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Common fragile sites
Cancer Letters 232 (2006) 4–12 www.elsevier.com/locate/canlet
Mini Review
Common fragile sites Thomas W. Glover* Department of Human Genetics, 4909 Buhl, Box 0618, 1241 E. Catherine Street, University of Michigan, Ann Arbor, MI 48109-0618, USA Received 24 August 2005; accepted 30 August 2005
Abstract Common fragile sites are regions showing site-specific gaps and breaks on metaphase chromosomes after partial inhibition of DNA synthesis. Common fragile sites are normally stable in somatic cells. However, following treatment of cultured cells with replication inhibitors, fragile sites display gaps, breaks, rearrangements and other features of unstable DNA. Studies showing that fragile sites and associated genes are frequently deleted or rearranged in many cancer cells have clearly demonstrated their importance in genome instability in cancer. Until recently, little was known about the molecular nature and mechanisms involved in fragile site instability. From studies conducted in many laboratories, it is now known that fragile sites extend over large regions, are associated with genes, exhibit delayed replication, and contain regions of high DNA flexibility. Recent findings from our laboratory showing that the key cell cycle checkpoint genes are important for genome stability at fragile sties have shed new light on these mechanisms and on the significance of these sites in cancer and normal chromosome structure. Since their discovery over two decades ago, much has been learned regarding their significance in chromosome structure and instability in cancer, but a number of key questions remain, including why these sites are ‘fragile’ and the impact of this instability on associated genes in cancer cells. These and other questions have been addressed by participants of this meeting, which highlighted instability at common fragile sites. This brief review is intended to provide background on common fragile sites that has led up to many of the studies presented in the accompanying reports in this volume and not to summarize the findings presented therein. Some aspects of this review were taken from Glover et al. (T.W. Glover, M.F. Arlt, A.M. Casper, S.G. Durkin, Mechanisms of common fragile site instability, Hum. Molec. Genet. 14 (in press). [1]). q 2005 Elsevier Ireland Ltd. All rights reserved.
1. Fragile sites in cultured cells Common fragile sites are seen in all individuals and, thus, represent a component of normal chromosome structure. They are conserved in the mouse and other mammals in which they have been investigated [2–13] and perhaps have counterparts in yeast [14,15]. * Tel.: C1 734 763-5222; fax: C1 734 763 3784. E-mail address: [email protected].
These fragile site regions are normally stable in cultured human cells. However, when cells are cultured under conditions of folate or thymidylate stress or with low doses of aphidicolin that only partially inhibit DNA synthesis, they are prone to forming chromosome gaps and breaks. With the cloning and characterization of common fragile sites, we know that these breaks can occur over broad regions extending up to a megabase or more. At present, over 80 common fragile sites are listed in
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T.W. Glover / Cancer Letters 232 (2006) 4–12
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the Genome Database (GDB). However, not all fragile sites form breaks at the same frequency, and a small number of sites in the genome are most prone to form lesions [16,17]. Gaps and breaks at just 20 fragile sites represent over 80% of all lesions observed in lymphocytes following treatment with low doses of aphidicolin [16]. FRA3B at 3p14.2 stands out as the most ‘fragile’ site in the genome and can be induced to break or form gaps in the majority of treated cells. Other highly expressed fragile sites in lymphocytes include those at 16q23 (FRA16D), 6q26 (FRA6E), 7q32.3 (FRA7H), and Xp22.3 (FRAXB) [16]. In addition to gaps and breaks, common fragile sites are ‘hot spots’ for increased sister chromatid exchange (SCE) [18] and show a high rate of translocations and deletions in somatic cell hybrid systems [19,20]. It has been demonstrated that fragile sites are preferred sites of recombination, or integration, with pSV2neoplasmid DNA transfected into cells pretreated with aphidicolin [21]. Perhaps related to this characteristic are reports of the coincidence of viral integration sites in tumors or tumor cell lines and fragile sites [22–25]. As discussed below, common fragile sites have also been implicated in intrachromosomal gene amplification events in cultured CHO cells and in cancer cells by leading to DNA strand breaks that trigger breakagefusion-bridge cycles [26].
was found to contain human cervical cancer HPV-16 integration sites [22], which led to studies of HPV integrations at fragile sites in cervical tumors [41]. Two other of the most frequently expressed fragile sites also lie within large genes. FRA16D lies within the large WWOX gene, and like FHIT, WWOX encodes a small 2.2kb transcript but extends over 1 Mb due to the presence of two very large introns [33,37]. Similarly, FRA6E lies within the large PARK2 gene, which extends over 500 kb [30]. Recently, the GRID2 gene, a hotspot for mutation and translocations in mouse and humans, has been associated with fragile site FRA4F [29]. GRID2, like FHIT, WWOX and PARK2, extends over more than a megabase but has a small kilobase transcript. The significance of the relationship of fragile sites to large genes is intriguing and could be a reflection of the base composition of large introns found in these genes or be associated with their transcription.
2. Genes at common fragile sites
Table 1 Genes associated with characterized, common fragile sites [taken from 1]
Eleven common fragile sites, FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA9E, FRA16D and FRAXB, have been cloned and characterized in various ways (Table 1). All have been found to extend over hundreds of kilobases as defined by the induction of gaps or breaks on metaphase chromosomes occurring throughout these regions. As might be predicted given their size, most either lie within, or span, known genes. FRA3B was the first fragile site to be mapped and cloned and was found to lie within the large FHIT gene [28,39,40]. FHIT spans approximately 900k and includes two large introns where FRA3B is centrally located but encodes only a small, 1.1kb transcript. There are no large di- or trinucleotide repeats within FRA3B, as are found associated with ‘rare fragile sites,’ such as the fragile X site, FRAXA. The region
3. Fragile site instability in tumor cells Although fragile sites regions are normally stable in cells, they often show rearrangements in tumor cells. The most common type of rearrangements appear to be one or more intra-locus deletions, as opposed to translocations or other rearrangements.
Fragile site
Location
Associated genes
Refs.
FRA2G
2q31
[27]
FRA3B FRA4F FRA6E
3p14.2 4q22 6q26
FRA6F
6q21
FRA7E FRA7G
7q21.11 7q31.2
FRA7H FRA9E
7q32.3 9q32-33.1
FRA16D FRAXB
16q23.3 Xp22.3
IGRP, RDHL, LRP2, others FHIT GRID2 PARKIN, MAP3K4, LPA, others REV3L, DIF13, FKHRL1, others LEP CAV1, CAV2, TESTIN, MET None identified PAPPA, ROD1, KLF4, others WWOX STS
[28] [29] [30]
[31] [32] [33,34] [35] [36] [33,37] [38]
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Most such studies have focused on FRA3B and FRA16D, due to the fact that they are the two most frequently expressed and best-characterized fragile sites, and both lie within large tumor suppressor genes. FRA3B frequently shows allelic loss or homozygous deletions in many tumor types, including lung, digestive tract, kidney, and breast [42–44]. The rearrangements are usually one or more large deletions of tens to hundreds of kilobases directly within the fragile site region. Investigation of chromosome 3 homologs from tumor cell lines has demonstrated multiple variable deletions within FRA3B, suggesting ongoing instability in the region [45]. Only a few translocations in cancer cells with breakpoints in FRA3B have been reported [46–48]. Whether this represents a form of selection or ascertainment bias, or is a true reflection of the frequency of translocations at fragile sites, is not known. The deletions at FRA3B inactivate the FHIT gene, and partly based on these deletions, FHIT was suggested to be a tumor suppressor gene. More recent functional studies from Kay Huebner and colleagues strengthen this hypothesis. FHIT-deficient mice have increased NMBA-induced gastric tumors [49], and this susceptibility can be rescued by introduction of a functional FHIT allele. In addition, overexpression of FHIT has been shown to suppress growth of different cancer cell lines both in vitro and in vivo [50]. Thus, loss of FHIT appears to play a role in mutagen sensitivity and cancer susceptibility. FRA16D maps within regions of frequent loss-ofheterozygosity (LOH) in breast and prostate cancers and is associated with homozygous deletions in various adenocarcinomas and with chromosomal translocations in multiple myeloma. These rearrangements inactivate the WWOX gene, defined as a tumor-suppressor gene by virtue of these large, intragenic, homozygous deletions and point mutations in cancers and its ability to suppress growth of mammary tumors in mice, possibly via a role in apoptosis pathways [37,51–54]. Similar patterns of deletions in cancer cells have also been shown for other common fragile sites, including FRA6E [30], FRA9E [36], and FRA7G [33,34]. It is, thus, clear from numerous studies that FRA3B, FRA16D and other common fragile sites are often associated with large, intra-locus deletions or translocations that frequently inactivate associated genes such as FHIT and WWOX in a variety of tumor
cells [55]. The type of mutations primarily seen in these genes in tumors, large deletions and translocations, as opposed to point mutations, supports the involvement of the associated fragile sites in the mutations. This raises the important question of whether the deletions are simply a result of chromosome instability at fragile sites, or selection for cells with loss of gene function, or a combination of both. A number of tumors have been identified with deletions in more than one fragile site, suggesting a common mechanism [38]. Fragile site instability in tumors appears not to be driven solely by associated genes, since FRAXB at Xp23.3 also shows frequent deletions in tumor cells, yet none of the genes identified at FRAXB are involved in tumor progression [38,56]. However, given the function of FHIT and WWOX in tumorigenesis, it seems likely that, at least in some tumors, selection for loss of function driven by fragile site instability plays a role in tumor progression. In addition to deletions, instability at fragile sites has also been implicated in DNA breakage associated with gene amplification. Coquelle et al. [26] and Debatisse et al. [57] have eloquently shown that breakage at fragile sites can initiate breakage-fusionbridge cycles, a mechanism responsible for accumulation of intrachromosomal amplicons, in drug-resistant mutant CHO cells. More recently, FRA7I and FRA7G have been identified at one boundary of the amplicons found in two tumor cell lines [58]. We have recently characterized the boundaries of amplicons involving the MET oncogene in six primary esophageal adenocarcinomas [59], and found all to have one boundary within FRA7G, consistent with the mechanisms proposed by Coquelle and Debatisee and colleagues. Why are fragile site regions unstable in these tumor cells but not normal cells? Casper et al. [60] suggested that breaks at fragile sites may serve as ‘signatures’ of stalled replication in tumor cells, caused by mutations or perturbations of the S-phase and G2/M checkpoints or associated repair genes during tumor progression. Two recent reports support this concept and experimentally tie together what is known about the mechanisms leading to fragile site expression (discussed below) and chromosomal rearrangements at fragile sites in tumors. Gorgoulis et al. [61] and Bartkova et al. [62] examined bladder, breast,
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colorectal, and lung tumors at various stages of progression for signs of a DNA damage response. Both found that early stages of cancer development are associated with an active DNA damage response, including phosphorylated ATR, ATM, CHK1, and CHK2 kinases and phosphorylated histone H2AX and p53. Associated with these events was a high frequency of LOH at known fragile site regions, including FRA3B. These findings suggest that in precancerous lesions, replication stress leads to stalled or collapsed replication forks, activation of the ATR, and with subsequent DNA DSBs, the ATM checkpoints. In cells that do not undergo apoptosis or cell cycle arrest, allelic imbalances will preferentially target fragile sites, since they are most sensitive to replication stress. Further mutation in p53 or other genes will release additional checkpoints and lead to progression. These findings suggest that lesions at common fragile sites are indicators of replication stress during early stages of tumorigenesis. They are also consistent with a role for some fragile siteassociated genes in cancer.
Late replication is a feature of rare fragile sites, as first shown for the fragile X site in FMR1 and later for FRAXE, FRA16B and FRA10B [64–69]. A likely explanation for the delayed replication at rare fragile sites is that the expansions found at these sites, CGG and AT repeats, can form secondary structures that inhibit progression of replication forks [70,71]. Late replication is also a feature of common fragile sites. LeBeau et al. [72] were the first to show that sequences at FRA3B replicate very late and that the addition of aphidicolin resulted in a further significant delay in replication with about 16.5% of FRA3B sites remaining unreplicated in G2 after the addition of aphidicolin. Replication timing studies of common fragile sites FRA16D and FRA7H indicate these fragile sites also may experience difficulty in replication fork progression [73,74]. These studies support a model in which common fragile site regions can initiate replication in early-mid S phase but are slow to complete replication, and the chromosomal breaks and gaps observed in metaphase cells result from unreplicated DNA [73].
4. DNA sequences and replication at fragile sites
5. Cell cycle checkpoint and repair pathways in fragile site stability
Why are fragile sites ‘fragile’? An obvious place to look for answers to this question is the sequence of these regions. All fragile sites cloned to date are relatively AT-rich [11,35,37,38,63] and have no expanded di- or trinucleotide repeats. Mishmar et al. [35] designed a program named FlexStab to measure local variation in the twist angle between bases and found that the FRA7H region contained more areas of high flexibility, termed ‘flexibility peaks,’ than nonfragile region. FRA2G, FRA3B, FRAXB, FRA7E and FRA16D have now been analyzed in this manner, and, like FRA7H, they all contain a high number of flexibility peaks relative to non-fragile regions [27,32,37,38]. These flexible sequences are composed of interrupted runs of AT-dinucleotides, and these sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B [32]. Such sequences have the potential to form secondary structures and, hence, may affect replication at fragile sites. Perhaps related to this sequence composition is the fact that common fragile sites are late replicating.
Cells have evolved cell cycle checkpoints to delay cell division in response to DNA lesions to allow for their repair. Our laboratory has been interested in elucidating those mechanisms that recognize fragile site lesions as a means of understanding the molecular nature of fragile sites and the cellular processes that affect their stability. Based on the appearance of chromosome gaps and breaks at fragile sites, and the methods used for their induction, the ATM and ATR kinases were primary candidates. ATM and ATR act in parallel, partially overlapping, checkpoint pathways in response to DNA double-strand breaks and replication stress [75,76]. Casper et al. [60] studied the effects of ATM and ATR on fragile site stability. No significant difference was found between ATM-deficient cells and normal control cells in spontaneous or aphidicolin-induced chromosome gaps or breaks, or breaks at specific fragile sites. However, ATR-deficient cells were very sensitive to aphidicolin and showed a highly significant increase in gaps and breaks at
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T.W. Glover / Cancer Letters 232 (2006) 4–12
fragile sites. Importantly, fragile sites were expressed in ATR-deficient cells even without the addition of replication inhibitors. Casper et al. [77] also found that cells from individuals with Seckel syndrome that have hypomorphic mutations in ATR are sensitive to aphidicolin and show increased instability at common fragile sites. Thus, ATR is necessary for the maintenance of stability at fragile sites during normal cell divisions, as well as in cells with replication stress. The ATR checkpoint was the first major pathway found to be associated with control of fragile site expression, and this discovery was important in linking cell cycle checkpoint function to fragile site stability. Subsequent investigations have focused on further delineation of these mechanisms, and a number of targets or modifiers of the ATR pathway have now been shown to influence fragile site stability, including BRCA1, the Fanconi anemia (FA) pathway proteins, and SMC1 [78–80]. While the lack of increased fragile site expression in ATMdeficient cells suggests that DSBs are not the initial or primary cause of fragile site expression, the ATM pathway may be important in subsequent events at fragile sites, particularly in the resolution of DSBs that must sometimes occur at fragile sites in giving rise to chromosomal rearrangements. Given the overlapping functions of these two proteins, it is also possible that ATR can compensate for ATM deficiency with regard to fragile site stability, but not vice versa. Little is known about how lesions at fragile sites are repaired. Homologous repair (HR) plays the major role in responding to DSBs and stalled or collapsed replication forks during S and G2, when the sister chromatid is present. Interestingly, Glover and Stein [18] reported that on average, 70% of all gaps and breaks at FRA3B after aphidicolin treatment had an SCE at that site. SCEs were also observed at FRA3B sites that were not broken, and the percent of unbroken FRA3B sites with an SCE doubled after aphidicolin treatment. The presence of SCEs at fragile sites is an indication that some type of repair has occurred at the site. The molecular basis for formation of SCEs in mammalian cells is not well understood, but it been hypothesized that SCEs are formed by the action of HR during replication repair. Cells with mutations in RAD51 and other members of the HR
pathway have been reported to have reduced spontaneous and MMC-induced SCE formation, although the reduction is not severe and may even be species-specific [81–87]. While these findings suggest a role for HR in repair at fragile sites, there is as of yet no direct evidence for any protein involved in HR being involved in the maintenance of fragile site stability.
6. Conclusions Data from a number of laboratories support a model for common fragile sites based on late or delayed replication, unusual sequence composition, and the role of checkpoint proteins in fragile site stability. They suggest that sequences at fragile sites present difficulties to replication that are further exacerbated by aphidicolin and certain other forms of replication stress. Incomplete replication at these sites can lead to chromosome gaps and breaks, or fragile site ‘expression’. These unreplicated regions can stimulate the activation of the S-phase and/or G2/M checkpoints in which ATR plays a key role. However, the appearance of fragile sites on metaphase chromosomes suggests that many of these lesions can escape these checkpoint controls. Deletions or translocations at fragile sites could result from DNA DSBs caused by breakage of already weakened, single-stranded regions or aberrant processing of Holliday junctions at damaged forks. Thus, the deletions seen in tumor cells are proposed to arise from unequal or faulty homologous repair of stalled forks and would be enhanced by mutations in the replication checkpoint or associated repair pathways. In this way, deletions at fragile sites in tumor cells are ‘signatures’ of replication stress. Due largely to the identification of frequent fragile site instability in tumor cells and the link to important cell cycle checkpoint pathways, the study of common fragile sites has become increasingly significant over the past few years. However, many questions remain regarding the mechanisms of fragile site instability and the significance of this instability and associated genes to cancer. These and related topics were one focus of this meeting on the ‘Fragilome’.
T.W. Glover / Cancer Letters 232 (2006) 4–12
Acknowledgements I thank Professor Manfred Schwab and all organizers of this meeting on the ‘Fragilome’. I thank Martin Arlt, Anne Casper, Niall Howlett and Ryan Ragland for help with this review and Cynthia Gaffney for assistance. TWG is supported by NIH grant CA43222.
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T.W. Glover / Cancer Letters 232 (2006) 4–12 [53] M. Mangelsdorf, K. Ried, E. Woollatt, S. Dayan, H. Eyre, M. Finnis, et al., Chromosomal fragile site FRA16D and DNA instability in cancer, Cancer Res. 60 (2000) 1683–1689. [54] A.J.W. Paige, K.J. Taylor, A. Stewart, J.G. Sgouros, H. Gabra, G.C. Sellar, et al., A 700-kb physical map of a region of 16q23.2 homozygously deleted in multiple cancers and spanning the common fragile site FRA16D, Cancer Res. 60 (2000) 1690–1697. [55] K. Huebner, C.M. Croce, FRA3B and other common fragile sites: the weakest links, Nat. Rev. Cancer 1 (2001) 214–221. [56] M. Finnis, S. Dayan, L. Hobson, G. Chenevix-Trench, K. Friend, K. Ried, et al., Common chromosomal fragile site FRA16D mutation in cancer cells, Hum. Mol. Genet. 14 (2005) 1341–1349. [57] M. Debatisse, A. Coquelle, F. Toledo, G. Buttin, Gene amplification mechanisms: the role of fragile sites, Recent Results Cancer Res. 154 (1998) 216–226. [58] A. Hellman, E. Zlotorynski, S.W. Scherer, J. Cheung, J.B. Vincent, D.I. Smith, et al., A role for common fragile site induction in amplification of human oncogenes, Cancer Cell 1 (2002) 89–97. [59] C.T. Miller, L. Lin, A.M. Casper, J. Lim, D.G. Thomas, M.B. Orringer, A.C. Chang, A.F. Chambers, T.J. Giordano, T.W. Glover, D.G. Beer, Genomic amplification of MET with boundaries within fragile site FRA7G and upregulation of MET signaling pathways in esophageal adenocarcinoma, (Submitted). [60] A.M. Casper, P. Nghiem, M.F. Arlt, T.W. Glover, ATR regulates fragile site stability, Cell 111 (2002) 779–789. [61] V.G. Gorgoulis, L.V. Vassiliou, P. Karakaidos, P. Zacharatos, A. Kotsinas, T. Liloglou, et al., Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions, Nature 434 (2005) 907–913. [62] J. Bartkova, Z. Horejsi, K. Koed, A. Kramer, F. Tort, K. Zieger, et al., DNA damage response as a candidate anticancer barrier in early human tumorigenesis, Nature 434 (2005) 864–870. [63] F. Boldog, R. Gemmill, J. West, M. Robinson, E. Li, Chromosome 3p14 homozygous deletions and sequence analysis of FRA3B, Hum. Mol. Genet. 6 (1997) 193–203. [64] H. Ohashi, A. Kuwano, M. Tsukahara, T. Arinami, T. Kajii, Replication patterns of the fragile X in heterozygous carriers: analysis by a BrdUrd antibody method, Am. J. Hum. Genet. 47 (1990) 988–993. [65] W.D. Yu, S.L. Wenger, M.W. Steele, X chromosome imprinting in fragile X syndrome, Hum. Genet. 85 (1990) 590–594. [66] R.S. Hansen, T.K. Canfield, M.M. Lamb, S.M. Gartler, C.D. Laird, Association of fragile X syndrome with delayed replication of the FMR1 gene, Cell 73 (1993) 1403–1409. [67] R.S. Hansen, T.K. Canfield, A.D. Fjeld, S. Mumm, C.D. Laird, S.M. Gartler, A variable domain of delayed replication in FRAXA fragile X chromosomes: X inactivation-like spread of late replication, Proc. Natl. Acad. Sci. USA 94 (1997) 4587–4592. [68] P.S. Subramanian, D.L. Nelson, A.C. Chinault, Large domains of apparent delayed replication timing associated with triplet
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repeat expansion at FRAXA and FRAXE, Am. J. Hum. Genet. 59 (1996) 407–416. O. Handt, E. Baker, S. Dayan, S.M. Gartler, E. Woollatt, R.I. Richards, R.S. Hansen, Analysis of replication timing at the FRA10B and FRA16B fragile site loci, Chromosome Res. 8 (2000) 677–688. X. Chen, S.V. Mariappan, P. Catasti, R. Ratliff, R.K. Moyzis, A. Laayoun, et al., Hairpins are formed by the single DNA strands of the fragile X triplet repeats: structure and biological implications, Proc. Natl. Acad. Sci. USA 92 (1995) 5199–5203. D.R. Hewett, O. Handt, L. Hobson, M. Mangelsdorf, H.J. Eyre, E. Baker, et al., FRA10B structure reveals common elements in repeat expansion and chromosomal fragile site genesis, Mol. Cell 1 (1998) 773–781. M.M. Le Beau, F.V. Rassool, M.E. Neilly, R. Espinosa III, T.W. Glover, D.I. Smith, T.W. McKeithan, Replication of a common fragile site, FRA3B, occurs late in S phase and is delayed further upon induction: implications for the mechanism of fragile site induction, Hum. Mol. Genet. 7 (1998) 755–761. A. Palakodeti, Y. Han, Y. Jiang, M.M. Le Beau, The role of late/slow replication of the FRA16D in common fragile site induction, Genes. Chromosomes Cancer 39 (2004) 71–76. A. Hellman, A. Rahat, S.W. Scherer, A. Darvasi, L.-C. Tsui, S. Kerem, Replication delay along FRA7H, a common fragile site on human chromosome 7, leads to chromosomal instability, Mol. Cell. Biol. 20 (2000) 4420–4427. R.T. Abraham, Cell cycle checkpoint signaling through the ATM, ATR kinases, Genes Dev. 15 (2001) 2177–2196. D. Durocher, S.P. Jackson, DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme?, Curr. Opin. Cell Biol. 13 (2001) 225–231. A.M. Casper, S.G. Durkin, M.F. Arlt, T.W. Glover, Chromosomal instability at common fragile sites in Seckel syndrome, Am. J. Hum. Genet. 75 (2004) 654–660. M.F. Arlt, B. Xu, S.G. Durkin, A.M. Casper, M.B. Kastan, T.W. Glover, BRCA1 is required for common fragile-site stability via its G2/M checkpoint function, Mol. Cell. Biol. 24 (2004) 6701–6709. N.G. Howlett, T. Taniguchi, S.G. Durkin, A.D. D’Andrea, T.W. Glover, The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability, Hum. Molec. Genet. 14 (2005) 693–701. A. Musio, C. Montagna, T. Mariani, M. Tilenni, M.L. Focarelli, L. Brait, et al., SMC1 involvement in fragile site expression, Hum. Molec. Genet. 14 (2005) 525–533. M.L.G. Dronkert, H.B. Beverloo, R.D. Johnson, J.H.J. Hoeijmakers, M. Jasin, R. Kanaar, Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange, Mol. Cell. Biol. 20 (2000) 3147–3156. L.F. Fuller, R.B. Painter, A Chinese hamster ovary cell line hypersensitive to ionizing radiation and deficient in repair replication, Mutat. Res. 193 (1988) 109–121. S. Lambert, B.S. Lopez, Role of RAD51 in sister-chromatid exchanges in mammalian cells, Oncogene 20 (2001) 6627–6631.
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[84] M.S. Sasaki, M. Takata, E. Sonoda, A. Tachibana, S. Takeda, Recombination repair pathway in the maintenance of chromosomal integrity against DNA interstrand crosslinks, Cytogenet. Genome Res. 104 (2004) 28–34. [85] J.M. Stark, P. Hu, A.J. Pierce, M.E. Moynahan, N. Ellis, M. Jasin, ATP hydrolysis by mammalian RAD51 had a key role during homology-directed DNA repair, J. Biol. Chem. 277 (2002) 20185–20194.
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Mini Review
Common fragile sites Thomas W. Glover* Department of Human Genetics, 4909 Buhl, Box 0618, 1241 E. Catherine Street, University of Michigan, Ann Arbor, MI 48109-0618, USA Received 24 August 2005; accepted 30 August 2005
Abstract Common fragile sites are regions showing site-specific gaps and breaks on metaphase chromosomes after partial inhibition of DNA synthesis. Common fragile sites are normally stable in somatic cells. However, following treatment of cultured cells with replication inhibitors, fragile sites display gaps, breaks, rearrangements and other features of unstable DNA. Studies showing that fragile sites and associated genes are frequently deleted or rearranged in many cancer cells have clearly demonstrated their importance in genome instability in cancer. Until recently, little was known about the molecular nature and mechanisms involved in fragile site instability. From studies conducted in many laboratories, it is now known that fragile sites extend over large regions, are associated with genes, exhibit delayed replication, and contain regions of high DNA flexibility. Recent findings from our laboratory showing that the key cell cycle checkpoint genes are important for genome stability at fragile sties have shed new light on these mechanisms and on the significance of these sites in cancer and normal chromosome structure. Since their discovery over two decades ago, much has been learned regarding their significance in chromosome structure and instability in cancer, but a number of key questions remain, including why these sites are ‘fragile’ and the impact of this instability on associated genes in cancer cells. These and other questions have been addressed by participants of this meeting, which highlighted instability at common fragile sites. This brief review is intended to provide background on common fragile sites that has led up to many of the studies presented in the accompanying reports in this volume and not to summarize the findings presented therein. Some aspects of this review were taken from Glover et al. (T.W. Glover, M.F. Arlt, A.M. Casper, S.G. Durkin, Mechanisms of common fragile site instability, Hum. Molec. Genet. 14 (in press). [1]). q 2005 Elsevier Ireland Ltd. All rights reserved.
1. Fragile sites in cultured cells Common fragile sites are seen in all individuals and, thus, represent a component of normal chromosome structure. They are conserved in the mouse and other mammals in which they have been investigated [2–13] and perhaps have counterparts in yeast [14,15]. * Tel.: C1 734 763-5222; fax: C1 734 763 3784. E-mail address: [email protected].
These fragile site regions are normally stable in cultured human cells. However, when cells are cultured under conditions of folate or thymidylate stress or with low doses of aphidicolin that only partially inhibit DNA synthesis, they are prone to forming chromosome gaps and breaks. With the cloning and characterization of common fragile sites, we know that these breaks can occur over broad regions extending up to a megabase or more. At present, over 80 common fragile sites are listed in
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.08.032
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the Genome Database (GDB). However, not all fragile sites form breaks at the same frequency, and a small number of sites in the genome are most prone to form lesions [16,17]. Gaps and breaks at just 20 fragile sites represent over 80% of all lesions observed in lymphocytes following treatment with low doses of aphidicolin [16]. FRA3B at 3p14.2 stands out as the most ‘fragile’ site in the genome and can be induced to break or form gaps in the majority of treated cells. Other highly expressed fragile sites in lymphocytes include those at 16q23 (FRA16D), 6q26 (FRA6E), 7q32.3 (FRA7H), and Xp22.3 (FRAXB) [16]. In addition to gaps and breaks, common fragile sites are ‘hot spots’ for increased sister chromatid exchange (SCE) [18] and show a high rate of translocations and deletions in somatic cell hybrid systems [19,20]. It has been demonstrated that fragile sites are preferred sites of recombination, or integration, with pSV2neoplasmid DNA transfected into cells pretreated with aphidicolin [21]. Perhaps related to this characteristic are reports of the coincidence of viral integration sites in tumors or tumor cell lines and fragile sites [22–25]. As discussed below, common fragile sites have also been implicated in intrachromosomal gene amplification events in cultured CHO cells and in cancer cells by leading to DNA strand breaks that trigger breakagefusion-bridge cycles [26].
was found to contain human cervical cancer HPV-16 integration sites [22], which led to studies of HPV integrations at fragile sites in cervical tumors [41]. Two other of the most frequently expressed fragile sites also lie within large genes. FRA16D lies within the large WWOX gene, and like FHIT, WWOX encodes a small 2.2kb transcript but extends over 1 Mb due to the presence of two very large introns [33,37]. Similarly, FRA6E lies within the large PARK2 gene, which extends over 500 kb [30]. Recently, the GRID2 gene, a hotspot for mutation and translocations in mouse and humans, has been associated with fragile site FRA4F [29]. GRID2, like FHIT, WWOX and PARK2, extends over more than a megabase but has a small kilobase transcript. The significance of the relationship of fragile sites to large genes is intriguing and could be a reflection of the base composition of large introns found in these genes or be associated with their transcription.
2. Genes at common fragile sites
Table 1 Genes associated with characterized, common fragile sites [taken from 1]
Eleven common fragile sites, FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA9E, FRA16D and FRAXB, have been cloned and characterized in various ways (Table 1). All have been found to extend over hundreds of kilobases as defined by the induction of gaps or breaks on metaphase chromosomes occurring throughout these regions. As might be predicted given their size, most either lie within, or span, known genes. FRA3B was the first fragile site to be mapped and cloned and was found to lie within the large FHIT gene [28,39,40]. FHIT spans approximately 900k and includes two large introns where FRA3B is centrally located but encodes only a small, 1.1kb transcript. There are no large di- or trinucleotide repeats within FRA3B, as are found associated with ‘rare fragile sites,’ such as the fragile X site, FRAXA. The region
3. Fragile site instability in tumor cells Although fragile sites regions are normally stable in cells, they often show rearrangements in tumor cells. The most common type of rearrangements appear to be one or more intra-locus deletions, as opposed to translocations or other rearrangements.
Fragile site
Location
Associated genes
Refs.
FRA2G
2q31
[27]
FRA3B FRA4F FRA6E
3p14.2 4q22 6q26
FRA6F
6q21
FRA7E FRA7G
7q21.11 7q31.2
FRA7H FRA9E
7q32.3 9q32-33.1
FRA16D FRAXB
16q23.3 Xp22.3
IGRP, RDHL, LRP2, others FHIT GRID2 PARKIN, MAP3K4, LPA, others REV3L, DIF13, FKHRL1, others LEP CAV1, CAV2, TESTIN, MET None identified PAPPA, ROD1, KLF4, others WWOX STS
[28] [29] [30]
[31] [32] [33,34] [35] [36] [33,37] [38]
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Most such studies have focused on FRA3B and FRA16D, due to the fact that they are the two most frequently expressed and best-characterized fragile sites, and both lie within large tumor suppressor genes. FRA3B frequently shows allelic loss or homozygous deletions in many tumor types, including lung, digestive tract, kidney, and breast [42–44]. The rearrangements are usually one or more large deletions of tens to hundreds of kilobases directly within the fragile site region. Investigation of chromosome 3 homologs from tumor cell lines has demonstrated multiple variable deletions within FRA3B, suggesting ongoing instability in the region [45]. Only a few translocations in cancer cells with breakpoints in FRA3B have been reported [46–48]. Whether this represents a form of selection or ascertainment bias, or is a true reflection of the frequency of translocations at fragile sites, is not known. The deletions at FRA3B inactivate the FHIT gene, and partly based on these deletions, FHIT was suggested to be a tumor suppressor gene. More recent functional studies from Kay Huebner and colleagues strengthen this hypothesis. FHIT-deficient mice have increased NMBA-induced gastric tumors [49], and this susceptibility can be rescued by introduction of a functional FHIT allele. In addition, overexpression of FHIT has been shown to suppress growth of different cancer cell lines both in vitro and in vivo [50]. Thus, loss of FHIT appears to play a role in mutagen sensitivity and cancer susceptibility. FRA16D maps within regions of frequent loss-ofheterozygosity (LOH) in breast and prostate cancers and is associated with homozygous deletions in various adenocarcinomas and with chromosomal translocations in multiple myeloma. These rearrangements inactivate the WWOX gene, defined as a tumor-suppressor gene by virtue of these large, intragenic, homozygous deletions and point mutations in cancers and its ability to suppress growth of mammary tumors in mice, possibly via a role in apoptosis pathways [37,51–54]. Similar patterns of deletions in cancer cells have also been shown for other common fragile sites, including FRA6E [30], FRA9E [36], and FRA7G [33,34]. It is, thus, clear from numerous studies that FRA3B, FRA16D and other common fragile sites are often associated with large, intra-locus deletions or translocations that frequently inactivate associated genes such as FHIT and WWOX in a variety of tumor
cells [55]. The type of mutations primarily seen in these genes in tumors, large deletions and translocations, as opposed to point mutations, supports the involvement of the associated fragile sites in the mutations. This raises the important question of whether the deletions are simply a result of chromosome instability at fragile sites, or selection for cells with loss of gene function, or a combination of both. A number of tumors have been identified with deletions in more than one fragile site, suggesting a common mechanism [38]. Fragile site instability in tumors appears not to be driven solely by associated genes, since FRAXB at Xp23.3 also shows frequent deletions in tumor cells, yet none of the genes identified at FRAXB are involved in tumor progression [38,56]. However, given the function of FHIT and WWOX in tumorigenesis, it seems likely that, at least in some tumors, selection for loss of function driven by fragile site instability plays a role in tumor progression. In addition to deletions, instability at fragile sites has also been implicated in DNA breakage associated with gene amplification. Coquelle et al. [26] and Debatisse et al. [57] have eloquently shown that breakage at fragile sites can initiate breakage-fusionbridge cycles, a mechanism responsible for accumulation of intrachromosomal amplicons, in drug-resistant mutant CHO cells. More recently, FRA7I and FRA7G have been identified at one boundary of the amplicons found in two tumor cell lines [58]. We have recently characterized the boundaries of amplicons involving the MET oncogene in six primary esophageal adenocarcinomas [59], and found all to have one boundary within FRA7G, consistent with the mechanisms proposed by Coquelle and Debatisee and colleagues. Why are fragile site regions unstable in these tumor cells but not normal cells? Casper et al. [60] suggested that breaks at fragile sites may serve as ‘signatures’ of stalled replication in tumor cells, caused by mutations or perturbations of the S-phase and G2/M checkpoints or associated repair genes during tumor progression. Two recent reports support this concept and experimentally tie together what is known about the mechanisms leading to fragile site expression (discussed below) and chromosomal rearrangements at fragile sites in tumors. Gorgoulis et al. [61] and Bartkova et al. [62] examined bladder, breast,
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colorectal, and lung tumors at various stages of progression for signs of a DNA damage response. Both found that early stages of cancer development are associated with an active DNA damage response, including phosphorylated ATR, ATM, CHK1, and CHK2 kinases and phosphorylated histone H2AX and p53. Associated with these events was a high frequency of LOH at known fragile site regions, including FRA3B. These findings suggest that in precancerous lesions, replication stress leads to stalled or collapsed replication forks, activation of the ATR, and with subsequent DNA DSBs, the ATM checkpoints. In cells that do not undergo apoptosis or cell cycle arrest, allelic imbalances will preferentially target fragile sites, since they are most sensitive to replication stress. Further mutation in p53 or other genes will release additional checkpoints and lead to progression. These findings suggest that lesions at common fragile sites are indicators of replication stress during early stages of tumorigenesis. They are also consistent with a role for some fragile siteassociated genes in cancer.
Late replication is a feature of rare fragile sites, as first shown for the fragile X site in FMR1 and later for FRAXE, FRA16B and FRA10B [64–69]. A likely explanation for the delayed replication at rare fragile sites is that the expansions found at these sites, CGG and AT repeats, can form secondary structures that inhibit progression of replication forks [70,71]. Late replication is also a feature of common fragile sites. LeBeau et al. [72] were the first to show that sequences at FRA3B replicate very late and that the addition of aphidicolin resulted in a further significant delay in replication with about 16.5% of FRA3B sites remaining unreplicated in G2 after the addition of aphidicolin. Replication timing studies of common fragile sites FRA16D and FRA7H indicate these fragile sites also may experience difficulty in replication fork progression [73,74]. These studies support a model in which common fragile site regions can initiate replication in early-mid S phase but are slow to complete replication, and the chromosomal breaks and gaps observed in metaphase cells result from unreplicated DNA [73].
4. DNA sequences and replication at fragile sites
5. Cell cycle checkpoint and repair pathways in fragile site stability
Why are fragile sites ‘fragile’? An obvious place to look for answers to this question is the sequence of these regions. All fragile sites cloned to date are relatively AT-rich [11,35,37,38,63] and have no expanded di- or trinucleotide repeats. Mishmar et al. [35] designed a program named FlexStab to measure local variation in the twist angle between bases and found that the FRA7H region contained more areas of high flexibility, termed ‘flexibility peaks,’ than nonfragile region. FRA2G, FRA3B, FRAXB, FRA7E and FRA16D have now been analyzed in this manner, and, like FRA7H, they all contain a high number of flexibility peaks relative to non-fragile regions [27,32,37,38]. These flexible sequences are composed of interrupted runs of AT-dinucleotides, and these sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B [32]. Such sequences have the potential to form secondary structures and, hence, may affect replication at fragile sites. Perhaps related to this sequence composition is the fact that common fragile sites are late replicating.
Cells have evolved cell cycle checkpoints to delay cell division in response to DNA lesions to allow for their repair. Our laboratory has been interested in elucidating those mechanisms that recognize fragile site lesions as a means of understanding the molecular nature of fragile sites and the cellular processes that affect their stability. Based on the appearance of chromosome gaps and breaks at fragile sites, and the methods used for their induction, the ATM and ATR kinases were primary candidates. ATM and ATR act in parallel, partially overlapping, checkpoint pathways in response to DNA double-strand breaks and replication stress [75,76]. Casper et al. [60] studied the effects of ATM and ATR on fragile site stability. No significant difference was found between ATM-deficient cells and normal control cells in spontaneous or aphidicolin-induced chromosome gaps or breaks, or breaks at specific fragile sites. However, ATR-deficient cells were very sensitive to aphidicolin and showed a highly significant increase in gaps and breaks at
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fragile sites. Importantly, fragile sites were expressed in ATR-deficient cells even without the addition of replication inhibitors. Casper et al. [77] also found that cells from individuals with Seckel syndrome that have hypomorphic mutations in ATR are sensitive to aphidicolin and show increased instability at common fragile sites. Thus, ATR is necessary for the maintenance of stability at fragile sites during normal cell divisions, as well as in cells with replication stress. The ATR checkpoint was the first major pathway found to be associated with control of fragile site expression, and this discovery was important in linking cell cycle checkpoint function to fragile site stability. Subsequent investigations have focused on further delineation of these mechanisms, and a number of targets or modifiers of the ATR pathway have now been shown to influence fragile site stability, including BRCA1, the Fanconi anemia (FA) pathway proteins, and SMC1 [78–80]. While the lack of increased fragile site expression in ATMdeficient cells suggests that DSBs are not the initial or primary cause of fragile site expression, the ATM pathway may be important in subsequent events at fragile sites, particularly in the resolution of DSBs that must sometimes occur at fragile sites in giving rise to chromosomal rearrangements. Given the overlapping functions of these two proteins, it is also possible that ATR can compensate for ATM deficiency with regard to fragile site stability, but not vice versa. Little is known about how lesions at fragile sites are repaired. Homologous repair (HR) plays the major role in responding to DSBs and stalled or collapsed replication forks during S and G2, when the sister chromatid is present. Interestingly, Glover and Stein [18] reported that on average, 70% of all gaps and breaks at FRA3B after aphidicolin treatment had an SCE at that site. SCEs were also observed at FRA3B sites that were not broken, and the percent of unbroken FRA3B sites with an SCE doubled after aphidicolin treatment. The presence of SCEs at fragile sites is an indication that some type of repair has occurred at the site. The molecular basis for formation of SCEs in mammalian cells is not well understood, but it been hypothesized that SCEs are formed by the action of HR during replication repair. Cells with mutations in RAD51 and other members of the HR
pathway have been reported to have reduced spontaneous and MMC-induced SCE formation, although the reduction is not severe and may even be species-specific [81–87]. While these findings suggest a role for HR in repair at fragile sites, there is as of yet no direct evidence for any protein involved in HR being involved in the maintenance of fragile site stability.
6. Conclusions Data from a number of laboratories support a model for common fragile sites based on late or delayed replication, unusual sequence composition, and the role of checkpoint proteins in fragile site stability. They suggest that sequences at fragile sites present difficulties to replication that are further exacerbated by aphidicolin and certain other forms of replication stress. Incomplete replication at these sites can lead to chromosome gaps and breaks, or fragile site ‘expression’. These unreplicated regions can stimulate the activation of the S-phase and/or G2/M checkpoints in which ATR plays a key role. However, the appearance of fragile sites on metaphase chromosomes suggests that many of these lesions can escape these checkpoint controls. Deletions or translocations at fragile sites could result from DNA DSBs caused by breakage of already weakened, single-stranded regions or aberrant processing of Holliday junctions at damaged forks. Thus, the deletions seen in tumor cells are proposed to arise from unequal or faulty homologous repair of stalled forks and would be enhanced by mutations in the replication checkpoint or associated repair pathways. In this way, deletions at fragile sites in tumor cells are ‘signatures’ of replication stress. Due largely to the identification of frequent fragile site instability in tumor cells and the link to important cell cycle checkpoint pathways, the study of common fragile sites has become increasingly significant over the past few years. However, many questions remain regarding the mechanisms of fragile site instability and the significance of this instability and associated genes to cancer. These and related topics were one focus of this meeting on the ‘Fragilome’.
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Acknowledgements I thank Professor Manfred Schwab and all organizers of this meeting on the ‘Fragilome’. I thank Martin Arlt, Anne Casper, Niall Howlett and Ryan Ragland for help with this review and Cynthia Gaffney for assistance. TWG is supported by NIH grant CA43222.
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