E2F AND APOPTOSIS

这项拟议研究的长期目标是确定增殖和凋亡之间正常关系的潜在分子机制，并了解在癌症等疾病中脱离这种联系的后果。越来越多的证据表明，Rb/E2F通路在细胞进入S期的过程中起核心作用，而P53通过诱导生长停滞或凋亡来限制该通路的增殖能力。为了支持这种关系，我们先前已经证明，E2F1的表达不仅导致S期的诱导，而且还导致P53依赖的细胞凋亡。这种凋亡是E2F1所特有的，并且与E2F1介导的P53蛋白积聚相一致。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p19ARF、P53缺失或MDM2/P53双缺失的小鼠胚胎成纤维细胞(MEF)中高效表达cDNA，我们现在有证据表明E2F1通过不同的途径传递细胞凋亡和P53积聚的信号。已有研究表明，E2F1通过激活p19ARF途径诱导P53积聚和细胞凋亡。然而，我们发现，虽然p19ARF在信号转导P53积聚方面位于E2F1的下游，但p19ARF对于E2F1介导的细胞凋亡并不是必需的。有趣的是，我们确实发现MDM2是E2F1介导的P53积聚和细胞凋亡所必需的。这意味着在MDM2影响P53功能的点上，P53的积累和凋亡途径是不同的。为了进一步研究E2F1诱导P53蛋白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，并分析诱导这一途径的后果；2)确定p19ARF非依赖途径中涉及的步骤，从而导致P53依赖的细胞凋亡；以及3)识别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.