Abstract
We have analysed cell cycle variations in thymidylate synthase (TS) protein in asynchronously growing NCl H630 and HT 29 colon cancer and MCF-7 breast cancer cell lines. Western immunoblot analysis using the TS 106 monoclonal antibody revealed a 14- to 24-fold variation in TS levels between the peak exponential and confluent growth phase in the three cell lines. Similar variations in TS levels and TS activity were detected using the 5-fluorodeoxyuridine monophosphate and deoxyuridine monophosphate biochemical assays. The percentage of cells in S-phase, which paralleled changes in TS levels, reached a maximum of 38-60% in asynchronous exponentially growing cells compared with 5-10% in confluent cells. In asynchronous exponential cells, analysis of TS levels in each cell cycle phase using two-parameter flow cytometric analysis revealed that TS protein levels were 1.3- to 1.5-fold higher in S than in G0/G1 phase cells, and 1.5- to 1.8-fold higher in G2/M than G0/G1 cells. Similar differences of 1.1- to 1.5-fold between G0/G1 and S-phase and 1.6- to 1.9-fold between G0/G1 and G2/M-phase were detected by Western immunoblot and biochemical assays. TS protein was not detectable by Western blot analysis, flow cytometry or biochemical analysis in the G0/G1 population of confluent cells. Twenty-six per cent of cells in this population were G0 cells compared with 2% in exponentially growing cells. In contrast to TS, a 4-fold difference in thymidine kinase (TK) was detected between G0/G1 and S-phase cells in exponentially growing MCF-7 cells. The level of TS enzyme is associated with cellular proliferation and the percentage of cells in S-phase; however, TS protein is not exclusively associated with S-phase in asynchronously growing cells. The variation in TS levels between exponentially growing and confluent cell population appears to be due to differences in TS levels between G0 and G1 cells.
Highlights
Other studies have shown that, while increased Thymidylate synthase (TS) activity correlates with the DNA synthetic phase, this increase is not blocked by inhibitors of DNA synthesis (Jenh et al, 1985)
This suggests that, while TS activity may be associated with proliferation, its regulation may be independent of DNA synthesis and cell cycle phase
The purpose of this study was to analyse cell cycle variations in TS levels during the various cell cycle phases and proliferation to determine its association with DNA synthesis and S-phase in asynchronously growing tumour cells
Summary
Other studies have shown that, while increased TS activity correlates with the DNA synthetic phase, this increase is not blocked by inhibitors of DNA synthesis (Jenh et al, 1985) This suggests that, while TS activity may be associated with proliferation, its regulation may be independent of DNA synthesis and cell cycle phase. We have demonstrated that increased TS protein levels predict for poor clinical outcome in patients with rectal cancer (Johnston et al, 1994) This may be the result of the association of TS protein levels with cellular proliferation. The purpose of this study was to analyse cell cycle variations in TS levels during the various cell cycle phases and proliferation to determine its association with DNA synthesis and S-phase in asynchronously growing tumour cells
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