We propose five specific aims that will elucidate the role of genetic alterations in pediatric rhabdomyosarcomas in influencing sensitivity to anticancer agents, and how the threshold for inducing a cytotoxic response may be modulated. Our long range goal is to establish new approaches to therapy of these tumors. The proposal will focus on the role of the p53 tumor suppressor gene in a unique series of pediatric alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) cell lines and xenografts established at SJCRH from both untreated and previously treated patients in determining sensitivity to agents that damage DNA. Our hypothesis is that drug sensitivity iwll depend upon the functional status pf p53, a functional apoptotic pathway determined by p53-regulatable survival factors of the Bcl-2 family, and the specific oncogene expressed. Firstly, utilizing an ARMS model system with inducible wtp53 expression, we will elucidate the spectrum of sensitization to six different classes of DNA damaging agents, since we have shown p53-dependent cytotoxicity for e.g. actinomycin-D and doxorubicin and p53-independence for the topoisomerase I and topisomerse II inhibitors topotecan and VP-16, respectively. Secondly, we will determine whether similar potentiation is extended to other ARMS and ERMS cell lines following drug treatment of cells transduced with wtp53 adenovirus. Our hypothesis is that the spectrum and degree of drug sensitization will be enhanced in the present of c-Myc, N-Myc or oncogenic K-Ras overexpression or reduced in the presence of MDM2 amplification. This will be tested in the third Specific Aim in stably transfected isogenic cell lines of ARMS and ERMS with specific alterations in the oncogene. Drug sensitization for N-Myc in the presence of wtp53 would assign a previously unidentified apoptotic role to N-Myce. Fourthly, we will determine whether principles derived in vitro can be applied to the cell lines as xenografts in vivo. Finally, the relationship between p53 functional status and the clinical response of RMS to chemotherapy will be determined. Ultimately it may be possible to select appropriate therapy based upon the genetic profile of the tumor.
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