ANALYSIS OF P73 FUNCTION AND REGULATION BY E2F 1

视网膜母细胞瘤(PRB)和P53肿瘤抑制蛋白的双重失活在癌变过程中经常发生。PRB的失活导致E2F转录因子家族的失调。E2F既促进细胞周期进程，又促进细胞凋亡。后者既涉及P53依赖机制，也涉及P53非依赖机制。我的初步数据表明，E2F-1可以诱导最近发现的名为p73的p53同源基因。至少在过量生产时，p73可以激活p53反应基因的转录，并通过诱导细胞凋亡来抑制细胞生长。由于p73的内源性水平非常低，而且与p53不同，导致p73激活的上游信号通路尚未阐明，有趣的是，E2F-1诱导p73水平显著升高。E2F/pRB通路的失调是大多数肿瘤细胞常见的现象。因此，了解p73在这一途径中的功能可能会为p73在肿瘤发生中的作用提供线索。具体目标1的目的是确定E2F-1诱导p73的机制。标准的蛋白质稳定性和定量RNA分析将被用来确定E2F-1对p73的影响是否由于转录、mRNA稳定性和/或翻译后修饰的变化。E2F-1对P53缺失细胞p73的诱导是否转化为P53靶基因的激活和细胞凋亡将构成特异性AIMS 2和3的基础。E2F-1诱导P73依赖的P53和P73靶基因反式激活的能力将通过生物化学和基于转录的功能分析来检测。在具体目标3中，将使用流式细胞术和免疫荧光技术来研究p73是否在E2F-1依赖的细胞凋亡中起作用。在这些检测中，将使用p73显性阴性蛋白来确定特异性。最后，在特定的目的4中，我们将研究p73在化疗药物诱导的E2F依赖的细胞凋亡中的作用。这些研究可能为调控p73的信号提供第一个线索，也可能有助于解释E2F对P53的非依赖性杀伤。最终，这项申请中提出的研究结果可能有助于开发新的基因疗法，在这种疗法中，E2F-1蛋白或可能是p73，可以用来诱导肿瘤细胞凋亡。

Dual inactivation of the retinoblastoma (pRb) and p53 tumor suppressor proteins occurs commonly during carcinogenesis. Inactivation of pRb leads to dysregulation of the E2F transcription factor family. E2F promotes both cell-cycle progression and apoptosis. The latter involves both p53- dependent and p53-independent mechanisms. My preliminary data suggests that E2F-1 can induce a recently identified p53 homolog called p73. p73 can, at least when overproduced, activate the transcription of p53-responsive genes and inhibit cell growth by inducing apoptosis. Since endogenous levels of p73 are very low and unlike p53, upstream signaling pathways leading to p73 activation have yet to be elucidated, it is intriguing that E2F-1 induces a significant increase in p73 levels. Dysregulation of the E2F/pRb pathway is common to most tumor cells. Therefore understanding the function of p73 within this pathway may provide clues as to the role of p73 in tumorigenesis. The goal of Specific Aim 1 is to determine the mechanism whereby E2F-1 induces p73. Standard protein stability and quantitative RNA assays will be employed to determine whether the effect of E2F-1 on p73 is due to changes in transcription, mRNA stability, and/or post-translational modifications. Whether the induction of p73 by E2F-1 in p53 nullizygous cells translates into activation of p53 target genes and apoptosis will form the basis of Specific Aims 2 and 3. The ability of E2F-1 to induce p73 dependent transactivation of p53 and p73 target genes will be assayed both biochemically and in transcription-based functional assays. In Specific Aim 3, flow cytometry and immunofluorescence techniques will be used to investigate whether p73 contributes to E2F-1 dependent apoptosis. p73 dominant negative proteins will be used to determine specificity in these assays. Finally, in Specific Aim 4 the role of p73 in E2F- dependent apoptosis induced by chemotherapeutic agents will be investigated. These studies may provide the first clues as to the signals that regulate p73 and may also help to explain p53 independent killing by E2F. Ultimately, the results of the studies proposed in this application may help in the development of novel gene therapies in which the E2F-1 protein, or perhaps, p73, can be used to induce tumor cell apoptosis.