Human breast tumors containing non-DNA-binding immunoreactive (67 kDa) estrogen receptor

Evidence to date indicates that structurally abnormal estrogen receptor (variant ER) can be detected in some human breast tumors. Based on in vitro ability to bind DNA sequences containing the cognate estrogen response element (ERE), these variant receptors may be categorized into DNA-binding ER (Type-1 variants) and non-DNA-binding ER (Type-2 variants). To look for Type-2 variants of normal size (67 kDa ER) that lack the ability to form immunoreactive ER-ERE complexes, a panel of 40 cryopreserved primary breast tumors were extracted and analyzed by enzyme immunoassay (ER-EIA), gel-shift, and Western blot techniques. For the 33 tumor extracts containing > or = 10 fmol/mg ER (by ER-EIA), the amount of 67 kDa ER detectable by D75 anti-ER monoclonal antibody under fully denatured and reduced assay conditions (Western blotting) did not correlate well with the presence or intensity of D75 immunoreactive ER-ERE bands seen under native conditions by gel-shift assay. Overall, 30% (10 of 33) of these extracts containing 67 kDa ER failed to produce immunoreactive ER-ERE complexes, with this frequency varying from over 40% in tumor samples with lower ER content (10-49 fmol/mg) to 11% in tumor samples with the highest ER content (> 100 fmol/mg). These results indicate that Type-2 variant receptors characterized as non-DNA-binding 67 kDa ER may be present in a significant fraction of ER-positive primary breast tumors; preliminary evidence suggests that further study of abnormalities in ER tertiary or quaternary structure, such as those produced by intracellular oxidation of ER thiol groups, is warranted.