Browsing by Subject "TCDD"
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Item Characterization of serum-induced CYP1A1 expression and activity in mouse embryo fibroblasts(2005-07-07) Carmen-Veronica Naira Obianwu; Cornelis Elferink Ph.D.; Xiaodong Cheng Ph.D.; Jonathan B. Ward Ph.D.The AhR is a ligand-activated transcription factor that mediates the toxic effects of environmental contaminants that include TCDD. Using a TCDD dose-response treatment in MEFs, we observed a super induction of CYP1A1 with newborn calf serum (NCS) in the presence (10nM/15nM) of TCDD. In addition to NCS, fetal bovine serum (FBS) also has the capability to yield a CYP1A1 super induction. These results suggest that components within the sera affect the activity of the AhR and consequent CYP1A1 expression. To pursue this idea, characterization of the serum factors were investigated. The findings indicated that serum factor(s) in both sera are heat sensitive at 50◦C, withstand removal from charcoal stripping sera and are ¡Ý 10,000 kDa in size. Using RT-PCR, we found that NCS factors only, could super induce CYP1A1 at the gene level. Moreover, MEFs are the only cells observed in this study that are susceptible to CYP1A1 super induction.Item Gene silencing in cancer cells using siRNA : genetic and functional studies(Texas A&M University, 2004-09-30) Abdel Rahim, Ma'en AhmadSequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17?-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 ? S phase progression. Surprisingly, TCDD alone induced G0/G1 ? S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 ? S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 ? S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.Item Inhibitory actions of Ah receptor agonists and indole-containing compounds in breast cancer cell lines and mouse models(Texas A&M University, 2005-08-29) Walker, Kelcey Manae BeckerThe aryl hydrocarbon receptor (AhR) binds synthetic and chemoprotective phytochemicals, and research in this laboratory has developed selective AhR modulators (SAhRMs) for treatment of breast cancer. Activation of the AhR through agonists such as TCDD inhibits hormone activation of several E2-responsive genes in breast cancer cell lines. In this study, inhibition of E2-induced proliferation and gene expression by TCDD has been investigated in the uterus of wildtype, ERKO and AhRKO mice. Cyclin D1, DNA polymerase ?, and VEGF mRNA levels are induced by E2 through ER? in the uterus as determined by in situ hybridization studies. TCDD down-regulated E2-induced cyclin D1 and DNA polymerase ? expression, but not E2-induced VEGF expression, in wild-type mice, but not AhRKO mice, confirming the role of the AhR. Furthermore, protein synthesis was not necessary for induction of cyclin D1 or DNA polymerase ?gene expression by E2 or inhibition of these responses by TCDD. Therefore, AhR-ER? crosstalk directly regulates the expression of genes involved in cell proliferation in vivo. AhR agonists induce down-regulation of ErbB family receptors in multiple tissues/organs suggesting possible inhibitory interactions with chemotherapeutic potential. Recently, it has been reported that the SAhRM 1,1??,2,2??-tetramethyldiindolylmethane inhibited DMBA-induced mammary tumor growth in rats and also inhibited MAPK and PI3-K pathways in human breast cancer cells. BT-474 and MDA-MB-453 cell lines are ErbB2-overexpressing breast cancer cells that express functional AhR and exhibit constitutive activation of MAPK and PI3-K pathways. Therefore, 1,1??,2,2??-tetramethyldiindolylmethane-induced inhibition of ErbB2 signaling was investigated in these cells lines and in the MMTV-c-neu mouse mammary tumor model, which overexpresses ErbB2 in the mammary gland. The growth of ErbB2 overexpressing cell lines and mammary tumors was inhibited by 1,1??,2,2??-tetramethyldiindolylmethane; however, modulation of MAPK or PI3-K pathways and cell cycle proteins nor induction of apoptosis by 1,1',2,2'-tetramethyldiindolylmethane was observed in the ErbB2overexpressing cell lines. Current studies are investigating mitochondrial effects of 1,1??,2,2??-tetramethyldiindolylmethane in the ErbB2-overexpressing cell lines, as well as continuing studies on gene expression profiles in the mammary glands of MMTV-c-neu mice to better understand and identify critical genes that are responsible for ErbB2-mediated transformation and growth of cancer cells/tumors.Item MOLECULAR INSIGHTS AND PHYSIOLOGICAL CONSEQUENCES OF ARYL HYDROCARBON RECEPTOR REGULATED PLASMINOGEN ACTIVATOR INHIBITOR-1 EXPRESSION(2013-06-03) Wilson, Shelly; Elferink, Cornelis; Barton, Michelle; Cicalese, Luca; Boor, Paul; Papaconstantinou, JohnThe aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, attenuates liver regeneration in vivo when activated by its prototypical agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) following 70% partial hepatectomy (PH). One reported target of the AhR that may account for suppression of the regenerative response is plasminogen activator inhibitor-1 (PAI-1), which negatively regulates the cleavage and activation of hepatocyte growth factor (HGF) from its latent form in the extracellular matrix. Once activated, HGF signalling through its receptor cMet is a crucial component early in regeneration. Recent studies identified a sequence distinct from the canonical AhR binding site, the ncXRE, which confers TCDD-inducible expression to the PAI-1 promoter. Since the ncXRE shares partial sequence homology with the Kruppel-like factor 6 (KLF6) consensus binding site; I hypothesize that the AhR interacts with KLF6 at the ncXRE, inducing transcription of PAI-1, suppressing HGF processing and its activation of cMet, inhibiting liver regeneration. To test this hypothesis, coimmunoprecipitation on liver nuclear extracts and recombinant proteins confirmed that KLF6 and the AhR interact, likely dependent on the C-terminus transactivating domain of AhR and the DNA binding domain of KLF6. Both proteins bind the ncXRE in vitro and deletion analyses revealed that the N-terminal 27 amino acids of hKLF6 were required for complex formation. Chromatin immunoprecipitation studies demonstrated that the AhR and KLF6 bind to the PAI-1 promoter in vivo. To assess the effects of AhR activation in vivo, C57BL/6 and PAI-1-/- mice were pretreated with TCDD, underwent PH, and liver samples and serum were collected at multiple time points post-PH to monitor PAI-1 expression, HGF processing, and cMet phosphorylation (activation) and DNA synthesis in the liver. I found that PAI-1 transcript and corresponding serum PAI-1 protein levels were markedly increased in TCDD-pretreated C57BL/6 mice, and this rise in PAI-1 levels inversely correlated to HGF processing and cMet phosphorylation. Hepatocytes in the periportal region of PAI-1-/- mice were able to overcome TCDD-mediated suppression of regeneration. The AhR-KLF6 interaction at the PAI-1 promoter, resulting in increased PAI-1 expression and decreased HGF processing and cMet activation, reveals a novel mechanism by which the AhR may contribute to liver homeostasis.Item Timing Matters: The Role of Circadian Clock Genes In Development and Toxin Responses(2009-05-15) Qu, XiaoyuMost members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.