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是细胞免疫反应的关键传感器。 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激活p53依赖性和非依赖性凋亡途径，而MC只激活p53依赖性途径。了解化疗药物如何向野生型p53发出信号对癌症治疗具有重要意义。确定可以向p53家族成员p73和p63发出信号的DNA损伤途径已经变得同样重要，最近发现这些蛋白质是协调调节DNA损伤凋亡反应所必需的。我们的假设是，DMC可以显着激活p53非依赖性凋亡途径，利用p73和p63。长期目标是确定DMC激活的p53非依赖性信号转导途径。目的1）在细胞系中，与其他DNA损伤药物相比，确定DMC的细胞毒性和特异性靶基因调控，以及检查点激酶激活，有和没有 第53页。细胞毒性和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表达将通过具有MC、DMC和一组充分表征的化疗剂的代表性细胞系的药物剂量曲线来确定。将使用实时RT-PCR监测多个经验证靶标（包括Bax、Fas、Puma、Noxa、Gadd 45、p21和mdm 2）的基因表达水平，并将通过磷酸特异性抗体监测靶标激酶激活。目的2）确定p53靶基因的差异激活是否与染色质上交替性多蛋白复合物的形成有关。利用p53特异性、p73特异性和MDM 2特异性抗体的染色质免疫沉淀-PCR（ChIP-PCR）将用于研究p53、p73和MDM 2在细胞凋亡和细胞周期调节靶基因的已知增强子结合位点处的核内相互作用。固定是ChlP-PCR方案的第一步，将保留较高分子量的复合物。目的3）通过亲和素DNA微阵列检测MC和DMC处理的细胞系中有和没有p53以及有和没有p53依赖性凋亡抑制剂WISP-1的基因表达。为了确定细胞凋亡是否可归因于特定的过表达基因，siRNA将用于功能分析。