Increased thymidylate synthase in L1210 cells possessing acquired resistance to N10-propargyl-5,8-dideazafolic acid (CB3717): development, characterization, and cross-resistance studies.
The properties are described of a mutant L1210 cell line (L1210:C15) with acquired resistance (greater than 200-fold) to the thymidylate synthase (TS) inhibitor N10-propargyl-5,8-dideazafolic acid. TS was overproduced 45-fold and was accompanied by a small increase in the activity of dihydrofolate reductase (2.6-fold). Both the level of resistance and enzyme activities were maintained in drug-free medium (greater than 300 generations). Failure of N10-propargyl-5,8-dideazafolic acid to suppress the [3H]-2'-deoxyuridine incorporation into the acid-precipitable material of the resistant line supported the evidence that TS overproduction was the mechanism of resistance; consequently the L1210:C15 cells were largely cross-resistant to another (but weaker) TS inhibitor, 5,8-dideazafolic acid. Minimal cross-resistance was observed to the dihydrofolate reductase inhibitors methotrexate and 5-methyl-5,8-dideazaaminopterin (5- and 2-fold, respectively). L1210 and L1210:C15 cells were, however, equally sensitive to 5-fluorodeoxyuridine (FdUrd), an unexpected finding since a metabolite, 5-fluorodeoxyuridine monophosphate, is a potent TS inhibitor; however, this cytotoxicity against the L1210:C15 cells was antagonized by coincubation with 5 microM folinic acid although folinic acid potentiated the cytotoxicity of FdUrd to the N10-propargyl-5,8-dideazafolic acid-sensitive L1210 line. Thymidine was much less effective as a FdUrd protecting agent in the L1210:C15 when compared with the L1210 cells; however, a combination of thymidine plus hypoxanthine was without any additional effect (compared with thymidine alone) against the sensitive line but effectively protected L1210:C15 cells such that the concentration of FdUrd necessary to reduce the cell count to 50% of control at 48 h was increased greater than 11,000-fold. We propose that the elevated TS levels result in sequestration of the reduced-folate pool (as N5,10-methylene tetrahydrofolic acid) into the TS ternary complex with 5-fluoro-2'-deoxyuridine 5'-monophosphate. Despite "free" TS, the de novo synthesis of thymidylate and purines is inhibited by substrate depletion. The fact that folinic acid is able to reverse the inhibition of [3H]-2'-deoxyuridine incorporation by FdUrd into the resistant cells supports this hypothesis.
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D003857:Deoxyuridine / Q000378:metabolism; D004351:Drug Resistance; D005467:Floxuridine / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D005492:Folic Acid / Q000378:metabolism / Q000031:analogs & derivatives; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D007700:Kinetics; D007939:Leukemia L1210 / Q000188:drug therapy / Q000201:enzymology*; D051379:Mice; D011799:Quinazolines / Q000378:metabolism / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000096:biosynthesis*; D014316:Tritium
D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D007042:Hypoxanthines; D011799:Quinazolines; D005467:Floxuridine; D014316:Tritium; D019271:Hypoxanthine; C031662:CB 3717; D005492:Folic Acid; D013940:Thymidylate Synthase; D005472:Fluorouracil; D003857:Deoxyuridine