Mitomycin C (MC) and 10-decarbamoyl MC Signaling to p53 and Family Members

丝裂霉素 C (MC) 和 10-去氨甲酰 MC 向 p53 及其家族成员发出信号

• 批准号: 6772180
• 负责人: Jill E. Bargonetti
• 金额: $ 13.7万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6772180
• 关键词: DNA damage; RNA interference; apoptosis; cell cycle; cell line; chromatin immunoprecipitation; cytotoxicity; dosage; enzyme activity; microarray technology; mitomycin C; p53 gene /protein; pharmacokinetics; protein kinase; protein protein interaction; small interfering RNA

DESCRIPTION (provided by applicant): The tumor suppressor p53 is a critical sensor of cellular DNA damage. Cellular stress is signaled to p53 by a number of different signal transduction pathways. We recently identified that DMC activates both p53 dependent and independent apoptotic pathways while MC only activates the p53 dependent pathway. Understanding how chemotherapeutic drugs signal to wild-type p53 is of central relevance to cancer treatment. Determining DNA damage pathways that can signal to p53 family members, p73 and p63 has emerged as equally important with the recent finding that these proteins are required to coordinately regulate the DNA damage apoptotic response. Our hypothesis is that DMC can significantly activate a p53-independent apoptotic pathway that utilizes p73 and p63. The long-range goal is to determine the p53-independent signal transduction pathway(s) activated by DMC. Aim 1) Identify the cytotoxicity and specific target gene regulation, and checkpoint kinases activated, by DMC as compared to other DNA damaging drugs in cell lines, with and without p53. The cytotoxicity and the expression of selected target genes known to associate with the induction of apoptosis and cell cycle regulation will be determined by drug dose curves of representative cell lines with MC, DMC and a battery of well characterized chemotherapeutics. Gene expression levels of multiple validated targets (including Bax, Fas, Puma, Noxa, Gadd45, p21 and mdm2) will be monitored using real-time RT-PCR and target kinase activation will be monitored by phosphor-specific antibodies. Aim 2) Determine if differential activation of p53 target genes associates with alternative multiprotein complex formation on chromatin. Chromatin immuno-precipitation-PCR (ChlP-PCR), utilizing p53 specific, p73 specific and MDM2 specific antibodies, will be used to investigate the nuclear in vivo interaction of p53, p73 and MDM2 at the known enhancer binding sites for the apoptotic and cell cycle regulating target genes. Fixation is the first step in the ChlP-PCR protocol and will preserve higher-molecular weight complexes. Aim 3) Examine gene expression by affymetrix DNA micro-arrays in MC and DMC treated cell lines with and without p53 as well as with and without the p53 dependent apoptosis inhibitor WISP-1. To determine whether apoptosis can be attributed to specific overexpressed genes, siRNA will be used for functional analysis.

描述（由申请人提供）：肿瘤抑制因子 p53 是细胞的关键传感器 DNA损伤。细胞应激通过许多不同的信号转导途径向 p53 发出信号。我们最近发现DMC同时激活p53依赖和独立的凋亡途径，而MC仅激活p53依赖的途径。了解化疗药物如何向野生型 p53 发出信号对于癌症治疗至关重要。确定向 p53 家族成员、p73 和 p63 发出信号的 DNA 损伤途径同样重要，最近发现这些蛋白质需要协调调节 DNA 损伤凋亡反应。我们的假设是，DMC 可以显着激活利用 p73 和 p63 的独立于 p53 的细胞凋亡途径。长期目标是确定 DMC 激活的独立于 p53 的信号转导途径。目标 1) 与细胞系中其他 DNA 损伤药物相比，确定 DMC 的细胞毒性和特定靶基因调节以及激活的检查点激酶（无论有或没有） 第 53 页。已知与诱导细胞凋亡和细胞周期调节相关的选定靶基因的细胞毒性和表达将通过具有 MC、DMC 和一系列充分表征的化疗药物的代表性细胞系的药物剂量曲线来确定。将使用实时 RT-PCR 监测多个已验证靶标（包括 Bax、Fas、Puma、Noxa、Gadd45、p21 和 mdm2）的基因表达水平，并通过磷特异性抗体监测靶标激酶激活。目标 2) 确定 p53 靶基因的差异激活是否与染色质上替代多蛋白复合物的形成相关。染色质免疫沉淀 PCR (ChlP-PCR)，利用 p53 特异性、p73 特异性和 MDM2 特异性抗体，将用于研究 p53、p73 和 MDM2 在凋亡和细胞的已知增强子结合位点上的核体内相互作用循环调节靶基因。固定是 ChlP-PCR 方案的第一步，将保留较高分子量的复合物。目标 3) 通过 affymetrix DNA 微阵列检查含有和不含有 p53 以及含有和不含有 p53 依赖性细胞凋亡抑制剂 WISP-1 的 MC 和 DMC 处理的细胞系中的基因表达。为了确定细胞凋亡是否归因于特定的过表达基因，将使用 siRNA 进行功能分析。