Chromosomal positions of common fragile sites differ in lymphoblasts and fibroblasts, with positions dependent on the epigenetically determined density of replication origins at these loci. Because rearrangement of fragile loci and associated loss of fragile gene products are hallmarks of cancers, we aimed to map common fragile sites in epithelial cells, from which most cancers derive. Among the five most frequently activated sites in human epithelial cells were chromosome bands 2q33 and Xq22.1, which are not among top fragile sites identified in lymphoblasts or fibroblasts. FRA16D at 16q23 was among the top three fragile sites in the human epithelial cells examined, as it is in lymphoblasts and fibroblasts, while FRA3B at 3p14.2, the top fragile locus in lymphoblasts, was not fragile in most epithelial cell lines tested. Epithelial cells exhibited varying hierarchies of fragile sites; some frequent epithelial cell fragile sites are apparently not frequently altered in epithelial cancers and sites that are frequently deleted in epithelial cancers are not necessarily among the most fragile. Since we have reported that loss of expression of the FRA3B-encoded FHIT protein causes increased replication stress-induced DNA damage, we also examined the effect of FHIT-deficiency on markers of genome instability in epithelial cells. FHIT-deficient cells exhibited increases in fragile breaks and in γH2AX and 53BP1 foci in G1 phase cells, confirming in epithelial cells that the FHIT gene and encompassing FRA3B, is a “caretaker gene” necessary for maintenance of genome stability.

The fragile histidine triad protein, Fhit, has a number of reported tumor suppressive functions which include signaling of apoptosis in cancer cells in vitro and in vivo, modulation of the DNA damage response, down-regulation of target oncogene expression, suppression of tumor growth in vivo, and suppression of cancer cell invasion and metastasis. Most of these functions of Fhit have been observed on exogenous re-expression of Fhit in Fhit-negative cancer cells. However, little is known about the tumorigenic changes that occur in normal or precancerous cells following loss of Fhit expression. Recently, we have shown that shortly after loss of Fhit expression, cells exhibit signs of DNA replication stress-induced DNA damage and develop genomic instability. Here, we extend these findings through investigation of different factors that affect Fhit function to prevent DNA damage. We found that Fhit activity is dependent upon a functional HIT domain and the tyrosine-114 residue, previously shown to be required for tumor suppression by Fhit. Furthermore, Fhit function was shown to be independent of exogenous and endogenous sources of oxidative stress. Finally, Fhit function was shown to be dependent upon Chk1 kinase activity, but independent of Atr or Atm kinases. Evidence suggests that Fhit and Chk1 kinase cooperate to prevent replication stress-induced DNA damage. These findings provide important and unexpected insights into the mechanism whereby loss of Fhit expression contributes to cell transformation.

SUMMARY A profoundly retarded, slightly dys-Case report morphic male was re-examined cytogenetically The patient by high resolution GTG banding and found to Caucasian r have a de novo interstitial direct duplication of of a 34-yea 1 5q. 36-year-old

A 10½/2 year old female with skeletal abnormalities was referred for genetic consultation because of learning disabilities and a suggestion of 'Turner-like' stigmata. Cytogenetic analysis revealed a paracentric inversion of an X(q13.1q26-1) chromosome.

Common chromosome fragile sites (CFSs) are highly sensitive to genotoxic stress and perturbation of replication, resulting in deletions within genes located at these sites. CFSs at 3p14.2, 16q23, 4q, 7q, and 6q are the most frequently expressed in lymphoblasts but CFSs have not yet been mapped in epithelial cells. We are defining CFSs in epithelial cells derived from breast tissue to determine if they represent the sites most frequently altered in cancer cells of epithelial organs from which most human cancers derive. We observed that in MCF10A noncancerous breast epithelial cells, chromosome regions 16q23, 2q, 12q, 1p most frequently exhibited chromosome breaks typical of CFS after aphidicolin treatment. This suggests that, as observed for cells of fibroblast origin, CFSs in epithelia are epigenetically determined. In parallel, we have focused on the role of Fhit protein, encoded at the CFS at 3p14.2, on chromosome stability and association with nucleotide pyrimidine pool imbalance. We investigated chromosome instability in epithelial cells deficient for Fhit expression vs Fhit‐expressing cells and observed increased aneuploidy, sister chromatid exchanges, chromosome breaks and gaps in Fhit‐deficient cells. Moreover, Fhit deficiency leads to thymidine kinase 1 down regulation, resulting in pyrimidine pool disequilibrium and accounting, in part, for genome instability.