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 gradua$ 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. M5OL3 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 ,uM (M50L3 derived) or 0.3 ,uM (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.

Inefficient growth arrest in response to dNTP starvation stimulates gene amplification through bridge-breakage-fusion cycles.

“…This undesired outcome can be minimized by making the initial aliquot very small, so that the probability of including a pre-existing mutant is correspondingly low. We can estimate the frequency of pre-existing mutant cells to be _10-5 from the following data: 100 Resistance to 5-FUdR is due to high levels of the target enzyme thymidylate synthetase (Rossana et al, 1982) (Table 8). Although amplification is the predominant mechanism of PALA resistance in HT29 cells Stark and Wahl, 1984;Schimke, 1988).…”

Stable inhibition of human thymidylate synthase expression following retroviral introduction of an siRNA gene

“…13,25 Trichloroacetic acid-insoluble material was collected by filtration through glass fiber filters and analyzed for radioactivity in a scintillation counter. In the indicated experiments, the amount of TS enzyme was normalized to the amount of total protein in the cell extract, which was determined by the DC Protein Assay (BioRad).…”

“…Finally, long-term exposure to TS inhibitors selects for cells in which TS is highly overproduced as a result of TS gene amplification. [13][14][15] The development of RNA interference technology has provided an alternative strategy to lower cellular TS enzyme activity by destroying the mRNA that encodes the enzyme. In mammalian cells, RNA interference involves the use of short interfering double-stranded RNA (siRNA) molecules that correspond to a specific mRNA that is targeted for destruction.…”

The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites.

“…Elimination of an upstream Spl element reduced expression threefold, whereas elimination of an adenovirus upstream stimulatory factor element had little effect. All of the upstream elements that are essential for promoter activity are located within 22 nucleotides of the first transcriptional initiation site.…”