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1.
Heart Vessels ; 37(2): 347-358, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34727208

RESUMO

Calcific aortic valve disease (CAVD) is the most common heart valve disease requiring intervention. Most research on CAVD has focused on inflammation, ossification, and cellular phenotype transformation. To gain a broader picture into the wide range of cellular and molecular mechanisms involved in this disease, we compared the total protein profiles between calcified and non-calcified areas from 5 human valves resected during surgery. The 1413 positively identified proteins were filtered down to 248 proteins present in both calcified and non-calcified segments of at least 3 of the 5 valves, which were then analyzed using Ingenuity Pathway Analysis. Concurrently, the top 40 differentially abundant proteins were grouped according to their biological functions and shown in interactive networks. Finally, the abundance of selected osteogenic proteins (osteopontin, osteonectin, osteocalcin, osteoprotegerin, and RANK) was quantified using ELISA and/or immunohistochemistry. The top pathways identified were complement system, acute phase response signaling, metabolism, LXR/RXR and FXR/RXR activation, actin cytoskeleton, mineral binding, nucleic acid interaction, structural extracellular matrix (ECM), and angiogenesis. There was a greater abundance of osteopontin, osteonectin, osteocalcin, osteoprotegerin, and RANK in the calcified regions than the non-calcified ones. The osteogenic proteins also formed key connections between the biological signaling pathways in the network model. In conclusion, this proteomic analysis demonstrated the involvement of multiple signaling pathways in CAVD. The interconnectedness of these pathways provides new insights for the treatment of this disease.


Assuntos
Estenose da Valva Aórtica , Calcinose , Valva Aórtica/metabolismo , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/cirurgia , Calcinose/metabolismo , Humanos , Osteogênese/fisiologia , Proteoma/metabolismo , Proteômica
2.
Am J Obstet Gynecol ; 224(3): 278.e1-278.e14, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32835719

RESUMO

BACKGROUND: Obesity is a well-known risk factor for endometrial cancer, but the mechanisms of obesity-related carcinogenesis are not well defined, particularly for premenopausal women. With the continuing obesity epidemic, increases in the incidence of endometrial cancer and a younger age of diagnosis are often attributed to a hyperestrogenic state created by hormone production in adipose tissue, but significant knowledge gaps remain. The balance of estrogen-responsive signals has not been defined in the endometrium of premenopausal women with obesity, where obesity may not create hyperestrogenism in the context of ovaries being the primary source of estrogen production. Obesity is associated with a state of low-grade, chronic inflammation that can promote tumorigenesis, and it is also known that hormonal changes alter the immune microenvironment of the endometrium. However, limited research has been conducted on endometrial immune-response changes in women who have an increased risk for cancer due to obesity. OBJECTIVE: Endometrial estrogen-regulated biomarkers, previously shown to be dysregulated in endometrial cancer, were evaluated in a cohort of premenopausal women to determine if obesity is associated with differences in the biomarker expression levels, which might reflect an altered risk of developing cancer. The expression of a multiplexed panel of immune-related genes was also evaluated for expression differences related to obesity. STUDY DESIGN: Premenopausal women with a body mass index of ≥30 kg/m2 (n=97) or a body mass index of ≤25 kg/m2 (n=33) were prospectively enrolled in this cross-sectional study, which included the assessment of serum metabolic markers and a timed endometrial biopsy for pathologic evaluation, hormone-regulated biomarker analysis, and immune response gene expression analysis. Medical and gynecologic histories were obtained. Endometrial gene expression markers were also compared across the body mass index groups in a previous cohort of premenopausal women with an inherited cancer risk (Lynch syndrome). RESULTS: In addition to known systemic metabolic differences, histologically normal endometria from women with obesity showed a decrease in gene expression of progesterone receptor (P=.0027) and the estrogen-induced genes retinaldehyde dehydrogenase 2 (P=.008), insulin-like growth factor 1 (P=.016), and survivin (P=.042) when compared with women without obesity. The endometrial biomarkers insulin-like growth factor 1, survivin, and progesterone receptor remained statistically significant in multivariate linear regression models. In contrast, women with obesity and Lynch syndrome had an increased expression of insulin-like growth factor 1 (P=.017). There were no differences in endometrial proliferation, and limited endometrial immune differences were observed. CONCLUSION: When comparing premenopausal women with and without obesity in the absence of endometrial pathology or an inherited cancer risk, the expression of the endometrial biomarkers does not reflect a local hyperestrogenic environment, but it instead reflects a decreased cancer risk profile that may be indicative of a compensated state. In describing premenopausal endometrial cancer risk, it may be insufficient to attribute a high-risk state to obesity alone; further studies are warranted to evaluate individualized biomarker profiles for differences in the hormone-responsive signals or immune response. In patients with Lynch syndrome, the endometrial biomarker profile suggests that obesity further increases the risk of developing cancer.


Assuntos
Estrogênios/sangue , Obesidade/sangue , Pré-Menopausa/sangue , Adulto , Biomarcadores/sangue , Estudos de Coortes , Estudos Transversais , Neoplasias do Endométrio/epidemiologia , Neoplasias do Endométrio/etiologia , Endométrio/metabolismo , Endométrio/patologia , Estrogênios/biossíntese , Feminino , Humanos , Obesidade/complicações , Fatores de Risco
3.
BMC Genomics ; 20(1): 852, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727022

RESUMO

BACKGROUND: Cleft lip (CL), one of the most common congenital birth defects, shows considerable geographic and ethnic variation, with contribution of both genetic and environmental factors. Mouse genetic studies have identified several CL-associated genes. However, it remains elusive how these CL-associated genes are regulated and involved in CL. Environmental factors may regulate these genes at the post-transcriptional level through the regulation of non-coding microRNAs (miRNAs). In this study, we sought to identify miRNAs associated with CL in mice. RESULTS: Through a systematic literature review and a Mouse Genome Informatics (MGI) database search, we identified 55 genes that were associated with CL in mice. Subsequent bioinformatic analysis of these genes predicted that a total of 33 miRNAs target multiple CL-associated genes, with 20 CL-associated genes being potentially regulated by multiple miRNAs. To experimentally validate miRNA function in cell proliferation, we conducted cell proliferation/viability assays for the selected five candidate miRNAs (miR-124-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p). Overexpression of miR-124-3p, but not of the others, inhibited cell proliferation through suppression of CL-associated genes in cultured mouse embryonic lip mesenchymal cells (MELM cells) isolated from the developing mouse lip region. By contrast, miR-124-3p knockdown had no effect on MELM cell proliferation. This miRNA-gene regulatory mechanism was mostly conserved in O9-1 cells, an established cranial neural crest cell line. Expression of miR-124-3p was low in the maxillary processes at E10.5, when lip mesenchymal cells proliferate, whereas it was greatly increased at later developmental stages, suggesting that miR-124-3p expression is suppressed during the proliferation phase in normal palate development. CONCLUSIONS: Our findings indicate that upregulated miR-124-3p inhibits cell proliferation in cultured lip cells through suppression of CL-associated genes. These results will have a significant impact, not only on our knowledge about lip morphogenesis, but also on the development of clinical approaches for the diagnosis and prevention of CL.


Assuntos
Fenda Labial/genética , Regulação da Expressão Gênica , Lábio/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Interferência de RNA , Animais , Proliferação de Células/genética , Células Cultivadas , Biologia Computacional/métodos , Desenvolvimento Embrionário/genética , Meio Ambiente , Epigênese Genética , Perfilação da Expressão Gênica , Camundongos , Mutação , Reprodutibilidade dos Testes
4.
Physiol Genomics ; 48(4): 281-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26884459

RESUMO

The intensification and concentration of animal production operations expose workers to high levels of organic dusts in the work environment. Exposure to organic dusts is a risk factor for the development of acute and chronic respiratory symptoms and diseases. Lung epithelium plays important roles in the control of immune and inflammatory responses to environmental agents to maintain lung health. To better understand the effects of organic dust on lung inflammatory responses, we characterized the gene expression profiles of A549 alveolar and Beas2B bronchial epithelial and THP-1 monocytic cells influenced by exposure to poultry dust extract by DNA microarray analysis using Illumina Human HT-12 v4 Expression BeadChip. We found that A549 alveolar and Beas2B bronchial epithelial and THP-1 cells responded with unique changes in the gene expression profiles with regulation of genes encoding inflammatory cytokines, chemokines, and other inflammatory proteins being common to all the three cells. Significantly induced genes included IL-8, IL-6, IL-1ß, ICAM-1, CCL2, CCL5, TLR4, and PTGS2. Validation by real-time qRT-PCR, ELISA, Western immunoblotting, and immunohistochemical staining of lung sections from mice exposed to dust extract validated DNA microarray results. Pathway analysis indicated that dust extract induced changes in gene expression influenced functions related to cellular growth and proliferation, cell death and survival, and cellular development. These data show that a broad range of inflammatory mediators produced in response to poultry dust exposure can modulate lung immune and inflammatory responses. This is the first report on organic dust induced changes in expression profiles in lung epithelial and THP-1 monocytic cells.


Assuntos
Quimiocinas/genética , Citocinas/genética , Poeira , Perfilação da Expressão Gênica/métodos , Pulmão/citologia , Animais , Quimiocinas/imunologia , Citocinas/imunologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Camundongos Endogâmicos C57BL , Pneumonia/genética , Pneumonia/imunologia , Aves Domésticas
5.
BMC Genomics ; 16: 984, 2015 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-26589571

RESUMO

BACKGROUND: Although extensive studies have investigated radiation-induced injuries in particular gastrointestinal (GI) segments, a systematic comparison among the different segments on the basis of mode, magnitude and mechanism has not been reported. Here, a comparative study of segment-specific molecular and cellular responses was performed on jejunum, ileum and colon obtained at three time points (4, 7 and 12 days after irradiation) from non-human primate (Rhesus macaque) models exposed to 6.7 Gy or 7.4 Gy total body irradiation (TBI). RESULTS: Pathway analysis on the gene expression profiles identified radiation-induced time-, dose- and segment-dependent activation of tumor necrosis factor α (TNFα) cascade, tight junction, apoptosis, cell cycle control/DNA damage repair and coagulation system signaling. Activation of these signaling pathways suggests that colon sustained the severest mucosal barrier disruption and inflammation, and jejunum the greatest DNA damage, apoptosis and endothelial dysfunction. These more pronounced alterations correlate with the high incidence of macroscopic pathologies that are observed in the colon after TBI. Compared to colon and jejunum, ileum was resistant to radiation injury. In addition to the identification a marked increase of TNFα cascade, this study also identified radiation induced strikingly up-regulated tight junction gene CLDN2 (196-fold after 7.4-Gy TBI), matrix degradation genes such as MMP7 (increased 11- and 41-fold after 6.7-Gy and 7.4-Gy TBI), and anoikis mediated gene EDA2R that mediate mucosal shedding and barrier disruption. CONCLUSIONS: This is the first systematic comparative study of the molecular and cellular responses to radiation injury in jejunum, ileum and colon. The strongest activation of TNFα cascades and the striking up-regulation of its down-stream matrix-dissociated genes suggest that TNFα modulation could be a target for mitigating radiation-induced mucosal barrier disruption.


Assuntos
Colo/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Íleo/metabolismo , Jejuno/metabolismo , Transcriptoma , Irradiação Corporal Total , Animais , Anoikis/genética , Apoptose/genética , Ciclo Celular , Análise por Conglomerados , Colo/imunologia , Íleo/imunologia , Imunidade nas Mucosas/genética , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos da radiação , Jejuno/imunologia , Macaca mulatta , Masculino , Doses de Radiação , Lesões Experimentais por Radiação , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos da radiação , Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
6.
J Biol Chem ; 288(50): 35940-51, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24163369

RESUMO

Genome-scale mapping suggests that the function of DNA methylation varies with genomic context beyond transcriptional repression. However, the use of DNA-demethylating agents (e.g. 5-aza-2'-deoxycytidine (5aza-dC)) to study epigenetic regulation often focuses on gene activation and ignores repression elicited by 5aza-dC. Here, we show that repression of NEK2, which encodes the never in mitosis A (NIMA)-related kinase, by 5aza-dC is context-specific as NEK2 transcript levels were reduced in HCT116 colon cancer cells but not in isogenic p53(-/-) cells. Bisulfite sequencing showed that DNA methylation was restricted to the distal region of the NEK2 promoter. Demethylation by 5aza-dC was associated with increased accessibility to micrococcal nuclease, i.e. nucleosome depletion. Conversely, methyltransferase accessibility protocol for individual templates (MAPit) methylation footprinting showed that nucleosome occupancy and DNA methylation at the distal promoter were significantly increased in p53(-/-) cells, suggesting dynamic regulation of chromatin structure at this region by p53 in HCT116 cells. Stabilization of endogenous p53 by doxorubicin or ectopic expression of p53, but not a p53 DNA-binding mutant, decreased NEK2 expression. Chromatin immunoprecipitation demonstrated direct and specific association of p53 with the distal NEK2 promoter, which was enhanced by doxorubicin. Luciferase reporters confirmed that this region is required for p53-mediated repression of NEK2 promoter activity. Lastly, modulation of p53 abundance altered nucleosome occupancy and DNA methylation at its binding region. These results identify NEK2 as a novel p53-repressed gene, illustrate that its repression by 5aza-dC is specific and associated with nucleosome reorganization, and provide evidence that identification of partially methylated regions can reveal novel p53 target genes.


Assuntos
Metilação de DNA , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HCT116 , Humanos , Quinases Relacionadas a NIMA , Nucleossomos/efeitos dos fármacos , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
7.
Gynecol Oncol ; 133(1): 83-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24680596

RESUMO

OBJECTIVE: Obesity-associated hyperestrogenism and hyperinsulinemia contribute significantly to the pathogenesis of endometrial cancer. We recently demonstrated that metformin, a drug long used for treatment of type 2 diabetes, attenuates both insulin- and estrogen-mediated proliferative signaling in the obese rat endometrium. In this study, we sought to identify tissue biomarkers that may prove clinically useful to predict tissue response for both prevention and therapeutic studies. We identified CGRRF1 (cell growth regulator with ring finger domain 1) as a novel metformin-responsive gene and characterized its possible role in endometrial cancer prevention. METHODS: CGRRF1 mRNA expression was evaluated by RT-qPCR in the endometrium of obese and lean rats, and also in normal and malignant human endometrium. CGRRF1 levels were genetically manipulated in endometrial cancer cells, and its effects on proliferation and apoptosis were evaluated by MTT and Western blot. RESULTS: CGRRF1 is significantly induced by metformin treatment in the obese rat endometrium. In vitro studies demonstrate that overexpression of CGRRF1 inhibits endometrial cancer cell proliferation. Analysis of human endometrial tumors reveals that CGRRF1 expression is significantly lower in hyperplasia, Grade 1, Grade 2, Grade 3, MMMT, and UPSC endometrial tumors compared to normal human endometrium (p<0.05), suggesting that loss of CGRRF1 is associated with the presence of disease. CONCLUSION: CGRRF1 represents a novel, reproducible tissue marker of metformin response in the obese endometrium. Furthermore, our preliminary data suggests that up-regulation of CGRRF1 expression may prove clinically useful in the prevention or treatment of endometrial cancer.


Assuntos
Endométrio/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Metformina/farmacologia , Obesidade/metabolismo , RNA Mensageiro/análise , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Hiperplasia Endometrial/genética , Hiperplasia Endometrial/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Exp Mol Pathol ; 94(1): 289-300, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22609242

RESUMO

It is now known that there are at least two basic patterns of cell injury progressing to cell death: cell injury with swelling, known as oncosis, and cell injury with shrinkage, known as apoptosis. Both types of cell death are "programmed" in the sense that the genetic information and many of the enzymes and other factors pre-exist in the cell. Previous investigation has pointed to cardiomyocyte ischemic injury evolving as the oncotic pattern of injury, although apoptosis has also been implicated. This study was designed, using a unique cell model system, to gain insight into the molecular events of anticancer agent-induced cardiomyocyte injury. Cardiomyocytes exposed for 2 h to 1.5 µg/ml sanguinarine consistently displayed the morphology of apoptosis in over 80% of cells, whereas a higher dose of 25 µg/ml at 2 h yielded the pattern of oncosis in over 90% of cells. Microarray analysis revealed altered expression of 2514 probes in sanguinarine-induced oncosis and 1643 probes in apoptosis at a level of significance of p<0.001. Some of the inductions such as perforin were found to be higher than 11-fold in oncosis. When perforin was blocked by perforin-specific siRNA we found a reduction in oncotic cell death. These results strengthen the notion that oncosis is not representative of nonspecific necrosis, but constitutes a genetically controlled form of "programmed cell death"; and also that oncosis might represent a pathogenetic mechanism of cardiomyocyte injury. This is also the first demonstration of the involvement of perforin in cardiomyocyte oncosis.


Assuntos
Apoptose , Morte Celular , Miócitos Cardíacos/fisiologia , Perforina/genética , Animais , Apoptose/genética , Benzofenantridinas/farmacologia , Morte Celular/genética , Linhagem Celular , Células-Tronco Embrionárias , Isoquinolinas/farmacologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno
9.
Physiol Genomics ; 43(7): 325-45, 2011 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-21224422

RESUMO

There is currently much interest in clinical applications of therapeutic hypothermia. Hypothermia can be a consequence of hypometabolism. We have recently established a procedure for the induction of a reversible deep hypometabolic state in mice using 5'-adenosine monophosphate (5'-AMP) in conjunction with moderate ambient temperature. The current study aims at investigating the impact of this technology at the gene expression level in a major metabolic organ, the liver. Our findings reveal that expression levels of the majority of genes in liver are not significantly altered by deep hypometabolism. However, among those affected by hypometabolism, more genes are differentially upregulated than downregulated both in a deep hypometabolic state and in the early arousal state. These altered gene expression levels during 5'-AMP induced hypometabolism are largely restored to normal levels within 2 days of the treatment. Our data also suggest that temporal control of circadian genes is largely stalled during deep hypometabolism.


Assuntos
Monofosfato de Adenosina/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Feminino , Hipotermia/induzido quimicamente , Hipotermia/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/metabolismo
10.
HPB (Oxford) ; 13(6): 369-76, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21609368

RESUMO

BACKGROUND: Epigenetics is a rapidly evolving field of genetic study applicable to nearly every aspect of genome-related research. The importance of epigenetics has been recognised in human hepatocellular carcinoma (HCC). Changes in DNA methylation patterns, including global hypomethylation and promoter hypermethylation, are thought to be early events in hepatocarcinogenesis. OBJECTIVES: This review aimed to summarise the role of epigenetics in HCC, to describe the mechanisms of epigenetic changes in HCC and to examine the clinical relevance of epigenetics in HCC. METHODS: This review examines the role of CpG-rich regions and DNA methylation, and describes an epigenetic model of cancer, tumour type-specific methylation, the relationships among methylation, cirrhosis and hepatocarcinogenesis, and the role of DNA methylation in HCC. The clinical implications of epigenetics in HCC are discussed. RESULTS: A multivariate predictor model based on traditional clinical factors and DNA methylation profile may have important applications in the early detection of neoplastic transformation in populations at high risk for HCC. CpG methylation may be valuable in HCC prognostics. DNA methylation profiles may enable clinical prediction in pre-therapy patient biopsies, paraffin-embedded samples or plasma DNA. CONCLUSIONS: Epigenetic changes and profiles may correlate to the biological behaviour of tumours and clinical outcome of HCC patients. The use of DNA methylation profiles as a surrogate biomarker remains an active area of clinical cancer research.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Metilação de DNA , Marcadores Genéticos , Neoplasias Hepáticas/genética , Animais , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Transformação Celular Neoplásica/genética , Ilhas de CpG , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Testes Genéticos , Humanos , Cirrose Hepática/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Fenótipo , Valor Preditivo dos Testes , Prognóstico
11.
J Lipid Res ; 51(7): 1704-18, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20173184

RESUMO

LDL mediates transfection with plasmid DNA in a variety of cell types in vitro and in several tissues in vivo in the rat. The transfection capacity of LDL is based on apo B100, as arginine/lysine clusters, suggestive of nucleic acid-binding domains and nuclear localization signal sequences, are present throughout the molecule. Apo E may also contribute to this capacity because of its similarity to the Dengue virus capsid proteins and its ability to bind DNA. Synthetic peptides representing two apo B100 regions with prominent Arg/Lys clusters were shown to bind DNA. Region 1 (0014Lys-Ser0160) shares sequence motifs present in DNA binding domains of Interferon Regulatory Factors and Flaviviridae capsid/core proteins. It also contains a close analog of the B/E receptor ligand of apo E. Region 1 peptides, B1-1 (0014Lys-Glu0054) and B1-2 (0055Leu-Ala0096), mediate transfection of HeLa cells but are cytotoxic. Region 2 (3313Asp-Thr3431), containing the known B/E receptor ligand, shares analog motifs with the human herpesvirus 5 immediate-early transcriptional regulator (UL122) and Flaviviridae NS3 helicases. Region 2 peptides, B2-1 (3313Asp-Glu3355), and B2-2 (3356Gly-Thr3431) are ineffective in cell transfection and are noncytotoxic. These results confirm the role of LDL as a natural transfection vector in vivo, a capacity imparted by the apo B100, and suggest a basis for Flaviviridae cell entry.


Assuntos
Apolipoproteína B-100 , DNA/metabolismo , Lipoproteínas LDL/metabolismo , Transfecção/métodos , Proteínas Virais , Sequência de Aminoácidos , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Linhagem Celular , DNA/genética , Feminino , Flaviviridae/genética , Flaviviridae/metabolismo , Genes Reporter , Humanos , Lipoproteínas LDL/genética , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
Anal Biochem ; 401(2): 288-94, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20227380

RESUMO

Wnts are secreted lipid-modified glycoproteins that carry out various signaling functions during development and in adult tissue. Wnt signaling is mediated by frizzled receptors (Fzds) at the cell surface and can be modulated by the secreted frizzled-related proteins (SFRPs) and other molecular antagonists. Abnormal Wnt signaling has been implicated in several diseases. However, due to the complexity of the Wnt signal and the lack of knowledge pertaining to the binding properties of different Wnt ligands, no therapeutic agents that target this pathway exist. Using a novel enzyme-linked immunosorbent assay (ELISA)-based technique, we were able to determine the first measurements of binding affinity for specific Wnt interactions. This study shows that purified Wnt3a, Wnt7a, and Wnt5a have different binding specificities for Fzds and SFRPs.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Receptores Frizzled/metabolismo , Proteínas Wnt/metabolismo , Linhagem Celular Tumoral , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , MAP Quinase Quinase 4/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismo
13.
Mol Cancer Res ; 6(6): 1017-28, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18567805

RESUMO

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.


Assuntos
Neoplasias do Endométrio/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Endométrio/patologia , Feminino , Receptores Frizzled/análise , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Comunicação Parácrina , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/análise , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismo
14.
Biochem Biophys Res Commun ; 368(2): 285-91, 2008 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-18230341

RESUMO

Wnts are secreted glycoproteins that regulate important cellular processes including proliferation, differentiation, and cell fate. In the beta-catenin/canonical pathway, Wnt interacts with Fzd receptors to inhibit degradation of beta-catenin and promote its translocation into the nucleus where it regulates transcription of a number of genes. Dysregulation of this pathway has been attributed to a host of diseases including cancer. As a result, components of the beta-catenin/canonical pathway have been gaining recognition as promising targets for the discovery of novel therapeutic agents. Here, we show, using an ELISA-based protein-protein binding assay that purified Wnt7a binds to the extracellular cysteine-rich domain of Fzd5 in the nanomolar range. We have developed a novel split eGFP complementation assay to visually detect Wnt7a-Fzd5 interactions and subsequent pathway activation in cells. These biological tools could help lead to a better understanding of Wnt-Fzd interactions and the identification of new modulators of Wnt signaling.


Assuntos
Receptores Frizzled/química , Receptores Frizzled/metabolismo , Proteínas de Fluorescência Verde , Mapeamento de Interação de Proteínas/métodos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/química , Proteínas Wnt/metabolismo , Humanos
15.
Blood Coagul Fibrinolysis ; 19(7): 709-18, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18832915

RESUMO

Inhibitors of Rho kinase (ROCK) are a relatively new class of drugs with potential benefits in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK inhibitors modulate many cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds that inhibit both isoforms of ROCK, ROCK1 and ROCK2. This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2 inhibitor SLx-2119 in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 h, after which total RNA was isolated and genome-wide gene-expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor, a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and connective tissue growth factor mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced connective tissue growth factor mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have antifibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Heptanoicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Sequência de Aminoácidos , Atorvastatina , Linhagem Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Músculo Liso/fisiologia , Reação em Cadeia da Polimerase/métodos
16.
Clin Cancer Res ; 12(21): 6373-8, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17085648

RESUMO

PURPOSE: Although there is considerable information on the molecular aberrations associated with endometrial cancer, very little is known of the changes in gene expression associated with endometrial hyperplasia. EXPERIMENTAL DESIGN: To address this, we have compared the level of expression of estrogen-regulated genes and components of the insulin-like growth factor I (IGF-I) signaling pathway in endometrial biopsies from subjects with normal endometrium, complex atypical endometrial hyperplasia, and endometrial adenocarcinoma (type I). RESULTS: There was a significant increase in the expression of the IGF-I receptor (IGF-IR) in biopsies from hyperplastic endometrium and endometrial carcinoma compared with the proliferative endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in endometrial hyperplasia and carcinoma, the downstream components of the IGF-IR pathway are activated, as reflected in increased Akt phosphorylation. Loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression in endometrial hyperplasia did not correlate with increased activation of IGF-IR. However, the simultaneous loss of PTEN expression and increased IGF-IR activation in hyperplasia was associated with an increased incidence of endometrial carcinoma. CONCLUSIONS: These results suggest that up-regulation of IGF-IR and loss of PTEN may be independent events that give rise to complementary activation of the IGF-I pathway and increase the probability of the development of cancer. These studies suggest that increased expression of IGF-IR may be an important contributor to the risk of endometrial hyperplasia and cancer.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Endométrio/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/biossíntese , Adenocarcinoma/patologia , Neoplasias do Endométrio/patologia , Ativação Enzimática/fisiologia , Feminino , Humanos , Hiperplasia , Imuno-Histoquímica , PTEN Fosfo-Hidrolase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
J Neurotrauma ; 34(6): 1175-1186, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-27750479

RESUMO

Spinal cord injury (SCI) results in devastating changes to almost all aspects of a patient's life. In addition to a permanent loss of sensory and motor function, males also will frequently exhibit a profound loss of fertility through poorly understood mechanisms. We demonstrate that SCI causes measureable pathology in the testis both acutely (24 h) and chronically up to 1.5 years post-injury, leading to loss in sperm motility and viability. SCI has been shown in humans and rats to induce leukocytospermia, with the presence of inflammatory cytokines, anti-sperm antibodies, and reactive oxygen species found within the ejaculate. Using messenger RNA and metabolomic assessments, we describe molecular and cellular changes that occur within the testis of adult rats over an acute to chronic time period. From 24 h, 72 h, 28 days, and 90 days post-SCI, the testis reveal a distinct time course of pathological events. The testis show an acute drop in normal sexual organ processes, including testosterone production, and establishment of a pro-inflammatory environment. This is followed by a subacute initiation of an innate immune response and loss of cell cycle regulation, possibly due to apoptosis within the seminiferous tubules. At 1.5 years post-SCI, there is a chronic low level immune response as evidenced by an elevation in T cells. These data suggest that SCI elicits a wide range of pathological processes within the testes, the actions of which are not restricted to the acute phase of injury but rather extend chronically, potentially through the lifetime of the subject. The multiplicity of these pathological events suggest a single therapeutic intervention is unlikely to be successful.


Assuntos
Traumatismos da Medula Espinal/complicações , Doenças Testiculares/etiologia , Doenças Testiculares/metabolismo , Animais , Modelos Animais de Doenças , Expressão Gênica/genética , Masculino , Metabolômica , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Doenças Testiculares/imunologia
18.
Clin Cancer Res ; 11(23): 8258-64, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16322283

RESUMO

PURPOSE: The identification of genes and pathways that are affected by estrogenization may shed light on the mechanisms of estrogen action. Here, we describe the expression pattern of a novel estrogen-induced gene, EIG121, in distinct types of endometrial cancer. EXPERIMENTAL DESIGN: EIG121 was identified by cDNA microarray analysis of endometrial RNA from women receiving either placebo or estrogen replacement therapy. The expression level of EIG121 was then measured by real-time quantitative reverse transcription-PCR in benign, hyperplastic, and malignant endometrial samples. A polyclonal antibody was used to detect EIG121 protein by immunohistochemistry. RESULTS: In postmenopausal endometrium, estrogen replacement therapy with Premarin and synthetic estrogen sulfate conjugates induced the expression of EIG121 2- and 3-fold, respectively. In premenopausal endometrium, the expression of EIG121 was higher in the estrogen-dominated proliferative phase than the secretory phase. In endometrial complex, hyperplasia, and endometrioid adenocarcinoma, neoplastic proliferations associated with estrogen excess, the expression of EIG121 was significantly elevated (on average 3.8-fold in hyperplasias and 21-fold in grade 1 tumors). Although the level of EIG121 mRNA in grade 3 endometrioid carcinoma was still 3.5-fold of that in benign endometrium, EIG121 expression tended to decline with increasing tumor grade and disease stage. Immunohistochemistry showed faint staining of normal endometrial epithelium, but intense staining of endometrioid tumors. In sharp contrast, EIG121 expression was significantly suppressed in both uterine papillary serous carcinoma and uterine malignant mixed mullerian tumor, two tumors not associated with estrogen exposure, to <5% of the level in benign endometrium. CONCLUSIONS: Our results suggest that EIG121 is a good endometrial biomarker associated with a hyperestrogenic state and estrogen-related type I endometrial adenocarcinoma.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Neoplasias do Endométrio/genética , Terapia de Reposição de Estrogênios , Estrogênios/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Hiperplasia Endometrial/genética , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Estrogênios Conjugados (USP)/uso terapêutico , Estrona/análogos & derivados , Estrona/uso terapêutico , Etiquetas de Sequências Expressas , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Proteínas de Membrana , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
PLoS One ; 8(1): e53711, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23341980

RESUMO

BACKGROUND: The intestinal mucosa is the compartment that sustains the most severe injury in response to radiation and is therefore of primary interest. The use of whole gut extracts for analysis of gene expression may confound important changes in the mucosa. On the other hand, laser capture microdissection (LCM) is hampered by the unstable nature of RNA and by a more complicated collection process. This study assessed, in parallel samples from a validated radiation model, the indications for use of LCM for intestinal gene expression analysis. METHODOLOGY/PRINCIPAL FINDINGS: RNA was extracted from mouse whole intestine and from mucosa by LCM at baseline and 4 h, 24 h, and 3.5 d after total body irradiation and subjected to microarray analysis. Among mucosal genes that were altered > = 2-fold, less than 7% were present in the whole gut at 4 and 24 h, and 25% at 3.5 d. As expected, pathway analysis of mucosal LCM samples showed that radiation activated the coagulation system, lymphocyte apoptosis, and tight junction signaling, and caused extensive up-regulation of cell cycle and DNA damage repair pathways. Using similar stringent criteria, regulation of these pathways, with exception of the p53 pathway, was undetectable in the whole gut. Radiation induced a dramatic increase of caspase14 and ectodysplasin A2 receptor (Eda2r), a TNFα receptor, in both types of samples. CONCLUSIONS/SIGNIFICANCE: LCM-isolated mucosal specimens should be used to study cellular injury, cell cycle control, and DNA damage repair pathways. The remarkable increase of caspase14 and Eda2r suggests a novel role for these genes in regulating intestinal radiation injury. Comparative gene expression data from complex tissues should be interpreted with caution.


Assuntos
Mucosa Intestinal/efeitos da radiação , Intestino Delgado/efeitos da radiação , Microdissecção e Captura a Laser/métodos , Animais , Apoptose/efeitos da radiação , Caspase 14/metabolismo , Ciclo Celular/efeitos da radiação , Citocinas/metabolismo , Reparo do DNA/efeitos da radiação , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/enzimologia , Intestino Delgado/metabolismo , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/efeitos da radiação , Fatores de Tempo , Transcriptoma/efeitos da radiação
20.
Cancer Prev Res (Phila) ; 6(8): 774-81, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23639481

RESUMO

Women with Lynch syndrome have a 40% to 60% lifetime risk for developing endometrial cancer, a cancer associated with estrogen imbalance. The molecular basis for endometrial-specific tumorigenesis is unclear. Progestins inhibit estrogen-driven proliferation, and epidemiologic studies have shown that progestin-containing oral contraceptives (OCP) reduce the risk of endometrial cancer by 50% in women at general population risk. It is unknown whether they are effective in women with Lynch syndrome. Asymptomatic women ages 25 to 50 with Lynch syndrome were randomized to receive the progestin compounds Depo-Provera (depo-MPA) or OCP for three months. An endometrial biopsy and transvaginal ultrasound were conducted before and after treatment. Endometrial proliferation was evaluated as the primary endpoint. Histology and a panel of surrogate endpoint biomarkers were evaluated for each endometrial biopsy as secondary endpoints. A total of 51 women were enrolled, and 46 completed treatment. Two of the 51 women had complex hyperplasia with atypia at the baseline endometrial biopsy and were excluded from the study. Overall, both depo-MPA and OCP induced a dramatic decrease in endometrial epithelial proliferation and microscopic changes in the endometrium characteristic of progestin action. Transvaginal ultrasound measurement of endometrial stripe was not a useful measure of endometrial response or baseline hyperplasia. These results show that women with Lynch syndrome do show an endometrial response to short-term exogenous progestins, suggesting that OCP and depo-MPA may be reasonable chemopreventive agents in this high-risk patient population.


Assuntos
Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Anticoncepcionais Orais/uso terapêutico , Neoplasias do Endométrio/prevenção & controle , Acetato de Medroxiprogesterona/uso terapêutico , Adulto , Biomarcadores Tumorais/genética , Neoplasias do Endométrio/etiologia , Neoplasias do Endométrio/metabolismo , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Mutação/genética , Prognóstico , Estudos Prospectivos
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