RESUMO
RATIONALE: BMP9 (bone morphogenetic protein 9) is a circulating endothelial quiescence factor with protective effects in pulmonary arterial hypertension (PAH). Loss-of-function mutations in BMP9, its receptors, and downstream effectors have been reported in heritable PAH. OBJECTIVES: To determine how an acquired deficiency of BMP9 signaling might contribute to PAH. METHODS: Plasma levels of BMP9 and antagonist soluble endoglin were measured in group 1 PAH, group 2 and 3 pulmonary hypertension (PH), and in patients with severe liver disease without PAH. MEASUREMENTS AND MAIN RESULTS: BMP9 levels were markedly lower in portopulmonary hypertension (PoPH) versus healthy control subjects, or other etiologies of PAH or PH; distinguished PoPH from patients with liver disease without PAH; and were an independent predictor of transplant-free survival. BMP9 levels were decreased in mice with PH associated with CCl4-induced portal hypertension and liver cirrhosis, but were normal in other rodent models of PH. Administration of ALK1-Fc, a BMP9 ligand trap consisting of the activin receptor-like kinase-1 extracellular domain, exacerbated PH and pulmonary vascular remodeling in mice treated with hypoxia versus hypoxia alone. CONCLUSIONS: BMP9 is a sensitive and specific biomarker of PoPH, predicting transplant-free survival and the presence of PAH in liver disease. In rodent models, acquired deficiency of BMP9 signaling can predispose to or exacerbate PH, providing a possible mechanistic link between PoPH and heritable PAH. These findings describe a novel experimental model of severe PH that provides insight into the synergy between pulmonary vascular injury and diminished BMP9 signaling in the pathogenesis of PAH.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Hipertensão Portal/metabolismo , Hipertensão Portal/fisiopatologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hepatopatias/metabolismo , Hepatopatias/fisiopatologia , Adulto , Biomarcadores/sangue , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Although biological effects of endocrine disrupting chemicals (EDCs) are often observed at unexpectedly low doses with occasional nonmonotonic dose-response characteristics, transcriptome-wide profiles of sensitivities or dose-dependent behaviors of the EDC responsive genes have remained unexplored. Here, we describe expressome analysis for the comprehensive examination of dose-dependent gene responses and its applications to characterize estrogen responsive genes in MCF-7 cells. Transcriptomes of MCF-7 cells exposed to varying concentrations of representative natural and xenobiotic estrogens for 48 h were determined by microarray and used for computational calculation of interpolated approximations of estimated transcriptomes for 300 doses uniformly distributed in log space for each chemical. The entire collection of these estimated transcriptomes, designated as the expressome, has provided unique opportunities to profile chemical-specific distributions of ligand sensitivities for large numbers of estrogen responsive genes, revealing that at low concentrations estrogens generally tended to suppress rather than to activate transcription. Gene ontology analysis demonstrated distinct functional enrichment between high- and low-sensitivity estrogen responsive genes, supporting the notion that a single EDC chemical can cause qualitatively distinct biological responses at different doses. Expressomal heatmap visualization of dose-dependent induction of Bisphenol A inducible genes showed a weak gene activation peak at a very low concentration range (ca. 0.1 nM) in addition to the main, strong gene activation peak at and above 100 nM. Thus, expressome analysis is a powerful approach to understanding the EDC dose-dependent dynamic changes in gene expression at the transcriptomal level, providing important information on the overall profiles of ligand sensitivities and nonmonotonic responses.
Assuntos
Disruptores Endócrinos/toxicidade , Estrogênios/toxicidade , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Compostos Benzidrílicos , Relação Dose-Resposta a Droga , Ontologia Genética , Humanos , Células MCF-7 , Análise em Microsséries , FenóisRESUMO
In Huntington's disease (HD), the size of the expanded HTT CAG repeat mutation is the primary driver of the processes that determine age at onset of motor symptoms. However, correlation of cellular biochemical parameters also extends across the normal repeat range, supporting the view that the CAG repeat represents a functional polymorphism with dominant effects determined by the longer allele. A central challenge to defining the functional consequences of this single polymorphism is the difficulty of distinguishing its subtle effects from the multitude of other sources of biological variation. We demonstrate that an analytical approach based upon continuous correlation with CAG size was able to capture the modest (â¼21%) contribution of the repeat to the variation in genome-wide gene expression in 107 lymphoblastoid cell lines, with alleles ranging from 15 to 92 CAGs. Furthermore, a mathematical model from an iterative strategy yielded predicted CAG repeat lengths that were significantly positively correlated with true CAG allele size and negatively correlated with age at onset of motor symptoms. Genes negatively correlated with repeat size were also enriched in a set of genes whose expression were CAG-correlated in human HD cerebellum. These findings both reveal the relatively small, but detectable impact of variation in the CAG allele in global data in these peripheral cells and provide a strategy for building multi-dimensional data-driven models of the biological network that drives the HD disease process by continuous analysis across allelic panels of neuronal cells vulnerable to the dominant effects of the HTT CAG repeat.
Assuntos
Expressão Gênica , Doença de Huntington/genética , Proteínas do Tecido Nervoso/genética , Repetições de Trinucleotídeos/genética , Idade de Início , Alelos , Linhagem Celular , Cerebelo/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Proteína Huntingtina , Doença de Huntington/diagnóstico , Doença de Huntington/metabolismo , Masculino , Modelos Genéticos , Polimorfismo Genético , Reprodutibilidade dos Testes , TranscriptomaRESUMO
Huntington's disease is initiated by the expression of a CAG repeat-encoded polyglutamine region in full-length huntingtin, with dominant effects that vary continuously with CAG size. The mechanism could involve a simple gain of function or a more complex gain of function coupled to a loss of function (e.g. dominant negative-graded loss of function). To distinguish these alternatives, we compared genome-wide gene expression changes correlated with CAG size across an allelic series of heterozygous CAG knock-in mouse embryonic stem (ES) cell lines (Hdh(Q20/7), Hdh(Q50/7), Hdh(Q91/7), Hdh(Q111/7)), to genes differentially expressed between Hdh(ex4/5/ex4/5) huntingtin null and wild-type (Hdh(Q7/7)) parental ES cells. The set of 73 genes whose expression varied continuously with CAG length had minimal overlap with the 754-member huntingtin-null gene set but the two were not completely unconnected. Rather, the 172 CAG length-correlated pathways and 238 huntingtin-null significant pathways clustered into 13 shared categories at the network level. A closer examination of the energy metabolism and the lipid/sterol/lipoprotein metabolism categories revealed that CAG length-correlated genes and huntingtin-null-altered genes either were different members of the same pathways or were in unique, but interconnected pathways. Thus, varying the polyglutamine size in full-length huntingtin produced gene expression changes that were distinct from, but related to, the effects of lack of huntingtin. These findings support a simple gain-of-function mechanism acting through a property of the full-length huntingtin protein and point to CAG-correlative approaches to discover its effects. Moreover, for therapeutic strategies based on huntingtin suppression, our data highlight processes that may be more sensitive to the disease trigger than to decreased huntingtin levels.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas Nucleares/biossíntese , Peptídeos/metabolismo , Expansão das Repetições de Trinucleotídeos , Alelos , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Estudo de Associação Genômica Ampla , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/terapia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genéticaRESUMO
Huntington's disease (HD) involves marked early neurodegeneration in the striatum, whereas the cerebellum is relatively spared despite the ubiquitous expression of full-length mutant huntingtin, implying that inherent tissue-specific differences determine susceptibility to the HD CAG mutation. To understand this tissue specificity, we compared early mutant huntingtin-induced gene expression changes in striatum to those in cerebellum in young Hdh CAG knock-in mice, prior to onset of evident pathological alterations. Endogenous levels of full-length mutant huntingtin caused qualitatively similar, but quantitatively different gene expression changes in the two brain regions. Importantly, the quantitatively different responses in the striatum and cerebellum in mutant mice were well accounted for by the intrinsic molecular differences in gene expression between the striatum and cerebellum in wild-type animals. Tissue-specific gene expression changes in response to the HD mutation, therefore, appear to reflect the different inherent capacities of these tissues to buffer qualitatively similar effects of mutant huntingtin. These findings highlight a role for intrinsic quantitative tissue differences in contributing to HD pathogenesis, and likely to other neurodegenerative disorders exhibiting tissue-specificity, thereby guiding the search for effective therapeutic interventions.
Assuntos
Cerebelo/patologia , Doença de Huntington/genética , Neostriado/patologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Ataxina-1 , Ataxinas , Biomarcadores/metabolismo , Cerebelo/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Doença de Huntington/patologia , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Neostriado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Transdução de Sinais/genéticaRESUMO
Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.
Assuntos
Aromatase/genética , Proteínas Aviárias/genética , Galinhas/genética , Animais , Embrião de Galinha , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Transtornos do Desenvolvimento Sexual/induzido quimicamente , Transtornos do Desenvolvimento Sexual/embriologia , Epigênese Genética/efeitos dos fármacos , Etinilestradiol/administração & dosagem , Feminino , Feminização/induzido quimicamente , Feminização/embriologia , Marcadores Genéticos , Masculino , Ovário/efeitos dos fármacos , Ovário/embriologia , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Processos de Determinação Sexual , Diferenciação Sexual/efeitos dos fármacos , Diferenciação Sexual/genética , Testículo/efeitos dos fármacos , Testículo/embriologiaRESUMO
Yin Yang 1 (YY1) is a critical transcription factor controlling cell proliferation, development and DNA damage responses. Retrotranspositions have independently generated additional YY family members in multiple species. Although Drosophila YY1 [pleiohomeotic (Pho)] and its homolog [pleiohomeotic-like (Phol)] redundantly control homeotic gene expression, the regulatory contributions of YY1-homologs have not yet been examined in other species. Indeed, targets for the mammalian YY1 homolog YY2 are completely unknown. Using gene set enrichment analysis, we found that lentiviral constructs containing short hairpin loop inhibitory RNAs for human YY1 (shYY1) and its homolog YY2 (shYY2) caused significant changes in both shared and distinguishable gene sets in human cells. Ribosomal protein genes were the most significant gene set upregulated by both shYY1 and shYY2, although combined shYY1/2 knock downs were not additive. In contrast, shYY2 reversed the anti-proliferative effects of shYY1, and shYY2 particularly altered UV damage response, platelet-specific and mitochondrial function genes. We found that decreases in YY1 or YY2 caused inverse changes in UV sensitivity, and that their combined loss reversed their respective individual effects. Our studies show that human YY2 is not redundant to YY1, and YY2 is a significant regulator of genes previously identified as uniquely responding to YY1.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Reação em Cadeia da Polimerase , Interferência de RNA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Raios Ultravioleta , Fator de Transcrição YY1/antagonistas & inibidores , Fator de Transcrição YY1/genéticaRESUMO
Emergence of antiestrogen-resistant cells in MCF-7 cells during suppression of estrogen signaling is a widely accepted model of acquired breast cancer resistance to endocrine therapy. To obtain insight into the genomic basis of endocrine therapy resistance, we characterized MCF-7 monoclonal sublines that survived 21-day exposure to tamoxifen (T-series sublines) or fulvestrant (F-series sublines) and sublines unselected by drugs (U-series). All T/F-sublines were resistant to the cytocidal effects of both tamoxifen and fulvestrant. However, their responses to the cytostatic effects of fulvestrant varied greatly, and their remarkably diversified morphology showed no correlation with drug resistance. mRNA expression profiles of the U-sublines differed significantly from those of the T/F-sublines, whose transcriptomal responsiveness to fulvestrant was largely lost. A set of genes strongly expressed in the U-sublines successfully predicted metastasis-free survival of breast cancer patients. Most T/F-sublines shared highly homogeneous genomic DNA aberration patterns that were distinct from those of the U-sublines. Genomic DNA of the U-sublines harbored many aberrations that were not found in the T/F-sublines. These results suggest that the T/F-sublines are derived from a common monoclonal progenitor that lost transcriptomal responsiveness to antiestrogens as a consequence of genetic abnormalities many population doublings ago, not from the antiestrogen-sensitive cells in the same culture during the exposure to antiestrogens. Thus, the apparent acquisition of antiestrogen resistance by MCF-7 cells reflects selection of preexisting drug-resistant subpopulations without involving changes in individual cells. Our results suggest the importance of clonal selection in endocrine therapy resistance of breast cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Moduladores de Receptor Estrogênico/farmacologia , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Mapeamento Cromossômico , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Citometria de Fluxo , Fulvestranto , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais , Análise de Sequência com Séries de Oligonucleotídeos , Tamoxifeno/farmacologiaRESUMO
Induction of mRNA for BIK proapoptotic protein by doxorubicin or gamma-irradiation requires the DNA-binding transcription factor activity of p53. In MCF7 cells, pure antiestrogen fulvestrant also induces BIK mRNA and apoptosis. Here, we provide evidence that, in contrast to doxorubicin or gamma-irradiation, fulvestrant induction of BIK mRNA is not a direct effect of the transcriptional activity of p53, although p53 is necessary for this induction. It is known that p53 up-regulated modulator of apoptosis (PUMA) mRNA is induced directly by the transcriptional activity of p53. Whereas gamma-irradiation induced both BIK and PUMA mRNA, only BIK mRNA was induced by fulvestrant. Whereas both fulvestrant and doxorubicin induced BIK mRNA, only doxorubicin enhanced the DNA-binding activity of p53 and induced PUMA mRNA. Small interfering RNA (siRNA) suppression of p53 expression as well as overexpression of dominant-negative p53 effectively inhibited the fulvestrant induction of BIK mRNA, protein, and apoptosis. Transcriptional activity of a 2-kb BIK promoter, which contained an incomplete p53-binding sequence, was not affected by fulvestrant when tested by reporter assay. Fulvestrant neither affected the stability of the BIK mRNA transcripts. Interestingly, other human breast cancer cells, such as ZR75-1, constitutively expressed BIK mRNA even without fulvestrant. In these cells, however, BIK protein seemed to be rapidly degraded by proteasome, and siRNA suppression of BIK in ZR75-1 cells inhibited apoptosis induced by MG132 proteasome inhibitor. These results suggest that expression of BIK in human breast cancer cells is regulated at the mRNA level by a mechanism involving a nontranscriptional activity of p53 and by proteasomal degradation of BIK protein.
Assuntos
Antineoplásicos Hormonais/farmacologia , Proteínas Reguladoras de Apoptose/biossíntese , Neoplasias da Mama/genética , Estradiol/análogos & derivados , Proteínas de Membrana/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Estradiol/farmacologia , Fulvestranto , Raios gama , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Leupeptinas/farmacologia , Proteínas de Membrana/genética , Proteínas Mitocondriais , Regiões Promotoras Genéticas , Inibidores de Proteassoma , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/biossíntese , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: The length of the huntingtin (HTT) CAG repeat is strongly correlated with both age at onset of Huntington's disease (HD) symptoms and age at death of HD patients. Dichotomous analysis comparing HD to controls is widely used to study the effects of HTT CAG repeat expansion. However, a potentially more powerful approach is a continuous analysis strategy that takes advantage of all of the different CAG lengths, to capture effects that are expected to be critical to HD pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: We used continuous and dichotomous approaches to analyze microarray gene expression data from 107 human control and HD lymphoblastoid cell lines. Of all probes found to be significant in a continuous analysis by CAG length, only 21.4% were so identified by a dichotomous comparison of HD versus controls. Moreover, of probes significant by dichotomous analysis, only 33.2% were also significant in the continuous analysis. Simulations revealed that the dichotomous approach would require substantially more than 107 samples to either detect 80% of the CAG-length correlated changes revealed by continuous analysis or to reduce the rate of significant differences that are not CAG length-correlated to 20% (nâ=â133 or nâ=â206, respectively). Given the superior power of the continuous approach, we calculated the correlation structure between HTT CAG repeat lengths and gene expression levels and created a freely available searchable website, "HD CAGnome," that allows users to examine continuous relationships between HTT CAG and expression levels of â¼20,000 human genes. CONCLUSIONS/SIGNIFICANCE: Our results reveal limitations of dichotomous approaches compared to the power of continuous analysis to study a disease where human genotype-phenotype relationships strongly support a role for a continuum of CAG length-dependent changes. The compendium of HTT CAG length-gene expression level relationships found at the HD CAGnome now provides convenient routes for discovery of candidates influenced by the HD mutation.
Assuntos
Bases de Dados Genéticas , Regulação da Expressão Gênica , Doença de Huntington/genética , Proteínas do Tecido Nervoso/genética , Expansão das Repetições de Trinucleotídeos/genética , Humanos , Proteína Huntingtina , Estatística como AssuntoRESUMO
Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.
Assuntos
Apoptose/efeitos dos fármacos , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Quinases da Família src/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteína Tirosina Quinase CSK , Resistencia a Medicamentos Antineoplásicos/genética , Estradiol/farmacologia , Estrogênios/metabolismo , Feminino , Fulvestranto , Humanos , Células MCF-7 , Paclitaxel/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Interferência de RNA , Tamoxifeno/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain. In addition to neuronal migration, a key process for normal cortical function is the regulation of neuronal morphogenesis. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments. We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.
Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/fisiologia , Eletroporação/métodos , Neurônios/citologia , Interferência de RNA , Animais , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Feminino , Camundongos , Inclusão em Parafina , GravidezRESUMO
Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from circulating estrogens and androgens into paracrine signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in morphological tissue development and sterol biosynthesis but suppress genes involved in growth factor signaling. Genes involved in cell adhesion were enriched among both up-regulated and down-regulated genes. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were enriched in the glycolytic pathway. At the highest dose (100 nM), E2 induced genes enriched for cell adhesion, steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF- ß signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas secreted growth factors and cytokines might play significant roles when estrogen level is high.
Assuntos
Estrogênios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Mesoderma/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Animais , Células Cultivadas , Análise por Conglomerados , Di-Hidrotestosterona/farmacologia , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Feminino , Ontologia Genética , Idade Gestacional , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Próstata/citologia , Próstata/embriologia , Próstata/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
To obtain insights into similarities and differences in the biological actions of related drugs or toxic agents, their transcriptomal signature profiles (TSPs) have been examined in a large number of studies. However, many such reports did not provide proper justification for the dosage criteria of each agent. Using a well characterized cell culture model of estrogen-dependent proliferation of MCF7 human breast cancer cells, we demonstrate how different approaches to dosage standardization exert critical influences on TSPs, leading to different and even conflicting conclusions. Using quantitative cellular response (QCR)-based dosage criteria, TSPs were determined by Affymetrix microarray when cells were proliferating at comparable rates in the presence of various estrogens. We observed that TSPs of the xenoestrogens (e.g., genistein or bisphenol A) were clearly different from the TSP of 17beta-estradiol; namely, the former strongly enhanced expression of genes involved in mitochondrial oxidative phosphorylation, whereas the latter showed minimal effects. In contrast, TSPs for genistein and 17beta-estradiol were indistinguishable by using the marker gene expression-based dosage criteria, conditions in which there was comparable expression of the mRNA transcripts for the estrogen-inducible WISP2 gene. Our findings indicate that determination and interpretation of TSPs in pharmacogenomic and toxicogenomic studies that examine the transcriptomal actions of related agents by microarray require a clear rationale for the dosage standardization method to be used. We suggest that future studies involving TSP analyses use quantitative and objective dosage standardization methods, such as those with quantitative cellular response or marker gene expression-based dosage criteria.
Assuntos
Estrogênios/farmacologia , Padrões de Referência , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Estradiol/metabolismo , Estrogênios/metabolismo , Perfilação da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/metabolismo , Transcrição GênicaRESUMO
To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed approximately 3-5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced approximately 1.5-2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, transforming growth factor alpha and stromal cell-derived factor 1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, depend on the E2 concentration, especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Expressão Gênica/efeitos dos fármacos , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CXCL12 , Quimiocinas CXC/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Ligantes , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Fator de Crescimento Transformador alfa/genéticaRESUMO
Evidence has been accumulating that some estrogen-dependent human breast cancers require estrogen for not only proliferation but also survival. To obtain insights into the molecular mechanisms of apoptosis of breast cancer cells subjected to estrogen starvation or exposed to antiestrogens, we characterized changes in the gene expression profile of MCF-7/BUS human breast cancer cells and revealed a strong induction of Bik, a member of the BH3-only proapoptotic proteins. The Bik mRNA transcript and protein were strongly induced by estrogen starvation or exposure to fulvestrant, a pure antiestrogen that competes with the natural estrogens for binding to the estrogen receptors. This Bik induction preceded apoptotic cell death, which was blocked by zVAD-fmk, a pancaspase inhibitor. Amounts of the Bcl-2-related proteins, such as Bcl-2, Bcl-XL, or Bax, showed only marginal changes in the presence or absence of estrogens or antiestrogens. Suppression of Bik expression by using the small interfering RNA effectively blocked the fulvestrant-induced breast cancer cell apoptosis. These results indicate that Bik is induced in MCF-7/BUS cells in the absence of estrogen signaling and plays a critical role in the antiestrogen-provoked breast cancer cell apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas Reguladoras de Apoptose , Sequência de Bases , Neoplasias da Mama , Linhagem Celular Tumoral , Primers do DNA , Feminino , Humanos , Cinética , Proteínas Mitocondriais , RNA Interferente Pequeno/genética , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
The CITED family proteins bind to CBP/p300 transcriptional integrators through their conserved C-terminal acidic domain and function as coactivators. The 21-kDa mouse Cited4 protein, a novel member of the CITED family, interacted with CBP/p300 as well as isoforms of the TFAP2 transcription factor, coactivating TFAP2-dependent transcription. The cited4 gene consisted of only a single exon located on chromosome 4 at 56.5-56.8 cM flanked by marker genes kcnq4 and scml1. Expression of Cited4 protein was strong and selective in embryonic hematopoietic tissues and endothelial cells. In adult animals, Cited4 showed strong milk cycle-dependent induction in pregnant and lactating mammary epithelial cells. Strong induction of Cited4 expression was also observed in SCp2 mouse mammary epithelial cells during their prolactin-dependent in vitro differentiation. These results implied possible roles for Cited4 in regulation of gene expression during development and differentiation of blood cells, endothelial cells, and mammary epithelial cells.