Checkpoint Response and Platinum Drug Sensitivity

描述(申请人提供)：尽管根据定义，癌细胞的细胞周期是不受调控的，但肿瘤通常通过p53依赖的p21的反式激活来保留完整的G1细胞周期检查点反应，然后p21抑制细胞周期依赖性激酶(CDK)并阻止G1/S转换。更重要的是，保留这种检查点反应的肿瘤对治疗药物也很敏感。事实上，在NCI-60细胞系肿瘤小组中，肿瘤细胞滞留在G1期的能力与对铂类药物(顺铂、四铂和奥沙利铂)的敏感性之间存在着强烈的正相关。这也与转导p21表达载体的突变型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-基因敲除诱导了对顺铂和一种新的旨在规避顺铂耐药性的类似物DAP的耐药性。在DNA损伤剂中，类似的DAP非常有趣，因为它只抑制G1期的CDK，选择性地诱导G1期停滞，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还没有明确的定义，可能是由于在S和G2期抑制CDK引起的信号干扰，这与细胞生存信号有关。在这两个阶段，DAP的使用不会抑制CDK，这为描绘这种关系提供了一个重要而及时的机会，从而可以出现合理的方法来改善治疗反应的频谱。在初步研究中，我们已经做了开创性的观察，在对照细胞中，被DAP诱导的p21抑制的G1期CDK复合体的大小明显大于相应的活性复合体。对被抑制的复合体的蛋白质组学分析确定了预期的p21，但揭示了增殖细胞核抗原是一种新的新药，它可能将抑制G1期CDK活性与铂介导的细胞死亡联系起来。因此，我们假设以铂为基础的药物对肿瘤细胞的死亡依赖于G1期CDK基因的p21功能，而这是通过增殖细胞核抗原的募集来促进的。我们将用三个具体的目标来解决这一假说，这将更好地定义我们对G1期抑制过程的理解，并将这一依赖p21的事件与细胞死亡联系起来。此外，与铂类化合物的比较研究将使我们能够理解顺铂耐药的机制，这种机制是非交叉耐药类似物可以绕过的。

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.