Checkpoint Response and Platinum Drug Sensitivity

描述（由申请人提供）：虽然根据定义，癌细胞具有失调的细胞周期，但肿瘤通常通过p53依赖性p21反式激活保留完整的G1细胞周期检查点反应，然后抑制细胞周期蛋白依赖性激酶（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也对治疗剂敏感。事实上，在NCI 60细胞系肿瘤组中，肿瘤细胞在G1期停滞的能力与对铂类药物（顺铂、四铂和奥沙利铂）的敏感性之间存在强正相关。这也与用p21表达载体转染的muplant-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基因敲除诱导耐药顺铂和一种新的类似物DAP，这是为了规避顺铂耐药。类似物DAP在DNA损伤剂中是非常有趣的，因为它仅抑制G1期Cdk以选择性地诱导G1停滞，并且这巩固了p21对G1期Cdk的抑制与细胞死亡之间的关系。然而，这种关系的基础尚未在文献中明确定义，可能是由于在S期和G2期抑制Cdk引起的串扰的信号干扰，这与细胞存活信号有关。DAP在这两个阶段中不抑制Cdk，它的可用性提供了一个重要而及时的机会来描述这种关系，从而可以出现合理的方法来改善治疗反应的谱。在初步研究中，我们已经取得了一个开创性的观察，G1期Cdk复合物抑制DAP诱导的p21的大小基本上大于相应的活性复合物在控制细胞。抑制复合物的蛋白质组学分析确定了预期的p21，但发现PCNA作为一种新的招募，这可能会抑制G1期Cdk活性铂介导的细胞死亡。因此，我们推测，肿瘤细胞死亡铂类药物依赖于p21功能在G1期Cdk基因座，这是促进增殖细胞核抗原的招聘。我们将通过三个具体目标来解决这个假设，这将更好地定义我们对G1期抑制过程的理解，并将这种p21依赖性事件与细胞死亡联系起来。此外，与铂络合物的比较研究将使我们能够理解非交叉耐药类似物可规避的顺铂耐药机制。

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.