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ABSTRACT: In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/2012; 729(1-2):90-9. · 2.85 Impact Factor
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ABSTRACT: Tetraploidy and aneuploidy can be caused by cell division errors and are frequently observed in many human carcinomas. We have recently reported delayed cytokinesis in primary human fibroblasts from BRCA2 mutation carriers, implying a function for the BRCA2 tumour suppressor in completion of cell division. Here, we address ploidy aberrations in breast tumours derived from BRCA2 germline mutation carriers. Ploidy aberrations were evaluated from flow cytometry histograms on selected breast tumour samples (n=236), previously screened for local BRCA mutations. The ploidy between BRCA2-mutated (n=71) and matched sporadic (n=165) cancers was compared. Differences in ploidy distribution were examined with respect to molecular tumour subtypes, previously defined by immunohistochemistry on tissue microarray sections. Tetraploidy was significantly 3 times more common in BRCA2 breast cancers than sporadic. However, no differences were found in the overall ploidy distribution between BRCA2-mutation carriers and non-carriers. In BRCA2 cancers, tetraploidy was associated with luminal characteristics. The increased frequency of tetraploidy in BRCA2 associated cancers may be linked to cell division errors, particularly cytokinesis. Additionally, tetraploidy emerges predominantly in BRCA2 breast cancers displaying luminal rather than triple-negative phenotypes.
European journal of cancer (Oxford, England: 1990) 11/2011; 48(3):305-10. · 4.12 Impact Factor
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ABSTRACT: Inherited mutations in the BRCA2 gene greatly increase the risk of developing breast cancer. Consistent with an important role for BRCA2 in error-free DNA repair, complex genomic changes are frequently observed in tumors derived from BRCA2 mutation carriers. Here, we explore the impact of DNA copy-number changes in BRCA2 tumors with respect to phenotype and clinical staging of the disease.
Breast tumors (n = 33) derived from BRCA2 999del5 mutation carriers were examined in terms of copy-number changes with high-resolution aCGH (array comparative genomic hybridization) containing 385 thousand probes (about one for each 7 kbp) and expression of phenotypic markers on TMAs (tissue microarrays). The data were examined with respect to clinical parameters including TNM staging, histologic grade, S phase, and ploidy.
Tumors from BRCA2 carriers of luminal and basal/triple-negative phenotypes (TNPs) differ with respect to patterns of DNA copy-number changes. The basal/TNP subtype was characterized by lack of pRb (RB1) coupled with high/intense expression of p16 (CDKN2A) gene products. We found increased proportions of Ki-67-positive cells to be significantly associated with loss of the wild-type (wt) BRCA2 allele in luminal types, whereas BRCA2wt loss was less frequent in BRCA2 tumors displaying basal/TNP phenotypes. Furthermore, we show that deletions at 13q13.1, involving the BRCA2wt allele, represents a part of a larger network of co-occurring genetic changes, including deletions at 6q22.32-q22.33, 11q14.2-q24.1, and gains at 17q24.1. Importantly, copy-number changes at these BRCA2-linked networking regions coincide with those associated with advanced progression, involving the capacity to metastasize to the nodes or more-distant sites at diagnosis.
The results presented here demonstrate divergent paths of tumor evolution in BRCA2 carriers and that deletion of the wild-type BRCA2 allele, together with co-occurring changes at 6 q, 11 q, and 17 q, are important events in progression toward advanced disease.
Breast cancer research: BCR 09/2011; 13(5):R95. · 5.24 Impact Factor
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Ferdinandos Skoulidis,
Liam D Cassidy,
Venkat Pisupati,
Jon G Jonasson,
Hordur Bjarnason, Jorunn E Eyfjord,
Florian A Karreth,
Michael Lim,
Lorraine M Barber,
Susan A Clatworthy,
Susan E Davies,
Kenneth P Olive,
David A Tuveson,
Ashok R Venkitaraman
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ABSTRACT: Inherited heterozygous BRCA2 mutations predispose carriers to tissue-specific cancers, but somatic deletion of the wild-type allele is considered essential for carcinogenesis. We find in a murine model of familial pancreatic cancer that germline heterozygosity for a pathogenic Brca2 truncation suffices to promote pancreatic ductal adenocarcinomas (PDACs) driven by Kras(G12D), irrespective of Trp53 status. Unexpectedly, tumor cells retain a functional Brca2 allele. Correspondingly, three out of four PDACs from patients inheriting BRCA2(999del5) did not exhibit loss-of-heterozygosity (LOH). Three tumors from these patients displaying LOH were acinar carcinomas, which also developed only in mice with biallelic Brca2 inactivation. We suggest a revised model for tumor suppression by BRCA2 with implications for the therapeutic strategy targeting BRCA2 mutant cancer cells.
Cancer cell 11/2010; 18(5):499-509. · 25.29 Impact Factor
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Cancer Epidemiology Biomarkers & Prevention 12/2007; 16(11):2517. · 4.12 Impact Factor
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ABSTRACT: In this study 759 breast cancer patients, including 9 BRCA1 and 98 BRCA2 mutation carriers, and 653 mutation-negative unaffected controls were genotyped for the AURKA 91T -->A polymorphism. Individuals homozygous for the 91A allele were found to be at increased risk of breast cancer compared to 91T homozygotes (OR=1.87; 95% CI=1.09-3.21). This association was strengthened when cases carrying BRCA mutations were excluded (OR=2.00; 95% CI=1.15-3.47). BRCA carrier cases differed from sporadic cases and their allele distribution was very similar to controls. These results show a statistically significant increased risk of sporadic breast cancer for individuals that are homozygous for the 91A allele but no effect in carriers of BRCA mutations. This may throw light on previously conflicting results.
Cancer Letters 06/2007; 250(2):206-12. · 4.24 Impact Factor
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ABSTRACT: Potential interaction of Aurora-A amplification and BRCA2 mutation was examined in breast tumours from BRCA2 999del5 mutation carriers (n=20) and non-carriers (n=41). Aurora-A amplification studied by FISH was significantly more common in breast tumours from BRCA2 mutation carriers (p=0.0005). Extensive Aurora-A amplification was also detected on metaphase chromosomes in three breast epithelial cell lines with the same BRCA2 mutation. In addition, significant association was found between Aurora-A amplification and TP53 mutations in non-BRCA2 mutation carrier tumours (p=0.007). These results suggest that breast tumours with mutations in BRCA2 or TP53 could be promising candidates for Aurora-A targeted treatment.
Cancer Letters 05/2007; 248(1):96-102. · 4.24 Impact Factor
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ABSTRACT: Lipid peroxidation of polyunsaturated fatty acids (PUFA) generates reactive products that may cause DNA damage. To examine the possible relationship between DNA damage in peripheral blood mononuclear cells (PBMC) and the concentration of PUFA in red blood cells (RBC), endogenous DNA strand breaks, formamidopyrimidine DNA glycosylase (FPG) sites, and hydrogen peroxide (H2O2) sensitive sites were evaluated by the comet assay in blood samples from 98 Icelandic women. Fatty acid composition of RBC was analyzed by gas chromatography. Endogenous DNA strand breaks in PBMC correlated positively with the concentration of total PUFA, total n-3 PUFA, docosahexaenoic acid, linoleic acid, oleic acid, and palmitic acid in RBC. However, there was no association between FPG sites or H(2)O(2) sensitive sites in DNA in PBMC and the concentration of total PUFA or total saturated fatty acid in RBC. As there was no association between oxidative DNA damage or sensitivity of DNA to oxidative stress and the concentration of PUFA in RBC, the positive association between endogenous DNA strand breaks in PBMC and the concentration of total PUFA in RBC is probably not related to oxidative stress.
Nutrition and Cancer 02/2007; 59(1):21-8. · 2.78 Impact Factor
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ABSTRACT: BRCA1 or BRCA2 germline mutations increase the risk of developing breast cancer. Tumour cells from germline mutation carriers have frequently lost the wild-type allele. This is predicted to result in genomic instability where cell survival depends upon dysfunctional checkpoint mechanisms. Tumorigenic potential could then be acquired through further genomic alterations. Surprisingly, somatic BRCA mutations are not found in sporadic breast tumours. BRCA1 methylation has been shown to occur in sporadic breast tumours and to be associated with reduced gene expression. We examined the frequency of BRCA1 methylation in 143 primary sporadic breast tumours along with BRCA1 copy number alterations and tumour phenotype.
Primary sporadic breast tumours were analysed for BRCA1alpha promoter methylation by methylation specific PCR and for allelic imbalance (AI) at BRCA1 and BRCA2 loci by microsatellite analysis and TP53 (also known as p53) mutations by constant denaturing gel electrophoresis. The BRCA1 methylated tumours were analysed for BRCA1 copy alterations by fluorescence in situ hybridisation and BRCA1 expression by immunostaining.
BRCA1 methylation was found in 13/143 (9.1%) sporadic breast tumours. The BRCA1 methylated tumours were significantly associated with estrogen receptor (ER) negativity (P = 0.0475) and displayed a trend for BRCA1 AI (P = 0.0731) as well as young-age at diagnosis (< or = 55; P = 0.0898). BRCA1 methylation was not associated with BRCA2 AI (P = 0.5420), although a significant association was found between BRCA1 AI and BRCA2 AI (P < 0.0001).Absent/markedly reduced BRCA1 expression was observed in 9/13 BRCA1 methylated tumours, most of which had BRCA1 deletion. An elevated TP53 mutation frequency was found among BRCA1 methylated tumours (38.5%) compared with non-methylated tumours (17.2%). The BRCA1 methylated tumours were mainly of tumour grade 3 (7/13) and infiltrating ductal type (12/13). Only one methylated tumour was of grade 1.
BRCA1 methylation is frequent in primary sporadic breast tumours. We found an indication for BRCA1 methylation to be associated with AI at the BRCA1 locus. Almost all BRCA1 methylated tumours with absent/markedly reduced BRCA1 expression (8/9) displayed BRCA1 deletion. Thus, epigenetic silencing and deletion of the BRCA1 gene might serve as Knudson's two 'hits' in sporadic breast tumorigenesis. We observed phenotypic similarities between BRCA1 methylated and familial BRCA1 tumours, based on BRCA1 deletion, TP53 mutations, ER status, young age at diagnosis and tumour grade.
Breast cancer research: BCR 01/2006; 8(4):R38. · 5.24 Impact Factor
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ABSTRACT: A founder mutation in the BRCA2 gene (BRCA2 999del5) accounts for 7-8% of female breast cancers and for 40% of male breast cancers in Iceland. If expressed, the mutant gene would encode a protein consisting of the first 256 amino acids of the BRCA2 protein. The purpose of this study was to determine whether this mutant protein is produced in heterozygous individuals and, if so, what might be the functional consequences of mutant protein production.
The presence of BRCA2 999del5 transcripts in fibroblasts from heterozygous individuals was assayed by cDNA synthesis and sequencing. The potential protein-coding portion of BRCA2 999del5 was cloned into the pIND(SP1)/V5-His vector and expressed in COS7 cells. The presence of the mutant protein in cell lysates from heterozygous fibroblasts and from COS7 cells was tested by a number of methods including immunoprecipitation, affinity purification with nickel-coated agarose beads, Western blotting and ELISA, using antibodies to the N-terminal end of BRCA2, antiserum specific for the 16 nonrelevant amino acids at the carboxyl end and antibodies to fusion partners of recombinant proteins.
The frequency of the BRCA2 999del5 transcript in heterozygous fibroblasts was about one-fifth of the wild-type transcript; however, no mutant protein could be detected. Overexpression of BRCA2 999del5 mRNA in COS7 cells failed to produce a mutant protein unless degradation by proteasomes was blocked.
Our results show that the protein product of BRCA2 999del5 is extremely unstable. Therefore, an increase in breast cancer risk in BRCA2 999del5 carriers is due to haploinsufficiency at the BRCA2 locus.
Breast cancer research: BCR 02/2004; 6(4):R284-90. · 5.24 Impact Factor
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ABSTRACT: Germline mutations in the BRCA genes dramatically increase the risk of breast cancer. In the general population, breast cancer risk is affected by age at menarche, by age at first birth, by the number of births and by the duration of breast feeding. Whether this is true for mutation carriers is not clear.
In a case-control study, nested in a population-based cohort of the Icelandic Cancer Detection Clinic, two groups of cases were defined, matched on year of birth, on age at diagnosis and on age when giving information on reproductive factors: 100 carriers of the Icelandic founder BRCA2 mutation 999del5, and 361 BRCA2-negative cases. The mean age at diagnosis was 48 years. There were 1000 women in a matched group of unaffected controls. Conditional logistic regression was used for the analysis.
An increased number of births was associated with a decreased risk of breast cancer in BRCA2-negative cases but not in BRCA2-positive cases. A negative association between risk and duration of breast feeding was observed only in the mutation carriers. These associations were not statistically significant, but the effects of the two variables differed significantly according to mutation status (P = 0.007 and P = 0.045 for interaction with number of births and with duration of breast feeding, respectively). This was maintained when limiting the analysis to women diagnosed older than the age of 40 years.
The association between breast cancer and the number of pregnancies and between breast cancer and the duration of breast feeding was not the same for carriers and noncarriers of a detrimental BRCA2 mutation. In the context of other epidemiological and laboratory studies, this may indicate that the product of the BRCA2 gene has a function relating to the differentiation of epithelial tissue in the breast.
Breast cancer research: BCR 02/2003; 5(5):R121-8. · 5.24 Impact Factor
