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[Western blot analysis and flow cytometric analysis of estrogen and progesterone receptors in fixed breast cancer cells]. 2004

De-Ding Tao, and Min Jiang, and Jian-Hong Wu, and Yong-Dong Feng, and Jian-Ping Gong
Molecular Medicine Center, Tongji Hospital Affiliated Tongji Medical College in Huazhong University of Science and Technology, Wuhan, Hubei, 430030, PR China.

OBJECTIVE Quantitative and qualitative detection of estrogen receptor (ER) and progesterone receptor (PR) levels are very important for predicting prognosis and evaluating the outcome of endocrine therapy of breast cancer patients. Western blot analysis and Flow cytometry (FCM) are important means for quantitative and qualitative analysis of proteins, but conventional Western blot analysis need to extract proteins from fresh cells. The present study was designed to establish Western blot analysis of human estrogen receptor (ER) and progesterone receptor (PR) in fixed breast cancer cells, and to explore the possibility of ER and PR analysis in fixed cells by flow cytometry and Western blot analysis simultaneously. METHODS Proteins extracted from fresh and fixed cells of different exponentially growing breast cancer cell lines were labelled using 1D5 and PgR636, which were monoantibodies to ERalpha and PR, respectively. The expression of ER and PR were determined using Western blot analysis, and the results were compared with that of fixed cells measured with flow cytometry. RESULTS Clear and correct ERalpha bands were observed with Western blot analysis in cell lines T-47d, MCF-7, and ZR-75-1, and the band density of fixed T-47d and ZR-75-1 was higher than that of fresh cells. The expression of ERalpha in MM231 cells was negative. Clear and correct PR bands were visible in T-47d and ZR-75-1 cells with Western blot analysis, and the band density of fixed cells was higher than that of fresh cells. And the expression of PR in MM231 and MCF-7 cells were negative. In addition, positive expression of ER and PR in different cell lines measured by flow cytometry were the same with that analyzed by Western blot analysis. CONCLUSIONS After fixed with 0.25% paraformaldehyde and 75% ethanol, breast cancer cells can be used for not only quantitative measurement of ER and PR, but also Western blot analysis of ER and PR.

UI MeSH Term Description Entries
D011960 Receptors, Estrogen Cytoplasmic proteins that bind estrogens and migrate to the nucleus where they regulate DNA transcription. Evaluation of the state of estrogen receptors in breast cancer patients has become clinically important. Estrogen Receptor,Estrogen Receptors,Estrogen Nuclear Receptor,Estrogen Receptor Type I,Estrogen Receptor Type II,Estrogen Receptors Type I,Estrogen Receptors Type II,Receptor, Estrogen Nuclear,Receptors, Estrogen, Type I,Receptors, Estrogen, Type II,Nuclear Receptor, Estrogen,Receptor, Estrogen
D011980 Receptors, Progesterone Specific proteins found in or on cells of progesterone target tissues that specifically combine with progesterone. The cytosol progesterone-receptor complex then associates with the nucleic acids to initiate protein synthesis. There are two kinds of progesterone receptors, A and B. Both are induced by estrogen and have short half-lives. Progesterone Receptors,Progestin Receptor,Progestin Receptors,Receptor, Progesterone,Receptors, Progestin,Progesterone Receptor,Receptor, Progestin
D001943 Breast Neoplasms Tumors or cancer of the human BREAST. Breast Cancer,Breast Tumors,Cancer of Breast,Breast Carcinoma,Cancer of the Breast,Human Mammary Carcinoma,Malignant Neoplasm of Breast,Malignant Tumor of Breast,Mammary Cancer,Mammary Carcinoma, Human,Mammary Neoplasm, Human,Mammary Neoplasms, Human,Neoplasms, Breast,Tumors, Breast,Breast Carcinomas,Breast Malignant Neoplasm,Breast Malignant Neoplasms,Breast Malignant Tumor,Breast Malignant Tumors,Breast Neoplasm,Breast Tumor,Cancer, Breast,Cancer, Mammary,Cancers, Mammary,Carcinoma, Breast,Carcinoma, Human Mammary,Carcinomas, Breast,Carcinomas, Human Mammary,Human Mammary Carcinomas,Human Mammary Neoplasm,Human Mammary Neoplasms,Mammary Cancers,Mammary Carcinomas, Human,Neoplasm, Breast,Neoplasm, Human Mammary,Neoplasms, Human Mammary,Tumor, Breast
D005260 Female Females
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D015153 Blotting, Western Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes. Immunoblotting, Western,Western Blotting,Western Immunoblotting,Blot, Western,Immunoblot, Western,Western Blot,Western Immunoblot,Blots, Western,Blottings, Western,Immunoblots, Western,Immunoblottings, Western,Western Blots,Western Blottings,Western Immunoblots,Western Immunoblottings

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