A 46-kDa antigen associated with estrogen receptor in human breast cancer.
A 65-kDa estrogen receptor (ER) protein has been demonstrated 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 anti-ER 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 [125I]-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 46-kDa 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.
Díaz-Chico
B N
BN
University of Texas, Health Science Center, San Antonio 78284.
Ogasawara
Y
Y
Chamness
G C
GC
Salman
M
M
McGuire
W L
WL
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D000951:Antigens, Neoplasm / Q000302:isolation & purification*; D001943:Breast Neoplasms / Q000032:analysis*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011960:Receptors, Estrogen / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm; D011960:Receptors, Estrogen