UCN-O1 Suppresses Thymidylate Synthase Gene Expression and Enhances 5-Fluorouracil-induced Apoptosis in a Sequence- dependent Manner’

UCN-01, a protein kinase C/cydin-dependent kinase inhibitor, suppressed thymidylate synthase (TS) protein expression in a dose-dependent manner with near complete suppression at 1 LM after a 24-h exposure in human gastric cancer cell line SK-GT5. Other protein kinase C/cydindependent kinase inhibitors, including fiavopiridol and safingol, had a similar effect on TS protein expression, but to a lesser degree. Moreover, UCN-01 repressed the induction of TS after 5-fluorouradil (FU) exposure by 90-95% and significantly enhanced the induction of apoptosis by FU from 4-8% with either Hi or UCN-Ol alone to 46 ± 1% (P < 0.005 versus either single drug, reverse sequence, or the combination) when UCN-01 was given after FU. The effect of UCN-0l on TS was associated with a dose-dependent suppression of the E2F-1 protein, a transcriptional activator of TS. Northern blot analysis revealed that TS mRNA levels decreased gradually as the concentration of UCN-01 increased, but that E2F-1 mRNA levels remained relatively unchanged. UCN-01 may provide a novel way to enhance cellular sensitivity toward FU by means of suppressing TS expression mediated mainly by down-regulation of E2F-1.