Alternative splicing of human NrCAM in neural and nonneural tissues

HEC1A endometrial cancer cells express the wild-type form of the estrogen receptor (ER) and 17beta-estradiol (E2) induces proliferation of these cells. In contrast, tamoxifen only causes a minimal increase (<20%) in cell proliferation. In HEC1A cells transiently transfected with the C3-Luc plasmid derived from the complement C3 gene, both E2 and tamoxifen exhibited ER agonist activity and tamoxifen was also a partial antagonist for this response. The relative ER agonist/antagonist activities of E2, tamoxifen and ICI 182,780 were also investigated in HEC1A1 cells transiently transfected with two E2-responsive plasmids, pCATHD-CAT and pCKB-CAT which contain 5'-promoter inserts from the cathepsin D and creatine kinase B genes, respectively. The results showed that E2 and tamoxifen induced reporter gene activity in cells transiently transfected with both constructs. ICI 182,780 exhibited partial ER agonist activity only in cells transiently transfected with pCKB-CAT and antagonized E2-induced reporter gene activity using both the CKB- and CATHD-derived constructs. These results demonstrate that HEC1A endometrial cancer cells are E2-responsive and represent a useful cell culture model for understanding hormone/antihormone-induced endometrial cell responses.     

Antiestrogenic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on 17 beta-estradiol-induced pS2 expression

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits a broad spectrum of antiestrogenic activities in rodents and mammalian cells in culture. The effects of TCDD on 17 beta-estradiol (E2)-induction of pS2, a prognostic marker for breast cancer, were investigated in MCF-7, ZR-75, HeLa, and Hepa 1c1c7 wild-type and mutant cells. These effects were compared to the suppressive activities of the congener, 2,8-dichlorodibenzo-p-dioxin, and the established antiestrogens, ICI 164,384 and tamoxifen, in order to determine the relative potency of TCDD and to distinguish the mechanism of action of Ah receptor-mediated antiestrogens. Treatment of MCF-7 cells with 10 nM TCDD decreased E2-induced secreted pS2 protein levels by 50% and the induction of the transiently transfected -1100 to -86 pS2 promoter-regulated reporter gene (pS2-LUC) by 57%. Comparable effects on PS2-LUC activity were observed in HeLa and ZR-75 cells. In contrast, TCDD had minimal effects on pS2ERE(-405 to -393)-LUC induction, whereas treatment with 10 nM ICI 164,384 caused a 60% decrease in luciferase activity. In Hepa 1c1c7 wild-type and clone 1 (C1) mutant cells, TCDD also reduced E2 induction of pS2-LUC activity but had little effect in clone 4 (C4) or clone 12 (C12) mutant cells. However, suppression was reestablished following transfection of the human Ah receptor nuclear translocator (ARNT) complementary DNA expression vector into C4 cells and the mouse Ah receptor (AhR) complementary DNA expression vector into C12 cells. Induction of pS2-LUC activity by the ligand-dependent and -independent chimeric estrogen receptors (HE15, HE19, ERcVP16, and ERGR) were also used to examine the role of E2 metabolism and the mechanism of TCDD-mediated antiestrogenic activity. Induction by HE15 and ERcVP16 was suppressed by 57 and 74%, respectively, following treatment with TCDD, whereas ICI 164,384 was significantly less effective (38 and 20%, respectively). These results demonstrate a role for the Ah receptor in TCDD-mediated suppression of E2-induced pS2 expression. Data is presented demonstrating that the effect requires sequences within the pS2 promoter other than the estrogen response element and is independent of E2 oxidative metabolism.     

Estrogen receptor does not directly regulate the murine Muc-1 promoter

Muc-1 is a heavily O-glycosylated, type 1 membrane glycoprotein present on the surface of polarized secretory uterine epithelial cells. Previous studies have shown that treatment of ovariectomized mice with 17-beta-estradiol (E2) strongly induces Muc-1 mRNA expression in an estrogen receptor (ER)-mediated fashion in the uterus. In this study, the 5.4 kb Muc-1 gene promoter has been isolated from a mouse genomic library and the proximal 1.85 kb region has been sequenced. Sequence analysis revealed the presence of one potential full estrogen response element (ERE) (GCTCGCGGTGACC) located at -748 to -735 bp in the Muc-1 promoter and several potential ERE half sites. Electrophoretic mobility shift assays (EMSA) showed that neither ERalpha nor ERbeta bind efficiently to this sequence. Transient cotransfection assays using constructs containing various deletion mutations of the 5' Muc-1 flanking sequences showed that E2 had no direct stimulation on promoter-driven reporter in NMuMG cells or primary mouse uterine epithelial cells, but did stimulate a consensus ERE CAT-reporter gene activity. In addition, E2-treatment of Weg-ER cells, a mouse uterine epithelial cell line stably expressing human ERalpha, did not restore endogenous Muc-1 expression or activate Muc-1 promoter-driven CAT activity. These results indicate that regions of the Muc-1 gene promoter within -1838 to +43 bp do not respond to E2 and ER stimulation and that ER alone is not sufficient to restore Muc1 gene expression. Deletion analyses also revealed that the sequence between -73 and +43 bp of the Muc-1 promoter is the minimal promoter region required for maximal Muc-1 promoter activity. Collectively, these results demonstrate that ER does not directly regulate the 1.85 kb murine Muc-1 gene promoter. Therefore, E2 control of uterine Muc-1 gene expression is likely to be indirect, i.e. mediated by stromal cell-derived factors.     

Comparison of estrogen receptor DNA binding in untreated and acquired antiestrogen-resistant human breast tumors

Preliminary studies have suggested that measuring the ability of immunoreactive 67-kDa estrogen receptor (ER) to bind DNA and form in vitro complexes with its cognate estrogen response element (ERE) might serve to identify breast tumors most likely to respond to antiestrogens like tamoxifen. Data from two different surveys of untreated primary breast tumors confirmed that only 67% (74 of 111) of ER-positive tumors express a receptor capable of forming ER-ERE complexes by gel-shift assay, with tumors of lower ER content having significantly reduced ER DNA-binding frequency (56%) relative to those of higher ER content (82%; P = 0.007). In contrast to these untreated tumors, a panel of 41 receptor-positive breast tumors excised after acquiring clinical resistance to tamoxifen during either primary (n = 26) or adjuvant therapy (n = 15) showed a significantly greater ER DNA-binding frequency, with nearly 90% capable of forming ER-ERE complexes (P < 0.02). To assess experimentally whether ER DNA-binding function is altered during the development of antiestrogen resistance, nude mouse MCF-7 tumor xenografts were analyzed before and after the acquisition of in vivo resistance to either tamoxifen or a pure steroidal antiestrogen, ICI 182,780. Tamoxifen-resistant MCF-7 tumors retained full expression of 67-kDa DNA-binding ER, and despite a markedly reduced ER content in the ICI 182,780-treated tumors, the expressed ER in these antiestrogen-resistant tumors exhibited full ability to form ER-ERE complexes. These findings indicate that breast tumors with acquired antiestrogen resistance continue to express ER of normal size and DNA-binding ability and suggest that the failure of antiestrogens to arrest tumor growth during emergence of clinical resistance results from an altered gene-regulatory mechanism(s) other than ER-ERE complex formation.     

Differential effects of heterogeneous nuclear ribonucleoprotein K on Sp1- and Sp3-mediated transcriptional activation of a neuronal nicotinic acetylcholine receptor promoter

The neuronal nicotinic acetylcholine receptor gene family consists of 11 members, alpha2-alpha9 and beta2-beta4. Three of the genes, those encoding the alpha3, alpha5, and beta4 subunits, are clustered tightly within the genome. These three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system. The genomic proximity of the three genes suggests a regulatory mechanism ensuring their coordinate expression. However, it is likely that gene-specific regulatory mechanisms are also functioning because the expression patterns of the three genes, although similar, are not identical. Previously we identified regulatory elements within the beta4 promoter region and demonstrated that these elements interact specifically with nuclear proteins. One of these elements, E1, interacts with the regulatory factor Puralpha as well as three other unidentified DNA-binding proteins with molecular masses of 31, 65, and 114 kDa. Another element, E2, interacts with Sp1 and Sp3. Because E1 and E2 are immediately adjacent to one another, we postulated that the proteins that bind to the elements interact to regulate beta4 gene expression. Here we report the identification of the 65-kDa E1-binding protein as heterogeneous nuclear ribonucleoprotein K and demonstrate that it affects the transactivation of beta4 promoter activity by Sp1 and Sp3 differentially.     

Identification of functional elements in the promoter region of the human gene for thymidylate synthase and nuclear factors that regulate the expression of the gene

To identify the essential motifs of the promoter of the human gene for thymidylate synthase (TS), we constructed a set of deletion mutants from the 5'-terminal region of the human TS gene. From the results of assays of the expression of chloramphenicol acetyltransferase (CAT), we identified two functional elements with positive effects on the promoter activity: a CACCC box (CCACACCC) and an Sp1-binding motif (GAGGCGGA) that was homologous to the Sp1-binding site in the mouse TS gene. In addition, negative regulatory sequences were identified between the two positive elements and in the region upstream of the CACCC box. The results of gel mobility shift analyses suggested that Sp1 binds to the Sp1-binding motif of the human TS gene promoter and that multiple nuclear factors that are related to Sp1 bind to the CACCC box. Furthermore, the binding of Sp1 to mutated Sp1-binding motifs in the promoter region of the human TS gene was correlated with the promoter activity, as measured by the CAT assay. Therefore, the Sp1 motif seems to be a major contributor to the basic promoter activity of the human TS gene, although multiple positive and negative regulatory elements are involved in the regulated expression of this gene.     

Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene

The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains an consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter was strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogated Sp1-dependent transactivation. In gel shift assays, the same mutations abolished Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter was dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells revealed that cell lines overexpressing Sp1 had increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bound Egr-1 in gel shift assays and Egr-1 competitively displaced bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppressed pgp2/mdr1b promoter activity and mutations in the Egr-1 site decreased Egr-1 binding and correlated with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreased Pgp expression and selectively increased vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells.     

Differential regulation by estrogens of growth and prolactin synthesis in pituitary cells suggests that only a small pool of estrogen receptors is required for growth

PR1 cells are a prolactin (PRL)-secreting cell line derived from a pituitary lactotroph tumor found in 17beta-estradiol-treated Fischer 344 rats. We examined the effect of estrogen on cell proliferation and PRL synthesis under various culture conditions. Estrogen, at extremely low concentrations, induces cell proliferation in this cell line, whereas antiestrogen inhibits proliferation. Interestingly, the proliferation response is much more sensitive than the PRL response because 0.01 pM estradiol or diethylstilbestrol induces half-maximal growth induction [ approximately 0.1% estrogen receptor (ER) occupancy is required], whereas 0.01 nM concentration is required for half-maximal PRL induction ( approximately 50% ER occupancy is required). The proliferation response is not as sensitive to antiestrogen as the PRL response, because 10 nM concentration of the pure antiestrogen ICI 182,780 could not inhibit 1 nM estradiol- or diethylstilbestrol-induced proliferation. The same concentration of ICI 182,780 decreased PRL secretion to 1% of estradiol- or diethylstilbestrol-induced prolactin secretion suggesting a possible dichotomy of ER control of proliferation and PRL synthesis. The Kd of ER binding in these cells is about 3 x 10(-11) M. These results with the PR1 cells extend previous studies in other estrogen- regulated systems and suggest that only a small pool of ER is required for cell proliferation in contrast with the regulation of expression of specific genes. They also raise questions as to how a dimeric receptor functions when only one ligand site is occupied or when both an estrogen and an antiestrogen occupy one dimer.