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来发挥作用，而非化疗药物 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性的。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，这些发现表明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，已经使用微阵列分析来筛选可以解释 E2F1如何以非P53依赖的方式诱导细胞凋亡。初步工作确定了两名成员 Bcl2家族Mcl-1和BOK/MTD是E2F1诱导细胞凋亡的新靶点和直接介导物。 具体地说，我们发现抗凋亡的Mcl-1基因在转录上受到E2F1的抑制，而PRO-1基因则在转录水平上受到抑制。 凋亡的BOK/MTD被E2F1的表达选择性地激活。我们提出了三个具体的目标来探索 E2F1水平升高可能通过非p53途径诱导细胞死亡的新假说 通过转录调控Mcl-1和BOK/MTD。 在特定目标1中提出的实验将确定E2F1抑制的机制 Mcl-1启动子的转录。因为只需要E2F1的DNA结合域来调节这一过程 假设E2F1通过干扰Mcl-1转录而阻断Mcl-1转录。 激活剂，如STAT3和CREB。在特定目标2中，BOK/MTD启动子包含的假设 E2F结合位点和E2F1通过直接结合和转录激活BOK启动子 将通过克隆和鉴定BOK/MTD启动子来测试激活情况。在具体目标3 a中 基因和药理学实验的结合将被用来确定相对贡献 Mcl-1下调和BoldMtd上调对E2F1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.