The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.
We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites. (+info
The effects of androgens and antiandrogens on hormone-responsive human breast cancer in long-term tissue culture.
We have examined five human breast cancer cell lines in continuous tissue culture for androgen responsiveness. One of these cell lines shows a 2- to 4-fold stimulation of thymidine incorporation into DNA, apparent as early as 10 hr following androgen addition to cells incubated in serum-free medium. This stimulation is accompanied by an acceleration in cell replication. Antiandrogens [cyproterone acetate (6-chloro-17alpha-acetate-1,2alpha-methylene-4,6-pregnadiene-3,20-dione) and R2956 (17beta-hydroxy-2,2,17alpha-trimethoxyestra-4,9,11-triene-1-one)] inhibit both protein and DNA synthesis below control levels and block androgen-mediated stimulation. Prolonged incubation (greater than 72 hr) in antiandrogen is lethal. The MCF- cell line contains high-affinity receptors for androgenic steroids demonstrable by sucrose density gradients and competitive protein binding analysis. By cross-competition studies, androgen receptors are distinguishable from estrogen receptors also found in this cell line. Concentrations of steroid that saturate androgen receptor sites in vitro are about 1000 times lower than concentrations that maximally stimulate the cells. Changes in quantity and affinity of androgen binding to intact cells at 37 degrees as compared with usual binding techniques using cytosol preparation at 0 degrees do not explain this difference between dissociation of binding and effect. However, this difference can be explained by conversion of [3H]-5alpha-dihydrotestosterone to 5alpha-androstanediol and more polar metabolites at 37 degrees. An examination of incubation media, cytoplasmic extracts and crude nuclear pellets reveals probable conversion of [3H]testosterone to [3H]-5alpha-dihydrotestosterone. Our data provide compelling evidence that some human breast cancer, at least in vitro, may be androgen dependent. (+info
Mrj encodes a DnaJ-related co-chaperone that is essential for murine placental development.
We have identified a novel gene in a gene trap screen that encodes a protein related to the DnaJ co-chaperone in E. coli. The gene, named Mrj (mammalian relative of DnaJ) was expressed throughout development in both the embryo and placenta. Within the placenta, expression was particularly high in trophoblast giant cells but moderate levels were also observed in trophoblast cells of the chorion at embryonic day 8.5, and later in the labyrinth which arises from the attachment of the chorion to the allantois (a process called chorioallantoic fusion). Insertion of the ROSAbetageo gene trap vector into the Mrj gene created a null allele. Homozygous Mrj mutants died at mid-gestation due to a failure of chorioallantoic fusion at embryonic day 8.5, which precluded formation of the mature placenta. At embryonic day 8.5, the chorion in mutants was morphologically normal and expressed the cell adhesion molecule beta4 integrin that is known to be required for chorioallantoic fusion. However, expression of the chorionic trophoblast-specific transcription factor genes Err2 and Gcm1 was significantly reduced. The mutants showed no abnormal phenotypes in other trophoblast cell types or in the embryo proper. This study indicates a previously unsuspected role for chaperone proteins in placental development and represents the first genetic analysis of DnaJ-related protein function in higher eukaryotes. Based on a survey of EST databases representing different mouse tissues and embryonic stages, there are 40 or more DnaJ-related genes in mammals. In addition to Mrj, at least two of these genes are also expressed in the developing mouse placenta. The specificity of the developmental defect in Mrj mutants suggests that each of these genes may have unique tissue and cellular activities. (+info
Isolation and purification of rat mammary tumor peroxidase.
7,12-Dimethylbenz(a)anthracene-induced rat mammary tumors often contain high levels of the enzyme perioxidase, a putative marker of estrogen dependence. This enzyme can be effectively extracted with 0.5 M CaCl2, giving rise to a soluble peroxidase with a molecular weight of about 50,000 as determined by gel filtration. This is the same size as the estrogen-induced peroxidase of rat uterus but smaller than other mammalian peroxidases. Further purification of the rat mammary tumor peroxidase by concanavalin A-Sepharose chromatography and hydrophobic interaction chromatography on phenyl Sepharose provides a 640-fold purification of the enzyme. (+info
Dominant activity of activation function 1 (AF-1) and differential stoichiometric requirements for AF-1 and -2 in the estrogen receptor alpha-beta heterodimeric complex.
Estrogenic responses are now known to be mediated by two forms of estrogen receptors (ER), ERalpha and ERbeta, that can function as homodimers or heterodimers. As homodimers the two have been recently shown to exhibit distinct transcriptional responses to estradiol (E2), antiestrogens, and coactivators, suggesting that the ER complexes are not functionally equivalent. However, because the three possible configurations of ER complexes all recognize the same estrogen response element, it has not been possible to evaluate the transcriptional properties of the ER heterodimer complex by transfection assays. Using ER subunits with modified DNA recognition specificity, we were able to measure the transcriptional properties of ERalpha-ERbeta heterodimers in transfected cells without interference from the two ER homodimer complexes. We first demonstrated that the individual activation function 1 (AF-1) domains act in a dominant manner within the ERalpha-ERbeta heterodimer: the mixed agonist-antagonist 4-hydroxytamoxifen acts as an agonist in a promoter- and cell context-dependent manner via the ERalpha AF-1, while activation of the complex by the mitogen-activated protein kinase (MAPK) pathway requires only the ERalpha- or ERbeta-responsive MAPK site. Using ligand-binding and AF-2-defective mutants, we further demonstrated that while the ERalpha-ERbeta heterodimer can be activated when only one E2-binding competent partner is present per dimer, two functional AF-2 domains are required for transcriptional activity. Taken together, the results of this study of a retinoid X receptor-independent heterodimer complex, the first such study, provide evidence of different stoichiometric requirements for AF-1 and -2 activity and demonstrate that AF-1 receptor-specific properties are maintained within the ERalpha-ERbeta heterodimer. (+info
Estrogen-dependent and independent activation of the P1 promoter of the p53 gene in transiently transfected breast cancer cells.
Loss of p53 function by mutational inactivation is the most common marker of the cancerous phenotype. Previous studies from our laboratory have demonstrated 17 beta estradiol (E2) induction of p53 protein expression in breast cancer cells. Although direct effects of E2 on the expression of p53 gene are not known, the steroid is a potent regulator of c-Myc transcription. In the present studies, we have examined the ability of E2 and antiestrogens to regulate the P1 promoter of the p53 gene which contains a c-Myc responsive element. Estrogen receptor (ER)-positive T47D and MCF-7 cells were transiently transfected with the P1CAT reporter plasmid and levels of CAT activity in response to serum, E2 and antiestrogens were monitored. Factors in serum were noted to be the dominant inducers of chloramphenicol acetyltransferase (CAT) expression in MCF-7 cells. The levels of CAT were drastically reduced when cells were maintained in serum free medium (SFM). However, a subtle ER-mediated induction of CAT expression was detectable when MCF-7 cells, cultured in SFM, were treated with E2. In serum-stimulated T47D cells, the CAT expression was minimal. The full ER antagonist, ICI 182 780 (ICI) had no effect. Treatment with E2 or 4-hydroxy tamoxifen (OHT) resulted in P1CAT induction; OHT was more effective than E2. Consistent with c-Myc regulation of the P1 promoter, E2 stimulated endogenous c-Myc in both cell lines. Two forms of c-Myc were expressed independent of E2 stimuli. The expression of a third more rapidly migrating form was E2-dependent and ER-mediated since it was blocked by the full ER antagonist, ICI, but not by the ER agonist/antagonist OHT. These data demonstrate both ER-mediated and ER-independent regulation of c-Myc and the P1 promoter of the p53 gene, and show differential effects of the two classes of antiestrogens in their ability to induce the P1 promoter of the p53 gene in breast cancer cells. (+info
Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentrations.
Daidzein and genistein glucuronides (DG and GG), major isoflavone metabolites, may be partly responsible for biological effects of isoflavones, such as estrogen receptor binding and natural killer cell (NK) activation or inhibition. DG and GG were synthesized using 3-methylcholanthrene-induced rat liver microsomes. The Km and Vmax for daidzein and genistein were 9.0 and 7.7 micromol/L, and 0.7 and 1.6 micromol/(mg protein. min), respectively. The absence of ultraviolet absorbance maxima shifts in the presence of sodium acetate confirmed that the synthesized products were 7-O-glucuronides. DG and GG were further purified by a Sephadex LH-20 column. DG and GG competed with the binding of 17beta-(3H) estradiol to estrogen receptors of B6D2F1 mouse uterine cytosol. The concentrations required for 50% displacement of 17beta-(3H) estradiol (CB50) were: 17beta-estradiol, 1.34 nmol/L; diethylstilbestrol, 1.46 nmol/L; daidzein, 1.6 micromol/L; DG, 14.7 micromol/L; genistein, 0.154 micromol/L; GG, 7.27 micromol/L. In human peripheral blood NK cells, genistein at <0.5 micromol/L and DG and GG at 0.1-10 micromol/L enhanced NK cell-mediated K562 cancer cell killing significantly (P < 0.05). At > 0.5 micromol/L, genistein inhibited NK cytotoxicity significantly (P < 0.05). The glucuronides only inhibited NK cytotoxicity at 50 micromol/L. Isoflavones, and especially the isoflavone glucuronides, enhanced activation of NK cells by interleukin-2 (IL-2), additively. At physiological concentrations, DG and GG were weakly estrogenic, and they activated human NK cells in nutritionally relevant concentrations in vitro, probably at a site different from IL-2 action. (+info
Cloning, sequencing, and localization of bovine estrogen receptor-beta within the ovarian follicle.
The potential role of estrogen receptor-beta (ERbeta) in normal ovarian folliculogenesis and in reproductive disorders such as ovarian follicular cysts has not been well defined. Therefore, we were interested in cloning, sequencing, and localizing ERbeta mRNA and protein within the bovine ovary. Bovine ERbeta (bERbeta) was amplified by reverse transcription-polymerase chain reaction (RT-PCR), then cloned and sequenced. Results showed that the open reading frame of bERbeta cDNA spanned 1584 nucleotides encoding a protein of 527 amino acids. The N-terminal region of bERbeta was found to be 80% homologous to human and mouse ERbeta and 79% homologous to rat ERbeta. Bovine ERbeta DNA-binding domain was 100% homologous to human, mouse, and rat ERbeta sequences. The C-terminal/ligand-binding domain of bERbeta was 89% homologous to human, 86% homologous to mouse, and 88% homologous to rat ERbeta. Human and bovine ERbeta amino acid sequences are similar in that their coding region extended farther 5' than initially reported for the published rat ERbeta sequence. Using in situ hybridization and immunohistochemistry, ERbeta mRNA and protein, respectively, were demonstrated to be present in granulosa cells of antral follicles in various stages of follicular growth. These findings suggest a role for bERbeta in ovarian follicular growth and maturation. (+info