| The p53 tumor suppressor protein functions as a central regulator of cellular responses to various stresses including DNA damage. Consistent with this role, p53 has been shown to inhibit genomic instability, including gene amplifications and deletions, which can be initiated by DNA double strand breaks. High-grade osteosarcoma is a cancer that is characterized by extremely high levels of genomic instability. Inactivation of p53 may be central to the pathogenesis of this tumor, as >50% of osteosarcomas are functionally inactivated for p53, and both humans and mice who inherit a mutant p53 allele develop osteosarcomas. The experiments presented in this thesis investigate the role of p53 in controlling the stability of the genome in primary osteosarcoma tumors. In addition, a CGH microarray approach was undertaken to characterize two of the most highly recurrent genomic amplifications in osteosarcoma, chromosomal regions 17p11.2–p12 and 6p12–p21, to search for putative oncogene candidates.; Thirty-two primary osteosarcoma tumors were analyzed for genomic instability by Comparative Genomic Hybridization (CGH), and for p53 pathway status by p53 gene mutation screening (SSCP and PCR/LDR) and HDM2 genomic amplification (Tagman real-time PCR). Mutation of p53 correlates with high-level instability of the genome in these tumors, with a mean of 16.6 total chromosomal aberrations detected in 9 p53 mutant tumors, as compared to 10.2 for all others (p = 0.0148). Surprisingly, genomic amplification of HDM2, the negative regulator of p53, does not correlate, with a mean of 8.8 aberrations detected in 5 HDM2 amplified tumors.; The two most common regions of amplification detected by CGH were 6p12–p21.1, at 47%, and 17p11.2–p12, at 44% of 32 tumors. These genomic regions are likely to harbor oncogenes that contribute to the osteosarcoma tumor phenotype. For chromosome 17p11.2–p12, three independent amplicons were mapped to this region by a combination of CGH microarray, Southern blotting, and quantitative PCR techniques. Several interesting candidate oncogenes occur within these amplified regions, including the p101 subunit of P13Kinase-γ gene, which is overexpressed in 17p11.2 amplified cell lines. Using a similar approach, the VEGF and HSP90β genes were determined to be amplified and overexpressed as a result of the 6p12–p21 amplification. |
