NEW APPROACH FOR VISUALIZING ESTROGEN-RECEPTORS IN TARGET-CELLS USING INHERENTLY FLUORESCENT LIGANDS AND IMAGE INTENSIFICATION

Abstract

The determination of steroid receptor concentrations in human breast tumor provides important prognostic information that is useful in selecting the most appropriate therapeutic strategy. Four fluorescent estrogen ligands were investigated as agents for visualization of estrogen receptors in cells: 2-(2,4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic acid .delta.-lactone (coumestrol) and 9(11)-dehydro-12-oxoestradiol [12-oxo-1,3,5-(10),9(11)-estratetraene-3,17.beta.-diol] (12-oxoestradiol), which are inherently fluorescent compounds; and tamoxifen {(Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenyl-1-butene} and 4-hydroxytamoxifen {(Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene}, which become maximally fluorscent only after UV irradiation. By conventional fluorescence techniques, these agents can be detected down to 10-8 M in water, but only to 10-6-10-7 M in protein solutions; however, by photon-counting spectrofluorimetry, coumestrol and 12-oxoestradiol can be detected in protein solutions down to 5 .times. 10-10 M. Three of these compounds have good affinity for the estrogen receptor: coumestrol (20%); 12-oxoestradiol (12%); and 4-hydroxytamoxifen (37%), relative to estradiol (100%). Under conditions where autoradiographic controls indicate that most of the estrogen receptor of MCF-7 human breast cancer cells is in the nucleus, nuclear fluorescence as demonstrated using 10-9 M concentrations of coumestrol, 12-oxoestradiol, and 4-hydroxytamoxifen. This nuclear fluorescence was abolished by 4-hydroxytamoxifen. This nuclear fluorescence was abolished by a 200-fold excess of diethylstilbestrol and could only be observed through a fluorescence microscope equipped with a microchannel image intensifier and a video camera detector that together provide a sensitivity enhancement of .apprx. 104. The estrogen receptor in breast cancer cells can be visualized by fluorescence techniques, provided that the visualizing ligands have adequate affinity and specificity for the receptor and appropriate flourescence characteristics, and provided that the fluorescence instrument has adequate sensitivity to observe fluorescence emission from cells treated with nM concentrations of the fluorescent agents.