E2F AND APOPTOSIS

这项研究的长期目标是确定增殖和细胞凋亡之间正常关系的分子机制，并了解在癌症等疾病中解耦这种联系的后果。越来越多的证据表明，Rb/E2F通路在细胞进入S期过程中发挥着核心作用，而p53则通过诱导生长停滞或细胞凋亡来限制该通路的增殖能力。为了支持这种关系，我们之前已经证明E2F1的表达不仅导致S期诱导，而且还导致p53依赖性的细胞凋亡。这种凋亡是E2F1特异性的，与E2F1介导的p53蛋白积累相吻合。这两个过程似乎都受到E2F1转录活性的刺激。利用重组腺病毒在小鼠胚胎成纤维细胞（mef）中高效表达p19ARF、p53或Mdm2/p53双零合子的cdna，我们现在有证据表明E2F1通过不同的途径信号细胞凋亡和p53积累。有研究表明，E2F1通过激活p19ARF通路诱导p53积累和凋亡。然而，我们发现尽管p19ARF在信号p53积累中位于E2F1的下游，但p19ARF并不是E2F1介导的细胞凋亡所必需的。有趣的是，我们确实发现Mdm2是e2f1介导的p53积累和细胞凋亡所必需的。这表明在Mdm2影响p53功能时，p53的积累和凋亡途径发生了分化。为了进一步研究E2F1诱导p53积累和凋亡的不同途径，我计划：1)表征E2F表达导致p53蛋白积累的途径，并分析诱导该途径的后果；2)确定p19arf独立通路导致p53依赖性凋亡的步骤；3)鉴定E2F1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.