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New approach for visualizing estrogen receptors in target cells using inherently fluorescent ligands and image
Abstract:
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 fluorescent only after ultraviolet irradiation. By conventional fluorescence techniques, these agents can be detected down to 10(-8) M in water, but only to 10(-6) to 10(-7) M in protein solutions; however, by photon-counting spectrofluorimetry, coumestrol and 12-oxoestradiol can be detected in protein solutions down to 5 X 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, we could demonstrate nuclear fluorescence using 10(-9) M concentrations of coumestrol, 12-oxoestradiol, and 4-hydroxytamoxifen. This nuclear fluorescence was abolished by a 200-fold excess of diethystilbestrol 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 approximately 10(4). These studies indicate that 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 fluorescence characteristics, and provided that the fluorescence instrument has adequate sensitivity to observe fluorescence emission from cells treated with nM concentrations of the fluorescent agents.
Insights
This study demonstrates that fluorescent ligands can visualize estrogen receptors in breast cancer cells. Advanced fluorescence microscopy techniques enable detection of these receptors at nanomolar concentrations.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Endocrinology
Background:
- Estrogen receptors (ERs) play a critical role in the development and progression of breast cancer.
- Visualizing ERs in cells is crucial for understanding their function and for developing targeted therapies.
- Current visualization methods have limitations in sensitivity and specificity.
Purpose of the Study:
- To investigate four fluorescent estrogen ligands for their ability to visualize estrogen receptors in cells.
- To evaluate the sensitivity and specificity of these ligands using advanced fluorescence techniques.
- To demonstrate the feasibility of visualizing nuclear estrogen receptors in breast cancer cells.
Main Methods:
- Synthesis and characterization of four fluorescent estrogen ligands: coumestrol, 12-oxoestradiol, tamoxifen, and 4-hydroxytamoxifen.
- Assessment of ligand detection limits in aqueous and protein solutions using conventional fluorescence and photon-counting spectrofluorimetry.
- Determination of ligand binding affinity for the estrogen receptor relative to estradiol.
- Visualization of nuclear estrogen receptors in MCF-7 human breast cancer cells using fluorescence microscopy with a microchannel image intensifier and video camera.
Main Results:
- Coumestrol and 12-oxoestradiol showed inherent fluorescence, while tamoxifen and 4-hydroxytamoxifen required UV irradiation.
- Photon-counting spectrofluorimetry significantly enhanced detection sensitivity for coumestrol and 12-oxoestradiol in protein solutions (down to 5 x 10(-10) M).
- Coumestrol (20%), 12-oxoestradiol (12%), and 4-hydroxytamoxifen (37%) exhibited good affinity for the estrogen receptor.
- Nuclear fluorescence in MCF-7 cells was successfully demonstrated using nanomolar concentrations of coumestrol, 12-oxoestradiol, and 4-hydroxytamoxifen.
- The observed nuclear fluorescence was specifically related to estrogen receptors, as it was abolished by excess diethylstilbestrol.
Conclusions:
- Fluorescent ligands with adequate affinity and specificity can visualize estrogen receptors in breast cancer cells.
- Advanced fluorescence instrumentation is essential for detecting low cellular concentrations of these receptors.
- This approach holds promise for studying estrogen receptor dynamics and guiding breast cancer therapy.