Expression of thymidylate synthase in human cells is an early G(1) event regulated by CDK4 and p16INK4A but not E2F.
Thymidylate synthase (TS) is the enzyme that catalyses the last step in de novo thymidylate synthesis. It is of interest clinically because it is an effective target for drugs such as 5-fluorouracil, often used in combination therapy. Despite a number of earlier reports indicating that TS is a cell cycle-dependent enzyme, this remains equivocal. Here, we show that in HCT116 cells synchronised by serum starvation, there is a clear dissociation between the expression of cyclin E (a well-characterised cell-cycle protein) and TS. Although both cyclin E and TS mRNA and protein increased during G(1), TS upregulation was delayed. Moreover, TS levels did not decrease following S-phase completion while cyclin E decreased sharply. Similarly, clear differences were seen between cyclin E and TS as asynchronously growing HCT116 cells were growth-inhibited by low-serum treatment. In contrast to previous reports using rodent cells, adenovirus-mediated over-expression of E2F1 and cyclin E in three human cell lines had no effect on TS. Cell-cycle progression was blocked by treatment of cells with pharmacological inhibitors of CDK2 and CDK4 and by ectopic expression of p16INK4A. Whereas CDK2 inhibition had no effect on TS levels, inhibition of CDK4 was associated with decreased TS protein levels. These results provide the first evidence that drugs targeting CDK4 may be useful with anti-TS drugs as combination therapy for cancer.
Le François
B G
BG
Department of Biochemistry Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
Maroun
J A
JA
Birnboim
H C
HC
D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D016895:Culture Media, Serum-Free; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050687:E2F1 Transcription Factor / Q000378:metabolism*; D005260:Female; D016193:G1 Phase / Q000502:physiology*; D045325:HCT116 Cells / Q000201:enzymology / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008297:Male; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013940:Thymidylate Synthase / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
D016895:Culture Media, Serum-Free; D019927:Cyclin E; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050687:E2F1 Transcription Factor; D013940:Thymidylate Synthase; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4