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Common fragile sites (CFSs) are large genomic regions present in all individuals that are highly unstable and prone to breakage and rearrangement, especially in cancer cells with genomic instability. Eight of the 90 known CFSs have been precisely defined and five of these span genes that extend from 700 kb to over 1.5 Mb of genomic sequence. Although these genes reside within some of the most unstable chromosomal regions in the human genome, they are highly conserved evolutionarily. These genes are targets for large chromosomal deletions and rearrangements in cancer and are frequently inactivated in multiple tumor types. There is also an association between these genes and cellular responses to stress. Based upon the association between large genes and CFSs, we began to systematically test other large genes derived from chromosomal regions that were known to contain a CFS. In this study, we demonstrate that the 730 kb retinoic acid receptor-related orphan receptor alpha (RORA) gene is derived from the middle of the FRA15A (15q22.2) CFS. Although this gene is expressed in normal breast, prostate and ovarian epithelium, it is frequently inactivated in cancers that arise from these organs. RORA was previously shown to be involved in the cellular response to hypoxia and here we demonstrate changes in the amount of RORA message produced in cells exposed to a variety of different cellular stresses. Our results demonstrate that RORA is another very large CFS gene that is inactivated in multiple tumors. In addition, RORA appears to play a critical role in responses to cellular stress, lending further support to the idea that the large CFS genes function as part of a highly conserved stress response network that is uniquely susceptible to genomic instability in cancer cells.

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Cell cycle checkpoints are essential for maintaining genomic integrity. Human topoisomerase II binding protein 1 (TopBP1) shares sequence similarity with budding yeast Dpb11, fission yeast Rad4/Cut5, and Xenopus Cut5, all of which are required for DNA replication and cell cycle checkpoints. Indeed, we have shown that human TopBP1 participates in the activation of replication checkpoint and DNA damage checkpoints, following hydroxyurea treatment and ionizing radiation. In this study, we address the physiological function of TopBP1 in S phase by using small interfering RNA. In the absence of exogenous DNA damage, TopBP1 is recruited to replicating chromatin. However, TopBP1 does not appear to be essential for DNA replication. TopBP1-deficient cells have increased H2AX phosphorylation and ATM-Chk 2 activation, suggesting the accumulation of DNA double-strand breaks in the absence of TopBP1. This leads to formation of gaps and breaks at fragile sites, 4N accumulation, and aberrant cell division. We propose that the cellular function of TopBP1 is to monitor ongoing DNA replication. By ensuring proper DNA replication, TopBP1 plays a critical role in the maintenance of genomic stability during normal S phase as well as following genotoxic stress.

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Exposure to corticosteroids increases the risks of avascular necrosis (AVN) of the bone after hematopoietic cell transplantation (HCT). However, whether this effect is dependent on the dose of corticosteroids is not well known. We conducted a case-controlled study, which included 74 recipients of autologous or allogeneic HCT with AVN and 147 controls without AVN that were matched by age, sex, and year of HCT to cases. Cases with AVN included 8 autologous HCT recipients, 58 myeloablative allogeneic HCT recipients, and 8 recipients of non-myeloablative allogeneic HCT. Corticosteroid exposure was expressed as cumulative doses of prednisone. Cases received higher cumulative doses of prednisone than controls, and among allogeneic HCT recipients, cases were more likely to have developed acute and chronic graft-versus-host disease (aGVHD, cGVHD). Cumulative dose of prednisone was an independent risk factor for AVN. Compared to no corticosteroid exposure, exposure to <3870 mg cumulative dose of prednisone was associated with 4.0 (95% confidence intervals, 1.5-11.2) times higher risk, 3870-9735 mg with 5.6 (2.1-15.2) times higher risk and >9735 with 8.6 (3.2-23.5) times higher risk of AVN. Exposure to higher doses of corticosteroids increases the risk of AVN in HCT recipients.

The common fragile sites are regions of profound genomic instability found in all individuals. The full size of each region of instability ranges from under one megabase (Mb) to greater than 10 Mbs. At least half of the CFS regions have been found to span extremely large genes that spanned from 600 kb to greater than 2.0 Mbs. The large CFS genes are also very interesting from a cancer perspective as several of them, including FHIT and WWOX, have already demonstrated the capacity to function as tumor suppressor genes, both in vitro and in vivo. We estimate that there may be 40–50 large genes localized in CFS regions. The expression of a number of the large CFS genes has been previously shown to be lost in many different cancers and this is frequently associated with a worse clinical outcome for patients. To determine if there was selection for the inactivation of different large CFS genes in different cancers, we examined the expression of 13 of the 20 known large CFS genes: FHIT, WWOX, PARK2, GRID2, NBEA, DLG2, RORA isoforms 1 and 4, DAB1, CNTNAP2, DMD, IL1RAPL1, IMMP2L and LARGE in breast, ovarian, endometrial and brain cancers using real-time RT-PCR analysis. Each cancer had a distinct profile of different large CFS genes that were inactivated. Interestingly, in breast, ovarian and endometrial cancers there were some cancers that had inactivation of expression of none or only one of the tested genes, while in other specimens there was inactivation of multiple tested genes. Brain cancers had inactivation of many of the tested genes, a number of which function in normal neurological development. We find that there is no relationship between the frequency that any specific CFS is expressed and the frequency that the gene from that region is inactivated in different cancers. Instead, it appears that different cancers select for the inactivation of different large CFS genes.