Replication stress induces tumor-like microdeletions in FHIT/FRA3B.
Common fragile sites (CFSs) are loci that preferentially exhibit metaphase chromosome gaps and breaks after partial inhibition of DNA synthesis. The fragile site FRA3B, which lies within the FHIT tumor-suppressor gene, is a site of frequent heterozygous and homozygous deletions in many cancer cells and precancerous lesions. The great majority of FHIT and other CFS-associated gene rearrangements in tumors are submicroscopic, intralocus deletions of hundreds of kilobases that often result in inactivation of associated genes. Although CFS instability leads to chromosome gaps and breaks and translocations, there has been no direct evidence showing that CFS instability or replication stress can generate large submicroscopic deletions of the type seen in cancer cells. Here, we have produced FHIT/FRA3B deletions closely resembling those in tumors by exposing human-mouse chromosome 3 somatic hybrid cells to aphidicolin-mediated replication stress. Clonal cell populations were analyzed for deletions by using PCR, array comparative genomic hybridization (aCGH), and FISH. Thirteen percent to 23% of clones exhibited submicroscopic FHIT deletions spanning approximately 200-600 kb within FRA3B. Chromosomes with FRA3B deletions exhibited significantly decreased fragility of this locus, with a 2- to 12-fold reduction in metaphase gaps and breaks compared with controls. Sequence analysis showed no regions of homology at breakpoints and suggests involvement of NHEJ in generating the deletions. Our results demonstrate that replication stress induces a remarkably high frequency of tumor-like microdeletions that reduce fragility at a CFS in cultured cells and suggests that similar conditions during tumor formation lead to intralocus deletion and inactivation of genes at CFSs and perhaps elsewhere in the genome.
Durkin
Sandra G
SG
Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-5618, USA.
Ragland
Ryan L
RL
Arlt
Martin F
MF
Mulle
Jennifer G
JG
Warren
Stephen T
ST
Glover
Thomas W
TW
D017766:Acid Anhydride Hydrolases / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D002875:Chromosomes / Q000648:ultrastructure; D004261:DNA Replication; D017353:Gene Deletion; D005786:Gene Expression Regulation; D015894:Genome, Human; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008957:Models, Genetic; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics*; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D009363:Neoplasm Proteins; D000099170:Bis(5'-adenosyl)triphosphatase