Mechanistic study of fragile site instability by investigating RET/PTC rearrangements, a common cause of papillary thyroid carcinoma

Chromosomal fragile sites are non-random regions of the genome with a predisposition to the formation of DNA breaks. The goal of this work is to investigate the mechanism of common fragile site breakage, the role it plays in the formation of cancer-causing chromosomal translocations, and how this knowledge can be utilized to tailor the treatment of patients.;To provide direct evidence for the role of fragile sites in the formation of oncogenic chromosomal translocations, RET/PTC rearrangements, a common cause of papillary thyroid carcinoma (PTC), were examined since the genes involved in the two most common subtypes, RET/PTC1 and RET/PTC3, are all located within common fragile sites. Furthermore, thyroid cancer rates are rapidly increasing, especially in women where it is the fastest growing cancer, and increased PTC incidences are almost entirely responsible for this upsurge. A large number of PTC tumors containing RET/PTC rearrangements are sporadic in nature, and may be the result of fragile site instability. The location of RET, CCDC6, and NCOA4 genes participating in RET/PTC1 and RET/PTC3 rearrangements, in fragile sites and their instability when exposed to fragile site-inducing chemicals was confirmed by Fluorescence In Situ Hybridization (FISH) and ligation mediation-PCR (LM-PCR). More importantly, treatment of thyroid cells with these chemicals resulted in the formation of RET/PTC1 rearrangements, providing direct evidence for fragile site involvement in the formation of a cancer-causing chromosomal translocation.;While a role for fragile sites in cancer development is strongly supported, the initial events leading to breakage at these sites are not well understood. Although no consensus sequence has been identified for common fragile sites, several characteristics are shared among many fragile sites studied to date, including the formation of stable DNA secondary structures, which are believed to impede replication fork progression and result in genomic instability. DNA topoisomerases I and II maintain chromosome structural integrity during replication and transcription by transiently inducing DNA breaks, and these enzymes have been shown to recognize and preferentially cleave at DNA secondary structures. Using the RET oncogene, initial events of fragile site breakage following treatment with the fragile site-inducing chemical aphidicolin (APH), were investigated. The location of DNA breaks within intron 11 of RET, the major breakpoint cluster region observed in PTC patients with RET/PTC rearrangements, was determined following treatment of thyroid cells with APH using LM-PCR. These breakpoints were located at or near predicted DNA topoisomerase I and/or II cleavage sites. Furthermore, these breakpoints were predicted to coincide with DNA structural features recognized by topoisomerases I and II. Using topoisomerase catalytic inhibitors in combination with APH treatment, the rate of APH-induced DNA breakage at RET and FHIT, also located at an APH-induced common fragile site, was significantly decreased, confirming the involvement of DNA topoisomerases I and II in initiating DNA breakage at these common fragile sites.;Overall, these results strongly support the role of fragile sites in the formation of cancer-causing chromosomal translocations, specifically the RET/PTC1 rearrangement in PTC. The location of fragile site breakage within the RET oncogene and the DNA secondary structure prediction analysis of chromosome 10, provide valuable insight into the initial events of common fragile site breakage, supporting a model of fragile site instability whereby the formation of stable DNA secondary structures impede replication fork progression and DNA topoisomerases I and II initiate DNA breakage at these sites through recognition of secondary structures. Furthermore, the ability of benzene and DEN to induce fragile site-specific breakage at the RET oncogene supports fragile site involvement in sporadic PTC tumors. Significant levels of double-strand DNA breakage at RET in normal thyroid tissue from RET/PTC-positive patients further supports this finding, suggesting this property could be used as a potential diagnostic assay to tailor the treatment of cancer patients with chemotherapeutic drugs or monitor cancer susceptibility in individuals exposure to high levels of external fragile site-inducing agents. (Abstract shortened by UMI.).