4-iodotamoxifen aziridine, a new affinity labeling agent for the rapid detection of estrogen receptor isoforms

We describe the simple and fast preparation of a new radioiodinated probe for the detection of the estrogen receptor (ER) and its isoforms. Iodotamoxifen aziridine was labeled with iodine 125 ([125I]TAZ) in position 4 of the alpha aromatic ring. The yield was high (>75%), the label was stable and the specific activity was near optimal (1900-2170 Ci/mmol). The apparent relative binding affinity of the probe to a recombinant human ER (hER) was high (RBA = 35 vs estradiol = 100). Electrophoretic studies (SDS-PAGE) with this hER indicated the high potency of [125I]TAZ at very low concentration (<1 nM) to reveal ER bands after a short exposure time (1-4 days). Competition between this probe and various compounds as well as chemical treatments of the ER with SH-reactive chemicals, demonstrated the labeling specificity. Analysis of cytosols from a panel of cell lines and various rat reproductive organs displayed characteristic ER bands (67, 50 and 37 kDa) suppressed by unlabeled E2. Detection in nonreproductive organs of 43 kDa E2-nondisplaceable peptide raised the question upon the presence of altered and/or variant ERs in many tissues. Data concerning human breast cancer cytosols were in complete accordance with those established with [3H]TAZ: high ER polymorphism in most ER-positive samples and peculiar forms (mainly 43 kDa) in ER-negative samples. Hence, [125I]TAZ appears especially useful for the detection of altered ER or related peptides in breast cancers.