Our initial goal was to develop cell culture systems in which PAX3-FOXO1 expression can be induced and in which the downstream effects of PAX3-FOXO1 can then be assessed at the gene expression and phenotypic levels. To measure PAX3-FOXO1 expression in the planned studies, a monoclonal antibody to the FOXO1 C-terminus was successfully used in western blot studies of RMS cell lines to detect expression of the wild-type FOXO1 and/or fusion protein. Quantitative RT-PCR assays were similarly validated for detection of the wild-type FOXO1 and PAX3-FOXO1 (or PAX7-FOXO1) mRNA's. To generate inducible expression in our planned studies, a tetracycline-inducible lentiviral expression vector was provided by Dr. Ji Luo of the Medical Oncology Branch. We subcloned the PAX3-FOXO1 cDNA into this expression vector and transduced this PAX3-FOXO1 expression construct into several cell lines. To validate the utility of this inducible construct, we first transduced this construct into murine NIH3T3 fibroblast cells and observed inducible increases in PAX3-FOXO1 mRNA and protein in dose-response studies. The induced level of PAX3-FOXO1 protein reached levels equivalent to and even greater than the high fusion expression level present in fusion-positive RMS cells. Based on our initial success in generating inducible PAX3-FOXO1 expression in NIH3T3 cells, we then introduced this construct into the immortalized human Dbt myoblast cell line. Using our western blot and RT-PCR assays, we found inducible expression of PAX3-FOXO1 in the transduced Dbt cells, from near undetectable levels in the uninduced state to high levels that mirror the high levels in fusion-positive RMS cells. Further use of qRT-PCR assays for FGFR4 and ABAT, two downstream PAX3-FOXO1 targets, revealed inducible increases in expression of both of these genes. We then proceeded with several cell culture assays of the phenotypic effects of PAX3-FOXO1 in these transduced Dbt cells. Similar to our previous published studies of constitutive PAX3-FOXO1 expression constructs, we analyzed phenotypic effects in cells with and without constitutive MYCN expression. The toxic effects of induced PAX3-FOXO1 expression were manifest by the finding of little or no growth of these cells when PAX3-FOXO1 expression was induced by doxycycline in contrast to the brisk growth of these cells in the absence of doxycycline. These toxic effects were seen in the presence or absence of exogenous MYCN expression. Despite these toxic effects, the cells with induced PAX3-FOXO1 expression (as well as exogenous constitutive MYCN expression) demonstrated a high level of focus formation, which is a measure of oncogenic transformation in cell culture. Similar to the cells without doxycycline induction, the cells with induced PAX3-FOXO1 but without exogenous MYCN expression did not demonstrate evidence of oncogenic transformation. To extend these findings to the in vivo setting, an animal protocol has been submitted to the Institutional Animal Care and Use Committee to assess the role of PAX3-FOXO1 (with or without MYCN) in contributing to and maintaining tumorigenicity in laboratory animals.
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