BRCA1 protein products: Antibody specificity…

Wilson, Christopher D.; Payton, Marc; Pekar, Susan K.; Zhang, Ke; Pacifici, Robert E.; Gudas, Jean L.; Thukral, S K; Calzone, Frank J.; Reese, David; Slamon, Dennis

doi:10.1038/ng0796-264

Cited by 52 publications

(37 citation statements)

References 15 publications

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“…This hypothesis was tested with a series of myc-tagged deletion mutants illustrated schematically in Figure 7 where BRCA1 and BRCA1-D11b are also represented. The hatched gray box spanning exons 2 ± 5 highlights the cysteine-rich ring ®nger motif, the two black bars in exon 11 mark the location of two putative NLS and the gray bars identify the granin motif (G) or an epitope (E) shared with the epidermal growth factor receptor (EGFR) and Her2 (Wilson et al, 1996). It should be noted that several clones depicted in Figure 7 contain amino acids substitutions relative to the GenBank sequence that represent either sequence polymorphisms or PCR ampli®cation errors as discussed in detail in Materials and methods.…”

Section: Deletion Mapping Of the Brca1 Nlsmentioning

confidence: 99%

“…However, the entire BRCA1 protein sequence is claimed to be present in both forms based on comparisons with recombinant protein produced in vitro or in vivo. We have shown that the analysis of BRCA1 subcellular localization has been complicated by the fact that a crucial antibody (C-20, Santa Cruz) crossreacts with the epidermal growth factor receptor (EGFR) and Her2 (Wilson et al, 1996) which are frequently overexpressed in human breast and ovarian cancers and malignant cell lines (Lewis et al, 1993;Earp et al, 1995). The level of EGFR expression in the MDA-MB-468 cells used to characterize BRCA1 secretion is particularly high (Wilson et al, 1996;Filmus et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

“…In this report we attempt to more fully characterize the BRCA1 proteins and address some of the controversial issues in the literature regarding this important molecule (Wilson et al, 1996;Chen et al, 1996;Scully et al, 1996;Koonin et al, 1996;Diamandis, 1996;Bradley and Sharan, 1996;Jensen et al, 1996b). We describe the isolation of an alternatively spliced BRCA1 message in which the majority of exon 11 (905 ± 4215) has been deleted.…”

Section: Introductionmentioning

confidence: 99%

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Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Δ11b

et al. 1997

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The mechanism of BRCA1 tumor suppression in human breast and ovarian cells is the focus of intense investigation. In this report, full length BRCA1 (230 kDa) introduced into cells with CMV promoter constructs was nuclear when transgene expression was low whereas high expression resulted in cytoplasmic accumulation, aberrant nuclear and cell morphology. A nuclear localization signal (NLS) was mapped to BRCA1 amino acid positions 262 ± 570. We describe a splice variant, BRCA1-D11b, missing the majority of exon 11 including the NLS. Exogenous BRCA1-D11b (110 kDa) was cytoplasmic and, unlike the full-length protein, overexpression of the protein encoded by the variant did not appear to be toxic. RNA probe titrations and RT ± PCR demonstrated that BRCA1 and D11b transcripts are coexpressed in a wide variety of cells and tissues. Interestingly, BRCA1-D11b message was greatly reduced or absent in several breast and ovarian tumor lines relative to exon 11 transcripts and a D9,10 splice variant. Taken together our results suggest that fulllength BRCA1 and BRCA1-D11b may have distinct roles in cell growth regulation and tumorigenesis.

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Section: Deletion Mapping Of the Brca1 Nlsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Δ11b

et al. 1997

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“…Controversy concerning the subcellular localisation of the BRCA1 protein has abounded, with it being reported as nuclear (Scully et al, 1996), nuclear in normal but cytoplasmic in breast and ovarian carcinoma cells , membrane associated on the cytoplasmic aspect of nuclear invaginations (Coene et al, 1997), or a secreted growth inhibitor Jensen et al, 1996). This debate has been fuelled by concerns regarding the specificity of the monoclonal antibodies available for BRCA1 detection (Wilson et al, 1996;Thakur et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies using anti-BRCA1 antibodies have described crossreactivity with various different molecules (Wilson et al, 1996). In this study, EGFR immunohistochemistry was performed in parallel with MS13 and MS110 immunohistochemistry.…”

Section: Discussionmentioning

confidence: 99%

A role for BRCA1 in sporadic breast cancer

et al. 2003

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To test the hypothesis that altered expression of BRCA1 protein may play an important role in sporadic breast cancer development, 50 randomly selected primary breast cancers (frozen sections, 5 years' median follow-up) were immunolabelled with two monoclonal BRCA1 antibodies (MS110 and MS13). MS110 labelling was exclusively nuclear showing no relation to outcome or tumour pathology. Western blotting demonstrated crossreactivity, suggesting antibody nonspecificity. MS13 labelling was predominantly cytoplasmic. Intense labelling predicted decreased overall survival (P ¼ 0.012), disease-free survival (P ¼ 0.029), oestrogen receptor negativity (P ¼ 0.0004) and c-erbB-2 overexpression (P ¼ 0.006). Western blotting detected a 110 kDa molecule consistent with BRCA1 D11b splice variant. BRCA1 protein is postulated to function as a tumour suppressor. We demonstrate cytoplasmic localisation in sporadic breast cancer suggesting excess D11b splice variant production, reduced production of full-length BRCA1 and thus postulate reduced tumour suppressor activity. BRCA1 protein appears to have a significant role in both sporadic and hereditary breast cancers.

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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

et al. 1997

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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 lineA recent publication of Wilson et al. (1996) reported on the specificity of rabbit polyclonal antibodies raised against a peptide corresponding to amino acids 1843-1862 mapping at the carboxy terminus of human BRCA 1 (C-20), commercially available from Santa Cruz Biotechnology (Santa Cruz, CA). Surprisingly, this antiserum cross-reacted with the human epidermal growth factor receptor (EGF-R) and HER 2.Here, we confirm the cross-reaction of C-20 antibodies with the human EGF-R (E 2645; Sigma, St Louis, MO) and we report cross-reaction of these antibodies with human EGF (E 1264, Sigma), using a quantitative method for determination of the BRCA 1 protein concentration in the MCF 10a human mammary epithelial cell line, previously developed in our laboratory (Bernard et al., 1985;1986).BRCA 1 proteins were quantified after internal cell protein labeling with [ 35 S]-methionine. All proteins were solubilized with a detergent (Nonidet P 40) and glycoproteins were isolated by affınity chromatography on Lentil-Lectin Sepharose 4B (Pharmacia, Uppsala, Sweden) (Fig. 1a). In this way, BRCA 1, which includes 21 predicted glycosylation sites (Jensen et al., 1996), was eluted with all cell glycoproteins. Specific immunoprecipitation was then performed with anti-BRCA 1 polyclonal antibodies (C-20). The immune complex consisting of BRCA 1 glycoproteins labeled with [ 35 S] bound to antibodies was isolated by affınity chromatography on Protein A-Sepharose CL 4B (Fig. 1b). A final quantification of BRCA 1 proteins via the immune complex was performed by chromatofocusing on PBE 9-4 gel (Pharmacia) because elution along a pH gradient 7.4 to 4 revealed one main labeled peak at pH 7.1, corresponding to immune complex elution, since the g-globulin pHi elutes between 7.3 and 6.3 (Cohn, 1948).Chromatofocusing of the immune complex BRCA 1/anti-BRCA 1 polyclonal antibodies, followed by counting of the radioactivity in the collected fractions, is shown in Figure 1c. A main labeled peak corresponding to [ 35 S]-labeled BRCA 1 proteins bound to anti-BRCA 1 polyclonal antibodies was obtained at pH 7.1 in fractions 12 to 16. In order to validate the technique, we used double-labeling, [ 131 I] for the proteins and [ 125 I] for the antibodies and we observed the main doublelabeled peak at pH 7.1 (data not shown).When the reaction between BRCA 1 proteins and the polyclonal antibodies against BRCA 1 (C-20) was performed in the presence of EGF (Fig. 1d) or EGF-R (Fig. 1e), the elution profile was similar, but the main peak at pH 7.1, meaning elution of labeled BRCA 1 glycoproteins, was markedly reduced in both cases due to cross-reaction of the antibodies with EGF or EGF-R, thus displacing the equilibrium between BRCA 1 and C-20 antibodies.The amount of BRCA 1 proteins obtained after chromatofocusing was expressed as a percentage, and corresponded to the ratio: Amount of labeled gl...

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BRCA1 protein products: Antibody specificity…

Cited by 52 publications

References 15 publications

Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Δ11b

Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Δ11b

A role for BRCA1 in sporadic breast cancer

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

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