The expression frequency of common fragile sites and genetic predisposition to colon cancer

The expression frequency of common fragile sites induced by aphidicolin (Apc), bromodeoxyuridine (BrdU), and caffeine was evaluated on prometaphase chromosomes obtained from the peripheral blood lymphocytes of 32 patients with colon cancer, 30 of their clinically healthy family members and 30 age-matched normal controls. The proportion of damaged cells (P < 0.001), the mean number of chromosomal aberrations and the expression frequencies of fragile sites were significantly higher in the patient and relative groups compared to the control group. Our findings show an increased genetic instability in patients with colon cancer and their first-degree relatives. In addition, common fragile sites can be used as a suitable marker for determining genetic predisposition to cancer.     

Investigation of genetic susceptibility to non-small cell lung cancer by fragile site expression

Fragile sites are non-staining gaps and breaks in specific points of chromosomes that are inducible by various culture conditions. Previous studies have shown that various clastogenic agents increase expression of fragile sites. In this study, the expression of common fragile sites induced by aphidicolin was evaluated on prometaphase chromosomes obtained from peripheral blood lymphocytes. Chromosomal aberrations and fragile site expression of 60 individuals, including 20 patients with non-small cell lung cancer (NSCLC), 20 of their clinically healthy family members, and 20 age-matched normal healthy controls without history of any cancer type were studied. Both the proportion of damaged cells (P < 0.001) and the mean number of gaps and breaks per cell (P < 0.001) were significantly higher in both the patients and relatives' groups when compared with the control group. However, they were insignificant when the patients were compared to their relatives (P > 0.05). We determined four aphidicolin type common fragile sites in our study. These sites in patients with NSCLC and relatives were the following: 1p21, 2q33, 3p14, and 16q23. In these fragile sites, 2q33, 3p14, and 16q23 sites were statistically significant when compared with control group (P < 0.001, P < 0.0005, and P < 0.05, respectively). Consequently, we believe that fragile site studies may be helpful to detection of cancer risk.     

Chromosomal fragile sites and relationship between genetic predisposition to small cell lung cancer.

Fragile sites are non-staining gaps and breaks on mammalian chromosomes. Several investigators have pointed out that these sites may act as factors that predispose to specific chromosomal rearrangements that are present in some cancer cases. The expression of common fragile sites induced by aphidicolin (Apc) was evaluated on prometaphase chromosomes obtained from the peripheral blood lymphocytes of 15 patients with lung cancer, 20 of their clinically healthy family members, and 20 age-matched normal controls. As a result of cytogenetic evaluation carried out by the High Resolution Banding (HRB) technique, 1q21, 2q33, 3p14, 7q32, 13q13, 16q23, 17q21, and 22q12 are defined as fragile sites in patients and relatives. The rate of total fragile sites and 2q33, 3p14, and 16q23 are statistically significant in both patients and relatives when compared with the control group. Therefore, our results showed that common fragile sites might be unstable factors in the human genome and they can be used as suitable markers for genetic predisposition to lung cancer.     

Common chromosomal fragile site FRA16D mutation in cancer cells

Neither the molecular basis for common fragile site DNA instability nor the contribution of this form of chromosomal instability to cancer is clearly understood. Fragile site FRA16D (16q23.2) is within regions of frequent loss-of-heterozygosity (LOH) in breast and prostate cancers, is associated with homozygous deletions in various adenocarcinomas and t(14;16) chromosomal translocations in multiple myeloma. The FOR (WWOX) gene spans FRA16D and encodes a partner of p53 that also has a role in apoptosis. Previously untested 53 cancer cell lines were screened for deletions within the FOR/WWOX gene. Deletions were detected in Co115, KM12C and KM12SM. Homozygous deletions in these and two previously identified tumour cell lines were intragenic on both alleles, indicating a distinct mutation mechanism from that causing LOH. Identical FRA16D deletions in two cell lines (one derived from the primary carcinoma and the other from a secondary metastasis) demonstrate that FRA16D DNA instability can be an early, transient event. Sequence analysis across one deletion locates one endpoint within a polymorphic AT-dinucleotide repeat and the other adjacent to an AT-rich mini-satellite repeat implicating AT-rich repeats in FRA16D DNA instability. Another deletion is associated with de novo repetition of the 9 bp AT-rich sequence at one of the deletion endpoints. FRA16D deleted cells retain cytogenetic fragile site expression indicating that the deletions are susceptible sites for breakage rather than regions that confer fragility. Most cell lines with FRA16D homozygous deletions also have FRA3B deletions, therefore common fragile sites represent highly susceptible genome-wide targets for a distinct form of mutation.     

Claspin inhibition leads to fragile site expression

Fragile sites are hot spots for sister chromatid exchanges, translocations, deletions, complex rearrangements, and gene amplification. It has been hypothesized that rearrangements at fragile sites derive from unreplicated regions resulting from stalled forks that escape the ATR replication checkpoint. In the present study, we investigated the role of the Claspin (CLSPN) gene, which codes for an adaptor protein in the ATR pathway, during DNA replication stress in human cells. We show that the inhibition of the CLSPN gene leads to both genome instability and fragile site expression. Following aphidicolin treatment, we found a transient increase of Claspin synthesis due to its requirement to checkpoint activation. However, Claspin synthesis decreased after a prolonged aphidicolin treatment. We propose that CLSPN modulation, following an extreme replication block, allows rare cells to escape checkpoint mechanisms and enter mitosis with a defect in genome assembly. Our observations provide the basis for a better understanding of cell cycle checkpoints deregulation in cancer. © 2009 Wiley‐Liss, Inc.     

Common fragile site expression in lymphocytes from an individual mosaic for trisomy 8

During the course of a survey of fragile site expression in lymphocytes from twins one member of a dizygotic pair was found to be mosaic for trisomy 8. One hundred fifty metaphases from this individual were analyzed (100 treated with aphidicolin and 50 untreated); 43% were 46,XY and 57% 46,XY, +8. No differences were observed between the treated and control cultures in either the proportions of normal and trisomic metaphases or the overall or specific fragile site expression in the normal and trisomic cells. © 1993 Wiley-Liss, Inc.     

Concordant loss of fragile gene expression early in breast cancer development

The FHIT and WWOX genes encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3, respectively. Reduced Fhit and Wwox expression has been reported in approximately two-thirds of invasive breast tumors. Expression of these fragile gene products, as well as ErbB2 and p53, were evaluated immunohistochemically in 44 pure and 31 adjacent-to-invasive ductal carcinoma in-situ (DCIS) cases. Reduced Fhit and Wwox expression were observed in (i) 70% and 68% of pure DCIS; (ii) 52% and 55% of DCIS adjacent-to-invasive tumor cases; and (iii) 20% and 50% of adjacent normal tissue in pure DCIS cases. Reduced Wwox expression in adjacent normal tissue was observed in 30% of cases in the DCIS adjacent-to-invasive group. Reduced Fhit and Wwox expression was observed in 61% of adjoining invasive tumors. In all normal, pure DCIS, and DCIS adjacent-to-invasive lesions, Fhit and Wwox expression was positively associated (P = 0.034, P = 0.042, P = 0.004, respectively) and in the invasive component there was a positive trend toward association (P = 0.075). Fhit and Wwox were more frequently reduced in high-grade lesions in the DCIS adjacent-to-invasive (P = 0.025, P = 0.004, respectively). In the pure DCIS group, there was a statistically significant negative association between Fhit and ErbB2 expression in DCIS (P = 0.035). In summary, reduced Fhit and Wwox expression in in-situ breast cancer was associated, which may contribute to the high-grade DCIS-invasive tumor pathway.     

SMC1 involvement in fragile site expression

Common fragile sites have been involved in neoplastic transformation, although their molecular basis is still poorly understood. Here, we demonstrate that inhibition of the SMC1 by RNAi is sufficient to induce fragile site expression. By investigating normal, ATM- and ATR-deficient cell lines, we provide evidence that the contribution of SMC1 in preventing the collapse of stalled replication fork is an Atr-dependent pathway. Using a fluorescent antibody specific for gamma-H2AX, we show that very rare discrete nuclear foci appear 1 and 2 h after exposure to aphidicolin and/or RNAi-SMC1, but became more numerous and distinct after longer treatment times. In this context, fragile sites might be viewed as an in vitro phenomenon originating from double-strand breaks formed because of a stalled DNA replication that lasted too long to be managed by physiological rescue acting through the Atr/Smc1 axis. We propose that in vivo, following an extreme replication block, rare cells could escape checkpoint mechanisms and enter mitosis with a defect in genome assembly, eventually leading to neoplastic transformation.     

Boveri at 100: Theodor Boveri and genetic predisposition to cancer

One hundred years have passed since the publication of Theodore Boveri's Zur Frage der Entstehung maligner Tumouren [Concerning the Origin of Malignant Tumours]. This prescient publication created the foundations for much of our understanding of the origins of cancer and in particular the genetic basis of some cancers. In his work, Boveri suggested that loss of key cellular attributes, now known as tumour suppressor genes, are a key driver event in the development of cancer and inheritance could play a role in cancer susceptibility. He also predicted that chromosomal (genomic) instability as a key hallmark of cancer. Whilst these key insights that still inform the practice of cancer genetics, they were not the main theme of Boveri's text, which was to describe the role of chromosomal abnormalities in the development of cancer. In making his case he also suggested that genetic information could be contained in distinct packages (genes) that are linearly arranged along chromosomes and that cancers arise from single cells. These remarkably accurate hypotheses add weight to the need to celebrate this landmark publication for its accurate prediction of so much that we take for granted. Here we focus on Boveri's contributions to our understanding of hereditary cancers, which, alongside the astute clinical observations of Paul Broca and Aldred Scott Warthin, were published decades before the field became respectable, yet could still inform anyone studying hereditary cancers. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Common fragile site instability in normal cells: Lessons and perspectives

Mechanisms and events related to common fragile site (CFS) instability are well known in cancer cells. Here, we argue that normal cells remain an important experimental model to address questions related to CFS instability in the absence of alterations in cell cycle and DNA damage repair pathways, which are common features acquired in cancer. Furthermore, a major gap of knowledge concerns the stability of CFSs during gametogenesis. CFS instability in meiotic or postmeiotic stages of the germ cell line could generate chromosome deletions or large rearrangements. This in turn can lead to the functional loss of the several CFS‐associated genes with tumor suppressor function. Our hypothesis is that such mutations can potentially result in genetic predisposition to develop cancer. Indirect evidence for CFS instability in human germ cells has been provided by genomic investigations in family pedigrees associated with genetic disease. The issue of CFS instability in the germ cell line should represent one of the future efforts, and may take advantage of the existence of sequence and functional conservation of CFSs between rodents and humans.     

Coincidence in fragile site expression with fluorodeoxyuridine and bromodeoxyuridine

Fragile site expression induced by 10 micrograms/ml or 20 micrograms/ml fluorodeoxyuridine (FudR) and 25 micrograms/ml or 50 micrograms/ml bromodeoxyuridine (BrdU) was studied in lymphocyte cultures of six healthy individuals. A significant decrease in mitotic indexes in respect to control cultures was observed with both FudR concentrations used. The cells showing chromosome aberrations and the total number of cytogenetic alterations were significantly increased both in FudR (p less than 0.001) and BrdU (25 micrograms/ml) (p less than 0.05) treated cultures with respect to the control culture. A site showing a gap or a break was defined as fragile if it appeared in 1% of the cells analyzed and in at least three of the six individuals studied with the same culture treatment. Using these criteria, fragile sites 4q31, 5q15, 6p22, 7p13, 7q32, 13q21, and 14q24 were induced in different proportions by both chemical agents. Although these drugs act via different mechanisms, they both substitute for thymidine in DNA. Our findings suggest that FudR is a more potent common fragile site inducer than BrdU.     

Common fragile site FRA11G and rare fragile site FRA11B at 11q23.3 encompass distinct genomic regions

Fragile sites are specific genomic loci that are particularly prone to chromosomal breakage. Based on their incidence in the human population, they are divided into rare fragile sites occurring in less than 5% of all individuals and common fragile sites being a constitutional feature of the genome of probably all individuals. In this study, cloning of unstable DNA sequences, which have been previously genetically tagged with a marker gene, was the basis for defining the genomic localization of the common fragile site FRA11G at 11q23.3. Mapping of the fragile site with six-color fluorescence in situ hybridization (FISH) resulted in the precise genomic localization of FRA11G to a 4.5 Mb region. The chromosomal subband 11q23.3 harbors both the common fragile site FRA11G and the rare fragile site FRA11B. Here, we show that FRA11G maps 0.8 Mb proximal to the genomic region previously defined to be affected by expression of FRA11B; thus, the common and the rare fragile sites at 11q23.3 encompass distinct genomic regions. The region of FRA11G is known to be involved in somatic and germline recurrent aberrations, and it is conceivable that genetic damage resulting from this fragile site might contribute to clinical phenotypes.     

Uptake of genetic testing for cancer predisposition.

Although there has been much debate about the uptake and effects of predictive testing for common cancers, such as breast and colon cancer, little has been published on the more classical tumour predisposing conditions, such as von Hippel-Lindau disease and familial adenomatous polyposis. Since 1990 the genetics departments in Manchester and Cambridge have had a genetic register for cancer predisposing syndromes and presymptomatic testing for these conditions has been offered once this has become possible. To investigate the factors that might influence uptake of genetic testing in familial cancer syndromes we have reviewed our experience. Demand for predictive testing has generally been high, but men had a lower uptake (77%) than a comparable group of women (93%) (p < 0.01).     

Deoxyuridine increases folate-sensitive fragile site expression in human lymphocytes

Deoxyuridine (dU) increases chromosome breakage at folic acid (FA)-sensitive common fragile sites (mostly 3p14 and 16q23) in human lymphocytes. This dU-related increase can be suppressed by thymidine or FA. These results suggest that the mechanism of fragile site expression in low FA medium involves misincorporation of dU into DNA.