Transcriptional Control of Mcl-1 and Bok/Mtd by E2F1

说明（由申请人提供）：大多数化疗药物通过诱导癌细胞凋亡而不影响非癌细胞来起作用。然而，在许多肿瘤中，p53途径的凋亡诱导活性已经丧失，并且所产生的肿瘤对凋亡疗法是无效的。与p53相反，活性E2 F1蛋白在癌细胞中经常过表达。此外，最近的工作表明，E2 F1可以诱导凋亡的p53非依赖性的方式。总之，这些发现表明E2 F1可能是一个重要的化疗靶点。该提案的长期目的是确定E2 F1诱导细胞凋亡的机制，以便这些信息可以用于开发更有效的化疗。为此，微阵列分析已被用于筛选靶点，解释E2 F1如何以p53非依赖性方式诱导细胞凋亡。初步工作已经确定Bcl-2家族的两个成员Mcl-1和Bok/Mtd是E2 F1的新靶点和E2 F1诱导的细胞凋亡的直接介质。具体而言，我们发现，抗凋亡的Mcl-1基因的转录抑制E2 F1，而促凋亡的博克/Mtd选择性激活E2 F1的表达。我们提出了三个具体的目的，以探讨新的假设，即E2 F1水平升高可能会通过一个p53非依赖性途径，通过转录控制Mcl-1和博克/Mtd诱导细胞死亡。具体目标1中提出的实验将确定E2 F1抑制Mcl-1启动子转录的机制。由于仅需要E2 F1的DNA结合结构域来介导这种作用，因此假设E2 F1通过干扰Mcl-1转录激活因子（例如STAT 3和CREB）来阻断Mcl-1转录。在特定目标2中，Bok/Mtd启动子含有E2 F结合位点并且E2 F1通过直接结合和克隆激活Bok启动子以及表征Bok/Mtd启动子的假设将测试转录激活。在具体目标3中，将使用遗传和药理学实验的组合来确定Mcl-1下调和BoldMtd上调对E2 F1诱导的细胞凋亡的相对贡献。

DESCRIPTION (provided by applicant): The majority of chemotherapeutic drugs work by inducing apoptosis of cancer cells, while sparing non-cancerous cells. However, in many tumors the apoptosis-inducing activity of the p53 pathway has been lost and the resulting tumors are recalcitrant to apoptotic therapies. In contrast to p53, active E2F1 protein is frequently over expressed in cancer cells. Furthermore, recent work has demonstrated that E2F1 can induce apoptosis in a p53-independent manner. Taken together these findings suggest that E2F1 could be an important chemotherapeutic target. The long-term purpose of this proposal is to determine the mechanism by which E2F1 induces apoptosis so that this information can be used to develop more effective chemotherapy. Toward this end, microarray analysis has been used to screen for targets that would explain how E2F1 induces apoptosis in a p53-independent manner. Preliminary work has identified two members of the Bcl-2 family, Mcl-1 and Bok/Mtd, as novel E2F1 targets and direct mediators of E2Fl-induced apoptosis. Specifically, we find that the anti-apoptotic Mcl-1 gene is transcriptionally repressed by E2F1, whereas proapoptotic Bok/Mtd is selectively activated by E2F1 expression. We propose three Specific Aims to explore the novel hypothesis that elevated levels of E2F1 may induce cell death via a p53-independent pathway through transcriptional control of Mcl-1 and Bok/Mtd. Experiments proposed in Specific Aim 1 will determine the mechanism by which E2F1 represses transcription of the Mcl-1 promoter. Since only the DNA binding domain of E2F1 is required to mediate this effect, it is hypothesized that E2F1 blocks Mcl-1 transcription by interfering with Mcl-1 transcriptional activators, such as STAT3 and CREB. In Specific Aim 2 the hypothesis that the Bok/Mtd promoter contains E2F binding sites and that E2F1 activates the Bok promoter through direct binding and cloning and characterizing the Bok/Mtd promoter will test transcriptional activation. In Specific Aim 3 a combination of genetic and pharmacological experiments will be used to determine the relative contribution of Mcl-1 down regulation and BoldMtd up regulation to E2Fl-induced apoptosis.