E2F AND APOPTOSIS

拟议研究的长期目标是确定增殖和凋亡之间正常关系的分子机制，并了解在癌症等疾病中解开这种联系的后果。 越来越多的证据表明，Rb/E2 F途径在细胞进入S期的过程中发挥着重要作用，p53通过诱导生长停滞或凋亡来限制该途径的增殖能力。 为了支持这种关系，我们以前已经表明，E2 F1的表达不仅导致S期诱导，而且导致p53依赖性细胞凋亡。 这种细胞凋亡是E2 F1特异性的，并且与E2 F1介导的p53蛋白积累相一致。 这两个过程似乎都受到E2 F1转录活性的刺激。 使用重组腺病毒在p19 ARF、p53或Mdm 2/p53双缺失的小鼠胚胎成纤维细胞（MEFs）中有效表达cDNA，我们现在有证据表明E2 F1通过不同的途径发出细胞凋亡和p53积累的信号。 研究表明，E2 F1通过激活p19 ARF通路诱导p53积聚和凋亡。然而，我们发现，虽然p19 ARF是下游的E2 F1在信号p53积累，p19 ARF是不必要的E2 F1介导的凋亡。 有趣的是，我们确实发现Mdm 2是E2 F1介导的p53积累和凋亡所必需的。这意味着p53积累和凋亡途径在Mdm 2影响p53功能的点处分叉。 为了进一步研究E2 F1诱导p53蛋白积聚和凋亡的不同途径，我计划1）表征E2 F表达导致p53蛋白积聚的途径并分析诱导该途径的后果：2）确定p19 ARF非依赖性途径导致p53依赖性凋亡的步骤; 3）鉴定E2 F1特异性诱导的凋亡基因。

The long-term objectives of the proposed research are to define the molecular mechanisms underlying the normal relationship between proliferation and apoptosis and to understand the consequences of uncoupling this connection in diseases like cancer. Mounting evidence points to central roles of the Rb/E2F pathway in the commitment of cells to S phase and for p53 to limit the proliferative capacity of this pathway by inducing growth arrest or apoptosis. In support of this relationship, we have previously shown that expression of E2F1 not only leads to S phase induction, but also to a p53-dependent apoptosis. This apoptosis is specific to E2F1 and coincides with an E2F1-mediated accumulation of p53 protein. Both of these processes appear to be stimulated by E2F1 transcriptional activity. Using recombinant adenoviruses to efficiently express cDNAs in mouse embryo fibroblasts (MEFs) that are nullizygous for p19ARF, p53 or double null for Mdm2/p53, we now have evidence to suggest that E2F1 signals apoptosis and p53 accumulation through separate pathways. It has been suggested that E2F1 induces p53 accumulation and apoptosis by activating the p19ARF pathway. However, we find that although p19ARF is downstream of E2F1 in signaling p53 accumulation, p19ARF is not necessary for E2F1- mediated apoptosis. Interestingly, we do find that Mdm2 is required for both E2F1-mediated p53 accumulation and apoptosis. This implies that the p53 accumulation and apoptosis pathways diverge at the point where Mdm2 affects p53 function. To further investigate the different pathways by which E2F1 induces p53 accumulation and apoptosis, I plan to 1) characterize the pathway by which E2F expression leads to p53 protein accumulation and analyze the consequences of inducing this pathway; 2) determine the steps involved in a p19ARF-independent pathway that leads to p53-dependent apoptosis; and 3) identify apoptosis genes that are specifically induced by E2F1.