Thymidylate Synthetase Overproduction in 5-Fluorodeoxyuridine-Resistant Mouse Fibroblasts

Abstract

We describe the isolation and characterization of a series of 5-fluorodeoxyuridine (FdUrd)-resistant mouse 3T6 cell lines that overproduce thymidylate synthetase (TS) by up to 50-fold compared with the parental cells. The resistant cells were selected by growing 3T6 cells or a methotrexate-resistant 3T6 cell line (M50L3, isolated previously in our laboratory) in gradually increasing concentrations of FdUrd. Uridine and cytidine were included in the culture medium to reduce toxicity from metabolic products of FdUrd. Cells that were resistant to the drug by virtue of loss of thymidine kinase activity were eliminated by selection in medium containing hypoxanthine, methotrexate, and thymidine. M50L3 cells were found to adapt to FdUrd more readily than 3T6 cells. A number of clones were isolated that were able to grow in the presence of 3 microM (M50L3 derived) or 0.3 microM (3T6 derived) FdUrd. Several were found to overproduce TS by 10 to 50-fold compared with normal 3T6 cells. All were found to have thymidine kinase activity, although the enzyme level was significantly reduced in some clones. The overproduced TS was inactivated by 5-fluorodeoxyuridylic acid at the same concentration as the enzyme from 3T6 cells. TS was purified from the LU3-7 clone (50-fold overproducer) by affinity chromatography on methotrexate-polyacrylamide. The monomer molecular weight was about 38,000, which was the same as the molecular weight of the monomer in 3T6 cells. The overproduction trait was gradually lost (half-life, 3 weeks) when LU3-7 cells were grown in the absence of FdUrd. The overproducing cells will provide an abundant supply of TS and (very likely) its mRNA and may serve as a convenient model system for detailed studies of the regulation of TS gene expression during the cell cycle.

Increased Mutational Rates in Chinese Hamster Ovary Cells Serially Selected for Drug Resistance

“…FP-1, FP-2, and FP-3 (resistant to 8-, 20-, and 1,000-fold concentration of FUdR, respectively) were all initially maintained in FUdR for many generations and screened before being transferred to nonselective medium. As reported by other laboratories (17,19), thymidylate synthetase (TS) activity was increased in each of the isolates 9to 11-fold, although it is clear that the increase in enzyme activity bears little relationship to the degree of resistance to FUdR in FP-2 or FP-3. The resistance was stable since transfer to nonselective medium did not alter drug sensitivity or TS activity.…”