Role of Cdc2 in HPV E6-mediated Apoptosis

描述（由申请人提供）：人乳头瘤病毒（HPV）感染可诱导疣，并与宫颈癌的发生密切相关。我们的长期目标是阐明HPV诱导的病变的分子基础，并开发消除HPV感染的治疗策略。细胞凋亡是一种遗传程序化的细胞破坏过程，可以消除病毒感染的细胞或肿瘤。病毒如HPV已经进化出特定的基因产物来调节细胞凋亡。诱导细胞凋亡是治疗HPV诱导的病变的理想方法。HPV E6基因是病毒复制所必需的，并诱导细胞凋亡。然而，E3介导细胞对凋亡敏感的机制仍有待确定。为了了解E6介导的细胞凋亡的分子基础，我们建立了表达HPV E6的人角质形成细胞，发现这些细胞对化疗药物诱导的细胞凋亡敏感。为了鉴定负责E6介导的细胞凋亡的基因，采用蛋白质组学方法。值得注意的是，Cdc2的水平和活性，细胞进入有丝分裂所需的细胞周期蛋白依赖性激酶，其异常激活已与细胞凋亡，在E6表达细胞凋亡增加。不能激活Cdc2的E6突变体在使细胞对凋亡敏感方面是有缺陷的。此外，用特异性抑制剂抑制Cdc2活性可减少细胞凋亡。我们推测Cdc2的激活在E6介导的细胞凋亡敏化中起重要作用。本提案的具体目的是通过以下方法测试这种可能性：（1）通过遗传方法检查Cdc2与E6介导的细胞致敏性对凋亡的相关性，以及（2）通过分子消融和异位表达确定Cdc2在E6介导的凋亡中的作用。这些研究将有助于理解E6介导的细胞凋亡机制，并有助于开发针对HPV诱导疾病的治疗方法。

DESCRIPTION (provided by applicant): Infection with the human papillomaviruses (HPV) induces warts and is strongly associated with the development of cervical cancer. Our long-term goal is to elucidate the molecular basis for HPV-induced lesions and to develop therapeutic strategies for the elimination of HPV infection, Apoptosis is a genetically programmed process of cellular destruction that can eliminate virally infected cells or tumors. Viruses such as HPV have evolved specific gene products to modulate apoptosis. Induction of apoptosis is a desirable approach in treating HPV-induced lesions. The HPV E6 gene is essential for viral replication and induces apoptosis. However, the mechanism by which E3 mediates cellular sensitization to apoptosis remains to be determined. To understand the molecular basis for E6-mediated apoptosis, we have established human keratinocytes expressing HPV E6 and found these cells to be susceptible to apoptosis induced by chemotherapeutic agents. To identify genes responsible for E6-mediated apoptosis, a proteomic approach was taken. Significantly, the level and activity of Cdc2, the cyclin-dependent kinase required for cells to enter mitosis and whose aberrant activation has been associated with apoptosis, was increased in E6-expressing cells undergoing apoptosis. An E6 mutant that is unable to activate Cdc2 is defective in sensitizing cells to apoptosis. Moreover, inhibition of Cdc2 activity with specific inhibitor reduced ap6ptosis. We hypothesize that activation of Cdc2 plays an important role in E6-mediated cellular sensitization to apoptosis. The specific aim of this proposal is designed to test this possibility with the following approaches: (1) to examine the relevance of Cdc2 to E6-mediated cellular sensitization to apoptosis through a genetic approach and (2) to determine the role of Cdc2 in E6-mediated apoptosis by molecular ablation and ectopic expression. These studies will help understand the mechanism of E6-mediated apoptosis and contribute to the development of therapeutic approaches against HPV-induced diseases.