FRA3B, at chromosomal band 3p14.2, is the most active common fragile site in the human genome. This fragile site lies within a chromosomal region frequently deleted in many solid tumors and very close to a translocation breakpoint observed in a family with inherited predisposition to renal cell carcinoma (RCC). In the last 8 years of funding on this project we have generated thousands of chromosome 3-specific cosmids, used these cosmids to assist in the cloning of several chromosome 3p translocation breakpoints, generated a large number of somatic cell hybrids with aphidicolin-induced breakage and localized each of the breakpoints including 17 within FRA3B. We have utilized a 1330 Kb YAC clone which crosses the familial RCC breakpoint and FRA3B to construct a 350 Kb cosmid contig from the RCC breakpoint presumably through all of FRA3B. The 17 aphidicolin-induced breakpoints within 3p14.2 were localized to two tight clusters, one 100 Kb and the other 300 Kb distal to the familial RCC breakpoint. We are proposing to continue and extend these studies to understand the molecular structure of FRA3B and determine why this region is particularly sensitive to aphidicolin. We will also characterize a large number of RCC's, squamous cell carcinomas of the head and neck, lung and breast cancers for chromosome 3p breakage using precisely localized highly polymorphic PCR-amplifiable markers. There are three specific aims of this competing renewal: (1) The molecular characterization of FRA3B; (2) The determination of proteins that bind to FRA3B sequences; and (3) The determination of chromosome 3p breakpoints in RCC, squamous cell carcinoma of the head and neck, and lung and breast cancer. This work will enable us to determine a potentially novel mechanism of chromosomal fragility distinct from expansion of a trinucleotide repeat. This work will also compare chromosome 3p breakpoints induced by aphidicolin to chromosome 3p breakpoints occurring in vivo in cancer patients. Thus this work should help determine the role that FRA3B plays in chromosome breakage and loss in solid tumors.
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