title: Initiation of genome instability and preneoplastic processes through loss of Fhit expression.
abstract: 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.
authors.affiliation: Biomedical Sciences Graduate Program, Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Joshua C
authors.initials: JC
authors.lastname: Saldivar
authors.forename: Satoshi
authors.initials: S
authors.lastname: Miuma
authors.forename: Jessica
authors.initials: J
authors.lastname: Bene
authors.forename: Seyed Ali
authors.initials: SA
authors.lastname: Hosseini
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Shibata
authors.forename: Jin
authors.initials: J
authors.lastname: Sun
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Wheeler
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Mathews
authors.forename: Kay
authors.initials: K
authors.lastname: Huebner
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D043283:Chromosome Fragile Sites; D053903:DNA Breaks, Double-Stranded; D015972:Gene Expression Regulation, Neoplastic; D042822:Genomic Instability; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism; D000099170:Bis(5'-adenosyl)triphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D009363:Neoplasm Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D013937:Thymidine Kinase; D000099170:Bis(5'-adenosyl)triphosphatase; C497207:Mdm2 protein, mouse; C092717:thymidine kinase 1