P53 AND WAF1 ARE INDUCED AND RB PROTEIN IS HYPOPHOSPHORYLATED DURING CELL-GROWTH INHIBITION BY THE THYMIDYLATE SYNTHASE INHIBITOR ZD1694 (TOMUDEX)

In a previous study, we found that treatment of HCT-8 cells with ZD169 4, a specific antifolate-based thymidylate synthase inhibitor, resulte d in DNA fragmentation. In this study, we have demonstrated the dose- and time-dependent induction of DNA fragmentation accompanied by eleva tion of p53 and WAF1 protein expression by ZD1694. WAF1 mRNA showed a time-dependent increase, whereas p53 mRNA was not found to be signific antly overexpressed. The initial increase in WAF1 mRNA was detected at 4 hp, but increased WAF1 protein expression was detected 8-24 hr afte r a 2-hr exposure. The amount of total and hypophosphorylated pRb seem s to be rising greatly after ZD1694 exposure. The effects of ZD1694 on the expression of E2F1 and formation of the E2F1-Rb complex were inve stigated after a 2-hr drug exposure (IC90). The results showed a time- dependent decrease in E2F1 mRNA and protein expression; an increase in the abundance of the E2F-Rb com; plex could he demonstrated beginning 4 br after drug exposure by a gel shift assay. Kinetic analysis showe d increased availability of hypophosphorylated pRb for inhibition of E 2F, which could indirectly result from WAF1-induced inhibition cyclin- dependent kinase activity. Whereas thymidylate synthase inhibition by ZD1694 was rapid in onset and maintained for at least 23 hr after drug treatment, drug-induced cellular growth inhibition was significant 24 hr after drug exposure. The increased abundance of hypophosphorylated pRb and binding to transcription factor E2F-1 is consistent with ZD16 94-induced cell growth inhibition in HCT-8 cells. Therefore, the obser ved effect on downstream events after effective inhibition of thymidyl ate synthase may offer the critical determinants of response to ZD1694 .