Effects of estrogens on MCF-7 cells: positive or negative regulation by the nature of the ligand-receptor complex.

The present study demonstrates that the nature of the binding of estrogens to the hormone-binding domain of the estrogen receptor (ER) modifies the responses of estrogen-dependent cells. We report here that 10 nM estradiol (E2) forms noncovalent associations with the ER, increases the level of ER and Progesterone Receptors (PR) in ER+ MCF-7 human breast cancer cells in culture following short-term or long-term exposure to E2. In contrast, 10 nM 16-alpha-hydroxyestrone (16 alpha-OHE1), a physiological metabolite of E2, in short-term cultures is equivalent to E2, but upon long-term incubation, 16 alpha-OHE1 forms covalent associations with the ER, produces a marked decrease in ER and PR levels reaching values similar to, or below to that of control cells.

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture.

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of estradiol 2- and 16-alpha-hydroxylase activities in MCF-7 human breast cancer cells in culture using radiometric analysis.

Using a radiometric assay the effects of estradiol upon the activity of estradiol 2- and 16 alpha-hydroxylases in MCF-7 human breast cancer cells in culture were studied. After 5 days of treatment and 36 h of withdrawal, incubation in the presence of either 2- or 16 alpha-tritiated substrate was carried out. Estradiol (10 nM) significantly increased 16 alpha-hydroxylase activity (21%, P less than 0.01), while no effects on 2-hydroxylase activity was observed. Treatment for 6 weeks caused a major increase in 16 alpha-hydroxylation (65%, P less than 0.001) and a significant reduction in 2-hydroxylation (21%, P less than 0.05). These effects were not observed in estrogen receptor-negative MDA-MB-231 human breast cancer cells. Our observations suggest that these two metabolic pathways in MCF-7 cells are independently regulated and that this regulation is affected via the estrogen receptor.

Enhancement of 2- and 16 alpha-estradiol hydroxylation in MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-P-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin exhibits antiestrogenic activity and induces cytochromes P-450 in estrogen-dependent MCF-7 human breast-cancer cells. To determine whether induction of 2- or 16 alpha-hydroxylation of 17 beta-estradiol has a role in this antiestrogenic activity, MCF-7 cells which were exposed to this xenobiotic for 72 hrs were incubated with either [2-3H] or [16 alpha-3H] 17 beta-estradiol and the extent of tritiated H2O formation, indicative of site-specific hydroxylation, was determined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-treated MCF-7 cultures showed an 8-fold increase in 2-hydroxylation and a 2-fold increase in 16 alpha-hydroxylation. These results support the suggestion that increased hydroxylation of 17 beta-estradiol may have a role in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells.

Covalent binding of the endogenous estrogen 16 alpha-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization.

The interactions of 16 alpha-hydroxyestrone (16 alpha-OHE1), a metabolite of estradiol (E2), with estrogen receptors (ERs) were compared in this study to the classic E2-receptor mechanism in human breast cancer cells MCF-7 in culture. When MCF-7 cells were incubated with radioinert 16 alpha-OHE1 or its 3H-labeled form for 4 weeks, the estrogen bound extensively and irreversibly in a time-dependent fashion to nuclear protein species that correspond to the ER. Here we show that the interactions of 16 alpha-OHE1 with the ER are different from those of E2 with the receptor. Dissociation of tritiated E2-ER or 16 alpha-OHE1-ER complexes, salt extraction, DNase and proteinase K digestion, and ethanol treatment demonstrated that the binding of 16 alpha-OHE1 to the ER corresponds to two different forms: a classical noncovalent interaction similar to that of E2, and a covalent adduct formation between the metabolite and the ER. These complexes localized preferentially in nuclear matrix components as revealed by cell fractionation and probing with a monoclonal anti-ER antibody. [3H]16 alpha-OHE1-ER complexes analyzed by polyacrylamide gel electrophoresis demonstrated a radiolabeled band at approximately 66 kDa that was absent when the exposure of cells was done in the presence of E2 in competition and that was also absent in [3H]E2 incubations. The present results when considered together with our previous findings of elevated activities of estrogen 16 alpha-hydroxylase, the enzyme responsible for the formation of 16 alpha-OHE1, in breast cancer patients and in women at enhanced risk for the disease, suggest that covalent modification of the ER may be one mechanism of malignant transformation in estrogen target tissues.

Metabolism of estradiol fatty acid esters in man.

Fatty acid esters of estradiol coupled at the C-17 position are naturally occurring metabolites of estradiol (E2). They have been recovered after incubation of E2 with human tissue and identified in human plasma. We investigated the primary metabolic transformation of E2, namely C-17 oxidation, of two representative fatty acid esters, in five normal subjects (four men and one woman), aged 25-55 yr, using a radiometric method. The transfer of tritium from the C-17 alpha position to tritium water after iv injection of free E2 was compared to that of E2-17 beta-stearate and E2-17 beta-arachidonate. Both esters were oxidized at the C-17 position to a greater extent than was free E2. In addition, the oxidation of the E2 fatty acid esters proceeded more slowly. Thus, the time necessary to reach half the maximal extent of reaction ranged from 30-45 min for the three E2-17 beta-arachidonate studies and from 2.5-4 h for the five E2-17 beta-stearate studies, while that for free E2 was less than 15 min in each instance. The disappearance of intact E2-17 beta-stearate from plasma after bolus injection was studied in two subjects (one of the five above and one additional subject). The t1/2 values calculated for the 0-60 min period were 24 and 16 min. The rate of disappearance E2-17 beta-stearate was slower than that of E2. The biological activity of E2 esters is thought to reside in their ability to be converted, by hydrolysis, to free E2. The E2 esters must undergo hydrolysis before the oxidation of free E2 to estrone can proceed. Since the oxidation reaction is extremely rapid, studying the rate of oxidation after injection of the E2 fatty acid esters provides an index of their in vivo hydrolysis and, thus, a measure of their subsequent biological activity as the free hormone. The unsaturated arachidonate ester of E2 was oxidized at a faster rate than the saturated stearate ester; this implies that the hydrolysis of the arachidonate ester was faster. Evidence of continuing oxidation of E2-17 beta-stearate at times well after its level in plasma markedly decreased indicates that the ester is removed from the circulation before its subsequent hydrolysis and oxidation. We conclude that in man, the C-17 oxidation of both E2-17 beta-arachidonate and E2-17 beta-stearate proceeds more slowly but to a greater extent than that of the free steroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific binding of 3H-estradiol to rat prostate nuclear matrix.

Specific estradiol binding activities can be demonstrated in nuclear matrix preparations obtained from intact rat prostate nuclei. Some of the characteristics of these in vitro binding activities to intranuclear components are presented and compared to those exhibited by purified nuclear fractions. Examination of the effects of exposure to castration and testosterone on the number of nuclear matrix binding sites revealed that the quantity and quality (Type) of receptors was modified. Furthermore, these changes are prevented when protein synthesis was inhibited.

Correlation of nuclear acceptor sites for oestrogen receptors with gene transcription in vitro.

The interaction of oestrogen receptors with their nuclear acceptor sites was studied to ascertain whether these acceptor sites are involved in the regulation of ovalbumin-gene expression in the chick oviduct. As previously described, two distinct oestrogen-receptor species exist, and both are translocated into the nucleus after oestrogen administration in vivo [Smith, Clarke, Zalta & Taylor (1979) J. Steroid Biochem.10, 31-35]. In the present investigation we observed that the tubular-gland-cell concentrations of cytoplasmic receptors (800-900/cell) do not vary with prolonged withdrawal, nor do the relative ratios of the two receptor types change; however, the nuclear accumulation and retention of these receptors after secondary oestrogen administration are attenuated in a time-dependent fashion. Chicks were withdrawn from oestrogen for 24-84h. Some animals were then restimulated with oestrogen and killed after 4h, when oviduct nuclei were isolated. These nuclei were assayed for nuclear receptor concentrations and for their capacity to synthesize ovalbumin mRNA in vitro. Although an equal number of cytoplasmic receptors appeared to be translocated, oestrogen-receptor occupancy within the nucleus was not equal, but was inversely proportional to the preceding length of withdrawal. This decrease in nuclear acceptor sites was accompanied by a similar decrease in the capacity of these same nuclei to transcribe ovalbumin mRNA in vitro. A statistical evaluation of nuclear oestrogen-receptor concentrations and ovalbumin-mRNA synthesis in vitro was made. Correlation analysis revealed a Pearson coefficient r=0.87 (P<0.001, n=17), indicating that a high degree of correlation exists between these two parameters. These results are consistent with the hypothesis that nuclear oestrogen-receptor-acceptor complexes may correspond to initiation sites for RNA polymerase II transcription of an oestrogen-regulated gene.

Effect of estrogen on gene expression in chicken oviduct: evidence for transcriptional control of ovalbumin gene.

The transcription of structural and intervening sequences of the chicken ovalbumin gene was studied in nuclei isolated from the oviduct, liver, and spleen of chickens in different states of estrogen simulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [(3)H]RNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (i) a method exists to assay synthesis of hormone-inducible specific eukaryotic [(3)H]mRNA in vitro; (ii) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (iii) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (iv) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription.

The ovalbumin gene: transcriptional regulation by estrogen.

De novo synthesis of RNA sequences corresponding to intervening as well as to structural sequences of the ovalbumin gene have been detected in isolated oviduct nuclei. Their presence in the nuclear transcripts and their time course of induction support the hypothesis that transcription of structural and intervening sequences of the natural ovalbumin gene are regulated by steroid hormones. These results are in agreement with out previous demonstration of high-molecular-weight species of ovalbumin RNA in nuclei that contain structural as well as intervening RNA sequences and are thus likely precursors to mature cytoplasmic mRNAov. Analysis of the size of in vivo nuclear RNA by gel electrophoresis under denaturing conditions, revealed that withdrawal of hormone depletes the level of high molecular weight ovalbumin RNA as well as that of nature mRNAov and that readministration of estrogen induces the accumulation of both species. These results are consistent also with transcriptional regulation of the ovalbumin gene. In addition, they rule out the possibility that the rapid accumulation of mature mRNAov after secondard stimulation results from processing of ovalbumin RNA precursors that might have been stored in the withdrawn oviduct. We conclude that steroid hormones exert a primary effect at the level of gene transcription. Following this event, a series of coordinated cellular responses may occur which involve RNA processing, mRNA transport to the cytoplasm, protein synthesis and mRNA degradation. The final consequence of this network of molecular reactions is the induced cellular function inherent to a specific steroid hormone.