RESUMO
Cystatin C (CST3) is a cysteine protease inhibitor that regulates lysosomal enzyme activity and is reported to be involved in the process of neurodegeneration. In the present study, we investigated whether CST3 interacts with other proteins and affects neurodegeneration in vitro and under disease conditions. We intended to identify any protein that interacts with CST3 by using a yeast two-hybrid system, and found prosaposin (PSAP) as a candidate protein. The binding of CST3 and PSAP was confirmed using an immunoprecipitation-based in vitro assay. An enzyme activity assay revealed that PSAP ameliorated CST3-mediated inhibition of cathepsin B activity. To investigate further, CST3 and PSAP were co-expressed in HeLa cells and in a human neuronal cell line (A1). Subsequent immunocytochemical studies demonstrated that they were co-localized mainly in the lysosomes. In spinal motor neurons of autopsy cases, both proteins showed a granular staining pattern. However, the staining intensities of CST3 and PSAP decreased in Bunina body-positive motor neurons of patients with amyotrophic lateral sclerosis (ALS). Further analysis with immunofluorescence staining revealed that CST3 was immunopositive in the inclusions of ALS motor neurons, where it was closely associated, and sometimes co-localized, with PSAP. CST3 immunoreactivity is recognized as a marker for Bunina bodies in ALS, suggesting that PSAP might also be included in Bunina bodies. The interaction of CST3 and PSAP may alter their functions, leading to motor neuron degeneration in ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Cistatina C/metabolismo , Neurônios/metabolismo , Saposinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/patologia , Western Blotting , Catepsina B/metabolismo , Células Cultivadas , Cistatina C/genética , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Lipofuscina/metabolismo , Masculino , Neurônios/patologia , Saposinas/genética , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Vascular remodeling plays an important role in the development of arteriosclerosis and any of the resulting white matter lesions in the brain. An imbalance between cysteine proteases and the cysteine protease inhibitor cystatin C (CST3) may exacerbate vascular remodeling through degradation of extracellular matrix proteins. Therefore, we evaluated the association between functional polymorphisms in the CST3 gene and the development of cerebral white matter lesions. METHODS: In a total of 2,676 participants, 3 CST3 genepolymorphisms were genotyped in 92 cases with severe deep white matter hyperintensity (DWMH), and 184 subjects were randomly selected age- and sex-matched controls without any signs of DWMH. The genetic effects of these polymorphisms on DWMH and plasma CST3 levels were examined. CST3 expression vectors were transfected into an astrocytoma cell line and the expression level of CST3 mRNA was analyzed by quantitative RT-PCR. Intracellular and secreted levels of CST3 in the cell culture were quantified by Western blot and ELISA, respectively. RESULTS: A significant association was found between one CST3 gene haplotype and DWMH (p = 0.002). This haplotype was also associated with lower plasma CST3 levels (p = 0.01). An in vitro transfection study revealed that the +148A allele, which is included in the risk haplotype, significantly reduced the secretion and increased the intracellular accumulation of CST3; however, it had no effect on the mRNA expression. CONCLUSIONS: Our study shows that polymorphisms in the CST3 gene are significantly associated with the likelihood of DWMH. Substitution of A for G at +148 of the CST3 gene decreased the extracellular availability of CST3 in vitro, which might result in the activation of protease activity.
Assuntos
Encefalopatias/genética , Cistatina C/genética , Leucoencefalopatias/genética , Polimorfismo de Nucleotídeo Único/genética , Idoso , Alelos , Encefalopatias/sangue , Estudos de Casos e Controles , Cistatina C/sangue , Feminino , Predisposição Genética para Doença/genética , Genótipo , Haplótipos/genética , Humanos , Leucoencefalopatias/sangue , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Cystatin C (CysC), an endogenous cysteine protease inhibitor, has been implicated in the apoptosis and differentiation processes of neuronal cells. In this study, we have investigated the pathway involved in the process. A human neuronal hybridoma cell line (A1 cell) was treated with CysC in both undifferentiated and retinoic acid (RA)-induced differentiated conditions, which decreased overall process length in both conditions. Also, CysC increased apoptotic cell number time-dependently, as revealed by TUNEL assay. Western blot analysis demonstrated that in differentiated A1 cells, CysC treatment decreased Bcl-2 and increased active caspase-9 protein level time-dependently. Immunocytochemistry results revealed that, CysC treatment significantly increased active form of Bax expressing cell number, which co-localized with mitochondria. Mitogen activated protein (MAP) kinase inhibition experiments showed that Bax mRNA induction and Bcl-2 mRNA inhibition by CysC treatment were c-Jun N-terminal kinase (JNK)-dependent. After RA-induced differentiation, choline acetyltransferase (ChAT) and neurofilament (NF) mRNA levels were increased in A1 cells. CysC treatment inhibited NF mRNA level in both undifferentiated and RA-differentiated, and increased TH mRNA in differentiated A1 neurons. Analysis of signal transduction pathway demonstrated that TH gene induction was also JNK-dependent. Thus, our results demonstrated the significance of JNK-dependent pathways on CysC-induced apoptosis and TH gene expression in neuronal cells, which might be an important target in the management of CysC dependent neurodegenerative processes.
Assuntos
Apoptose/efeitos dos fármacos , Cistatina C/farmacologia , Regulação Enzimológica da Expressão Gênica/fisiologia , MAP Quinase Quinase 4/metabolismo , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Linhagem Celular , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Neurônios/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Cystatin C Leu68Gln variant is known to induce amyloid deposition in cerebral arterioles, resulting in Icelandic type cerebral amyloid angiopathy (CAA). Wild-type cystatin C is also observed in solitary CAA involving amyloid beta protein (Abeta), and accelerates the amyloidogenicity of Abeta in vitro. In neurological inflammatory diseases and leptomeningeal metastasis, low cystatin C levels are accompanied with high activities of cathepsins in the cerebrospinal fluid. Among the cells in CNS, astrocytes appear to secrete cystatin C in response to various proteases and cytokines. Co-localization of Abeta and cystatin C in the brains of Alzheimer's disease (AD) led to the hypothesis that cystatin C is involved in the disease process. We demonstrated that cystatin C microinjection into rat hippocampus induced neuronal cell death in dentate gyrus. Furthermore, apoptotic cell death was observed in neuronal cells treated with cystatin C in vitro. Up-regulation of cystatin C was observed in glial cells with neuronal cell death in vivo. These findings indicate the involvement of cystatin C in the process of neuronal cell death.
Assuntos
Doenças do Sistema Nervoso Central/fisiopatologia , Cistatinas/fisiologia , Substituição de Aminoácidos , Amiloidose/genética , Amiloidose/patologia , Morte Celular , Doenças do Sistema Nervoso Central/patologia , Angiopatia Amiloide Cerebral/genética , Angiopatia Amiloide Cerebral/patologia , Proteínas do Líquido Cefalorraquidiano/fisiologia , Cistatina C , Cistatinas/líquido cefalorraquidiano , Cistatinas/genética , Variação Genética , Humanos , Inflamação/prevenção & controleRESUMO
OBJECTIVE: To determine whether oestrogen enhances platinum sensitivity, and if promoter CpG methylation of the oestrogen receptor-alpha (ER-alpha) gene determines the potential of cisplatin-induced apoptosis in prostate cancer, as the high-mobility group 1 (HMG1) preferentially binds to cisplatin-modified DNA and is up-regulated after oestrogen treatment in breast cancer cell line MCF-7. MATERIALS AND METHODS: The study comprised prostate cancer cell lines (LNCaP and PC-3), 156 pathologically confirmed 156 radical prostatectomy samples and eight hormone-refractory prostate cancer (HRPC) samples (from needle biopsy). Expression of HMG1 in cell lines was analysed by Western blotting or differential reverse-transcription-polymerase chain reaction (PCR). The methylation status of ER-alpha was analysed by methylation-specific PCR using bisulphite DNA as a template or bisulphite DNA sequencing. RESULTS: In LNCaP cells, treatment with oestrogen increased HMG1 expression and co-treatment with cisplatin and oestrogen reduced cell viability by accelerating apoptosis, compared with cisplatin alone. However, in PC-3, oestrogen did not up-regulate HMG1 or accelerate the cisplatin-induced apoptosis. Although ER-beta was expressed in both LNCaP and PC-3, ER-alpha was expressed only in LNCaP. Bisulphite DNA sequencing of the ER-alpha promoter showed partial methylation in LNCaP but complete methylation in PC-3. ER-alpha AS transfection diminished the cisplatin-induced apoptosis in oestrogen-treated LNCaP cells. In clinical samples there was ER-alpha hypermethylation in 40% of prostate cancers this correlated with Gleason score (GS, 31% for GS < 7, 50% for GS = 7 and 56% for GS > 7). In addition, five of eight HRPC samples showed ER-alpha hypermethylation. CONCLUSION: These findings suggest that HMG1 induction as an enhancer of platinum sensitivity is mediated through interaction between oestrogen and ER-alpha. As CpG hypermethylation of the ER-alpha promoter is a frequent event in aggressive prostate cancer, negative conversion of ER-alpha methylation is essential to achieve the most beneficial effect when combined chemotherapy of cisplatin with oestrogen is used in patients with prostate cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Receptor alfa de Estrogênio/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Idoso , Western Blotting , Cisplatino/administração & dosagem , Estrogênios/administração & dosagem , Humanos , Masculino , Metilação , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Arginine-specific ADP-ribosylation is one of the posttranslational modifications of proteins by transferring one ADP-ribose moiety of NAD to arginine residues of target proteins. This modification, catalyzed by ADP-ribosyltransferase (Art), is reversed by ADP-ribosylarginine hydrolase (AAH). In this study, we describe a new method combining an anti-ADP-ribosylarginine antibody (alphaADP-R-Arg Ab) and AAH for detection of the target protein of ADP-ribosylation. We have raised alphaADP-R-Arg Ab with ADP-ribosylated histone and examined the reactivity of the antibody with proteins treated by Art and/or AAH, as well as in situ ADP-ribosylation system with mouse T cells. Our results indicate that the detection of ADP-ribosylated protein with alphaADP-R-Arg Ab and AAH is a useful tool to explore the target proteins of ADP-ribosylation. We applied the method to search endogenously ADP-ribosylated protein in the rat, and detected possible target proteins in the skeletal muscle, which has high Art activity.
Assuntos
Adenosina Difosfato Ribose/análise , Adenosina Difosfato Ribose/metabolismo , Anticorpos/imunologia , Arginina/análise , Arginina/metabolismo , N-Glicosil Hidrolases/metabolismo , Adenosina Difosfato Ribose/imunologia , Animais , Arginina/imunologia , Galinhas , Camundongos , RatosRESUMO
PURPOSE: Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the pathogenesis of several human cancers. Wnt inhibitory factor-1 (Wif-1) was identified as one of the secreted antagonists that can bind Wnt protein. We hypothesize that Wif-1 plays an important role in bladder cancer pathogenesis. EXPERIMENTAL DESIGN: To test this hypothesis, epigenetic and genetic pathways involved in the Wif-1 gene modulation and expression of Wnt/beta-catenin-related genes were analyzed in 4 bladder tumor cell lines and 54 bladder tumor and matched normal bladder mucosa. RESULTS: Wif-1 mRNA expression was significantly enhanced after 5-aza-2'-deoxycytidine treatment in bladder tumor cell lines. Wif-1 promoter methylation level was significantly higher and Wif-1 mRNA expression was significantly lower in bladder tumor samples than in bladder mucosa samples. In the total bladder tumor and bladder mucosa samples, an inverse correlation was found between promoter methylation and Wif-1 mRNA transcript levels. However, loss-of-heterozygosity at chromosome 12q14.3 close to the Wif-1 gene loci was a rare event (3.7%). Nuclear accumulation of beta-catenin was significantly more frequent in bladder tumor than in bladder mucosa and inversely correlated with Wif-1 expression. In addition, known targets of the canonical Wnt/beta-catenin signaling pathway, such as c-myc and cyclin D1, were up-regulated in bladder tumor compared with bladder mucosa, and this up-regulation was associated with reduced Wif-1 expression at both mRNA and protein levels. Furthermore, transfection of Wif-1 small interfering RNA into bladder tumor cells expressing Wif-1 mRNA transcripts had increased levels of c-myc and cyclin D1 and accelerated cell growth. CONCLUSION: This is the first report showing that CpG hypermethylation of the Wif-1 promoter is a frequent event in bladder tumor and may contribute to pathogenesis of bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway. The present study elucidates novel pathways that are involved in the pathogenesis of bladder cancer.
Assuntos
Proteínas de Transporte/genética , Epigênese Genética/fisiologia , Proteínas Repressoras/genética , Transdução de Sinais , Neoplasias da Bexiga Urinária/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND: Aberrant CpG methylation profiles of gene promoters and their correlation with advanced pathologic features have been well investigated in prostate carcinoma (PC). Several case-control and prospective studies have revealed a positive association between current smoking and PC. The authors hypothesized that smoking influences both progression and prognosis of PC through CpG hypermethylation of related genes. METHODS: A total of 164 PC patients (52 current, 30 former, and 82 never smokers) and 69 benign prostatic hyperplasia (BPH) patients were examined by methylation-specific PCR (MSP) for 3 genes: adenomatous polyposis coli (APC), glutathione S-transferase pi (GSTP1), and multidrug resistance one (MDR1). The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The newly defined methylation score (M-score) of each sample is the sum of the corresponding log hazard ratio (HR) coefficients derived from multivariate logistic regression analysis for pathology (BPH vs. PC), and was related to clinical and pathologic outcome including smoking status. RESULTS: The M-score was significantly higher in the current smokers than in never smokers (P = 0.008). Spearman rank correlation test demonstrated a significant correlation between pack-years smoked and M-score in PCs (P = 0.039). Significant correlation of the M-score methylation was observed with high pT category (P < 0.001), high Gleason sum (P < 0.001), high preoperative prostate-specific antigen (PSA) (P = 0.041), and advanced pathologic features. In addition, Gleason sum was significantly associated with PSA failure-free probability as a poor outcome (P = 0.020). CONCLUSIONS: This is the first study to demonstrate significant correlation of the methylation status of multigenes with smoking status in PC. Smoking status may influence both progression and prognosis of PC through CpG hypermethylation of related genes.
Assuntos
Ilhas de CpG , Metilação de DNA , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Fumar/efeitos adversos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Regiões Promotoras Genéticas , Hiperplasia Prostática/patologia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Análise de RegressãoRESUMO
Cystatin C, a cysteine protease inhibitor, is implicated in pathogenesis of late-onset Alzheimer's disease and other neurological disorders. Our recent study showed that cystatin C injection into rat hippocampus induced neuronal cell death in granule cell layer of dentate gyrus in vivo. We further confirmed that cystatin C neurotoxicity was inhibited by simultaneous coapplication of cathepsin B, a cysteine protease. In vitro cytotoxicity was also studied in cultures of human CNS neurons, mixed cultures with astrocytes and A1 human hybrid neurons. Cystatin C induced neuronal cell death in a dose-dependent manner, which accompanied increased number of TUNEL (+) cells, up-regulation of active caspase-3 and DNA ladder. The results of the present study indicate that cystatin C participates in the process of apoptotic neuronal cell death in experimental conditions by means of inhibitory activity of cysteine proteases, and that cystatin C might be involved in the pathogenesis in human neurological disorders including Alzheimer's disease.
Assuntos
Catepsina B/farmacologia , Sistema Nervoso Central/citologia , Cistatinas/farmacologia , Neurônios/fisiologia , Animais , Apoptose , Astrócitos/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Morte Celular , Sobrevivência Celular , Células Cultivadas , Sistema Nervoso Central/efeitos dos fármacos , Cistatina C , Cistatinas/antagonistas & inibidores , DNA/biossíntese , DNA/genética , Hipocampo , Humanos , Células Híbridas , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Microinjeções , Ratos , Ratos Sprague-Dawley , Sais de Tetrazólio , TiazóisRESUMO
In atherosclerosis, abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role to form fibroproliferative lesions and platelet-derived growth factor (PDGF)-BB is one of the most potent chemoattractants and proliferative factors for VSMCs. Taurine, sulfur-containing beta-amino acid, has been considered to prevent the development of atherosclerosis, although the molecular mechanism remains obscure. Previously, we demonstrated that taurine significantly suppressed PDGF-BB-induced cell proliferation, DNA synthesis, immediate-early gene expressions and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in VSMCs. The present study was aimed at elucidating the precise molecular mechanism of taurine in PDGF-BB signaling pathway. We showed that taurine significantly suppressed PDGF-BB-induced phosphorylation of PDGF-beta receptor and activation of its downstream signaling molecules such as Ras, MAPK/ERK kinase (MEK)1/2 and Akt. Because taurine did not attenuate phorbol 12-myristate 13-acetate (PMA)-induced PDGF-beta receptor-independent ERK1/2 phosphorylation, we further investigated the suppressive mechanism of taurine in PDGF-beta receptor level. Although taurine did not directly affect PDGF receptor autophosphorylation in vitro, taurine promoted PDGF-beta receptor dephosphorylation and restored PDGF-BB-induced suppression of protein tyrosine phosphatase (PTPase) activity. Taken together, we propose that taurine could prevent or delay the progression of atherosclerosis by PTPase-mediated suppression of PDGF-beta receptor phosphorylation, and by decreasing the activation of its downstream signaling molecules in VSMCs.
Assuntos
Aterosclerose/metabolismo , Músculo Liso Vascular/citologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Taurina/farmacologia , Animais , Becaplermina , Western Blotting , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Endogâmicos WKY , Transdução de Sinais/efeitos dos fármacosRESUMO
Heparanase plays a critical role in the degradation of extracellular matrix and cell membrane and is frequently upregulated in malignant tumors. Transcription factor, early growth response 1 (EGR1), is closely associated with inducible transcription of the heparanase gene. We hypothesized that promoter CpG hypomethylation with increased EGR1 expression could determine heparanase expression during the pathogenesis of bladder cancer. Bladder cancer cell lines (J82, T24 and transitional cell carcinoma) significantly restored heparanase expression after 5-Aza-dC treatment. Transfection of EGR1 siRNA with T24 bladder cancer cell line significantly downregulated heparanase expression compared to the control siRNA transfection. In 54 bladder cancer and paired normal bladder samples, heparanase expression was significantly higher in bladder cancer than in normal bladder (P<0.01). We performed methylation-specific PCR targeting the CpG sites within the core-binding consensus motifs of EGR1 (GGCG) and Sp1 (GGGCGG). Methylation prevalence was significantly higher in normal bladder than in bladder cancer (P<0.05) and inversely correlated with heparanase expression (P=0.055). In the total series of bladder cancer and normal bladder samples, the combination of promoter CpG methylation and EGR1 expression regulated heparanase expression in a stepwise manner, where heparanase expression was the lowest in methylation-positive and EGR1-negative samples and the highest in methylation-negative and EGR1-positive samples. To our knowledge, this is the first study demonstrating that increased heparanase expression during the pathogenesis of bladder cancer is due to promoter hypomethylation and transcription factor EGR1.
Assuntos
Ilhas de CpG , Metilação de DNA , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Glucuronidase/metabolismo , Regiões Promotoras Genéticas , Sequência de Bases , Linhagem Celular Tumoral , DNA de Neoplasias , Proteína 1 de Resposta de Crescimento Precoce/genética , Glucuronidase/genética , Humanos , Reação em Cadeia da Polimerase , Interferência de RNA , RNA Interferente Pequeno/genética , Bexiga Urinária/enzimologiaRESUMO
TGF-beta1 is a well-known immunosuppressive cytokine that inhibits inducible nitric oxide synthase (iNOS) gene expression in various cells including macrophages. In this study, we investigated the suppressive mechanisms of TGF-beta1 on IFN-gamma-induced iNOS gene expression using the murine macrophage-like cell line RAW 264.7. TGF-beta1 decreased iNOS protein amount through enhanced degradation, although TGF-beta1 did not affect IFN-gamma-induced iNOS mRNA level or stability. In addition, the enhancement of iNOS protein degradation by TGF-beta1 treatment was almost completely blocked by MG132, a proteasome inhibitor. Furthermore, TGF-beta1 enhanced the trypsin-like activity of proteasomes in the presence of IFN-gamma, although did not enhance the peptidylglutamyl-peptide hydrolyzing and chymotrypsin-like activities of proteasomes. The level of ubiquitinated iNOS protein was not significantly altered by IFN-gamma or IFN-gamma plus TGF-beta1 treatment. Because MG132 inhibited iNOS protein degradation and IFN-gamma plus TGF-beta1 treatment increased the trypsin-like activity of proteasomes, we hypothesized that TGF-beta1 might enhance iNOS protein degradation via the ubiquitin-proteasome pathway in the presence of IFN-gamma. We propose that these mechanisms of TGF-beta1 in the posttranslational regulation of iNOS gene expression may contribute to suppression of excess nitric oxide during inflammatory processes.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Óxido Nítrico Sintase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Leupeptinas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Complexos Multienzimáticos , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta1 , Ubiquitina/metabolismoRESUMO
Mono-ADP-ribosylation is a post-translational modification that regulates the functions of target proteins or peptides by attaching an ADP-ribose moiety. Here we report the purification, molecular cloning, characterization and tissue-specific distribution of novel arginine-specific Arts (ADP-ribosyltransferases) from chicken. Arts were detected in various chicken tissues as GPI (glycosylphosphatidylinositol)-anchored forms, and purified from the lung membrane fraction. By molecular cloning based on the partial amino acid sequence using 5'- and 3'-RACE (rapid amplification of cDNA ends), two full-length cDNAs of chicken GPI-anchored Arts, cgArt1 (chicken GPI-anchored Art1) and cgArt2, were obtained. The cDNA of cgArt1 encoded a novel polypeptide of 298 amino acids which shows a high degree of identity with cgArt2 (82.9%), Art6.1 (50.2%) and rabbit Art1 (42.1%). In contrast, the nucleotide sequence of cgArt2 was identical with that of Art7 cloned previously from chicken erythroblasts. cgArt1 and cgArt2 proteins expressed in DT40 cells were shown to be GPI-anchored Arts with a molecular mass of 45 kDa, and these Arts showed different enzymatic properties from the soluble chicken Art, Art6.1. RNase protection assays and real-time quantitative PCR revealed distinct expression patterns of the two Arts; cgArt1 was expressed predominantly in the lung, spleen and bone marrow, followed by the heart, kidney and muscle, while cgArt2 was expressed only in the heart and skeletal muscle. Thus GPI-anchored Arts encoded by the genes cgArt1 and cgArt2 are expressed extensively in chicken tissues. It may be worthwhile determining the functional roles of ADP-ribosylation in each tissue.
Assuntos
ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Arginina/química , Glicosilfosfatidilinositóis/química , ADP Ribose Transferases/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Clonagem Molecular , Regulação da Expressão Gênica , Dados de Sequência Molecular , Especificidade por Substrato , Distribuição TecidualRESUMO
Gamma-catenin is a cell adhesion molecule and a candidate mediator of Wnt signal transduction. We hypothesized that impaired regulation of gamma-catenin through genetic and epigenetic pathways is associated with the pathogenesis of prostate cancer. To test this hypothesis, cytosine-phosphate-guanine methylation, loss of heterozygosity (LOH), and mutation status of the gamma-catenin gene were analyzed in cultured prostate cancer cell lines, 180 localized prostate cancers, 69 benign prostatic hyperplasias, and 11 hormone refractory prostate cancers (HRPC). In prostate cancer cell lines (DuPro, LNCaP, ND-1, and PC3), gamma-catenin mRNA transcripts were increased after 5-aza-2'-deoxycytidine treatment. In localized prostate cancer, gamma-catenin expression was lower but prevalence of gamma-catenin methylation was higher compared with benign prostatic hyperplasia. However, gamma-catenin methylation did not correlate with Gleason sum, pT category, or capsular penetration. Among localized prostate cancers with positive gamma-catenin methylation, the presence of LOH at chromosome 17q21 was closely related to down-regulation of gamma-catenin mRNA expression. The gamma-catenin mutations were not found in localized prostate cancers, whereas six mutations were found in five HRPCs within or close to the GSK-3beta consensus motif phosphorylation site, among which four HRPCs showed strong nuclear gamma-catenin accumulation. In these four HRPCs, Bcl-2 expression was increased, whereas the target of the Wnt signal, c-myc, was only expressed in one HRPC. Therefore, although epigenetic gamma-catenin methylation is an early event in the development of prostate cancer, simultaneous events of epigenetic cytosine-phosphate-guanine methylation and genetic LOH may be responsible for functional loss of gamma-catenin. The gamma-catenin mutation related to Bcl-2 overexpression has a significant effect on the pathogenesis of HRPC. This is the first report to characterize the epigenetic and genetic regulation of gamma-catenin in human prostate cancer.
Assuntos
Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias da Próstata/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/deficiência , Metilação de DNA , Desmoplaquinas , Regulação para Baixo , Humanos , Perda de Heterozigosidade , Masculino , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Hiperplasia Prostática/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , gama CateninaRESUMO
PURPOSE: Gamma-catenin is a cell adhesion protein, and its functional loss is associated with tumor invasion and metastasis. We hypothesize that (1) promoter CpG methylation regulates the expression and function of the gamma-catenin gene in renal cell carcinoma (RCC) and (2) methylation of the gamma-catenin gene is associated with poor prognosis of RCC. To test these hypotheses, we analyzed the CpG methylation status of the gamma-catenin gene and its correlation with clinical outcome in RCC. EXPERIMENTAL DESIGN: Genomic DNA and total RNA were extracted from three renal cancer cell lines (A498, Caki-1, and Caki-2) and 54 RCC tissue samples with their corresponding normal kidney tissue samples. Expression of gamma-catenin gene was analyzed by reverse transcription-PCR and immunostaining. Promoter methylation was analyzed by two different methylation-specific PCR (MSP-A and MSP-B), and the results were verified by DNA sequencing. RESULTS: The demethylating agent (5-aza-2'-deoxycytidine) increased levels of mRNA transcript of the gamma-catenin gene in three renal cancer cell lines. Gamma-catenin mRNA and protein expression were significantly reduced in RCC samples compared with normal kidney samples, respectively (P < 0.05). MSP-A and MSP-B bands were detected in 45 of 54 (83.3%) and 49 of 54 (90.7%) RCC samples, respectively. In normal kidney, weak products of MSP-A and MSP-B were detected in 5 of 54 (9.3%) and 6 of 54 (11.1%) samples, respectively. Likewise, both MSP-A and MSP-B ratios were significantly higher in RCC samples compared with normal kidney samples, respectively (P < 0.01). Multivariate analysis revealed that the MSP-B ratio was a powerful and independent predictor superior to nuclear grade and Robson stage with respect to survival and disease progression (P = 0.029 and 0.0071, respectively). No mutations in the NH(2)-terminal region of gamma-catenin were found in this study. CONCLUSION: Expression of gamma-catenin is regulated by promoter CpG methylation, and the balance between methylated and unmethylated RCC cell populations could determine its functional role. Because the conventional nuclear grade and/or staging system have some limitations to predict precise clinical outcome, this is the first report demonstrating that promoter CpG methylation of gamma-catenin can be an independent and superior predictor for survival and disease progression.
Assuntos
Azacitidina/análogos & derivados , Carcinoma de Células Renais/genética , Ilhas de CpG/genética , Proteínas do Citoesqueleto/genética , Metilação de DNA , Neoplasias Renais/genética , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Azacitidina/farmacologia , Carcinoma de Células Renais/metabolismo , DNA de Neoplasias/análise , Decitabina , Desmoplaquinas , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Rim/metabolismo , Rim/patologia , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/genética , Prognóstico , Taxa de Sobrevida , gama CateninaRESUMO
PURPOSE: Heparanase degrades heparan sulfate and has been implicated in tumor invasion and metastasis. The transcription factor, early growth response 1 (EGR1), is associated with the inducible transcription of the heparanase gene. We hypothesize that CpG hypomethylation in the heparanase promoter coupled with up-regulation of EGR1 levels may induce heparanase expression in human prostate cancer. EXPERIMENTAL DESIGN: Cultured prostate cancer cell lines (Du145, DuPro, LNCaP, and PC-3) with and without 5'-aza-2-deoxycytidine treatment, 177 prostate cancer samples, and 69 benign prostatic hyperplasia (BPH) samples were used. The frequency and level of heparanase promoter methylation were analyzed by methylation-specific primers which covered the core binding motif of EGR1 (GGCG) or SP1 (GGGCGG) or both. RESULTS: In cultured Du145, DuPro, LNCaP, and PC-3 cell lines, mRNA transcripts of heparanase were significantly increased after 5'-aza-2-deoxycytidine treatment, suggesting that promoter methylation was involved in the regulation of heparanase mRNA transcript. Significantly higher methylation was found in BPH samples than in prostate cancer samples (P < 0.0001), whereas mRNA transcripts of the heparanase gene were inversely lower in BPH samples than in prostate cancer samples (P < 0.01). EGR1 expression in prostate cancer tissues was significantly higher than in BPH tissues (P < 0.001) and correlated with heparanase expression (P < 0.0001). Moreover, multiple regression analysis revealed that up-regulation of EGR1 contributed significantly more to heparanase expression than did promoter CpG hypomethylation in prostate cancer samples (P < 0.0001). CONCLUSIONS: To our knowledge this is the first comprehensive study demonstrating that increased heparanase expression in prostate cancer tissues is due to promoter hypomethylation and up-regulation of transcription factor EGR1.
Assuntos
Azacitidina/análogos & derivados , Metilação de DNA , Proteínas de Ligação a DNA/genética , Glucuronidase/genética , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Idoso , Idoso de 80 Anos ou mais , Azacitidina/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Proteína 1 de Resposta de Crescimento Precoce , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Hiperplasia Prostática/enzimologia , Hiperplasia Prostática/genética , Hiperplasia Prostática/patologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Regulação para Cima/genéticaRESUMO
Recently, survival benefit by chemotherapy using paclitaxel (PTX) and the induction of thymidine phosphorylase (TP) by PTX have been reported in several solid tumors. On the other hand, TP confers antiapoptotic effect on tumor cells through inhibition of caspase-8 activation in vitro. On the basis of these previous observations, we hypothesized that (a) TP can be induced after PTX treatment in human prostate cancer (PC) and (b) blockade of PTX-induced TP expression can enhance the apoptotic processes in human PC cells. PTX was used to find TP expression in all eight hormone-refractory PC cases after chemotherapy; however, cleaved caspase-8 was not expressed after chemotherapy in the six hormone-refractory PC cases with strong TP expression. In PC cell lines (PC-3, DU 145, and LNCaP), TP expression after PTX treatment was clearly up-regulated in a dose-dependent manner. Cell viability of PC cell lines treated with PTX and TP antisense was significantly reduced in a time-dependent and dose-dependent manner compared with the PTX treatment alone. Likewise, apoptotic index of PC cells treated with PTX and TP antisense was significantly increased in comparison with PTX alone. After complete blockade of PTX-induced TP translation by TP antisense transfection, cleaved form of caspase-3 and poly(ADP-ribose) polymerase was increased, and this exaggeration of apoptosis also ran parallel with caspase-8 activation in a PTX dose-dependent manner. However, in PC cell lines treated with TP antisense alone, neither caspase-3 nor poly(ADP-ribose) polymerase was cleaved despite caspase-8 activation. These results indicate that PTX-induced TP up-regulation is associated with decreased caspase-8 activation. This study is the first report showing that blockade of PTX-induced TP expression could exaggerate the processing of apoptosis in PC cells treated with PTX. Our results provide preclinical evidence that TP could be a new molecular target for enhancing the potency of PTX-mediated apoptosis in PC cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Paclitaxel/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Timidina Fosforilase/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Fitogênicos/antagonistas & inibidores , Apoptose/fisiologia , Caspase 3 , Caspase 8 , Caspases/biossíntese , Indução Enzimática/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Paclitaxel/antagonistas & inibidores , Poli(ADP-Ribose) Polimerases/biossíntese , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Timidina Fosforilase/biossíntese , Timidina Fosforilase/metabolismoRESUMO
Multidrug resistance 1 (MDR1) gene encodes for P-glycoprotein (P-gp), a Mr 170,000 transmembrane calcium-dependent efflux pump that is inactivated in prostate cancer. We hypothesize that inactivation of the MDR1 gene through CpG methylation contributes to the pathogenesis and progression of prostate cancer. To test this hypothesis, CpG methylation status of the MDR1 promoter and its correlation with clinicopathological findings were evaluated in 177 prostate cancer samples and 69 benign prostate hypertrophy (BPH) samples. Cellular proliferation index and apoptotic index were determined by proliferating cell nuclear antigen (PCNA) and single-strand DNA immunostaining, respectively. After 5-aza-2'-deoxycytidine treatment, increased expression of MDR1 mRNA transcript was found in prostate cancer cell lines (DU145, DuPro, and ND1). MDR1 methylation frequency was significantly higher in prostate cancer samples compared with BPH samples (54.8 versus 11.6%, respectively, P < 0.001). Logistic regression analysis revealed that PC patients are 11.5 times more likely to have MDR1 methylation than BPH patients (95% confidence interval 4.87-27.0) and that MDR1 methylation is independent of the age. Significant correlation of MDR1 methylation was observed with high pT category (P < 0.001), high Gleason sum (P = 0.008), high preoperative prostate-specific antigen (P = 0.01), and advancing pathological features. In addition, PCNA-labeling index were significantly higher in methylation-specific PCR (MSP)-positive than in MSP-negative prostate cancer samples (P = 0.048). In contrast, no significant difference in apoptotic index was found between MSP-positive and -negative prostate cancer samples. These findings suggest that CpG hypermethylation of MDR1 promoter is a frequent event in prostate cancer and is related to disease progression via increased cell proliferation in prostate cancer cells.
Assuntos
Azacitidina/análogos & derivados , Metilação de DNA , Regulação Neoplásica da Expressão Gênica/genética , Genes MDR/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Azacitidina/farmacologia , Sequência de Bases , Divisão Celular/genética , Ilhas de CpG/genética , Decitabina , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Genes MDR/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Platelet-derived growth factor (PDGF) plays an important role in the pathogenic course of atherosclerosis, pulmonary fibrosis, and glomerulonephritis, and increased activity of the PDGF signaling pathway has been implicated as a contributing factor in the progression of the diseases. Taurine may be a prophylactic amino acid for atherosclerosis not only by decreasing plasma cholesterol level, but also by inhibiting the cell proliferation-signaling pathway. To elucidate how taurine affects the signaling pathway, we investigated the effect of taurine on the expression of immediate-early genes and activation of mitogen-activated protein kinases (MAPKs) in NIH/3T3 cells as standard mesenchymal cells. Taurine inhibited PDGF-BB-induced c-fos and c-jun mRNA expressions dose-dependently, although structural analogues of taurine did not. Taurine decreased the PDGF-induced p44/p42 ERK (extracellular signal-regulated kinase) phosphorylation state dose-dependently, although no phosphorylation was observed on JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) and p38 MAPK. Further, PDGF-BB-induced tyrosine phosphorylation of the PDGF-beta receptor was not influenced by treatment with taurine, indicating that taurine never affects ligand-receptor interaction, and may act downstream of the PDGF receptor. Thus, the inhibitory mechanism of taurine on PDGF-induced c-fos and c-jun mRNA expressions may depend on the p44/p42 ERK pathway, but not on PDGF-beta receptor tyrosine phosphorylation, JNK/SAPK or p38 MAPK pathway. These results suggest that taurine may suppress the cell proliferation-signaling pathway through the inhibition of ERK activity and immediate-early gene expression.