ANALYSIS OF P73 FUNCTION AND REGULATION BY E2F 1

E2F 1对P73功能和调控的分析

• 批准号: 6198873
• 负责人: MEREDITH S IRWIN
• 金额: $ 13.24万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6198873
• 关键词: apoptosis; cis platinum compound; doxorubicin; etoposide; flow cytometry; gene induction /repression; genetic transcription; immunofluorescence technique; messenger RNA; northern blottings; posttranslational modifications; protein structure function; tissue /cell culture; transcription factor; tumor suppressor proteins; western blottings

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.

视网膜母细胞瘤 (pRb) 和 p53 肿瘤抑制蛋白的双重失活通常发生在癌发生过程中。 pRb 失活会导致 E2F 转录因子家族失调。 E2F 促进细胞周期进程和细胞凋亡。后者涉及 p53 依赖性和 p53 独立机制。我的初步数据表明 E2F-1 可以诱导最近鉴定的 p53 同源物，称为 p73。 p73 至少在过量产生时可以激活 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 的内源水平非常低并且与 p53 不同，导致 p73 激活的上游信号通路尚未阐明，因此有趣的是 E2F-1 诱导 p73 水平显着增加。 E2F/pRb 通路失调对于大多数肿瘤细胞来说是常见的。因此，了解 p73 在该途径中的功能可能为了解 p73 在肿瘤发生中的作用提供线索。具体目标 1 的目标是确定 E2F-1 诱导 p73 的机制。将采用标准蛋白质稳定性和定量 RNA 测定来确定 E2F-1 对 p73 的影响是否是由于转录、mRNA 稳定性和/或翻译后修饰的变化所致。 p53 无效细胞中 E2F-1 对 p73 的诱导是否转化为 p53 靶基因的激活和细胞凋亡，将构成具体目标 2 和 3 的基础。将通过生化和基于转录的功能测定来测定 E2F-1 诱导 p73 依赖性 p53 和 p73 靶基因反式激活的能力。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 是否有助于 E2F-1 依赖性细胞凋亡。 p73 显性失活蛋白将用于确定这些测定中的特异性。最后，在具体目标 4 中，将研究 p73 在化疗药物诱导的 E2F 依赖性细胞凋亡中的作用。这些研究可能提供有关调节 p73 的信号的第一个线索，也可能有助于解释 E2F 独立杀伤 p53 的现象。最终，本申请中提出的研究结果可能有助于开发新型基因疗法，其中 E2F-1 蛋白或可能 p73 可用于诱导肿瘤细胞凋亡。