Trends in Genetics
ReviewThe complex basis underlying common fragile site instability in cancer
Section snippets
The involvement of CFSs in cancer
CFSs were first described in 1984 as gaps and constrictions in metaphase chromosomes of cells grown under mild replication stress conditions [1]. The expression level of a CFS is measured by the frequency of the gaps and constrictions at that site on metaphase chromosomes. CFSs are present in all individuals and are considered to be part of the normal chromosomal structure. For some CFSs in humans, an ortholog has been found in mice 2, 3, 4, 5 and primates [6]. Several studies suggested that
Sequence characteristics of CFSs
For many years, no specific DNA sequences characterizing CFSs were found. However, examination of the DNA sequence and structural characteristics showed that they are enriched in sequences with high DNA helix flexibility in the twist angle 27, 28. These flexible sequences were found to comprise interrupted AT-dinucleotide repeats (AT-rich) with a potential to form stable DNA secondary structures that might lead to inhibition of DNA replication [29] (Box 1). Recent studies described below in
Replication timing studies
Numerous mechanisms underlying CFS sensitivity to replication stress have been proposed years ago, including late timing of replication, widely separated origins and DNA sequences that are unusually difficult to replicate [36]. Analyzing the unique replication pattern of CFSs is important for understanding the replication features leading to their sensitivity to replication stress, which results in their instability. The replication pattern of several CFSs was initially analyzed using
FRA3B
FRA3B is mapped over a large genomic region exceeding 3 Mb. Within this region lies the large tumor suppressor gene, fragile histidine triad (FHIT), extending over 1.5 Mb. DNA combing analysis of 1.6 Mb overlapping FHIT demonstrated that the replication rate and fork stalling frequency along this site are similar to that along the whole genome [45]. A correlation between the origin pattern and the expression level of FRA3B was found. Analysis in lymphocyte cells, where FRA3B is highly
FRA16C
A recent DNA combing study analyzing the replication pattern along 600 kb within the CFS FRA16C identified four AT-rich flexibility sequences over a length of 400 bp [35]. The analysis revealed a slower replication rate and shorter origin distance at the fragile site region compared with the whole genome under normal growth conditions, indicating that additional origins are activated. Because dormant origins are normally activated following replication stress, the replication pattern of FRA16C
FRA6E
FRA6E harbors the large parkinson protein 2 (PARK2) gene, which extends over 1.3 Mb. The replication pattern of approximately 1 Mb of FRA6E containing part of PARK2 was analyzed using DNA combing [49]. The analyzed region of this fragile site contains relatively long AT-rich sequences (400–1383 bp), along which the replication rate of the fragile site was slower, the origin distance was shorter and a higher frequency of fork arrests was found compared with the whole genome. A comparison between
Mechanisms leading to CFS instability
Several lines of evidence suggest that no single mechanism can account for the instability of all CFSs. Different potential characteristics of CFSs have been proposed over the years, but none are common to all of the cloned CFSs (Figure 1).
One characteristic of CFSs contributing to instability is colocalization with very large genes. Some of the large CFS genes [such as FHIT and WW domain containing oxidoreductase (WWOX)] play important roles in cancer development. It has been proposed that
Genome instability of CFSs during cancer development
Molecular mapping of fragile sites reveals that genomic instability in CFSs alters the expression of important cancer genes in different cancer types. FRA3B, the most studied CFS, maps to intron 5 of the tumor suppressor gene FHIT [52]. This CFS is altered in a variety of human cancers, including lung, breast, head and neck, stomach and pancreatic carcinomas [53]. Interestingly, integrations of human papilloma virus (HPV) in cervical cancer cells were also found in FRA3B [54]. FRA16D also
CFSs are preferentially unstable during early stages of cancer development
To shed light on the events leading to genomic instability in cancer, several studies focused on the early stages of cancer development. Large-scale LOH and comparative genome hybridization analyses during early stages of different cancers revealed that the instability in CFSs precedes the instability in other genomic regions 25, 59. Similar results were obtained in genome-wide analyses in precancerous experimental models [75]. These findings suggest a new model in which replication stress is
Concluding remarks
Recent advances in technologies enabling whole-genome instability and DNA replication analyses have led to a breakthrough in understanding the mechanisms leading to the instability of CFSs during the early stages of cancer development. These analyses revealed that replication stress that occurs during cancer development leads to preferential instability along the replication-sensitive CFS regions. Instability in CFSs in cancer may lead to deletions, translocations, amplifications and
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These authors contributed equally to this manuscript.