Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.

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.

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Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit -/- MEFs escape senescence to become immortal more rapidly than Fhit +/+ MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a ‘mutator’ phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.

Aim:  Sleep is closely related to physical and mental health. Sleep disturbance is reported in patients without encephalopathy. We examined the relationship among cirrhotic symptoms, laboratory data and sleep disturbances. Next, we examined the influence of a branched chain amino acid (BCAA) supplement on sleep disturbance in cirrhotic patients. Methods:  We investigated a total of 21 patients at Nagasaki University Hospital from January to June 2009. We constructed questionnaire items for the evaluation of cirrhotic symptoms. The items, as major symptoms of cirrhotic patients, were as follows: hand tremor, appetite loss, muscle cramp of foot, fatigue, decreased strength, anxiety, abdominal fullness, abdominal pain and a feeling of low energy. We used the Epworth Sleepiness Scale (ESS) for the evaluation of daytime hypersomnolence. Energy supplementation with a BCAA snack was performed as a late evening snack (LES). All patients were assessed at the time of entry into the study, and at 4 and 8 weeks. Results:  It was found that BCAA snack, taken p.o. as an LES, improved the ESS for cirrhotic patients without encephalopathy. This beneficial result was recognized in the short term, 4 weeks after beginning of treatment. This study demonstrated the utility of BCAA supplementation for cirrhotic patients with sleep disturbance. However, the cirrhotic symptom‐related score was positively relation with the Child–Pugh score at the time of patient entry, and we were unable to identify the item that related to ESS. Conclusion:  A BCAA snack is a useful drug for cirrhotic patients who do not have any overt encephalopathy, but who suffered from sleep disturbance.