The Senior Investigator moved to the National Cancer Institute during FY2011 and has been setting up the laboratory during the remaining time. Therefore no research studies were performed on this project during FY2011. However, in previous studies at the University of Pennsylvania, the Senior Investigator made the following observations. We have utilized a human myogenic system to study the PAX3-FOXO1 fusion in a cellular environment more relevant to alveolar rhabodmyosarcoma (ARMS). Starting with a human myoblast line immortalized with BMI1 and TERT, transduction with PAX3-FOXO1 and MYCN resulted in transforming and tumorigenic activities. The far majority of transduced cells expressed low PAX3-FOXO1 levels and did not have a transformed or tumorigenic phenotype. In contrast, only a small number of transduced cells expressed high PAX3-FOXO1 levels, and were transformed and tumorigenic. These findings are consistent with the toxicity of high PAX3-FOXO1 expression and the occurrence of cell autonomous changes allowing rare cells to circumvent the toxic effects of high-level expression. To investigate the relationship between phenotype and fusion protein level in a more dynamic system, we developed a 4-hydroxytamoxifen inducible system in which PAX3-FOXO1 is joined to a modified estrogen receptor ligand-binding domain. Using myoblasts transduced with MYCN and this inducible construct, we found that PAX3-FOXO1 induction results in transforming and tumorigenic activities, but also causes increasing growth suppression. Therefore, the inducible system will be useful for our studies of the mechanism of PAX3-FOXO1-induced toxicity.
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