p53 mutations have been detected in many tumors and interpreted as a late event in tumorigenesis, probably involved in tumor progression rather than tumor development. Recent studies have demonstrated the presence of germline p53 mutations in some Li-Fraumeni pedigrees and hence a role of p53 mutations in hereditary susceptibility to human cancer. Rhabdomyosarcoma, a common soft tissue sarcoma in the families with the Li- Fraumeni syndrome, was found to have p53 mutations. However, the frequency of such mutations in pediatric tumors in general remains unknown. We will study the presence of p53 mutations in established Ewing's sarcoma cell lines in comparison with existing data from rhabdomyosarcoma cell lines. In addition, p53 mutations will be studied in paraffin embedded tissues from rhabdomyosarcoma tumors to investigate the validity of paraffin embedded tissues for such studies. The detection of mutations will be performed on PCR fragments spanning exons 4, 5, 6, 7, and 8 of the p53 gene. Two methods will be compared: the standard single stranded conformational polymorphism (SSCP) method, and a non-radioactive method, based on conformational differences in double stranded molecules (heteroduplex analysis) and using non-denaturing electrophoresis in mutation detection enhancement (MDE) gels.
