Le Francois, Brice2013-11-082013-11-0820082008Source: Dissertation Abstracts International, Volume: 70-08, Section: B, page: 4821.http://hdl.handle.net/10393/29734http://dx.doi.org/10.20381/ruor-19884Thymidylate synthase (TS) catalyzes the last step of the only de novo synthesis pathway for deoxythymidine monophosphate (dTMP), a nucleotide required for DNA synthesis and repair. The central role of TS in nucleic acid metabolism has made it an attractive target for antineoplastic drugs. 5-fluorouracil is a prototypic drug targeting TS activity, which blocks the enzyme by forming a stable inhibitory complex with the protein. Depletion of dTMP following TS inhibition leads to repeated cycles of incorporation of uridine into DNA, causing single strand breaks, and ultimately triggering apoptosis. TS is commonly believed to be a S-phase dependent enzyme, an assumption largely based on a score of studies performed in rodent cells. Recent reports do not support this view and suggest that TS is a cell proliferation marker rather than a S-phase enzyme. The mechanisms controlling TS expression in human cells are poorly understood and could potentially provide new therapeutics approaches to decrease TS levels in cancer cells and to sensitize them to anti-TS drugs. We showed that, in human cells synchronized by serum starvation or subjected to serum deprivation, the expression patterns of TS and cyclin E (a well characterized E2F target) were significantly different. Ectopic expression of E2F1 or cyclin E had no effect on TS levels. However, inhibition of CDK4 but not CDK2 was associated with a clear decrease in TS levels in human cells. We also demonstrated that inhibition of MEK1 was associated with a decrease in TS levels, but this event was independent of ERK2 activity. Using reporter assays, we showed that MEK1 controlled TS promoter transcription, and that deletion of the Sp1 site from the essential promoter region greatly reduced the ability of MEK1 to stimulate the TS promoter. Surprisingly, unlike in rodent cells, LSF and GABP failed to increase TS levels in human cells. Taken together, our data demonstrate that TS levels in human cells are largely independent of S-phase and are primarily controlled by CDK4 and MEK1 activity during G1. Furthermore, we provide strong evidence that the mechanisms controlling TS expression in human cells significantly differ from those of rodent cells.153 p.enChemistry, Biochemistry.Thesis