Replication Stress Induced Tumor-like Deletions of FRA3B in Human/Mouse Cell Hybrids. S.G. Durkin, R.L. Ragland, T.W. Glover. Dept Human Genetics, Univ Michigan, Ann Arbor, MI.
   Common fragile sites (CFS) are regions of the genome that form gaps and breaks on metaphase chromosomes when DNA synthesis is partially inhibited. While normally stable in somatic cells, CFS and associated genes are frequently rearranged in cancer cells and may be indicators of DNA replication stress early in tumorigenesis. We and others have previously reported the induction of large scale chromosome rearrangements, including translocations and terminal deletions with breakpoints at CFS in normal cells in vitro. However, the great majority of rearrangements observed at CFS in cancer cells are not these types of rearrangements, but are intra-locus deletions spanning tens to hundreds of kb. Although it has been assumed that these tumor deletions are due to replication stress-induced CFS destabilization, it has been unclear if replication stress and CFS breaks in vitro can also result in tumor-like deletions. To this end, we have utilized human/mouse cell hybrids harboring a single human chromosome 3 that contains the most frequently broken human CFS, FRA3B. To induce replication stress, these cells were exposed to a low-dose of aphidicolin, which induces CFS breaks, for five days followed by one day of recovery. Seventy-four resulting clonal cell populations were analyzed by PCR spanning FRA3B at approximately 10-30 kb marker intervals to detect deletions. Thirty clones (40%) were detected to have deletions within the CFS critical region, previously defined to lie between intron 3 to intron 7 of FHIT. Ten of the 30 deletion clones were shown to have contiguous deletions within this region ranging from approximately 300 to 800 kb, similar in size and location to those deletions observed in esophageal adenocarcinomas, renal carcinomas and other cancers while other clones exhibited smaller deletions. We are currently cloning the specific breakpoints of these deletions to gain insight on the sequences involved in these rearrangements and how they are repaired, and are analyzing deletion clones for CFS breakage. These findings support a direct mechanistic relationship between replication stress and CFS breaks in vitro, and genome rearrangements in cancer.