The interactions of 16a-hydroxyestrone (16a-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 16a-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 weshow that the interactions of 16a-OHE, with the ER are different from those of E2 with the receptor. Dissociation of tritiated E2-ER or 16ci-OHE,-ER complexes, salt extraction, DNase and proteinase K digestion, and ethanol treatment demonstrated that the binding of 16a-OHE, 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]16a-OHEj-ER complexes analyzed by polyacrylamide gel electrophoresis demonstrated a radiolabeled band at %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 PH]E2incubations. The present results when considered together with our previous findings of elevated activities of estrogen 16a-hydroxylase, the enzyme responsible for the formation of 16a-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.The pathway of receptor-mediated estrogen actions in target tissues has been studied extensively (1)(2)(3)(4)(5). Evidence that target tissues for estradiol (E2) respond variably to its metabolites raises questions whether the estrogen receptor (ER) bound to these metabolites might exert unusual functions at the nuclear level. We have previously shown that the natural estrogen metabolite 16a-hydroxyestrone (16a-OHE1), whose role in biological processes has not been established, binds covalently to primary amino groups of proteins by a nonenzymatic process. In vitro and in vivo covalent interactions of 16a-OHE1 with proteins occur between the steroid carbonyl and the E-amino group of lysine residues in many human tissues (6-9). Our group has been studying the role of steroid metabolites and their covalent interactions in the induction of human breast cancer, since we have found that the activity of estrogen 16a-hydroxylase, the enzyme involved in the formation of 16a-OHE,, is increased in a population of women with breast cancer and in women at risk for the disease (10, 11).The objective of the present study has been to test directly the hypothesis that 16a-OHE, binds covalently to nuclear regulatory proteins, specifically the ER, in estrogen target cells. We now report that covalent binding of 16a-OHE1 to specific nuclear proteins is extensive and that ERs at ...

The transcription of structural and intervening se uences of the chicken ovalbumin gene was studied in nuclei isoated from the oviduct, liver, and spleen of chickens in different states of estrogen stimulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [3HJRNA 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[3H]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.Previous studies on the regulation of gene expression in eukaryotes have shown that steroid hormones induce synthesis of specific proteins in vivo by increasing the concentration of their mRNAs (1-3). Injections of estrogen into chickens that had been prestimulated with estrogen and then withdrawn from all hormone (secondary stimulation) induced the accumulation of ovalbumin mRNA (mRNAOv) (4-7). Recently, it has been shown that the structural sequences of the ovalbumin gene (8-10) and globin gene are interrupted by multiple regions of nonstructural intervening sequences. In addition, the 15S precursor to globin mRNA was shown to contain both structural and intervening sequences of the globin gene (11). Similarly, transcripts of both types of ovalbumin sequences accumulated in vivo in oviduct nuclei after stimulation with estrogen (12).In addition, we have demonstrated the existence of high-mo-The publication costs of this article were def...

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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.