TY - JOUR T1 - p53 and WAF1 Are Induced and Rb Protein Is Hypophosphorylated during Cell Growth Inhibition by the Thymidylate Synthase Inhibitor ZD1694 (Tomudex) JF - Molecular Pharmacology JO - Mol Pharmacol SP - 630 LP - 636 DO - 10.1124/mol.51.4.630 VL - 51 IS - 4 AU - Ming-Biao Yin AU - Wieland Voigt AU - Angeles Panadero AU - Udo Vanhoefer AU - Cheryl Frank AU - Sanja Pajovic AU - Jane Azizkhan AU - Youcef M. Rustum Y1 - 1997/04/01 UR - http://molpharm.aspetjournals.org/content/51/4/630.abstract N2 - In a previous study, we found that treatment of HCT-8 cells with ZD1694, a specific antifolate-based thymidylate synthase inhibitor, resulted in DNA fragmentation. In this study, we have demonstrated the dose- and time-dependent induction of DNA fragmentation accompanied by elevation of p53 and WAF1 protein expression by ZD1694. WAF1 mRNA showed a time-dependent increase, whereas p53 mRNA was not found to be significantly overexpressed. The initial increase in WAF1 mRNA was detected at 4 hr, but increased WAF1 protein expression was detected 8–24 hr after a 2-hr exposure. The amount of total and hypophosphorylated pRb seems to be rising greatly after ZD1694 exposure. The effects of ZD1694 on the expression of E2F1 and formation of the E2F1-Rb complex were investigated 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 complex could be demonstrated beginning 4 hr after drug exposure by a gel shift assay. Kinetic analysis showed increased availability of hypophosphorylated pRb for inhibition of E2F, 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 24 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 ZD1694-induced cell growth inhibition in HCT-8 cells. Therefore, the observed effect on downstream events after effective inhibition of thymidylate synthase may offer the critical determinants of response to ZD1694. ER -