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 regi on in order to assess estrogen receptor (ER) structural integrity in h uman breast tumor samples. The antibodies (Abs) reacted specifically w ith the native (undenatured) ER from various species. The synthetic pe ptides competed effectively for ER binding to the Abs, suggesting site -specificity. The Abs recognized the activated (4S) and transformed (5 S) but not the unactivated, untransformed, molybdate-stabilized (8S) E R, suggesting that the epitope is inaccessible in the 8S form. Some of these Abs reacted with ER bound to its responsive elements, as determ ined 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-bindin g domain; and c) site-directed MAb to the N-terminal transactivation d omain. Analysis of ER from 29 human breast rumors revealed that 10 out of 29 tumors (35%) contained ER with intact hormone-, DNA-, and N-ter minal domains. Thirteen out of 29 tumors (similar to 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 (similar to 10 %) contained ER defective only in the N-terminal domain. Another subgr oup of tumors (3/29; similar to 10%) had ER with normal hormone bindin g domain but were defective in both the DNA-binding and the N-terminal activation domains. These observations suggest the potential usefulne ss of site-directed MAb to ER in identification of structurally defect ive ER in human breast tumors.