Conditions that partially inhibit DNA replication induce expression of common fragile sites. These sites form gaps and breaks on metaphase chromosomes and are deleted and rearranged in many tumors. Yet, the mechanism of fragile site expression has been elusive. We demonstrate that the replication checkpoint kinase ATR, but not ATM, is critical for maintenance of fragile site stability. ATR deficiency results in fragile site expression with and without addition of replication inhibitors. Thus, we propose that fragile sites are unreplicated chromosomal regions resulting from stalled forks that escape the ATR replication checkpoint. These findings have important implications for understanding both the mechanism of fragile site instability and the consequences of stalled replication in mammalian cells.

The study of common fragile sites has its roots in the early cytogenetic investigations of the fragile X syndrome. Long considered an interesting component of chromosome structure, common fragile sites have taken on novel significance as regions of the genome that are particularly sensitive to certain forms of replication stress, which are frequently rearranged in cancer cells. In recent years, much has been learned about the genomic structure at fragile sites and the cellular checkpoint functions that monitor their stability. Recent findings suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early stages of tumorigenesis. Thus, the study of common fragile sites can provide insight not only into the nature of fragile sites, but also into the broader consequences of replication stress on DNA damage and cancer. However, despite recent advances, many questions remain regarding the normal functional significance of these conserved regions and the basis of their fragility.

Esophageal adenocarcinoma (EA) is characterized by a poor prognosis making the identification of clinically targetable proteins essential for improving patient outcome. We report the involvement of multiple alterations of the MET pathway in EA development and progression. Microarray analysis of Barrett's metaplasia, dysplasia, and EA revealed overexpression of the MET oncogene in EAs but only those with MET gene amplification. STSamplification mapping revealed that the boundary of the MET amplicon in these EAs is defined by fragile site FRA7G. We also identified an amplicon at 11p13 that resulted in amplification and overexpression of CD44, a gene involved in MET autophosphorylation upon HGF stimulation. Tissue microarrays with phospho-METspecific antibodies demonstrated a uniformly high abundance of MET activation in primary EA and cells metastatic to lymph nodes but to a lesser extent in a subset of metaplastic and dysplastic Barrett's samples. Increased expression of multiple genes in the MET pathway associated with invasive growth, for example, many MMPs and osteopontin, also was found in EAs. Treatment of EA-derived cell lines with geldanamycin, an inhibitor for tyrosine kinases including MET receptor kinase, reduced cell migration and induced EA cell apoptosis. The data indicate that upregulation of the MET pathway may contribute to the poor outcome of EA patients and that therapeutic agents targeting this pathway may help improve patient survival.

Aphidicolin-induced common fragile sites are site-specific gaps or breaks seen on metaphase chromosomes after partial inhibition of DNA synthesis. These fragile sites were first recognized during the early studies of the fragile X syndrome and are induced by the same conditions of folate or thymidylate stress used to induce the fragile X site. Common fragile sites are normally stable in cultured human cells. However, following induction with replication inhibitors, they display a number of characteristics of unstable and highly recombinogenic DNA. From the many studies that have cloned and characterized fragile sites, it is now known that these sites extend over large regions, are associated with genes, exhibit late or delayed replication, and contain regions of high flexibility but are otherwise unremarkable in sequence. Studies showing that fragile sites and their associated genes are frequently deleted or rearranged in cancer cells have clearly demonstrated their importance in genome instability in tumorigenesis. Yet until recently, very little was known about the molecular mechanisms involved in their stability. Recent findings showing that the key checkpoint genes ATR and BRCA1 are critical for genome stability at fragile sites have shed new light on these mechanisms and on the biological significance of common fragile sites.

Common fragile sites are loci that form chromosome gaps or breaks when DNA synthesis is partially inhibited. Fragile sites are prone to deletions, translocations, and other rearrangements that can cause the inactivation of associated tumor suppressor genes in cancer cells. It was previously shown that ATR is critical to fragile-site stability and that ATR-deficient cells have greatly elevated fragile-site expression (A. M. Casper, P. Nghiem, M. F. Arlt, and T. W. Glover, Cell 111:779-789, 2002). Here we demonstrate that mouse and human cells deficient for BRCA1, due to mutation or knockdown by RNA interference, also have elevated fragile-site expression. We further show that BRCA1 functions in the induction of the G 2 /M checkpoint after aphidicolininduced replication stalling and that this checkpoint function is involved in fragile-site stability. These data indicate that BRCA1 is important in fragile-site stability and that fragile sites are recognized by the G 2 /M checkpoint pathway, in which BRCA1 plays a key role. Furthermore, they suggest that mutations in BRCA1 or interacting proteins could lead to rearrangements at fragile sites in cancer cells.Common fragile sites are loci that exhibit site-specific gaps and breaks on metaphase chromosomes when cells are grown under conditions that partially inhibit DNA synthesis, such as folate deficiency or treatment with aphidicolin (11). These fragile sites extend over hundreds of kilobases, with gaps and breaks occurring throughout the regions. Following aphidicolin treatment, 80% of all gaps and breaks are seen at just 20 fragile sites, with FRA3B (3p14.2) and FRA16D (16q23) being the most frequently broken, or expressed, fragile sites (11). Whereas rare fragile sites, such as FRAXA within the FMR1 gene, arise from mutation at di-or trinucleotide repeats, common fragile sites are found in all individuals and represent a normal component of chromosome structure.Fragile sites are so-called hot spots for sister chromatid exchanges, translocations, deletions, and plasmid integration in cultured cells following replication stress (12,14,32,40). Numerous studies have also shown that common fragile sites are prone to deletions and rearrangements in many cancers (1,17,21,28,33), and they may play a role in some gene amplification and viral integration events (6,16,26,41). Some fragile sites lie within putative tumor suppressor genes, such as FHIT at FRA3B and WWOX at FRA16D (30, 33), leading to the model that fragile-site instability is a contributing factor in tumorigenesis.Determining the mechanisms of fragile-site instability is important in understanding normal chromosome structure and DNA replication as well as the instability found at fragile sites in tumor cells. Sequence analysis of common fragile sites has not revealed why they are unstable. However, all sites studied to date are relatively AT rich and contain more areas of high flexibility than non-fragile-site regions (22,(24)(25)(26)(27). Studies examining replication timing at common fragile sites have shown...