Effects of DNA Damaging Agents on G2 Phase Checkpoint

描述(由申请人提供)：DNA损伤剂 放射和化疗药物通常用于治疗人类 癌症。了解细胞对DNA损伤的反应对于 提高这些治疗方式的治疗效果。它是 确定治疗引起的DNA损伤不会杀死癌细胞 直接去吧。相反，它们最初激活一个或多个细胞周期 检查站，随后导致细胞程序性死亡。积累 证据表明，G2期检查点响应与 DNA损伤剂对细胞的杀伤作用及G2期的消除 检查站可能会改善化疗的结果。顺铂是一种应用广泛的 化疗中的DNA损伤剂。中国的主要问题之一 顺铂为主的化疗是后天获得性的。抗药性。DACH-乙酸铂 DAP是一种新型顺铂类似物，具有很大的临床应用潜力 癌细胞中的顺铂耐药。为了更有效地使用这部小说 在未来的药物化疗中，皮？S小组一直在进行研究 阐明这种新制剂引起的细胞反应，并鉴定 对其独特的抗肿瘤活性至关重要的机制。 在他们之前的研究中，他们比较了DAP和顺铂对 细胞周期检查点。这些研究清楚地表明，DAP和 顺铂对G2期检查点有明显不同的影响。而当 顺铂主要抑制G2期进展，DAP对G2期影响不大 它。这种差异增加了使G2阶段静默的可能性 检查点反应可能有助于独一无二的抗肿瘤活性 DAP。他们最近发现DAP是一种高度有效的诱导剂(-100倍 增加)通用CDK抑制剂p21 Wafl/Cip1(P21)通过 P53依赖机制。然而，虽然诱导的p21结合了Gi期和 G_2期CDK复合体，仅抑制G_I期CDK活性。这 提示DAP可能激活了一种尚未确定的机制，该机制可以防止 诱导p21抑制02期CDK活性。在他们的努力中 揭开这一新机制后，他们发现DAP似乎能诱导 在与已有的不同的位置上去磷酸化CDc2激酶 已知可以调节CDC2激酶的活性。相反，他们发现顺铂 似乎诱导了这些位点的磷酸化，同时抑制了 Cdc2激酶活性。这些新奇的初步发现引导他们 假说(I)未特定化的Cdc2蛋白 位点是诱导p21抑制CDc2激酶活性的先决条件； (Ii)DAP诱导的cdc2激酶在这些位点的去磷酸化可防止 DNA损伤诱导的G2期检查点反应；以及 顺铂诱导的CDc2激酶在这些位点上的磷酸化促进 顺铂诱导的G2期检查点反应。为了检验这些假说，有三个 提出了具体的目标。首先，Cdc2中未确定的位置是 DAP处理的细胞中低磷酸化和高磷酸化 顺铂处理的细胞将通过质谱学和 通过定点突变得到确认。第二，该蛋白的激酶活性 这些部位的磷酸化的cdc2将从粗细胞裂解物中鉴定出来。 生物化学分级及其对细胞内CDC2激酶活性的影响 DAP处理的细胞将被确定。第三，新型的Cdc2磷酸化 位点将突变为不可磷酸化的氨基酸残基及其影响 对顺铂诱导的G2期检查点反应的突变体中 通过使用转基因细胞进行检测。拟议的研究将推进我们的 了解DAP和顺铂的分子药理学。此外, 它们可能填补了我们对参与其中的分子途径的理解的空白 DNA损伤诱导的G2期检查点反应，并提供新的战略 消除G2期检查点响应的方法。

DESCRIPTION (provided by applicant): DNA damaging agents in the form of radiation and chemotherapeutic drugs are commonly used for treatment of human cancers. Understanding the cellular response to DNA damage is crucial for improving the therapeutic effects of these treatment modalities. It is established that the treatment-induced DNA damage does not kill cancer cells directly. Instead, they initially activate one or multiple cell cycle checkpoints and subsequently induce programmed cell death. Accumulating evidence indicates that the G2 phase checkpoint response is antagonistic to the cell killing effect of DNA damaging agents and that abrogation of the G2 phase checkpoint may improve the outcome of chemotherapy. Cisplatin is a widely used DNA damaging agent in chemotherapy. One of the major problems in cisplatin-based chemotherapy is the acquired. drug resistance. DACH-acetato-Pt (DAP) is a novel cisplatin analog with great clinical potential to overcome cisplatin resistance in cancer cells. For a more effective use of this novel drug in future chemotherapy, PI?s group have been undertaking studies to elucidate the cellular responses this new agent elicits and to identify the mechanisms crucial for its unique antineoplastic activity. In their previous studies, they compared effects of DAP and cisplatin on the cell cycle checkpoints. These studies clearly demonstrated that DAP and cisplatin have dramatically different effects on the G2 phase checkpoint. While cisplatin predominantly inhibits G2 phase progression, DAP has little effect on it. This difference raises the possibility that silencing the G2 phase checkpoint response may contribute to the unique antineoplastic activity of DAP. They recently discovered that DAP is a highly potent inducer (-100 fold increases) of the universal Cdk inhibitor p2lWafl/Cipl (p21) via a p53-dependent mechanism. However, while the induced p21 binds both Gi phase and G2 phase Cdk complexes, it only inhibits G I phase Cdk activities. This suggests that DAP may activate an as yet unidentified mechanism that prevents the induced p21 from inhibiting 02 phase Cdk activities. In their effort to uncover this novel mechanism, they discovered that DAP appeared to induce dephosphorylation of Cdc2 kinase at sites that are distinct from those already known to regulate Cdc2 kinase activity. In contrast, they found that cisplatin appeared to induce phosphorylation of these sites along with its inhibition of the Cdc2 kinase activity. These novel preliminary findings lead them to hypothesize (i) that phosphorylation of Cdc2 kinase at the uncharacterized sites is a prerequisite for the induced p21 to inhibit Cdc2 kinase activity; (ii) that DAP-induced dephosphorylation of Cdc2 kinase at these sites prevents the DNA damage-induced G2 phase checkpoint response; and (iii) that cisplatin-induced phosphorylation of Cdc2 kinase at these sites promotes the cisplatin-induced G2 phase checkpoint response. To test these hypotheses, three specific aims are proposed. First, the uncharacterized sites in Cdc2 that are hypophosphorylated in DAP-treated cells and hyperphosphorylated in cisplatin-treated cells will be identified through mass spectrometry and confirmed through site directed mutagenesis. Second, the kinase activity that phosphorylates Cdc2 at these sites will be identified from crude cell lysates by biochemical fractionations and its effect on the Cdc2 kinase activity from DAP treated cells will be determined. Third, the novel Cdc2 phosphorylation sites will be mutated to nonphosphorylatable amino acid residues and the effect of the mutants on cisplatin-induced G2 phase checkpoint response will be examined by using transfected cells. The proposed studies will advance our understanding of the molecular pharmacology of DAP and cisplatin. In addition, they may fill a gap in our understanding of the molecular pathways involved in DNA damage-induced G2 phase checkpoint response, and provide new strategic approaches to abrogate G2 phase checkpoint response.