Stem Cell Factor (SCF) is the ligand for KIT, a receptor tyrosine kinase. We hypothesize that KIT activation can affect the expression of MAGE genes, which are usually only expressed in developing sperm and in tumors. We also hypothesize that MAGE proteins promote tumor survival by suppressing p53 dependant apoptosis. The goals of this proposal are to determine the relationship between KIT activation and MAGE induced apoptosis and to develop means of treating MAGE positive malignancies by interfering with MAGE protein expression in vivo. These goals will be achieved by the following specific aims:
Specific Aim 1 : will test the hypothesis that KIT activation directly affects MAGE gene expression by determining whether KIT controls MAGE gene promoter methylation, and will determine whether MAGE protein expression is common in mastocytosis by immunohistochemistry and reverse transcription-PCR.
Specific Aim 2 : will define mechanisms by which MAGE proteins regulate apoptosis by critically testing the requirement for p53 using inducible lentivirus based suppression of p53 and by determining the effect of MAGE gene expression on p53 metabolism including: p53 phosphorylation and acetylation by immunoblot analysis, p53 transcription by real time PCR and nuclear run on analysis; p53 function by p53 target gene activation assays; and p53 degradation by ubiquitin immunoblot and pulse chase analysis. We will then test the hypothesis that multiple MAGE proteins regulate apoptosis via Kap1 binding to the MAGE common homology domain using tagged MAGE and Kap1 proteins expressed from nested MAGE cDNAs in immunoprecipitation and mammalian two hybrid assays.
Specific Aim 3. will test the hypothesis that mastocytosis and other tumors may be treated by inhibiting MAGE expression in vivo using human cell lines xenografted onto nu/nu mice and treated systemically with MAGE siRNA. Public Health Relevance: These studies will improve health by causing a major shift of the current clinical paradigm that envisions the therapeutic use of tumor specific MAGE proteins as targets for immunologic attack, rather than as targets for functional manipulation. The results will impact multiple aspects of health by developing novel therapies for malignant tumors and increasing our understanding of the development of sperm from spermatogonial stem cells. ? ? ? ?
