Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator.
Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator.
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发表时间：
2001-11
期刊：
影响因子：
11.2
通讯作者：
Yu-li Wang;Jun Yu Li;R. Booher;A. Kraker;T. Lawrence;W. Leopold;Yi Sun
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作者：
Yu-li Wang;Jun Yu Li;R. Booher;A. Kraker;T. Lawrence;W. Leopold;Yi Sun
许多癌细胞中缺乏功能性p53为治疗提供了一个治疗靶点。缺乏p53的细胞不会表现出G(1)检查点，而是依赖于G(2)检查点来允许DNA在进行有丝分裂之前进行修复。我们假设G(2)检查点废除子可以通过去除唯一的检查点来优先杀死p53非活性癌细胞，该检查点保护这些细胞免受DNA损伤的过早有丝分裂。由于Wee1激酶通过Cdc2的抑制磷酸化在维持G(2)阻滞中起着至关重要的作用，我们开发了一种高通量的大规模筛选试验，并用它来筛选Wee1抑制剂的化学文库。发现一种吡啶类分子PD0166285在纳摩尔浓度下对Wee1具有抑制作用。在细胞水平上，0.5微米PD0166285显著抑制辐照诱导的Cdc2 tyr15和Thr-14磷酸化。生化标记和荧光激活细胞分选分析显示，PD0166285消除了辐射诱导的G(2)阻滞，并显著增加了有丝分裂细胞的数量。生物学上，PD0166285作为放射增敏剂使细胞对辐射诱导的细胞死亡增敏，根据标准克隆测定，其敏感性增强比为1.23。这种放射增敏活性依赖于p53，在p53无活性细胞中具有更高的功效。因此，G(2)检查点抑制剂代表了一类新的抗癌药物，通过诱导过早有丝分裂来增强传统癌症治疗的细胞杀伤作用。
The lack of functional p53 in many cancer cells offers a therapeutic target for treatment. Cells lacking p53 would not be anticipated to demonstrate a G(1) checkpoint and would depend on the G(2) checkpoint to permit DNA repair prior to undergoing mitosis. We hypothesized that the G(2) checkpoint abrogator could preferentially kill p53-inactive cancer cells by removing the only checkpoint that protects these cells from premature mitosis in response to DNA damage. Because Wee1 kinase is crucial in maintaining G(2) arrest through its inhibitory phosphorylation of Cdc2, we developed a high-throughput mass screening assay and used it to screen chemical library for Wee1 inhibitors. A pyridopyrimidine class of molecule, PD0166285 was identified that inhibited Wee1 at a nanomolar concentration. At the cellular level, 0.5 microM PD0166285 dramatically inhibits irradiation-induced Cdc2 phosphorylation at the Tyr-15 and Thr-14 in seven of seven cancer cell lines tested. PD0166285 abrogates irradiation-induced G(2) arrest as shown by both biochemical markers and fluorescence-activated cell sorter analysis and significantly increases mitotic cell populations. Biologically, PD0166285 acts as a radiosensitizer to sensitize cells to radiation-induced cell death with a sensitivity enhancement ratio of 1.23 as shown by standard clonogenic assay. This radiosensitizing activity is p53 dependent with a higher efficacy in p53-inactive cells. Thus, G(2) checkpoint abrogators represent a novel class of anticancer drugs that enhance cell killing of conventional cancer therapy through the induction of premature mitosis.