RESUMO
MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.
Assuntos
Colo/patologia , Neoplasias do Colo/patologia , Neoplasias Hepáticas/secundário , Fígado/patologia , Mucinas/análise , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Estudos de Coortes , Colo/metabolismo , Neoplasias do Colo/genética , Citoplasma/metabolismo , Citoplasma/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Mucinas/genética , RNA/genética , Análise Serial de Tecidos , Regulação para CimaRESUMO
BACKGROUND: O-linked N-acyl-glycosylation may regulate protein function by competing with phosphorylation of serine residues. Availability of substrate for this process is regulated, in part, by N-Acyl-D-glucosamine 2-epimerase (NAGE), which interconverts N-acetyl-glucosamine (GlcNAc) and N-acetylmannosamine (ManNAc). NAGE is also a putative renin-binding protein. This study tested the hypothesis that NAGE is present in the human heart and that NAGE expression is increased in the failing human heart. METHODS AND RESULTS: Ribonuclease protection assays (RPAs) demonstrated increased NAGE gene expression in failing hearts from subjects with idiopathic dilated and ischemic cardiomyopathies compared with nonfailing hearts. In situ reverse transcriptase-polymerase chain reaction, using primers designed to localize NAGE mRNA, demonstrated that, in nonfailing hearts, NAGE gene expression was restricted to endothelial cells and not detectable in cardiac myocytes. However, in failing human hearts NAGE gene expression was selectively activated in cardiac myocytes, but not endothelial cells. Immunohistochemistry confirmed that the pattern of NAGE protein expression corresponded to the pattern of gene expression. CONCLUSIONS: NAGE gene and protein expression were selectively activated in left ventricular myocytes from end-stage failing human hearts.
Assuntos
Carboidratos Epimerases/biossíntese , Cardiomiopatia Dilatada/metabolismo , Proteínas de Transporte/biossíntese , Adolescente , Adulto , Carboidratos Epimerases/genética , Proteínas de Transporte/genética , Ativação Enzimática/fisiologia , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Variação Genética/genética , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Hipertrofia Ventricular Esquerda/metabolismo , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatística como Assunto , Disfunção Ventricular Esquerda/metabolismoRESUMO
OBJECTIVES: We examined the prevalence, genotype-phenotype correlation, and natural history of lamin A/C gene (LMNA) mutations in subjects with dilated cardiomyopathy (DCM). BACKGROUND: Mutations in LMNA have been found in patients with DCM with familial conduction defects and muscular dystrophy, but the clinical spectrum, prognosis, and clinical relevance of laminopathies in DCM are unknown. BACKGROUND: A cohort of 49 nuclear families, 40 with familial DCM and 9 with sporadic DCM (269 subjects, 105 affected), was screened for mutations in LMNA using denaturing high-performance liquid chromatography and sequence analysis. Bivariate analysis of clinical predictors of LMNA mutation carrier status and Kaplan-Meier survival analysis were performed. RESULTS: Mutations in LMNA were detected in four families (8%), three with familial (R89L, 959delT, R377H) and one with sporadic DCM (S573L). There was significant phenotypic variability, but the presence of skeletal muscle involvement (p < 0.001), supraventricular arrhythmia (p = 0.003), conduction defects (p = 0.01), and "mildly" DCM (p = 0.006) were predictors of LMNA mutations. The LMNA mutation carriers had a significantly poorer cumulative survival compared with non-carrier DCM patients: event-free survival at the age of 45 years was 31% versus 75% in non-carriers. CONCLUSIONS: Mutations in LMNA cause a severe and progressive DCM in a relevant proportion of patients. Mutation screening should be considered in patients with DCM, in particular when clinical predictors of LMNA mutation are present, regardless of family history.
Assuntos
Cardiomiopatia Dilatada/epidemiologia , Cardiomiopatia Dilatada/genética , Predisposição Genética para Doença , Mutação de Sentido Incorreto , Lâmina Nuclear/genética , Sequência de Aminoácidos , Cardiomiopatia Dilatada/diagnóstico , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Seguimentos , Genótipo , Humanos , Masculino , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Prevalência , Probabilidade , Medição de Risco , Índice de Gravidade de Doença , Estatísticas não Paramétricas , Taxa de SobrevidaRESUMO
INTRODUCTION: Hypoplastic left heart syndrome (HLHS) is the term used to describe a group of congenital malformations characterized by marked underdevelopment of the left side of the heart. HLHS accounts for nearly 25% of cardiac deaths in the first year of life. Although much has been reported regarding diagnosis, gross morphology and surgical treatment, no information on gene expression in HLHS myocytes is available. METHODS: We examined heart tissue from patients with HLHS using routine histology, immunohistochemistry, quantitative polymerase chain reaction (PCR), two-dimensional (2-D) gel electrophoresis and protein identification by mass spectrometry. RESULTS: Histologic examination of right and left ventricles from HLHS patients revealed characteristic features of myocyte differentiation, including striations and intercalated disc formation. Immunohistochemical staining using antibody to N-cadherin demonstrated clear development of intercalated discs between myocytes. However, many of the myocytes contained scant cytoplasm and were grouped in small, disorganized bundles separated by abundant connective tissue and dilated, thin-walled vessels. Quantitative PCR analysis demonstrated that both left and right ventricular tissue from HLHS hearts expressed the fetal or "heart failure" gene expression pattern. Two-dimensional gel electrophoresis and protein identification by mass spectrometry also confirmed that myocytes from HLHS ventricles were differentiated but expressed the fetal isoform of some cardiac specific proteins. However, HLHS myocytes in all of the heart samples (n=21) were inappropriately expressing platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), a member of the cell adhesion molecule (CAM) family that has a primary role in the regulation of tissue morphogenesis. These findings indicate that myocytes from HLHS syndrome patients, while differentiated, have a unique gene expression pattern.
Assuntos
Síndrome do Coração Esquerdo Hipoplásico/genética , Miócitos Cardíacos/fisiologia , Adolescente , Adulto , Western Blotting , Caderinas/biossíntese , Diferenciação Celular/fisiologia , Criança , Pré-Escolar , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Humanos , Síndrome do Coração Esquerdo Hipoplásico/metabolismo , Síndrome do Coração Esquerdo Hipoplásico/patologia , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Espectrometria de Massas , Miócitos Cardíacos/patologia , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseRESUMO
BACKGROUND: Endothelin signaling is activated in failing human hearts, and may contribute to progressive myocardial dysfunction and remodeling. However, the behavior of endothelin receptor systems (ET(A) and ET(B)) in failing human hearts is not well understood. METHODS AND RESULTS: (125)[I]-endothelin-1 binding assays conducted in the presence of a non-hydrolyzable guanine nucleotide to uncouple agonist binding demonstrated that membranes prepared from nonfailing left ventricles (LVs) exhibit a mixed pattern of ET(A) ( approximately 60%) and ET(B) ( approximately 40%) receptor protein expression. Chronic LV failure from either idiopathic dilated (IDC) or ischemic (ISC) cardiomyopathy was accompanied by a significant (P<0.001) increase in ET(A) receptor density, to approximately 80% of the total population, and a significant (P<0.02) decrease in ET(B) receptor density. Ribonuclease protection assays demonstrated an increase in ET(A) mRNA abundance in IDC and ISC LVs, and a significant (P<0.04) increase in ET(B) mRNA abundance in ISC LVs. Enzyme-linked immunoabsorbent assays demonstrated a significant increase in tissue immunoreactive endothelin-1 concentration in IDC (P=0.01) and in IDC+ISC LVs (P=0.02), but receptor subtype protein or mRNA level was not significantly correlated with tissue ET-1 across all LVs. In situ reverse-transcription polymerase chain reaction in LV sections demonstrated that in both failing and nonfailing LVs the ET(A) gene is expressed in cardiac myocytes, vascular smooth muscle and endothelium; the ET(B) gene is expressed in cardiac myocytes, fibroblasts and endothelium; and the prepro-endothelin-1 gene is expressed in myocytes and interstitial cells. CONCLUSIONS: In chronically failing human LVs, ET(A) receptor density is increased to become the dominant subtype while ET(B) receptor density is decreased. The ET(A), but not the ET(B) density change is accompanied by cognate regulation of mRNA abundance. Both receptor genes and prepro-endothelin-1 are expressed in cardiac myocytes. Finally, based on a lack of correlation with endothelin-1 tissue levels, it is unlikely that the failure-related changes in ET(A) and ET(B) receptor protein and mRNA expression result from homologous regulation by agonist exposure.