MCF-7 Mammary Epithelial Cells Research Articles

GIRK1 triggers multiple cancer-related pathways in the benign mammary epithelial cell line MCF10A

Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

Decreased All- trans Retinoic Acid-Induced Expression of Sodium-Iodide Transporter in Mammary Epithelial Cells Caused by Conjugated Linoleic Acid Isomers.

Expression of sodium-iodide symporter (NIS) is stimulated by sterol-regulatory-element-binding transcription factors (SREBFs) in mammary epithelial MCF-7 cells. Because conjugated linoleic acid (CLA) isomers have been shown to inhibit transcriptional activity of SREBFs in the mammary gland, the hypothesis was tested that CLA isomers inhibit NIS expression induced by all- trans retinoic acid (ATRA) in MCF-7 cells through inhibiting SREBF activity. c9t11-CLA and t10c12-CLA decreased ATRA-induced NIS-mRNA expression from 1.00 (ATRA alone) to 0.80 ± 0.12 (200 μM c9t11-CLA, P < 0.05) and 0.62 ± 0.10 (200 μM t10c12-CLA, P < 0.05), NIS-protein expression from 1.00 (ATRA alone) to 0.77 ± 0.08 (200 μM c9t11-CLA, P < 0.05) and 0.63 ± 0.05 (200 μM t10c12-CLA, P < 0.05), and NIS-promoter activity from 1.00 (ATRA alone) to 0.74 ± 0.13 (200 μM c9t11-CLA, P < 0.05) and 0.76 ± 0.13 (200 μM t10c12-CLA, P < 0.05); however, c9t11-CLA and t10c12-CLA increased the mRNA levels of SREBF isoforms and their target genes. In contrast, the mRNA expression of peroxisome-proliferator-activated receptor γ (PPARG) was strongly induced by ATRA alone but decreased by CLA isomers from 1.00 (ATRA alone) to 0.80 ± 0.06 (200 μM c9t11-CLA, P < 0.05) and 0.86 ± 0.06 (200 μM t10c12-CLA, P < 0.05). Overexpression of PPARγ in MCF-7 cells increased basal NIS-promoter activity, and treatment with the PPARγ ligand troglitazone stimulated ATRA-induced NIS-promoter activity. In conclusion, the results suggest that CLA isomers exert their effect on the expression of NIS by decreasing PPARG expression in MCF-7 cells.

Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells

The sodium/iodide symporter (NIS), which is essential for iodide concentration in the thyroid, is reported to be transcriptionally regulated by sterol regulatory element-binding proteins (SREBP) in rat FRTL-5 thyrocytes. The SREBP are strongly activated after parturition and throughout lactation in the mammary gland of cattle and are important for mammary epithelial cell synthesis of milk lipids. In this study, we tested the hypothesis that the NIS gene is regulated also by SREBP in mammary epithelial cells, in which NIS is functionally expressed during lactation. Regulation of NIS expression and iodide uptake was investigated by means of inhibition, silencing, and overexpression of SREBP and by reporter gene and DNA-binding assays. As a mammary epithelial cell model, the human MCF-7 cell line, a breast adenocarcinoma cell line, which shows inducible expression of NIS by all-trans retinoic acid (ATRA), and unlike bovine mammary epithelial cells, is widely used to investigate the regulation of mammary gland NIS and NIS-specific iodide uptake, was used. Inhibition of SREBP maturation by treatment with 25-hydroxycholesterol (5 µM) for 48h reduced ATRA (1 µM)-induced mRNA concentration of NIS and iodide uptake in MCF-7 cells by approximately 20%. Knockdown of SREBP-1c and SREBP-2 by RNA interference decreased the mRNA and protein concentration of NIS by 30 to 50% 48h after initiating knockdown, whereas overexpression of nuclear SREBP (nSREBP)-1c and nSREBP-2 increased the expression of NIS in MCF-7 cells by 45 to 60%, respectively, 48h after initiating overexpression. Reporter gene experiments with varying length of NIS promoter reporter constructs revealed that the NIS 5'-flanking region is activated by nSREBP-1c and nSREBP-2 approximately 1.5- and 4.5-fold, respectively, and activation involves a SREBP-binding motif (SRE) at -38 relative to the transcription start site of the NIS gene. Gel shift assays using oligonucleotides spanning either the wild-type or the mutated SRE at -38 of the NIS 5'-flanking region showed that in vitro-translated nSREBP-1c and nSREBP-2 bind only the wild-type but not the mutated SRE at -38 of NIS. Collectively, the present results from cell culture experiments with human mammary epithelial MCF-7 cells and from genetic studies show for the first time that the NIS gene and iodide uptake are regulated by SREBP in cultured human mammary epithelial cells. Future studies are necessary to clarify if the regulation of NIS expression and iodide uptake by SREBP also applies to the lactating bovine mammary epithelium.

Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells.

Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane-disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals.

Dynamic Transcription Factor Networks in Epithelial-Mesenchymal Transition in Breast Cancer Models

The epithelial-mesenchymal transition (EMT) is a complex change in cell differentiation that allows breast carcinoma cells to acquire invasive properties. EMT involves a cascade of regulatory changes that destabilize the epithelial phenotype and allow mesenchymal features to manifest. As transcription factors (TFs) are upstream effectors of the genome-wide expression changes that result in phenotypic change, understanding the sequential changes in TF activity during EMT provides rich information on the mechanism of this process. Because molecular interactions will vary as cells progress from an epithelial to a mesenchymal differentiation program, dynamic networks are needed to capture the changing context of molecular processes. In this study we applied an emerging high-throughput, dynamic TF activity array to define TF activity network changes in three cell-based models of EMT in breast cancer based on HMLE Twist ER and MCF-7 mammary epithelial cells. The TF array distinguished conserved from model-specific TF activity changes in the three models. Time-dependent data was used to identify pairs of TF activities with significant positive or negative correlation, indicative of interdependent TF activity throughout the six-day study period. Dynamic TF activity patterns were clustered into groups of TFs that change along a time course of gene expression changes and acquisition of invasive capacity. Time-dependent TF activity data was combined with prior knowledge of TF interactions to construct dynamic models of TF activity networks as epithelial cells acquire invasive characteristics. These analyses show EMT from a unique and targetable vantage and may ultimately contribute to diagnosis and therapy.

Cu(GlyGlyHis) effects on MCF7 cells: Copper uptake, reactive oxygen species generation and membrane topography changes

Elevated levels of copper have been detected in various types of human cancer cells, such as breast cancer cells, and a number of mechanisms have been proposed to explain the action and influence of copper on tumor progress. In this work, we found that stimulating the proliferation of mammary epithelial MCF7 cells with the high-redox-potential copper complex Cu (GlyGlyHis) is associated with the copper-induced intracellular generation of reactive oxygen species (ROS) that induces lipid peroxidation and causes increased roughness of external cell membranes, which leads to the formation of larger cell domes. The results presented herein provide new insights into the molecular link between copper and the proliferation of breast cancer cells and, consequently, into the mechanism by which changes in redox balance and ROS accumulation regulates cell membrane roughness.

P2.28 Antioxdant and Cytoprotective Effect of Plants Medicinals from Morrocco

ABSTRACT Production of reactive oxygen species (ROS) is heavily involved in the mechanisms of toxicity of chemotherapy drugs and thus in the generation of their side effects. The use of protective agents coupled to chemotherapy is therefore the object of intensive research for optimal use of anticancer drugs. The objective of this work is to select and isolate from natural substances, new molecules, with an antioxidant, protecting cells from non-cancerous, but do not affect the effectiveness of anticancer agents vis-a-vis tumor cells. The screening was used to select plants with potential antioxidant and antiradical: Delphinium Staphysagria, Berberis hispanica and Aristolochia Longa. These activities were evaluated by in vitro methods FRAP (Ferric Reducing Ability of Plasma) and DPPH (2,2-diphenyl-1-picrylhydrazyl) in comparison with reference molecules such as vitamin E (TroloxR) and BHT (butylated hydroxytoluene). The results indicate that extracts of three selected plants have high antioxidant properties, Berberis Hispanica in the range of 18·61 nmol Fe / mg of dry matter for the aqueous extract, 0·791 nmol Fe / mg of dry matter for ethyl acetate extract and 32·62 nmol Fe / mg of dry matter. This led us to fractionate the crude extracts of this plant. The phytochemical analysis and the use of flash chromatography on organic extracts were allowed to separate active and inactive fractions. Subsequently, the cytotoxicity of extracts of Berberis Hispanica was tested on cells of mammary adenocarcinoma MCF-7 mammary epithelial cells and noncancerous immortalized hTERT-hme1 using MTT assay. The three extracts (aqueous, ethyl acetate and methanol) have cytotoxicity comparable with IC50 of the order of 297·5 mg MS / mL, MS 8190mg / mL and 308 mg of MS / mL, respectively, for MCF-7,IC50 is on the order of 150mg of MS / mL, 8190 mg DM / mL and 300 mg MS / ml respectively for hTERT-hme1. The evaluation of the cytoprotective effect of extracts on the rate of intracellular ROS through the probe H2DCF-DA by flow cytometry to reveal that the methanol extract exerts a protective effect on the non-cancerous cell line hTERT-HEM1 against the oxidative stress induced by t-BHP with a decrease of 50·12 % of ROS.

Abstract LB-74: The estrogen receptor regulates the transcription of p53 target genes in breast cancer cells

Abstract Breast Cancer is a disease that affects thousands of women every year. A large subset of breast cancer is attributed to estrogen receptor (ER) dysregulation. Previously, our lab and others have determined the estradiol-stimulated ER cistrome in the MCF7 mammary epithelial cell line. To determine the relationship between ER and the apoptotic response, we compared our ER cistrome to a published p53 dataset. The p53 transcription factor is a tumor suppressor protein that is responsible for cell-cycle control and apoptosis. Our lab generated a doxorubicin-stimulated p53 cistrome in MCF7 cells, and we employed an integrative analysis approach examining the p53 and ER cistromes and gene expression microarray data to determine the genes coordinately regulated by both transcription factors. We identified a number of genes that are involved in the cell cycle, apoptosis, and DNA damage repair. By treating MCF7 cells with both stimuli, we found that the ER could inhibit the expression of a p53 target gene subset, thus diminishing the efficacy of the p53 program. These genes correlate with poor breast cancer clinical outcome. Moreover, we found that treatment of breast cancer cells with a combination of the anti-estrogen fulvestrant with p53 stimuli (i.e., doxorubicin and nutlin-3) leads to the synergistic enhancement of pro-apoptotic genes. Together, these data point to a set of genes essential for breast cell survival in ER-mediated breast cancer and suggest an improved paradigm for ER-positive breast cancer treatment i.e., the addition of fulvestrant with a p53 stabilizing drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-74. doi:1538-7445.AM2012-LB-74

P4-02-08: Obesity-Induced Aromatase Expression in the Breast Microenvironment Promotes Estrogen Receptor Activity Independent of Circulating Estradiol Levels.

Abstract Epidemiological studies indicate that obesity increases the risk of postmenopausal breast cancer by approximately 50%. In the past, researchers have hypothesized that elevated estrogen synthesis by the peripheral adipose tissue may be the principal mediator of breast tumorigenesis in this population, which primarily develops estrogen receptor alpha (ERα) positive breast cancer. However, obesity is also accompanied by an elevation in growth factor and cytokine signaling, and these pathways have been linked to tumorigenesis. In addition, certain growth factor and cytokine family members can promote aromatase expression in both the epithelial and stromal tumor compartments. Consequently, we hypothesized that obesity increases the risk of postmenopausal breast cancer via elevated aromatase expression and/or activity in the local mammary tissue. To test our hypothesis, we investigated how ERa activity in mammary epithelial cells was influenced by adipose stromal cells (ASC) cultured under obesity-associated conditions, including high cell density and exposure to elevated levels of circulating growth factors and cytokines. For the latter condition, sera was obtained from postmenopausal women, pooled by BMI category (lean: 18.5−24.9; obese: ≥30), and applied to the ASC, which were originally derived from women undergoing reduction mammoplasty. High ASC density was achieved via the protocol previously published by Dr. Li. Preliminary data indicated that both elevated cell density and sera from obese postmenopausal women induces greater aromatase expression in ASC, indicating that multiple factors may be contributing to the increased local aromatase expression seen with obesity. We are currently exploring the signaling pathways responsible for obesity's upregulation of ASC aromatase expression and will present these results at the meeting. Intriguingly, exposure to conditioned media from both the high density and obese sera-exposed ASC enhanced ERa activity in MCF-7 mammary epithelial cells, independent from exogenous estradiol but dependent on the presence of androgens, suggesting an important role for the aromatase enzyme in this observation. To expand on this finding, we plan to investigate the effect of ASC conditioned media on different markers of cancer aggression, including proliferation and survival, and assess the degree to which these effects depend on estradiol. Through further examination of obesity's impact on signaling pathways in both the epithelial and stromal tumor compartments, we ultimately hope to identify more effective chemopreventive and therapeutic regimens for the high-risk obese postmenopausal population. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-08.

Effects of sodium arsenite on the PI3K/AKT signaling pathway, PTEN and proliferation in MCF-7 mammary epithelial cells

Effects of sodium arsenite on the PI3K/AKT signaling pathway, PTEN and proliferation in MCF-7 mammary epithelial cells

Plasma Membrane Cholesterol Content Affects Nitric Oxide Diffusion Dynamics and Signaling

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO approximately 20-40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser(239) induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.

Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-foot-printing" demonstrated that CPD formation within a long run of Ts (T(18)-tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.

Dynamic and Differential in Vivo Modifications of the Isoform HMGA1a and HMGA1b Chromatin Proteins

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.

Inhibition of NMU-induced mammary tumorigenesis by dietary soy

We previously demonstrated that female Sprague–Dawley rats fed AIN-93G diets containing soy protein isolate (SPI +) had lower DMBA-induced mammary tumor incidence than those fed diets containing casein (CAS), due partly to altered Phase I metabolism with soy. Here, we evaluated the tumor protective effects of these same diets to the direct-acting carcinogen N-methyl-nitrosourea (NMU). Tumor incidence was reduced and tumor latency was enhanced, in NMU-administered female rats lifetime exposed to SPI +, relative to the CAS group. Tumor multiplicity did not differ with diet, while tumor grade tended to be more advanced with SPI +. Normal mammary glands of CAS and SPI + tumor-bearing rats had comparable proliferative and apoptotic status. However, mammary expression of HER-2/neu and progesterone receptor (PR) genes was higher for SPI + rats. Moreover, tumored SPI + rats had lower serum progesterone levels than those fed CAS, while serum estrogen did not differ. Serum from tumored SPI + rats had higher apoptotic activity towards mammary epithelial MCF-7 cells, than CAS serum. Thus, dietary soy protects against mammary tumorigenesis induced by a direct-acting carcinogen and alters signaling pathways involving PR and HER-2/neu.

Cell-Type-Dependent Effect of Transforming Growth Factor β, a Major Cytokine in Breast Milk, on Human Immunodeficiency Virus Type 1 Infection of Mammary Epithelial MCF-7 Cells or Macrophages

Breastfeeding plays a substantial role in mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1). Mammary epithelial cells, as well as macrophages and lymphocytes, are thought to serve as sources of the virus in breast milk. Soluble factors in breast milk exert various biological functions, including immune tolerance or immune modulation, and may influence milk-borne infection with HIV-1. In this study we show that transforming growth factor beta (TGF-beta), a major cytokine in breast milk, inhibited HIV-1 infection of mammary epithelial MCF-7 cells but enhanced that of macrophages. TGF-beta downregulated the HIV-1 long terminal repeat (LTR) promoter in MCF-7 cells but upregulated it in macrophages. Stimulation with TGF-beta suppressed NF-kappaB binding to the HIV-1 LTR in MCF-7 cells, at least in part by downregulating induced IkappaB kinase expression. Cell type-dependent effects of TGF-beta on HIV-1 expression may play a role in milk-borne infection with HIV-1.

Brca1 inactivation induces p27(Kip1)-dependent cell cycle arrest and delayed development in the mouse mammary gland.

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin-cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.

ZEB negatively regulates the lytic-switch BZLF1 gene promoter of Epstein-Barr virus.

Epstein-Barr virus (EBV) is a human herpesvirus capable of establishing a latent state in B lymphocytes. The product of the immediate-early BZLF1 gene, Zta, is a transcriptional transactivator essential for viral DNA amplification and virion production. Previously, we identified a negative cis-acting element within the BZLF1 promoter termed ZV. ZV contains the sequence 5'-CAGGTA-3' located at nucleotides -17 to -12 relative to the transcription initiation site. It sequence specifically binds a cellular factor, ZVR. Based on sequence binding specificity, we postulated that ZVR may be zinc finger E-box binding factor (ZEB) or a related zinc finger/homeodomain family member. We show here by immunoshift assays that ZVR and human ZEB specifically cross-react with an antibody to deltaEF1, the chicken homolog of ZEB. Competition electrophoretic mobility shift assays confirmed that ZEB binds to the ZV element with the same binding specificity as ZVR. Overexpression of ZEB in either B-lymphocytic DG75 cells or mammary epithelial MCF-7 cells repressed Zta-induced activation of the BZLF1 promoter four- to fivefold via the ZV site. Thus, we conclude that the previously identified cellular repressor ZVR is, in fact, ZEB. We also present evidence that other cellular factors likely affect the transcriptional activity of ZEB. Lastly, we identify a ZEB-binding site within the promoter of the lytic BRLF1 gene of EBV. We postulate that ZEB likely plays an important role in regulating the life cycle of EBV.

Expression of uterine and cervical epithelial cadherin during relaxin-induced growth in pigs

Epithelial cadherin (E-cadherin), a member of the cadherin family of calcium-dependent adhesion molecules, is present in reproductive tissues. Relaxin, a hormone important for uterine and cervical growth in pigs, increases the expression of E-cadherin in the MCF-7 mammary epithelial cell line. The objective of this study was to characterize the expression of E-cadherin during relaxin-induced growth of the uterus and cervix in an immature pig model, independent of high circulating steroids. After administration of relaxin to prepubertal gilts, the uterus and cervix were collected. E-cadherin mRNA and protein were measured by northern and western blot analysis, respectively. A 120 kDa protein band, corresponding to E-cadherin, was detected in all tissues examined. Relaxin significantly (P < 0.05) increased the amount of E-cadherin protein in the uterus (P < 0.05), whereas no significant changes were observed in E-cadherin protein in the cervix. A 4.2 kb E-cadherin transcript was detected in all tissues and E-cadherin mRNA was significantly higher (P < 0.05) in uteri from relaxin-treated gilts compared with control gilts. E-cadherin was localized by immunocytochemistry to the epithelial cells of the uterine and cervical lumen, and the uterine glandular epithelium. Quantitative analysis revealed that administration of relaxin significantly increased (P < 0.05) the height of the uterine luminal epithelium compared with that of the controls. This is the first report of the expression of E-cadherin in the uterus and cervix of pigs. The findings from this study indicate that relaxin increases the expression of uterine E-cadherin in the reproductive tract of pigs. Administration of relaxin to prepubertal gilts in vivo increased uterine epithelial cell growth independent of circulating steroids, with a concomitant increase in E-cadherin expression.

Expression of uterine and cervical epithelial cadherin during relaxin-induced growth in pigs

Epithelial cadherin (E-cadherin), a member of the cadherin family of calcium-dependent adhesion molecules, is present in reproductive tissues. Relaxin, a hormone important for uterine and cervical growth in pigs, increases the expression of E-cadherin in the MCF-7 mammary epithelial cell line. The objective of this study was to characterize the expression of E-cadherin during relaxin-induced growth of the uterus and cervix in an immature pig model, independent of high circulating steroids. After administration of relaxin to prepubertal gilts, the uterus and cervix were collected. E-cadherin mRNA and protein were measured by northern and western blot analysis, respectively. A 120 kDa protein band, corresponding to E-cadherin, was detected in all tissues examined. Relaxin significantly (P < 0.05) increased the amount of E-cadherin protein in the uterus (P < 0.05), whereas no significant changes were observed in E-cadherin protein in the cervix. A 4.2 kb E-cadherin transcript was detected in all tissues and E-cadherin mRNA was significantly higher (P < 0.05) in uteri from relaxin-treated gilts compared with control gilts. E-cadherin was localized by immunocytochemistry to the epithelial cells of the uterine and cervical lumen, and the uterine glandular epithelium. Quantitative analysis revealed that administration of relaxin significantly increased (P < 0.05) the height of the uterine luminal epithelium compared with that of the controls. This is the first report of the expression of E-cadherin in the uterus and cervix of pigs. The findings from this study indicate that relaxin increases the expression of uterine E-cadherin in the reproductive tract of pigs. Administration of relaxin to prepubertal gilts in vivo increased uterine epithelial cell growth independent of circulating steroids, with a concomitant increase in E-cadherin expression.

Identification of Activating Transcription Factor 4 (ATF4) as an Nrf2-interacting Protein: IMPLICATION FOR HEME OXYGENASE-1 GENE REGULATION

Nrf2 regulates expression of genes encoding enzymes with antioxidant (e.g. heme oxygenase-1 (HO-1)) or xenobiotic detoxification (e.g. NAD(P)H:quinone oxidoreductase, glutathione S-transferase) functions via the stress- or antioxidant-response elements (StRE/ARE). Nrf2 heterodimerizes with small Maf proteins, but the role of such dimers in gene induction is controversial, and other partners may exist. By using the yeast two-hybrid assay, we identified activating transcription factor (ATF) 4 as a potential Nrf2-interacting protein. Association between Nrf2 and ATF4 in mammalian cells was confirmed by co-immunoprecipitation and mammalian two-hybrid assays. Furthermore, Nrf2.ATF4 dimers bound to an StRE sequence from the ho-1 gene. CdCl(2), a potent inducer of HO-1, increased expression of ATF4 in mouse hepatoma cells, and detectable induction of ATF4 protein preceded that of HO-1 (30 min versus 2 h). A dominant-negative mutant of ATF4 inhibited basal and CdCl(2)-stimulated expression of a StRE-dependent/luciferase fusion construct (pE1-luc) in hepatoma cells but only basal expression in mammary epithelial MCF-7 cells. A dominant mutant of Nrf2 was equally inhibitory in both cell types in the presence or absence of CdCl(2). These results indicate that ATF4 regulates basal and CdCl(2)-induced expression of the ho-1 gene in a cell-specific manner and possibly in a complex with Nrf2.