Sequence-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. 17β-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.

Most 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.

Most 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.

Sequence-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. 17β-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.

TiPARP is a PARP-like mART that is induced by and negatively regulates AHR transactivation. Despite these insights, not much is known about TiPARP. This study aimed to characterize the regulation of TiPARP by AHR, PDGFR, and ERα, and investigate potential receptor interplay. Gene expression studies revealed that coactivation of AHR and PDGFR can enhance TiPARP expression after 3 h relative to activation of either receptor pathway alone. Gene expression and ChIP studies demonstrated that while co-activation of AHR and ER enhanced AHR, ARNT, and ERα recruitment to the regulatory region of TiPARP, TiPARP mRNA levels were not potentiated by co-activation relative to activation of either pathway. Dissection of the 5’ regulatory region of TiPARP using reporter gene assays revealed that a putative AHRE cluster and an ERE half-site were functional. Lastly, overexpression of TiPARP with an estrogen-responsive reporter revealed that TiPARP can repress ERα signalling and requires its catalytic activity.

TiPARP is a PARP-like mART that is induced by and negatively regulates AHR transactivation. Despite these insights, not much is known about TiPARP. This study aimed to characterize the regulation of TiPARP by AHR, PDGFR, and ERα, and investigate potential receptor interplay. Gene expression studies revealed that coactivation of AHR and PDGFR can enhance TiPARP expression after 3 h relative to activation of either receptor pathway alone. Gene expression and ChIP studies demonstrated that while co-activation of AHR and ER enhanced AHR, ARNT, and ERα recruitment to the regulatory region of TiPARP, TiPARP mRNA levels were not potentiated by co-activation relative to activation of either pathway. Dissection of the 5’ regulatory region of TiPARP using reporter gene assays revealed that a putative AHRE cluster and an ERE half-site were functional. Lastly, overexpression of TiPARP with an estrogen-responsive reporter revealed that TiPARP can repress ERα signalling and requires its catalytic activity.

Orientador: Flávia Karina Delella / Resumo: Células tronco mesenquimais (CTMs) possuem papel relevante na manutenção da homeostase e reparação em casos de lesão tecidual através da renovação do estoque celular. Tais células são implicadas na medicina regenerativa e terapia celular como fontes promissoras, sendo o tecido adiposo uma rica e relevante fonte dessas células. Nesse sentido, as células tronco mesenquimais derivadas de tecido adiposo (CTMs- TA) surgem como alternativa segura em estudos pré-clínicos e clínicos. As chamadas “Boas Práticas em Cultura Celular” (do inglês Good Manufacturing Practices - GMPs) tem sido utilizadas como uma nova abordagem para obtenção de controle de qualidade para fins terapêuticos. Neste contexto, o controle toxicológico dessas células surgem como uma questão relevante. Considera-se que os disruptores endócrinos mimetizam a ação de certos hormônios endógenos. Dessa forma, o BPA é conhecido por mimetizar o receptor de estrógeno e também causar alterações no processo de adipogênese. Já o TCDD possui afinidade com o receptor aril hidrocarboneto (AhR), assim como interfere através das vias de sinalização de estrógenos e andrógenos. Desta maneira, o presente estudo teve como objetivo geral testar o potencial de diferenciação das CTMs- TA humanas e de rato na presença dos compostos BPA e TCDD. Os objetivos específicos foram: a quantificação de proteínas relacionadas ao fenótipo de células- tronco, bem como de receptores específicos de ligação desses compostos, testes para verificação do po... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mesenchymal stem cells (CTMs) plays a relevant role in the maintenance of homeostasis and repair in cases of tissue injury through cell stock renovation. Such cells are implicated in regenerative medicine and cell therapy as promising sources, been the adipose tissue a rich and relevant source. In this sense, mesenchymal stem cells derived from adipose tissue (ASCs) appears to be a safe alternative in preclinical and clinical studies. The so- called Good Manufacturing Practices (GMPs) have been used as a new approach to obtaining quality control for therapeutic purposes. In this context, the toxicological control approach becomes a relevant issue. Endocrine disrupting chemicals are thought to mimic the action of certain endogenous hormones. Thus, BPA is known to mimic the estrogen receptor and also cause changes in the process of adipogenesis. TCDD, on the other hand, has affinity with the aryl hydrocarbon receptor (AhR), as well as interfering with the estrogen and androgen signaling pathways. In this way, the present study had the rationale to test the differentiation potencial of human and rat MSCs in the presence of BPA and TCDD. The specific objectives were: quantification of proteins related to the stem cell phenotype, as well as specific binding receptors of these compounds, verification of apoptotic and genotoxic profile of these substances and also the quantification of the potential of adipogenic and osteogenic differentiation of these cells when exposed. First, ASC... (Complete abstract click electronic access below) / Doutor

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The anteroventral periventricular nucleus (AVPV) is a sexually dimorphic preoptic structure that is nearly three times larger in the female rat. Estradiol (E2) exposure during development decreases AVPV volume through apoptosis, a process which normally occurs preferentially in male. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disrupter that interferes with E2-dependent sexual differentiation of the AVPV. Whole-genome microarrays were used to identify sex-specific genes regulated by E2 and TCDD in postnatal day 2 (P2) AVPV punches from untreated males and females, males treated with TCDD or vehicle, and females treated with E2 or vehicle. Trio emerged as a gene target regulated by E2 and TCDD, and this gene is essential for neurite outgrowth which is sexually dimorphic in the AVPV. My goal was to verify microarray data that Trio was expressed in the P2 AVPV and to test whether expression was affected by sex, E2 and TCDD. The microarray did not discriminate among Trio splice variants, 9S, 9L and Duet, so I mapped each of these in the P2 AVPV using in situ hybridization. I used quantitative real-time PCR to examine the effects of sex, E2 and TCDD on mRNA levels encoding each splice variant. I found that the AVPV expressed 9S and 9L at moderate levels and Duet at low levels. The expression of 9S and 9L mRNA was increased by E2 in females. However, only 9S expression differed between sexes and it was lower in males. TCDD had no effect on expression of any of the splice variants. This work provides the first evidence that Trio splice variants are independently regulated and that Trio may mediate effects of E2 in the developing AVPV.

No description available.