Preferential allelic expression can lead to reduced expression of BRCAI in sporadic breast cancers

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
International Journal of Cancer (Impact Factor: 5.09). 08/1998; 77(1):1-6. DOI: 10.1002/(SICI)1097-0215(19980703)77:1<1::AID-IJC1>3.0.CO;2-Y
Source: PubMed


BRCA1 is considered to be a tumor-suppressor gene, yet mutations in this gene are uncommon in sporadic breast tumors. We investigated whether mechanisms other than DNA mutations that affect the coding region might be involved in breast carcinogenesis. Since loss of expression of the BRCA1 gene would lead to lack of protein, we evaluated the level of BRCA1 mRNA in 21 normal epithelial specimens and in 74 breast carcinomas using quantitative reverse-transcription-polymerase-chain-reaction (RT-PCR). All normal breast epithelial samples expressed BRCA1 mRNA. On the other hand, the tumor specimens exhibited approximately 10-fold range of levels of BRCA1, with some specimens expressing barely detectable amounts of BRCA1 mRNA. The distribution in levels was significantly higher in normal breast epithelial cells than in tumor specimens (p = 0.004). Examination of the BRCA1 locus indicated that deletion of the BRCA1 gene may account for low levels of BRCA1 in a number of specimens. In addition, analysis of samples with relatively reduced levels of BRCA1 expression revealed preferential allele-specific expression in a number of cases, suggesting the presence of regulatory mutations. Our data suggest that the BRCA1 gene may be involved in sporadic breast carcinogenesis through a reduction in gene expression.

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    • "The breast cancer susceptibility gene, BRCA1, is involved in the development of a significant proportion of familial breast and ovarian cancers and may also play a role in the development of sporadic breast cancer [1,2]. The BRCA1 protein has an amino terminal RING finger domain, potentially involved in ubiquitination, and two BRCA1 C-terminal domains (BRCT), which interact with a number of proteins involved in the transactivation of genes. "
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    ABSTRACT: The breast cancer susceptibility gene, BRCA1, is implicated in multiple cellular processes including DNA repair, the transactivation of genes, and the ubiquitination of proteins; however its precise functions remain to be fully understood. Identification and characterization of BRCA1 protein interactions may help to further elucidate the function and regulation of BRCA1. Additionally, detection of changes in the expression levels of BRCA1 and its interacting proteins in primary human breast tumors may further illuminate their role in the development of breast cancer. We performed a yeast two-hybrid study to identify proteins that interact with exon11 of BRCA1 and identified Protein Phosphatase 1beta (PP1beta), an isoform of the serine threonine phosphatase, PP1. GST-pull down and co-immunoprecipitation assays were performed to further characterize this interaction. Additionally, Real-Time PCR was utilized to determine the expression of BRCA1, PP1alpha, beta and gamma in primary human breast tumors and normal breast tissue to identify alterations in the expression of these genes in breast cancer. PP1 and BRCA1 co-immunoprecipitate and the region within BRCA1 as well as the specific PP1 interacting domain mediating this interaction were identified. Following mRNA expression analysis, we identified low levels of BRCA1 and variable levels of PP1alpha and beta in primary sporadic human breast tumors. Furthermore, BRCA1, PP1beta and PP1gamma were significantly higher in normal tissue specimens (BRCA1 p = 0.01, PP1beta: p = 0.03, PP1gamma, p = 1.9 x 10(-6)) compared to sporadic breast tumor samples. Interestingly, we also identified that ER negative tumors are associated with low levels of PP1alpha expression. The identification and characterization of the interaction of BRCA1 with PP1 and detection of changes in the expression of PP1 and genes encoding other BRCA1 associated proteins identifies important genetic pathways that may be significant to breast tumorigenesis. Alterations in the expression of genes, particularly phosphatases that operate in association with BRCA1, could negatively affect the function of BRCA1 or BRCA1 associated proteins, contributing to the development of breast cancer.
    Full-text · Article · Feb 2007 · BMC Cancer
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    • "Germline mutations within the BRCA1 gene are responsible for familial cancer and reduced expression of the BRCA1 gene is frequently observed in sporadic breast [12,16,20] and ovarian tumours [23]. Various mechanisms such as methylation of the CpG islands within the promoter region [2,6,10,13], allelic deletion of the BRCA1 locus and sequence alterations identified outside the BRCA1 coding region, especially within the positive regulatory region (PRR) of the BRCA1 promoter, can modulate the level of BRCA1 expression [17,19]. "
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    ABSTRACT: The 5' region of BRCA1 contains multiple regulatory sequences flanking the two alternative promoters alpha and beta and two alternative, non-coding exons, 1a and 1b. Aberrations within the 5' region BRCA1 (encompassing two alternative promoters alpha and beta and exons 1a and 1b) may be associated with an increased risk of breast and ovarian cancer. In this study we screened 150 patients for polymorphism and mutations in this region of BRCA1. All probands came from familial breast and/or ovarian cancer that had been found to be mutation-negative in a previous search for founder mutations in BRCA1 (185delAG, C61G, 4153delA, 5382insC) or BRCA2 (6174delT, 9631delC). In our study we found several sequence alterations within the non-coding region of BRCA1 by using direct DNA sequencing and allele-specific PCR amplification. Three families with a polymorphic deletion in BRCA1 exon 1b (2223delAAAAA, Acc. U37574) were found. Moreover, two linked nucleotide substitutions (2642A>T, 2743T>C, Acc. U37574) in BRCA1 intron 1 were detected in 16 patients. In order to assess the functional significance of these two sequence variants, we constructed a reporter vector encoding firefly luciferase under the transcriptional and translational control of wild type and altered BRCA1 promoter region. The reporter assay was performed using a lung cancer cell line (NCI-H1299) and a breast cancer cell line (MCF7). We have demonstrated that the analysed sequence variants have no functional significance in our experimental system. However, we have found that the BRCA1 promoter has lower relative activity in the breast cancer cell line compared with the lung cancer cell line. Based on the results of our functional experiments we conclude that the polymorphic deletion 2223delAAAAA and two linked substitutions 2642A>T and 2743T>C do not significantly alter BRCA1 expression and are probably not disease-causing mutations.
    Full-text · Article · Jan 2006 · Hereditary Cancer in Clinical Practice
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    • "For this reason, in order to avoid contamination, extreme precautions were taken for the handling of the BRCA1 amplicon as in the case of a nested PCR methodology. cDNA was synthesized from total RNA extracted from MCF-7 cells and then amplified for BRCA1 by conventional PCR with primers and the cycling program described before [17] in a PTC-200 DNA Engine cycler (MJ Research, USA). The primers selected (B1-F and B-4R) are located in different exons (12 and 13, respectively) so they are targeting solely cDNA. "
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    ABSTRACT: To develop a real-time quantitative RT-PCR method for BRCA1 mRNA and then use it for the study of BRCA1 gene expression in human MCF-7 breast cancer cells after their exposure to antineoplastic agents and gamma irradiation. The developed QRT-PCR method is based on the real-time monitoring of a fluorescein-labeled TaqMan probe, specific for BRCA1 mRNA, during PCR in the LightCycler. A BRCA1 PCR amplicon was purified, quantitated and used as a standard of known concentration for the development and analytical evaluation of the assay. The method was applied to study the alteration of BRCA1 gene expression after exposure to taxol, doxorubicin, 5-fluorouracil, etoposide or gamma irradiation in human MCF-7 breast cancer cells. The developed method is quantitative, highly specific for mRNA and highly sensitive (detection limit of 4 BRCA1 copies per mug of total RNA). We observed a reduction of BRCA1 expression for all antineoplastic agents used, while the gamma irradiated MCF-7 cells had an increase of expression with a peak at the 10 Gy dose. The developed BRCA1 QRT-PCR method is quantitative, highly sensitive and specific. The proposed method is rapid, automated, and cost effective and can be used to study BRCA1 expression in a variety of clinical samples.
    Full-text · Article · Feb 2005 · Clinical Biochemistry
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