BRCA1 Gene Expression in Breast Cancer : A Correlative Study between Real-time RT-PCR and Immunohistochemistry
Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait
Correspondence to: Dr. Fahd Al-Mulla, Department of Pathology, Molecular Pathology Laboratory, Faculty of Medicine, Kuwait University, PO Box 24923, Safat 13110, Kuwait. E-mail: fahd{at}al-mulla.org
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: BRCA1 breast cancer immunohistochemistry mRNA protein real-time RT-PCR
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are major discrepancies concerning the usefulness of various antibodies in detecting BRCA1 protein expression and localization. Different studies have generated conflicting results related to BRCA1 expression and the subcellular localization of this protein. This controversy is even more pronounced when paraffin-embedded tissues are used (Wilson et al. 1999). Thus, although some researchers conclude that the specificity of some of the BRCA1 antibodies is adequate to consider immunohistochemistry (IHC) as a valuable screening method (Yoshikawa et al. 1999
), others believe that commercially available BRCA1 antibodies lack the specificity required to identify the BRCA1 protein (Perez-Valles et al. 2001
).
BRCA1 has been claimed to be exclusively a nuclear protein in both normal and cancer cells (Scully et al. 1996; Thomas et al. 1996
; Thakur et al. 1997
; Wilson et al. 1999
), a nuclear protein in normal cells but an aberrantly localized cytoplasmic protein in breast and ovarian tumor cells (Chen et al. 1995
; Lee et al. 1999
), or a cytoplasmic protein found in tube-like structures that invaginate the nucleus (Coene et al. 1997
). The role of BRCA1 in DNA repair is becoming clearer: BRCA1 protein is involved in several DNA repair pathways and also has an effect on global genomic repair, involving transcriptional regulation of nucleotide excision repair genes (Hartman and Ford 2002
,2003
; Zhang et al. 2004
). Also, it has recently been reported that BRCA1 expression influences the choice of chemotherapeutic agents used in the treatment of breast cancer (Egawa et al. 2003
; Zhou et al. 2003
). Therefore, it is now becoming clear that the knowledge of BRCA1 expression in breast cancer has important clinical ramifications.
This study focused on BRCA1 expression status in paraffin-embedded breast cancer tissues by using RT-PCR and IHC and correlated the results of the two. Such a comparison could be useful in assessing the specificity of commercially available BRCA1 antibodies.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry
To study BRCA1 protein expression, four antibodies were applied to formalin-fixed paraffin-embedded breast cancer sections (Table 1). Breast cancer sections were deparaffinized, then rehydrated through three different concentrations of alcohol and 0.3% H2O2 for 30 min to block endogenous peroxidase. Epitope retrieval was carried out in 0.1 M citrate buffer at 95C in water bath for 20 min. Nonspecific binding was blocked using rabbit anti-mouse serum before monoclonal antibodies (AB-1, AB-8F7), and swine anti-rabbit serum before polyclonal antibodies (AB-D20, AB-C-terminus) for 30 min. After overnight incubation, biotinylated antibody (link antibody) was added, followed by streptavidin (dilution 1:500). Diaminobenzene was used as chromogen. Sections from MCF-7 breast cancer cell line paraffin blocks were used as positive control. For negative controls, the antibodies were substituted with the corresponding serum. It is worthy of note that several buffers and antigen retrieval methods were tested before this optimal protocol was achieved. These methods include microwave treatment at 90C for 10 min and at 95C for 20 min.
|
RNA Extraction and Real-time RT-PCR
Of the 48 breast cancer tissues studied using IHC, 29 samples had enough tissue for successful RNA extraction and RT-PCR amplification of GAPDH. To control for RNA degradation, GAPDH RNA had to be amplified in RNA samples extracted from breast tissue sections before BRCA1 expression in that tissue was deemed negative. Macrodissection of tumor tissue from several paraffin sections was used to minimize the influence of surrounding normal tissues. In these samples, 70% or more of the tissue sections contained tumor. The sections were rehydrated and scraped off the glass slide, using a needle, into an Eppendorf tube containing RNase inhibitor, proteinase K buffer (pH 8.3), 1% Tween surfactant, and proteinase K (10 mg/ml). The tubes were kept at 60C in water bath overnight, then mixed with 400 µl Trizol and chloroform to precipitate the RNA. There was no need to treat the samples with Dnase, because the BRCA1 probe and GAPDH primers spanned an exonexon junction. RT-PCR master mixes were prepared according to the Invitrogen ThermoScript Platinum Quantitative One-Step RT-PCR System (Invitrogen; Carlsbad, CA) BRCA1 primers (Egawa et al. 2001) (forward, 5'-ACAGCTGTGTGGTGCTTCTGTG-3', reverse 5'-CATTGTCCTCTGTCCAGGCATC-3') and BRCA1 probe labeled with FAM (Egawa et al. 2001
) (5'-CATCATTCACCCTTGGCACAGGTGT-3') were used to amplify and detect BRCA1 mRNA. GAPDH primers (forward 5'-TCATTGACCTCAACTACATGGTTT-3', reverse 5'-GAAGATGGTGATGGGATTTC-3') and GAPDH probes labeled with JOE [TaqMan] (JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA) were used as internal control for RT-PCR. Samples for no-RT and no-template were also included in each test to detect any DNA or RNA contamination. The Applied BioSystems ABI7000 real-time sequence detection system (Applied Biosystems, Foster City, CA) was used to detect amplifications. Amplification conditions were 60C for 60 min for RT, followed by 95C for 5 min, 45 PCR cycles at 95C for 15 sec, and 60C for 1 min. The cut-off value for BRCA1 mRNA negativity was when there was no specific or significant reduction in BRCA1 signals detected even after 45 cycles of amplification. Each sample was assayed in triplicate in independent reactions.
Statistical Analysis
The data were analyzed using the Statistical Package for Social Sciences (SPSS version 11.01) software. The association between BRCA1 mRNA and protein expression was analyzed using the 2 test. A p-value less than 0.05 was considered significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Polyclonal Antibody against N-terminus, AB-D20
BRCA1 protein staining with polyclonal antibody against N-terminus, AB-D20 was positive in 62.5% of cases, and staining was localized in the cytoplasm in all these cases (Figure 1C).
Polyclonal Antibody against C-terminus, AB-C-terminus
Polyclonal antibody against AB-C-terminus showed positive staining for BRCA1 protein in 33.3% of cases. In 69% of these cases, staining was localized only in the cytoplasm. Staining in the remaining 31% was localized in both the nucleus and cytoplasm (Figure 1D).
Cytoplasmic staining appeared to be the most common pattern, with or without nuclear staining, and none of the antibodies demonstrated nuclear staining only (Tables 2 and 3).
Real-time RT-PCR
Given the generally poor quality of RNA extracted from formalin-fixed paraffin-embedded tissues, several steps were implemented to ensure the accuracy of the results. First, only samples that had successful amplification of GAPDH, a housekeeping gene, were considered for subsequent BRCA1 amplifications. This step ensured that samples with only high-quality RNA were considered and represented a control for RNA degradation. Second, to ensure amplification of the less common transcript, namely, BRCA1, the number of amplification cycles was increased to 45. Third, the MCF7 cell line and a paraffin-embedded tissue known to express BRCA1 were used in all reactions as positive controls. Finally, to ensure reproducibility, three independent RT-PCR reactions were performed for each sample. Such stringent criteria reduced our analyzable tissues to 29 of the 48 for which IHC was successful. Of 29 breast cancer tissues analyzed for BRCA1 mRNA expression using real-time RT-PCR, only 6 (21%), showed detectable BRCA1 mRNA. Twenty-three breast cancer tissues (79.3%) showed no BRCA1 mRNA amplification, even after 45 cycles. The amplification curves of RNA extracted from the breast cancer MCF-7 cell line and paraffin-embedded breast cancer tissues using BRCA1 primers are demonstrated in Figure 2.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the major tasks of this study was to choose the appropriate and reliable antibody for BRCA1 protein IHC. There is a major controversy about the usefulness and specificity of different BRCA1 antibodies. This controversy is even more pronounced when paraffin-embedded tissues are used. In the present study, four anti-BRCA1 antibodies against different BRCA1 epitopes were used. The results obtained were compared to determine which antibody is most reliable for detecting BRCA1 protein on paraffin-embedded breast cancers. To evaluate the specificity and sensitivity of the four anti-BRCA1 antibodies, the RT-PCR results were considered as a standard. Our results demonstrate a significant relationship between BRCA1 mRNA and BRCA1 protein expression with only one of the antibodies, monoclonal AB-1. Almost all breast cancers that had no or significantly reduced BRCA1 mRNA expression in this study were also negative for BRCA1 protein expression with AB-1 (21/23, 91%; p=0.002). Accordingly, AB-1 has the best combined specificity (91.3%) and sensitivity (66.6%) in detecting BRCA1 protein (Table 5). In a study using four commercially available anti-BRCA1 antibodies on paraffin-embedded breast cancers (including AB-1 and AB-D20 ), Perez-Valles et al. (2001) reported that available BRCA1 antibodies lack the specificity required to identify the BRCA1 protein. Others, however, have used AB-1 for immunolocalization of BRCA1 on breast cancer and have reported that this antibody is the most reliable antibody for detecting BRCA1 protein (Lee et al. 1999
; Wilson et al. 1999
; Niwa et al. 2000
). Our data are consistent with this finding. IHC results using the other three antibodies have shown no statistically significant relationship with BRCA1 mRNA. Of all the antibodies, AB-8F7 had the best sensitivity, but its specificity was low (30.4%). Results reported by Yoshikawa et al. (1999)
suggest that N-terminus AB-1 could be useful in prescreening tumors for BRCA1 mutations because of a high detection rate (7 of 19, 37%) of alterations in the BRCA1 gene product in breast cancer. Our data are also consistent with the findings that BRCA1 loss is at mRNA level (Russell et al. 2000
; Baldassarre et al. 2003
). Interestingly, we have found that some of the breast cancers with positive BRCA1 mRNA expression had no BRCA1 protein expression with AB-1. This finding supports the hypothesis that there might be other mechanisms by which BRCA1 expression is controlled at translational or posttranslational levels (Miyamoto et al. 2002
; Sobczak and Krzyzosiak 2002
). Nevertheless, loss of both mRNA and BRCA1 protein could indicate that the genetic control of BRCA1 expression in the breast cancer analyzed is at transcriptional level.
The subcellular localization of BRCA1 has been also been controversial. BRCA1 has been claimed to be an exclusively nuclear protein in both normal and cancer cells (Scully et al. 1996; Thomas et al. 1996
; Thakur et al. 1997
; Wilson et al. 1999
), a nuclear protein in normal cells but an aberrantly localized cytoplasmic protein in breast and ovarian tumor cells (Chen et al. 1995
; Lee et al. 1999
), and a cytoplasmic protein found in tubelike structures that invaginate the nucleus (Coene et al. 1997
). The variation in the subcellular localization of BRCA1 might be attributable to several causes, including the specificity of the antibodies used to localize the protein, antibody cross-reactivity (Smith et al. 1996
; Bernard-Gallon et al. 1997
; Wilson et al. 1999
), and the presence of splice variant isoforms (Thakur et al. 1997
; Wilson et al. 1997
). The present study confirms that BRCA1 is expressed as both nuclear and cytoplasmic antigen in breast cancer tissues. Cytoplasmic staining was a consistent feature in the breast cancers positive for BRCA1, but the nuclear expression of BRCA1 protein ranged from 37% to 83% of breast cancers (depending on the type of antibody used) and was absent in a subset of them. AB-1 monoclonal N-terminus antibody, which showed a nuclear dot pattern both in normal and cancer cell lines in other studies (Scully et al. 1996
,1997
; Wilson et al. 1999
; Hsu et al. 2001
), resulted in only 16.7% positive staining for BRCA1. In 87.5% of these cases, staining was located in the cytoplasm only; in the remaining 12.5%, staining was in both the cytoplasm and nuclei. Differences in subcellular localization of the BRCA1 protein can affect the interpretation of the IHC results (Chen et al. 1995
). Wilson et al. (1997)
compared 19 BRCA1 antibodies and detected nuclear foci in both a normal breast epithelial cell line and a breast malignancyderived cell line. In accord with Wilson's study are the studies of Thomas et al. (1996)
, Ruffner and Verma (1997)
, and Zhang et al. (1997)
, which have also reported the nuclear subcellular localization of BRCA1 protein on cell lines. On the other hand, Chen et al. (1995)
have reported that BRCA1 is a nuclear protein in normal cells and becomes aberrantly localized in cytoplasm of breast and ovarian tumor cells. In the present study, none of the breast cancer tissues showed BRCA1 nuclear localization alone. It can be argued that breast cancer cell lines, not archival paraffin-embedded breast cancer tissues, were used in the majority of these studies. Subcellular localization of proteins in cell lines cannot be compared with formalin-fixed, paraffin-embedded tissue, because several factors affect the protein localization in the tissue. The staining pattern in the paraffin-embedded tissues could be altered by changes in tissue fixation conditions and antigen retrieval methods; differences in immunostaining methodology, antibody concentrations, specificity of the antibodies used to localize the protein, and antibody cross-reactions (Scully et al. 1996
; Smith et al. 1996
; Bernard-Gallon et al. 1997
; Wilson et al. 1997
,1999
); the presence of splice variant isoforms (Lu et al. 1996
; Wang et al. 1997
; Orban and Olah 2001
; Fabbro et al. 2002
); and the problems of working with preserved archival tissue (Scully et al. 1996
; Thomas et al. 1996
; Bernard-Gallon et al. 1997
; Wilson et al. 1999
). According to Yoshikawa et al. (1999)
, results obtained by using C-terminus antibody should be evaluated carefully because of false-positive immunostaining demonstrated. BRCA1 protein localization is a complex issue, but the antibodies used in this study were chosen against different BRCA1 protein epitopes (N-terminus, exon 11, and C-terminus), which enabled us to recognize different protein isoforms. In addition, the real-time probe used for BRCA1 detects all known mRNA variants (because it hybridizes to the junction of exon 22exon 23).
In conclusion, the clinical benefits of establishing BRCA1 expression status and its effects on breast cancer treatment, prophylaxis, and prognosis are obvious (Lafarge et al. 2001; Egawa et al. 2003
; Zhou et al. 2003
). Thus, IHC can be a valuable preliminary test for detecting the reduction in BRCA1 protein expression. Among the different available BRCA1 antibodies, we consider AB-1 to be the best anti-BRCA1 antibody that can be applied on formalin-fixed paraffin-embedded tissues, with 91.3% specificity and 66.6% sensitivity. Nevertheless, AB-8F7 detected six of six tumors positive for BRCA1 mRNA, making it highly sensitive. The antibody, however, also produced signals in another 16 tumors that were negative for BRCA1 mRNA, which indicates low specificity if one considers, as we did, RT-PCR to be the reference standard. We acknowledge that the small number of positive RT-PCR values constitutes a limitation of the study. A further, larger study focusing on identifying various BRCA1 splice variants and their correlation with a range of antibodies raised against a larger number of epitopes is now warranted.
![]() |
Acknowledgments |
---|
We wish to thank Dr. Josley George, Dr. Shirley George, Mrs. Bency John, and Mrs. Tessy Saji for their technical support.
![]() |
Footnotes |
---|
Received for publication October 5, 2004; accepted December 9, 2004
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baldassarre G, Battista S, Belletti B, Thakur S, Pentimalli F, Trapasso F, Fedele M, et al. (2003) Negative regulation of BRCA1 gene expression by HMGA1 proteins accounts for the reduced BRCA1 protein levels in sporadic breast carcinoma. Mol Cell Biol 23:22252238
Bernard-Gallon DJ, Crespin NC, Maurizis JC, Bignon YJ (1997) Cross-reaction between antibodies raised against the last 20 C-terminal amino acids of BRCA 1 (C-20) and human EGF and EGF-R in MCF 10a human mammary epithelial cell line. Int J Cancer 71:123126[CrossRef][Medline]
Chen CF, Li S, Chen Y, Chen PL, Sharp ZD, Lee WH (1996) The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor. J Biol Chem 271:3286332868
Chen Y, Chen CF, Riley DJ, Allred DC, Chen PL, Von Hoff D, Osborne CK, et al. (1995) Aberrant subcellular localization of BRCA1 in breast cancer. Science 270:789791[Abstract]
Coene E, Van Oostveldt P, Willems K, van Emmelo J, De Potter CR (1997) BRCA1 is localized in cytoplasmic tube-like invaginations in the nucleus. Nat Genet 16:122124[CrossRef][Medline]
Dalton LW, Page DL, Dupont WD (1994) Histologic grading of breast carcinoma. A reproducibility study. Cancer 73:27652770[Medline]
Dobrovic A, Simpfendorfer D (1997) Methylation of the BRCA1 gene in sporadic breast cancer. Cancer Res 57:33473350[Abstract]
Easton DF, Ford D, Bishop DT (1995) Breast and ovarian cancer incidence in BRCA1-mutation carriers. Breast Cancer Linkage Consortium. Am J Hum Genet 56:265271
Egawa C, Miyoshi Y, Taguchi T, Tamaki Y, Noguchi S (2001) Quantitative analysis of BRCA1 and BRCA2 mRNA expression in sporadic breast carcinomas and its relationship with clinicopathological characteristics. Jpn J Cancer Res 92:624630[Medline]
Egawa C, Motomura K, Miyoshi Y, Takamura Y, Taguchi T, Tamaki Y, Inaji H, et al. (2003) Increased expression of BRCA1 mRNA predicts favorable response to anthracycline-containing chemotherapy in breast cancers. Breast Cancer Res Treat 78:4550[CrossRef][Medline]
Fabbro M, Rodriguez JA, Baer R, Henderson BR (2002) BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export. J Biol Chem 277:2131521324
Futreal PA, Liu Q, Shattuck-Eidens D, Cochran C, Harshman K, Tavtigian S, Bennett LM, et al. (1994) BRCA1 mutations in primary breast and ovarian carcinomas. Science 266:120122.[Medline]
Hartman AR, Ford JM (2002) BRCA1 induces DNA damage recognition factors and enhances nucleotide excision repair. Nat Genet 32:180184[CrossRef][Medline]
Hartman AR, Ford JM (2003) BRCA1 and p53: compensatory roles in DNA repair. J Mol Med 81:700707[CrossRef][Medline]
Hill AD, Doyle JM, McDermott EW, O'Higgins NJ (1997) Hereditary breast cancer. Br J Surg 84:13341339[CrossRef][Medline]
Hsu LC, Doan TP, White RL (2001) Identification of a gamma-tubulin-binding domain in BRCA1. Cancer Res 61:77137718
Jacquemier J, Eisinger F, Birnbaum D, Sobol H (1995) Histoprognostic grade in BRCA1-associated breast cancer. Lancet 345:1503[Medline]
Khoo US, Ozcelik H, Cheung AN, Chow LW, Ngan HY, Done SJ, Liang AC, et al. (1999) Somatic mutations in the BRCA1 gene in Chinese sporadic breast and ovarian cancer. Oncogene 18:46434646[CrossRef][Medline]
Lafarge S, Sylvain V, Ferrara M, Bignon YJ (2001) Inhibition of BRCA1 leads to increased chemoresistance to microtubule-interfering agents, an effect that involves the JNK pathway. Oncogene 20:65976606[CrossRef][Medline]
Lakhani SR, Gusterson BA, Jacquemier J, Sloane JP, Anderson TJ, van de Vijver MJ, Venter D, et al. (2000) The pathology of familial breast cancer: histological features of cancers in families not attributable to mutations in BRCA1 or BRCA2. Clin Cancer Res 6:782789
Lee WY, Jin YT, Chang TW, Lin PW, Su IJ (1999) Immunolocalization of BRCA1 protein in normal breast tissue and sporadic invasive ductal carcinomas: a correlation with other biological parameters. Histopathology 34:106112[CrossRef][Medline]
Lu M, Conzen SD, Cole CN, Arrick BA (1996) Characterization of functional messenger RNA splice variants of BRCA1 expressed in nonmalignant and tumor-derived breast cells. Cancer Res 56:45784581[Abstract]
Lynch HT, Lynch J, Conway T, Watson P, Feunteun J, Lenoir G, Narod S, et al. (1994) Hereditary breast cancer and family cancer syndromes. World J Surg 18:2131[CrossRef][Medline]
Mancini DN, Rodenhiser DI, Ainsworth PJ, O'Malley FP, Singh SM, Xing W, Archer TK (1998) CpG methylation within the 5' regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site. Oncogene 16:11611169[CrossRef][Medline]
Merajver SD, Pham TM, Caduff RF, Chen M, Poy EL, Cooney KA, Weber BL, et al. (1995) Somatic mutations in the BRCA1 gene in sporadic ovarian tumours. Nat Genet 9:439443[CrossRef][Medline]
Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, et al. (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266:6671[Medline]
Miyamoto K, Fukutomi T, Asada K, Wakazono K, Tsuda H, Asahara T, Sugimura T, et al. (2002) Promoter hypermethylation and post-transcriptional mechanisms for reduced BRCA1 immunoreactivity in sporadic human breast cancers. Jpn J Clin Oncol 32:7984
Niwa Y, Oyama T, Nakajima T (2000) BRCA1 expression status in relation to DNA methylation of the BRCA1 promoter region in sporadic breast cancers. Jpn J Cancer Res 91:519526[Medline]
Orban TI, Olah E (2001) Expression profiles of BRCA1 splice variants in asynchronous and in G1/S synchronized tumor cell lines. Biochem Biophys Res Commun 280:3238[CrossRef][Medline]
Perez-Valles A, Martorell-Cebollada M, Nogueira-Vazquez E, Garcia-Garcia JA, Fuster-Diana E (2001) The usefulness of antibodies to the BRCA1 protein in detecting the mutated BRCA1 gene. An immunohistochemical study. J Clin Pathol 54:476480
Rio PG, Maurizis JC, Peffault de Latour M, Bignon YJ, Bernard-Gallon DJ (1999) Quantification of BRCA1 protein in sporadic breast carcinoma with or without loss of heterozygosity of the BRCA1 gene. Int J Cancer 80:823826[CrossRef][Medline]
Ruffner H, Verma IM (1997) BRCA1 is a cell cycle-regulated nuclear phosphoprotein. Proc Natl Acad Sci USA 94:71387143
Russell PA, Pharoah PD, De Foy K, Ramus SJ, Symmonds I, Wilson A, Scott I, et al. (2000) Frequent loss of BRCA1 mRNA and protein expression in sporadic ovarian cancers. Int J Cancer 87:317321[CrossRef][Medline]
Scully R, Chen J, Ochs RL, Keegan K, Hoekstra M, Feunteun J, Livingston DM (1997) Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90:425435[CrossRef][Medline]
Scully R, Ganesan S, Brown M, De Caprio JA, Cannistra SA, Feunteun J, Schnitt S, et al. (1996) Location of BRCA1 in human breast and ovarian cancer cells. Science 272:123126[Medline]
Shen SX, Weaver Z, Xu X, Li C, Weinstein M, Chen L, Guan XY, et al. (1998) A targeted disruption of the murine Brca1 gene causes gamma-irradiation hypersensitivity and genetic instability. Oncogene 17:31153124[CrossRef][Medline]
Smith TM, Lee MK, Szabo CI, Jerome N, McEuen M, Taylor M, Hood L, et al. (1996) Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1. Genome Res 6:10291049[Abstract]
Sobczak K, Krzyzosiak WJ (2002) Structural determinants of BRCA1 translational regulation. J Biol Chem 277:1734917358
Sourvinos G, Spandidos DA (1998) Decreased BRCA1 expression levels may arrest the cell cycle through activation of p53 checkpoint in human sporadic breast tumors. Biochem Biophys Res Commun 245:7580[CrossRef][Medline]
Thakur S, Zhang HB, Peng Y, Le H, Carroll B, Ward T, Yao J, et al. (1997) Localization of BRCA1 and a splice variant identifies the nuclear localization signal. Mol Cell Biol 17:444452[Abstract]
Thomas JE, Smith M, Rubinfeld B, Gutowski M, Beckmann RP, Polakis P (1996) Subcellular localization and analysis of apparent 180-kDa and 220-kDa proteins of the breast cancer susceptibility gene, BRCA1. J Biol Chem 271:2863028635
Thompson ME, Jensen RA, Obermiller PS, Page DL, Holt JT (1995) Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression. Nat Genet 9:444450[CrossRef][Medline]
Vaughn JP, Davis PL, Jarboe MD, Huper G, Evans AC, Wiseman RW, Berchuck A, et al. (1996) BRCA1 expression is induced before DNA synthesis in both normal and tumor-derived breast cells. Cell Growth Differ 7:711715[Abstract]
Vissac C, Peffault De Latour M, Communal Y, Bignon YJ, Bernard-Gallon DJ (2002) Expression of BRCA1 and BRCA2 in different tumor cell lines with various growth status. Clin Chim Acta 320:101110[CrossRef][Medline]
Wang H, Shao N, Ding QM, Cui J, Reddy ES, Rao VN (1997) BRCA1 proteins are transported to the nucleus in the absence of serum and splice variants BRCA1a, BRCA1b are tyrosine phosphoproteins that associate with E2F, cyclins and cyclin dependent kinases. Oncogene 15:143157[CrossRef][Medline]
Wilson CA, Payton MN, Elliott GS, Buaas FW, Cajulis EE, Grosshans D, Ramos L, et al. (1997) Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-delta11b. Oncogene 14:116[CrossRef][Medline]
Wilson CA, Ramos L, Villasenor MR, Anders KH, Press MF, Clarke K, Karlan B, et al. (1999) Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas. Nat Genet 21:236240[CrossRef][Medline]
Yoshikawa K, Honda K, Inamoto T, Shinohara H, Yamauchi A, Suga K, Okuyama T, et al. (1999) Reduction of BRCA1 protein expression in Japanese sporadic breast carcinomas and its frequent loss in BRCA1-associated cases. Clin Cancer Res 5:12491261
Zhang HT, Zhang X, Zhao HZ, Kajino Y, Weber BL, Davis JG, Wang Q, et al. (1997) Relationship of p215BRCA1 to tyrosine kinase signaling pathways and the cell cycle in normal and transformed cells. Oncogene 14:28632869[CrossRef][Medline]
Zhang J, Willers H, Feng Z, Ghosh JC, Kim S, Weaver DT, Chung JH, et al. (2004) Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair. Mol Cell Biol 24:708718
Zhou C, Smith JL, Liu J (2003) Role of BRCA1 in cellular resistance to paclitaxel and ionizing radiation in an ovarian cancer cell line carrying a defective BRCA1. Oncogene 22:23962404[CrossRef][Medline]