produced by iododeoxyuridine (IdUrd), a halogenated analogue of thymidine, in a murine tumor cell line (L1210) and three human tumor cell lines (HL-60, HT-29, and MCF-7). This increased growth inhibition was associated with increased incorporation of IdUrd into DNA. Consistent with previous reports, IdUrd (as iododeoxyuridine monophosphate) inhibited thymidylate synthase (TS) and was dehalogenated intracellularly by TS to generate thymidine nucleotides. In all four cell lines, LV decreased the dehalogenation of IdUrd, producing a 3-fold increase in the labeled iododeoxyuridine triphosphate/dTTP ratios in cytoplasm and labeled IdUrd/thymidine in DNA derived from tritiated IdUrd. In intact L1210 cells, apparent TS activity was inhibited 50% by 3 microM IdUrd alone and 75% by the combination of 3 microM IdUrd and 20 microM LV. Apparent TS activity was unchanged with 20 microM LV alone. In all cell lines except HL-60, the ratios of labeled iododeoxyuridine triphosphate/dTTP derived from tritiated IdUrd were 3-fold lower than the labeled IdUrd/thymidine ratios in DNA. This observation suggests that replicative DNA polymerases preferentially incorporate iododeoxyuridine triphosphate into DNA compared to the endogenous substrate dTTP. This preferential incorporation was independent of the effect of LV. These novel findings suggested that a potential mechanism for the effects of LV on IdUrd was increased inhibition of TS analogous to the interaction between fluoropyrimidines and LV. Enzyme inhibition studies using L1210 cell extracts showed that iododeoxyuridine monophosphate was a weak inhibitor of TS (Ki greater than 10 microM) when compared to 5-fluorodeoxyuridine monophosphate (Ki less than 10 nM). Despite the major differences in potency of these two halogenated pyrimidines, LV appears to modulate the activity of IdUrd as well as 5-fluorodeoxyuridine. LV may provide a clinically useful approach to improve the radiosensitizing and/or cytotoxic properties of IdUrd.
Office of Research Resources, Food and Drug Administration, Rockville, Maryland 20857.
R F
RF
Greene
J M
JM
Collins
D004247:DNA / Q000378:metabolism*; D004357:Drug Synergism; D006801:Humans; D007065:Idoxuridine / Q000378:metabolism / Q000494:pharmacology*; D002955:Leucovorin / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000032:analysis / Q000037:antagonists & inhibitors; D013942:Thymine Nucleotides / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects
D013942:Thymine Nucleotides; D004247:DNA; D013940:Thymidylate Synthase; D007065:Idoxuridine; D002955:Leucovorin; C024157:thymidine 5'-triphosphate