p53 and WAF1 are induced and Rb protein is hypophosphorylated during cell growth inhibition by the thymidylate synthase inhibitor ZD1694 (Tomudex).
p53 and WAF1 are induced and Rb protein is hypophosphorylated during cell growth inhibition by the thymidylate synthase inhibitor ZD1694 (Tomudex).
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DOI：
10.1124/mol.51.4.630
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发表时间：
1997-04
影响因子：
3.6
通讯作者：
M. Yin;W. Voigt;A. Panadero;U. Vanhoefer;C. Frank;S. Pajovic;J. Azizkhan;Y. Rustum
中科院分区：
文献类型：
--
作者：
M. Yin;W. Voigt;A. Panadero;U. Vanhoefer;C. Frank;S. Pajovic;J. Azizkhan;Y. Rustum
在以前的研究中，我们发现用ZD 1694（一种特异性抗叶酸胸苷酸合成酶抑制剂）处理HCT-8细胞会导致DNA片段化。在本研究中，我们证明了ZD 1694诱导DNA断裂的剂量和时间依赖性，并伴有p53和WAF 1蛋白表达升高。WAF 1 mRNA呈时间依赖性增加，而p53 mRNA未被发现显着过表达。在4小时时检测到WAF 1 mRNA的初始增加，但在2小时暴露后8-24小时检测到WAF 1蛋白表达增加。暴露于ZD 1694后，总pRb和低磷酸化pRb的量似乎大幅上升。在药物暴露2小时后，研究了ZD 1694对E2 F1表达和E2 F1-Rb复合物形成的影响（IC 90）。结果显示E2 F1 mRNA和蛋白表达呈时间依赖性下降;凝胶迁移试验显示药物暴露后4小时开始E2 F-Rb复合物丰度增加。动力学分析表明，低磷酸化的pRb抑制E2 F的可用性增加，这可能间接导致WAF 1诱导的抑制细胞周期蛋白依赖性激酶活性。然而，ZD 1694对胸苷酸合成酶的抑制作用起效迅速，并在药物处理后维持至少24小时，而药物诱导的细胞生长抑制作用在药物暴露后24小时显著。在HCT-8细胞中，低磷酸化pRb的丰度增加以及与转录因子E2 F-1的结合与ZD 1694诱导的细胞生长抑制一致。因此，有效抑制胸苷酸合成酶后观察到的对下游事件的影响可能是ZD 1694应答的关键决定因素。
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.