Nuclear Uptake of a 17 β-Estradiol-Fluorescein Derivative as a Marker of Estrogen Dependence

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Abstract
17-Fluorescein-labeled estradiol was prepared by linkage of fluorescein-isothiocyanate through a succinamide-ethyl-amine chain to the 17 position of 17β-estradiol. This compound, N-fluoresceino-N’Ll7β-(estradiol-hemisuccinamido)-ethyl]-thiourea, displaced tritiated estradiol from purified estrogen-receptor protein and readily crossed intact cellular membranes. In female rats, in-vivo injection was followed by nuclear labeling of endometrium, whereas nuclear labeling of non-target tissue was absent. Temperature-dependent nuclear transfer could be visualized directly by fluorescent microscopy when human estradiol target tissue (mammary and proliferative endometrium) was incubated in vitro with the compound. Temperature-dependent nuclear labeling was not present in human non-target tissue, including skeletal muscle, skin, stomach, colon, appendix, and lung, after in-vitro incubation. Temperature-dependent nuclear labeling with the compound was inhibited by estradiol and mixtures of natural and synthetic estrogens. A significant positive correlation of the presence of nuclear labeling in human mammary cancer was obtained with standard dextrancoated charcoal radioligand assay for estradiol receptor. Unlike radioligand assay and cytochemical assays, nuclear uptake of the fluorescein-labeled estradiol appears to require an intact estradiol-receptor mechanism.