Identification of structurally altered estrogen receptors in human breast cancer by site-directed monoclonal antibodies.
We have developed and characterized site-directed monoclonal (MAb) and polyclonal antibodies to a specific domain in the N-terminal A/B region in order to assess estrogen receptor (ER) structural integrity in human breast tumor samples. The antibodies (Abs) reacted specifically with the native (undenatured) ER from various species. The synthetic peptides competed effectively for ER binding to the Abs, suggesting site-specificity. The Abs recognized the activated (4S) and transformed (5S) but not the unactivated, untransformed, molybdate-stabilized (8S) ER, suggesting that the epitope is inaccessible in the 8S form. Some of these Abs reacted with ER bound to its responsive elements, as determined by gel mobility shift assay. To evaluate the structural integrity of ER in breast cancer, we have utilized a) ligand binding analysis for the hormone binding domain; b) site-directed MAb to the DNA-binding domain; and c) site-directed MAb to the N-terminal transactivation domain. Analysis of ER from 29 human breast tumors revealed that 10 out of 29 tumors (35%) contained ER with intact hormone-, DNA-, and N-terminal domains. Thirteen out of 29 tumors (approximately 45%) contained ER with intact hormone binding and N-terminal domains but were defective only in the DNA-binding domain. Three out of 29 tumors (approximately 10%) contained ER defective only in the N-terminal domain. Another subgroup of tumors (3/29; approximately 10%) had ER with normal hormone binding domain but were defective in both the DNA-binding and the N-terminal activation domains.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D001666:Binding Sites, Antibody; D001943:Breast Neoplasms / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D011960:Receptors, Estrogen / Q000032:analysis* / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D004247:DNA