Immunological detection of the oestradiol receptor protein in cell lines derived from the lymphatic system and the haematopoietic system: variability of specific hormone binding in vitro.
Extracts of human MCF 7 mammary carcinoma cells, the human lymphoblastoid cell lines AEH 1 and IM 9, T-cell derived CCRF cells, HL 60 myeloic leukaemia cells and murine myeloma cells SP 0 and NS I were analysed for immunoreactivity with polyclonal goat antibodies raised against homogeneous preparations of C-terminal fragments (32 kDa) of porcine uterine oestradiol receptor (ER). Whole cells and low speed cytosols were analysed for specific oestradiol-binding activity. ERs were enriched from cell extracts by either fractionated ethanol precipitation (0-25% (v/v) ethanol) and/or microscale-immunoaffinity chromatography. Immunoreactive proteins of identical molecular weight (approximately 65 kDa) were detected in all cell lines examined. Whole cell binding assays showed specific oestradiol-binding activity in MCF 7, IM 9 and CCRF cells. Borderline binding was found in HL 60 myeloid cells. No specific binding could be detected in AEH 1, NS I and SP 0 cells. Identical results were obtained using agar-electrophoresis after dextran-coated charcoal treatment. Immunoaffinity purified ERs from MCF 7, AEH 1 and HL 60 cells were subjected to limited proteolysis, where identical tryptic fragments were generated. In conclusion, we have confirmed by immunological methods that ERs are expressed in a variety of cell lines derived from the immune system and the haematopoietic system. The lack of specific hormone binding or very low-affinity hormone binding in some of the cells examined may be due to post-translational events or point mutations.
Jakob
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F
Medizinische Poliklinik, University of Würzburg, Germany.
Tony
H P
HP
Schneider
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Thole
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HH
D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004958:Estradiol / Q000378:metabolism; D006413:Hematopoietic System / Q000737:chemistry*; D006801:Humans; D007106:Immune Sera; D016130:Immunophenotyping / Q000379:methods; D008208:Lymphatic System / Q000737:chemistry*; D011485:Protein Binding / Q000502:physiology; D011959:Receptors, Estradiol / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism
D007106:Immune Sera; D011959:Receptors, Estradiol; D004958:Estradiol