Effects of DNA Damaging Agents on G2 Phase Checkpoint

描述（由申请人提供）：DNA损伤剂，形式为 放射和化学治疗药物通常用于治疗人类 癌的了解细胞对DNA损伤的反应对于 改善这些治疗方式的治疗效果。是 证实治疗诱导的DNA损伤不会杀死癌细胞 直接.相反，它们最初激活一个或多个细胞周期， 检查点，随后诱导程序性细胞死亡。积累 有证据表明，G2期检查点反应是拮抗的， DNA损伤剂的细胞杀伤作用和G2期的消除 检查点可以改善化疗的结果。顺铂是一种广泛使用的 化疗中的DNA损伤剂。的主要问题之一 以顺铂为基础的化疗是后天性的。耐药性DACH-乙酰氨基铂 (DAP)是一种新顺铂类似物，具有很大的临床潜力， 顺铂耐药的癌细胞。为了更有效地利用这本小说 在未来的化疗药物，PI？的小组一直在进行研究， 阐明这种新药物引起的细胞反应，并确定 其独特的生物活性至关重要的机制。 在他们以前的研究中，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， 顺铂对G2期检查点具有显著不同的作用。而 顺铂主要抑制G2期进展，DAP对G2期进展几乎没有影响。 了这种差异增加了G2期沉默的可能性， 检查点反应可能有助于独特的生物活性， DAP。他们最近发现DAP是一种高效诱导剂（约100倍 增加）的通用Cdk抑制剂p21 Wafl/Cipl（p21）， p53依赖机制。然而，虽然诱导的p21结合G1期和 G2期Cdk复合物，仅抑制G1期Cdk活性。这 表明DAP可能激活一种尚未识别的机制， 诱导的p21抑制02期Cdk活性。在努力 发现这种新的机制，他们发现DAP似乎诱导 Cdc 2激酶在不同于那些已经 已知调节Cdc 2激酶活性。相反，他们发现顺铂 似乎诱导这些位点的磷酸化沿着抑制 Cdc 2激酶活性。这些新的初步发现使他们 假设（i）Cdc 2激酶在未表征 位点是诱导p21抑制Cdc 2激酶活性的先决条件; (ii)DAP诱导的Cdc 2激酶在这些位点的去磷酸化， DNA损伤诱导的G2期检查点反应;和（iii） 顺铂诱导的Cdc 2激酶在这些位点的磷酸化， 顺铂诱导的G2期检查点反应。为了验证这些假设， 提出了具体目标。首先，Cdc 2中未表征的位点， 在DAP处理的细胞中低磷酸化， 顺铂处理的细胞将通过质谱鉴定， 通过定点诱变证实。第二，激酶活性， 将从粗细胞裂解物中鉴定这些位点的磷酸化Cdc 2 及其对Cdc 2激酶活性的影响 将测定DAP处理的细胞。第三，新的Cdc 2磷酸化 位点将突变为不可磷酸化的氨基酸残基， 顺铂诱导的G2期检查点反应的突变体将是 通过使用转染的细胞检测。建议的研究将促进我们的 了解DAP和顺铂的分子药理学。此外，本发明还提供了一种方法， 它们可能填补了我们对参与这些疾病的分子途径的理解的空白。 DNA损伤诱导的G2期检查点反应，并提供新的策略 消除G2期检查点反应的方法。

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.