Development of gel staining techniques for detecting the secretion of procathepsin D (52-kDa protein) in MCF-7 human breast cancer cells.
17 beta-Estradiol stimulates the secretion of the 34- and 52-kDa protein (i.e., cathepsin D and procathepsin D, respectively) from MCF-7 human breast cancer cells and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits this estrogen-stimulated response. A comparison of the effects of 17 beta-estradiol, TCDD, and their combinations on the secretion of these two proteins was determined using four different assay procedures, namely autoradiographic analysis of the 35S-labeled proteins (from [35S]methionine) separated by polyacrylamide gel electrophoresis (PAGE), densitometric analysis of the silver- and double-stained proteins separated by PAGE, and radioimmunoassay of the proteins using commercially available antibodies to the 52-kDa protein. The results showed that the autoradiographic, staining, and radioimmunoassay procedures gave comparable results with only a few minor differences in the relative amounts of the 52-kDa detected in the various treatment groups. In the medium obtained from 17 beta-estradiol-treated cells that was serially diluted, there was an excellent linear correlation for the relative concentrations of the 52-kDa protein using the double-staining/densitometric procedure and the radioimmunoassay. These results indicate that the double- or silver-staining method may be a useful and rapid method for screening new compounds as antiestrogens in MCF-7 cells.
Krishnan
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Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843-4466.
Narasimhan
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Safe
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D001345:Autoradiography; D001943:Breast Neoplasms / Q000378:metabolism*; D002402:Cathepsin D / Q000032:analysis / Q000378:metabolism*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000032:analysis / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006801:Humans; D008970:Molecular Weight; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D012834:Silver; D013194:Staining and Labeling / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D004792:Enzyme Precursors; D000072317:Polychlorinated Dibenzodioxins; D012834:Silver; D004958:Estradiol; C058345:procathepsin D; D002402:Cathepsin D