Studies with Mutant L1210 Cell Lines that have Acquired Resistance to CB 3717
The development of resistance to antimetabolites is often associated with either overproduction of the target enzyme or deletion of the enzymes responsible for activation of the drug. Cell lines possessing such defects may be useful for the elucidation of metabolic pathways and the effects of inhibitors. In addition, cells that overproduce enzyme can be a useful source of large quantities of enzyme for primary structure analysis and associated studies. CB 3717 is a quinazoline analogue of folic acid that competitively inhibits thymidylate synthetase (TS) (Ki = 4nM). Its single locus of action and lack of requirement for metabolic activation made it useful in raising several monoclonal cell lines which overproduce the target enzyme TS (>30 fold). Thymidine kinase is not elevated. This acquired resistance to CB 3717 (>100-fold) is stable in the absence of the drug (gt;9 months). Studies with TS purified from one of these cell lines suggest that it is identical to that of the sensitive line and future work will determine whether gene amplification is the cause of resistance. Two cell lines also have a small increase in DHFR (>5-fold), and are slightly cross-resistant to MTX. 5-fluorouracil (FU) and 5-fluorodeoxyuridine (FUdR) may inhibit TS through their active metabolite, 5-fluorodeoxyuridylate, but can also be incorporated into nucleic acids. Unexpectedly no cross-resistance was observed to either compound. However 10µM thymidine failed to give protection from the cytotoxicity of FU and FUdR in contrast to the sensitive L1210 line. Current work should determine which factors influence the cytotoxic loci of these fluorinated pyrimidines in sensitive and resistant cell lines.