Localization of BRCA1 protein in human breast cancer cells.
There is still an ongoing debate concerning the cellular localization of BRCA1 protein in breast cancer. To address this question, we compared the localization of BRCA1 protein using several monoclonal (Ab-1) or polyclonal (C20, D20, I20) antibodies under different technical conditions on human breast cancer cell lines. We worked on the fixation and permeabilization conditions in order to preserve the morphological structures of the cells, as confirmed by transmission electron microscopy studies. As expected from the gene sequence analysis and the biochemical features, both nucleus and cytoplasmic BRCA1 protein staining were detected in cells fixed for 60 min in 4% paraformaldehyde and permeabilized with either 0.3% saponin or 0.02% Triton. In these conditions, the same results were obtained: (i) with the four antibodies tested, (ii) with several dilutions (up to tenfold) of the monoclonal antibody, and (iii) in all the tested breast cancer cell lines. In addition, we validated the functionality of these conditions by quantifying the effects of estrogens and their antagonists on the regulation of BRCA1 protein expression in the MCF7 cell line.
Chambon
Monique
M
Université de Montpellier 1, Montpellier, France. chambon@bacchus.montp.inserm.fr
Nirdé
Philippe
P
Gleizes
Michel
M
Roger
Pascal
P
Vignon
Françoise
F
D000911:Antibodies, Monoclonal; D019313:BRCA1 Protein / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D002277:Carcinoma / Q000378:metabolism* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D019313:BRCA1 Protein; D004958:Estradiol