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Estradiol Decreases Retention of Rhodamine 123 Fluorescence in GH₄C₁ Pituitary Tumor Cells

Department of Pharmacology, Yale University School of Medicine New Haven, Connecticut 06510
Yale Comprehensive Cancer Center, Yale University School of Medicine New Haven, Connecticut 06510

Address requests for reprints to: Priscilla S. Dannies, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticutt 06510.

Abstract

Rhodamine 123 is a lipophilic cationic fluorescent dye that localizes in mitochondria. We found that 17β-estradiol changes the ability of GH₄C₁ cells, clonal rat pituitary tumor cells, to retain rhodamine 123. Cells incubated with 10 µg/ml rhodamine 123 for 30 min at 37 C took up about equal amounts of rhodamine 123, as determined by fluorescence microscopy, regardless of whether they had been treated with estradiol. After three 5-min washes at 37 C, cells treated with 1 nM estradiol for 7 days before incubation with rhodamine 123 had lost more fluorescence than untreated cells. We further characterized the effect by flow cytometry. The difference in fluorescence between control and treated cells ranged from 50- to 500-fold. The effect of estradiol was maximal at 10^–10 M and took a week to develop fully. The effect is specific for estradiol, because estradiol and diethylstilbestrol reduced retention of rhodamine 123 fluorescence at 10^–10 M, but the same concentrations of dihydrotestosterone, progesterone, dexamethasone, and cholesterol did not. To test if the effect on rhodamine 123 fluorescence was caused by activation of the multidrug resistance transport system, we examined the effect of estradiol on the retention of daunomycin, a known substrate of the transport system. Estradiol treatment caused a 3-fold decrease in daunomycin fluorescence. We isolated clones resistant to estradiolinduced loss of rhodamine 123 fluorescence by flow cytometry and found that two clones still showed an estradiol-induced decrease in daunomycin fluorescence equivalent to that of the parent line. The estradiol effect on daunomycin, therefore, can be separated from the effect on rhodamine 123, indicating that estradiol does not affect rhodamine 123 fluorescence by a general activation of the multidrug resistance system. The estradiol-induced loss of rhodamine 123 fluorescence may be caused by activation of efflux specific for rhodamine 123 and not daunomycin, by inhibition of a mechanism by which cells retain the dye, or by metabolism of the dye. The ability to use flow cytometry to select variants that have lost responsiveness to estradiol will be a useful tool for investigating estrogen actions on pituitary cells.

FOOTNOTES

This work was supported by USPHS Grants HD-11487 from the NICHHD and CA-16359 from the NCI.

Received for publication July 27, 1989. Revision received October 4, 1989. Accepted for publication October 23, 1989.