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

说明书(申请人提供)：大多数化疗药物通过诱导癌细胞凋亡而起作用，同时保留非癌细胞。然而，在许多肿瘤中，P53通路的凋亡诱导活性已经丧失，由此产生的肿瘤对凋亡治疗是顽固的。与P53不同，活性E2F1蛋白在癌细胞中经常过度表达。此外，最近的研究表明，E2F1可以以不依赖于P53的方式诱导细胞凋亡。综上所述，这些发现提示E2F1可能是一个重要的化疗靶点。这项建议的长期目的是确定E2F1诱导细胞凋亡的机制，以便利用这一信息开发更有效的化疗。为此，微阵列分析已经被用来筛选可以解释E2F1如何以非p53依赖的方式诱导细胞凋亡的靶点。初步研究发现，Bcl2家族的两个成员Mcl-1和Bok/MTD是E2F1诱导细胞凋亡的新靶点和直接介导物。具体来说，我们发现抗凋亡的Mcl-1基因被E2F1转录抑制，而促凋亡的BOK/MTD被E2F1的表达选择性地激活。我们提出了三个特定的目标来探索这一新的假设，即E2F1水平的升高可能通过转录控制Mcl-1和BOK/MTD的P53非依赖性途径诱导细胞死亡。在特定目标1中提出的实验将确定E2F1抑制Mcl-1启动子转录的机制。由于只需要E2F1的DNA结合域来介导这一作用，因此假设E2F1通过干扰Mcl-1转录激活剂，如STAT3和CREB来阻止Mcl-1的转录。在特定目的2中，假设BOK/MTD启动子包含E2F结合位点，并且E2F1通过直接结合和克隆激活BOK启动子，并表征BOK/MTD启动子，将测试转录激活。在具体目标3中，将结合遗传学和药理学实验来确定Mcl-1下调和BoldMtd上调在E2F1诱导的细胞凋亡中的相对贡献。

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.