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.
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