Checkpoint Response and Platinum Drug Sensitivity

描述（由申请人提供）：尽管根据定义，癌细胞具有不受调节的细胞周期，但肿瘤通常通过p53依赖性p21的转激活保持完整的G1细胞周期检查点反应，然后抑制周期蛋白依赖性激酶（Cdk）并阻止G1/S转变。更重要的是，保留这种检查点反应的肿瘤对治疗剂也很敏感。事实上，在NCI 60细胞系肿瘤组中，肿瘤细胞在G1期阻滞的能力与对铂类药物（顺铂、四铂和奥沙利铂）的敏感性之间存在很强的正相关。这也与p21表达载体转染的突变p53肿瘤细胞对顺铂敏感的发现一致。此外，我们的初步数据表明，p21-敲除可诱导对顺铂和一种新型类似物DAP的耐药，而DAP被设计用于规避顺铂耐药。类似物DAP在dna损伤剂中非常有趣，因为它只抑制G1期Cdk来选择性地诱导G1期阻滞，这巩固了p21抑制G1期Cdk与细胞死亡之间的关系。然而，这种关系的潜在基础在文献中尚未明确定义，可能是由于抑制Cdk在S期和g2期引起的串扰信号干扰，这两个阶段与细胞存活信号传导有关。DAP在这两个阶段不抑制Cdk，这为描述这种关系提供了重要和及时的机会，从而可以出现合理的方法来改善治疗反应的范围。在初步研究中，我们进行了开创性的观察，发现dap诱导的p21抑制g1期Cdk复合物的大小明显大于对照细胞中相应的活性复合物。抑制复合物的蛋白质组学分析确定了预期的p21，但显示PCNA是一个新的招募，可能将g1期Cdk活性的抑制与铂介导的细胞死亡联系起来。因此，我们假设基于铂类药物的肿瘤细胞死亡依赖于g1期Cdk位点的p21功能，而PCNA的募集促进了这一点。我们将以三个具体目标来解决这一假设，这将更好地定义我们对g1期抑制过程的理解，并将这种依赖于p21的事件与细胞死亡联系起来。此外，与铂配合物的比较研究将使我们能够了解顺铂耐药的机制，而这种机制是由非交叉耐药类似物规避的。

DESCRIPTION (provided by applicant): Although, by definition, cancer cells have a deregulated cell cycle, tumors often retain an intact G1 cell cycle checkpoint response via p53-dependent transactivation of p21, which then inhibits cyclin-dependent kinases (Cdk) and prevents G1/S transition. More importantly, tumors retaining this checkpoint response are also sensitive to therapeutic agents. Indeed, a strong positive correlation exists between the ability of tumor cells to arrest in G1 and sensitivity to platinum-based agents (cisplatin, tetraplatin and oxaliplatin) in the NCI 60- cell line tumor panel. This is also consistent with the finding that mutant-p53 tumor cells transfected with a p21 expression vector are sensitized to cisplatin. Moreover, our preliminary data demonstrate that p21- knockout induces resistance to cisplatin and to a novel analog DAP, which was designed to circumvent cisplatin resistance. The analog DAP is highly interesting among DNA-damaging agents in that it only inhibits G1-phase Cdk to selectively induce G1 arrest, and this consolidates the relationship between G1-phase Cdk inhibition by p21 and cell death. However, the underlying basis for this relationship has not been clearly defined in the literature, possibly due to signaling interference from the cross talk caused by inhibition of Cdk in S- and G2-phases, which are linked to cell-survival signaling. The availability of DAP, which does not inhibit the Cdk in these two phases, provides an important and timely opportunity to delineate this relationship so that rational approaches could emerge to improve the spectrum of therapeutic response. In preliminary studies, we have made a seminal observation that G1-phase Cdk complexes inhibited by DAP-induced p21 are substantially larger in size than the corresponding active complexes in control cells. Proteomic analysis of the inhibited complex identified the expected p21, but revealed PCNA as a novel recruit, which potentially links inhibition of G1-phase Cdk activity to platinum-mediated cell death. Therefore, we hypothesize that tumor cell death by platinum-based agents is dependent on p21 function at the G1-phase Cdk locus and this is facilitated by PCNA recruitment. We will address this hypothesis with three specific aims, which will better define our understanding of the process of G1-phase inhibition, and link this p21-dependent event to cell death. Moreover, comparative studies with platinum complexes will allow us to appreciate a mechanism of cisplatin resistance that is circumventable by non-cross-resistant analogs.