|Title:||A 46-kDa antigen associated with estrogen receptor in human breast cancer||Authors:||Díaz-Chico, B. N.
Chamness, G. C.
McGuire, W. L.
|UNESCO Clasification:||32 Ciencias médicas
|Issue Date:||1988||Journal:||Journal of Steroid Biochemistry||Abstract:||A 65-kDa estrogen receptor (ER) protein has been demonst~ted both by sucrose gradient analysis and by immunoblot, using anti-ER monoclonal antibodies (MAbs). Since the ER is denatured in many experimental situations, such as formaldehyde fixing of samples for histochemistry and electroimmunoblotting studies, in this work we used a denatured 60-70-kDa ER-rich protein preparation as antigen for mice immunization in order to raise antiER MAbs. That material was obtained by affinity purification on an allyl-estradiol matrix of the MCF-7 cytosolic ER, followed by further isolation and enrichment by PAGE. NS-1 myeloma cells and spleen lymphocytes from the immunized mice were fused, and resultant hybridoma colonies were screened by -estradiol-labelled nuclear ER immunoprecipitation. The isolated MAb, E476, shows a moderate ability to precipitate ER and reacts strongly with a 46-kDa antigen in Western blot assay. The 46kDa antigen was not detectable in native cytosol but became reactive after 50% ammonium sulfate precipitation of cytosolic proteins. The 46-kDa antigen appeared concentrated in the NaSCN plus estradiol eluate of the affinity column used for cytosolic ER purification. Freshly prepared 60-70-kDa material from the preparative gel electrophoresis did not show any E476 reactivity. However, when the 60-70-kDa proteins were frozen, thawed and speed vacuum concentrated, the 46-kDa antigen became detectable. Storage increased the reactivity of the 60-70-kDa material with the E476 MAb. The 46 kDa antigen was present only in the ER positive cell lines, and was absent in all negative cell lines tested. The 46-kDa protein is also present in the ER positive human breast cancer specimens. We conclude that the 46-kDa protein identified with the E476 MAb in human breast cancer is probably a naturally occurring ER fragment.||URI:||http://hdl.handle.net/10553/50474||ISSN:||0022-4731||DOI:||10.1016/0022-4731(88)90114-8||Source:||Journal of Steroid Biochemistry[ISSN 0022-4731],v. 30, p. 315-320|
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