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

大多数化疗药物通过诱导癌细胞凋亡而起作用，同时保留非肿瘤细胞。 癌细胞然而，在许多肿瘤中，p53通路的促凋亡活性已经丧失 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蛋白是 经常在癌细胞中过度表达。此外，最近的研究表明，E2 F1可以 以p53非依赖性方式诱导细胞凋亡。这些发现表明，E2 F1可能是 重要的化疗靶点。本提案的长期目的是确定 E2 F1诱导细胞凋亡的机制，以便该信息可用于开发更有效的 化疗为此，微阵列分析已被用于筛选靶点， E2 F1如何以p53非依赖性方式诱导细胞凋亡。初步工作已经确定了两名成员， Bcl-2家族，Mcl-1和Bok/Mtd，作为E2 F1新靶点和E2 F1诱导的细胞凋亡的直接介质。 具体来说，我们发现抗凋亡Mcl-1基因被E2 F1转录抑制，而促凋亡Mcl-1基因被E2 F1转录抑制。 凋亡的Bok/Mtd被E2 F1表达选择性激活。我们提出了三个具体目标，以探讨 E2 F1水平升高可能通过p53非依赖性途径诱导细胞死亡的新假说 通过Mcl-1和Bok/Mtd的转录调控。 具体目标1中提出的实验将确定E2 F1抑制 Mcl-1启动子的转录。由于仅需要E2 F1的DNA结合结构域来介导这一过程， E2 F1可能通过干扰Mcl-1的转录而阻断Mcl-1的转录。 活化剂，如STAT 3和CREB。在特异性目的2中，假设Bok/Mtd启动子含有 E2 F结合位点并且E2 F1通过直接结合和转录激活Bok启动子 通过克隆和表征Bok/Mtd启动子来测试激活。具体目标3a 遗传学和药理学实验的组合将用于确定相对贡献 Mcl-1下调和BoldMtd上调对E2 F1诱导的细胞凋亡的影响。

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 pro- apoptotic 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 transcriptional activation will be tested by cloning and characterizing the Bok/Mtd promoter. 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.