Mechanisms of Common Fragile Site Instability and Cancer.

Common fragile sites (CFSs) are loci that are especially prone to forming gaps and breaks on metaphase chromosomes under conditions of replication stress. While much has been learned about the cellular responses to gaps and breaks at CFSs, less is known about what makes these sites inherently unstable. CFS sequences are highly conserved in mammalian evolution and contain a number of sequence motifs that are hypothesized to contribute to their instability. To examine the role of CFS sequences in chromosome breakage, we stably transfected two BACs containing FRA3B sequences and two non-CFS control BACs containing similar sequence content into HCT116 cells and isolated cell clones with BACs integrated at ectopic sites. Integrated BACs were present at just a few to several hundred contiguous copies. Cell clones containing integrated FRA3B BACs showed a significant, three to seven fold increase in aphidicolin-induced gaps and breaks at the integration site as compared to control BACs. Furthermore, many FRA3B integration sites displayed additional chromosome rearrangements associated with CFS instability. Clones were examined for replication 1 This chapter has previously been published: Ragland RL, Glynn MW, Arlt MF, Glover TW, (2008) Stably transfected common fragile site sequences exhibit instability at ectopic sites. Genes Chromosomes and Cancer. Oct;47(10):860-72. 42 timing and it was found that the integrated FRA3B sequences were not dependent on late replication for their fragility. This is the first direct evidence in human cells that introduction of CFS sequences into ectopic non-fragile loci is sufficient to recapitulate the instability found at CFSs. These data support the hypothesis that sequences at CFSs are inherently unstable, and are a major factor in the formation of replication stress induced gaps and breaks at CFSs. Introduction Common fragile sites (CFSs) are chromosomal loci that are especially prone to forming cytogenetically visible gaps and breaks on metaphase chromosomes under conditions of replication stress such as treatment with low concentrations of the DNA polymerase inhibitor aphidicolin (APH) (reviewed in (Durkin and Glover, 2007)). These sites span several hundred kilobases to over a megabase in size with gaps and breaks occurring throughout the region, and many are located within or span large genes. In addition to cytogenetically identifiable gaps and breaks, CFSs exhibit a number of other characteristics of unstable DNA in cultured cells including gross chromosome deletions (Wang et al., 1993), smaller submicroscopic deletions (Durkin et al., 2008), translocations (Glover and Stein, 1988), intrachromosomal gene amplifications (Coquelle et al., 1997), and sister chromatid exchanges (Glover and Stein, 1987). In cells pretreated with APH, CFSs are also preferred sites for the integration of transfected DNA (Rassool et al., 1991). Despite their instability, CFSs are found in all individuals and are a normal component of the human genome. Furthermore, CFSs are conserved in mammalian evolution(Soulie and De Grouchy, 1981; Elder and Robinson, 1989; Smeets and van de