RESUMO
We hypothesize that dysregulated expression levels of the developmental regulatory genes in the adult body result in tumor development and malignant progression. PAX genes discovered as human orthologous genes of Drosophila 'paired' encode transcription factors, which control the expression of target genes to go on along the program of development. In this study, we first quantified expression of 9 PAX genes in human nevus pigmentosus tissues, melanoma tissues and melanoma cell lines by the real-time reverse transcription-PCR method. As a result, we found that the expression levels of PAX4 and PAX9 were extremely low in melanoma tissues and cell lines compared to nevus pigmentosus tissues. We then established melanoma cells overexpressing PAX4 and examined roles of PAX4 in cell growth. PAX4-overexpression reduced in vitro cell growth of human melanoma C8161 and MeWo cells. BrdU-uptake assay and cell cycle analysis by flow cytometry indicated that the retardation of cell proliferation by PAX4-overexpression was due to decreased DNA synthesis and cell cycle arrest at the G0/G1 phase. Furthermore, treatment of C8161 and MeWo cells with 5-azacytidine, a DNA demethylating agent, induced the expression of PAX4, suggesting that DNA methylation repressed the PAX4 gene expression in human melanoma. These results suggest that PAX4 functions as a potent tumor suppressor.
Assuntos
Proteínas de Homeodomínio/metabolismo , Melanoma/metabolismo , Nevo Pigmentado/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Neoplasias Cutâneas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Azacitidina/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA/efeitos dos fármacos , Replicação do DNA , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Nevo Pigmentado/genética , Nevo Pigmentado/patologia , Fatores de Transcrição Box Pareados/genética , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/genéticaRESUMO
OBJECTIVE: Epstein-Barr virus (EBV) has been implicated in the genesis of a variety of human cancers. We aimed to confirm the presence and define the role of EBV in bladder cancer. METHODS: A total of 39 bladder cancer specimens were analyzed. Ten urinary bladder tissues obtained at autopsy were used as a normal control. EBV-encoded RNA (EBER) was evaluated by in situ hybridization (ISH). Frozen material available from 18 EBER-positive cases was analyzed by using reverse transcription-polymerase chain reaction for BZLF1, an early lytic gene product. The expression of CD20, CD3, ZEBRA (BZLF1 product) and transforming growth factor beta1 (TGFbeta-1) was assessed using an immunohistochemical technique. RESULTS: Infiltration of EBER-expressing lymphocytes was detected in 26 of 39 bladder cancer cases (66.7%). A small fraction of the tumor cells as well as the infiltrating lymphocytes were positive in two cases. All normal urinary bladder specimens showed negative results. The incidence of EBV-positive lymphocyte infiltration was significantly higher for advanced stage cancers than those in earlier stages (Ta-152% vs T2-4 93%, P = 0.013). The presence of BZLF1 mRNA was demonstrated in seven out of the 18 EBER-positive cases. CONCLUSIONS: Infiltration of EBV-harboring lymphocytes occurs in a large subset of bladder cancers cases. It is more frequently associated with advanced stages. EBV infection in tumor cells is very limited. Our findings suggest that EBV-positive lymphocytes might play a role in bladder cancer progression.
Assuntos
Herpesvirus Humano 4 , Linfócitos do Interstício Tumoral/virologia , Neoplasias da Bexiga Urinária/patologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-IdadeRESUMO
Dominant negative (DN) mutations of tumor suppressor p53 (TP53) are clinically associated with cancer progression and metastasis of endometrial malignancy. To investigate the DN effect on tumor migration and invasion, we generated cells that stably co-expressed wild-type (wt) and R273H DN mutant TP53 (273H cells), and wt and R213Q recessive mutant TP53 (213Q cells), by transfection in endometrial cancer cells HHUA that expressed wt p53. R273H, but not R213Q, repressed wt p53-stimulated transcription of p21, Bax, and MDM2. 273H cells also showed markedly increased in vitro invasion and migration potentials, and displayed reduced Maspin, PAI-1, and KAI1 mRNA expressions as compared with 213Q and wt cells. The induction of wt p53 function by use of Adriamycin resulted in the inhibition of the invasion/migration capacity in association with the up-regulation of p53 target genes to a far greater degree in 213Q and wt cells than in 273H cells. R273H expression in p53-null cancer cell SK-OV-3 and Saos-2 did not significantly affect cell invasion and migration activities. Taken together, these results suggest that transdominance of R273H mutant over wt p53 rather than a gain-of-function promotes tumor metastasis by increasing invasion and migration in HHUA cells.
Assuntos
Neoplasias do Endométrio/patologia , Genes p53 , Mutação , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Neoplasias do Endométrio/genética , Feminino , Humanos , Reação em Cadeia da Polimerase , Transcrição Gênica/genética , TransfecçãoRESUMO
It has been reported that an endogenous matrix metalloproteinase (MMP) inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), is able to inhibit tumour angiogenesis, invasion, and metastasis through inhibition of MMP-2, MMP-9, and membrane type-1 (MT1)-MMP (MMP-14) secretion and activity. In this study, using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR), we have analysed RECK expression levels in resected non-small-cell lung cancer (NSCLC) tissue and compared these data with the clinicopathological features of these patients to investigate the role of RECK in NSCLC. We have also analysed the expression of MMP-2, MMP-9, and MMP-14 and compared the data with those for RECK expression. Tissue samples of primary lung cancers were obtained from a total of 83 patients [46 with adenocarcinomas (ADC) and 37 with squamous cell carcinomas (SCC)] who underwent curative resection. The samples were taken from 83 tumours and 20 matched normal lung tissue samples as controls. Expressions of RECK in ADC and SCC were significantly lower than in the control. In ADC tissue, the expression of RECK was higher in stage IA than in stage IB-IIIA. There was no such a correlation in SCC. In ADC, univariate analysis for relapse-free survival using Cox regression analysis identified low RECK expression (p=0.036), low MMP-14 expression (p=0.038), and tumour T2 (p=0.034) as significant negative prognostic predictors. However, in SCC, none of the clinicopathological factors assessed, including RECK expression, had prognostic value. In conclusion, our study suggests that suppression of RECK expression is involved in the progression of ADC of the lung and that RECK expression in resected ADC of the lung is a favorable predictor of patients' prognosis.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/cirurgia , Idoso , Intervalo Livre de Doença , Feminino , Proteínas Ligadas por GPI , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Masculino , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Glicoproteínas de Membrana/genética , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/patologia , Neoplasias de Células Escamosas/cirurgia , Taxa de Sobrevida , Fatores de TempoRESUMO
Homeobox genes function as master regulators in embryonic morphogenesis. We hypothesized that homeobox genes are essential to maintain tissue- or organ-specificity even in adult body and that the dysregulated expression of homeobox genes results in tumor development and progression. To better understand the roles of homeobox genes in development and progression of esophageal cancer, we analyzed the expression patterns of 39 HOX genes and 4 ParaHOX (CDX1, CDX2, CDX4 and PDX1) genes in esophageal squamous cell carcinoma (ESCC) and normal esophageal mucosa tissues. A total of 48 primary ESCC tissues and 7 normal esophageal mucosa tissues were resected from patients who underwent radical surgery without any preoperative chemotherapy or radiotherapy. The expression of HOX and ParaHOX genes were analyzed by a quantitative real-time RT-PCR method and immunohistochemistry. The expression levels of 24 HOX genes, CDX1, CDX2 and PDX1 were significantly higher in ESCC compared to normal mucosa (p<0.01, Mann-Whitney U test). The Immunohistochemical study revealed that HOXA5 and D9 proteins were more cytoplasmic in ESCC than normal mucosa cells. Our data indicate that the disordered expression of HOX and ParaHOX genes are involved in the development of ESCC or its malignancy.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/química , Neoplasias Esofágicas/patologia , Feminino , Proteínas de Homeodomínio/análise , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mucosa/química , Mucosa/metabolismo , Mucosa/patologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
We have previously shown that transduction of HOXD3, one of homeobox genes, into human lung cancer A549 cells enhances cell motility, invasion and metastasis. In the present study, we examined the roles of integrin beta3 which was up-regulated by HOXD3-overexpression in the HOXD3-induced motility of A549 cells. We first established integrin beta3-transfectants and compared their motile activity to those of the HOXD3-transfected, control-transfected and parental cells by three different assays. The integrin beta3-transfectants as well as the HOXD3-transfectants formed heterodimer with integrin alphav subunit, and showed highly motile activities assessed by haptotaxis or phagokinetic track assay compared to the control transfectants or parental cells. In vitro wound-healing assay revealed that migratory activities were graded as the HOXD3-transfectants > the integrin beta3-transfectants > the control transfectants or parental cells. E-cadherin was expressed in the integrin beta3-transfectants but not expressed in the HOXD3-transfectants. An addition of function-blocking antibody to E-cadherin into the wound-healing assay promoted the migratory activity of the integrin beta3-transfectants, suggesting that E-cadherin prevented the cells from dissociating from the wound edges. These results indicate that increased expression of integrin alphav beta3 and loss of E-cadherin by HOXD3-overexpression are responsible for the enhanced motility and dissociation.
Assuntos
Caderinas/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Homeodomínio/fisiologia , Integrina alfaVbeta3/fisiologia , Neoplasias Pulmonares/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Humanos , Camundongos , Metástase Neoplásica , Transdução de Sinais , Fatores de Transcrição , Fator de Crescimento Transformador beta/farmacologia , CicatrizaçãoRESUMO
Human HOX genes consist of 39 genes and encode transcription factors that function as master developmental regulators. We hypothesized that the misexpression of HOX genes was associated with carcinogenesis and malignant progression. The expression levels of 39 HOX genes in 31 human oral squamous cell carcinoma (SCC), 11 dysplasia, and 10 normal mucosa tissues were quantified by the real-time RT-PCR method. The expression levels of 18 HOX genes in the SCC tissues were significantly higher than those in the normal mucosa tissues. The dysplasia tissues showed higher expression of HOXA2, A3, B3, and D10 than normal mucosa tissues whereas they showed lower expression of HOXA1, B7, B9, and C8 than SCC. The SCC with lymph node metastasis showed high expression of HOXC6 compared to the SCC without it. These results suggest that misexpressions of particular HOX genes are implicated in the development of oral dysplasia and SCC.
Assuntos
Carcinoma de Células Escamosas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Genes Homeobox/genética , Neoplasias Bucais/genética , Humanos , Linfonodos/metabolismo , Metástase Linfática/patologia , Mucosa Bucal/metabolismo , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
We hypothesized that the disordered tissue architecture in cancer results from the cells executing the program designed during ontogeny in a spatio-temporally inappropriate manner. HOX genes are known as master regulators of embryonic morphogenesis, and encode transcription factors which regulate the transcription of the downstream genes to realize the program of body plan. In this study, we quantified the expression levels of 39 HOX genes in 41 human non-small cell lung cancer (non-SCLC) and non-cancerous lung tissues by a comprehensive analysis system based on the real-time RT-PCR method. We found that the expression levels of HOXA1, A5, A10 and C6 in squamous cell carcinoma tissues (and HOXA5 and A10 in adenocarcinoma tissues) were significantly higher than those in the non-cancerous tissues. Comparison of HOX gene expressions between adenocarcinoma and squamous cell carcinoma tissues showed higher expressions of HOXA1, D9, D10 and D11 in squamous cell carcinoma tissues than in adenocarcinoma tissues. Immunohistochemical analysis revealed that HOXA5 and A10 proteins were localized in the cytoplasm of tumor cells in both adenocarcinoma and squamous cell carcinoma tissues. These results suggest that the disordered patterns of HOX gene expressions were involved not only in the development of non-SCLC but also in the histologically aberrant diversity such as adenocarcinoma and squamous cell carcinoma.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/análise , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Homeobox genes regulate sets of genes that determine cellular fates in embryonic morphogenesis and maintenance of adult tissue architecture by regulating cellular motility and cell-cell interactions. Our previous studies showed that a specific member, HOXD3, when overexpressed, upregulates integrin beta3 expression in human erythroleukemia HEL cells and lung cancer A549 cells, and enhances their motility and invasiveness. We performed a microarray study of over 7075 genes to determine the mechanisms underlying the HOXD3-enhanced motility and invasiveness in A549 cells. RT-PCR-based tracking gene analyses highlighted a set of TGF-beta-upregulated genes, which included matrix metalloproteinase-2, syndecan-1, CD44, and TGF-beta-induced 68 kDa protein. Exogenous TGF-beta also caused this pattern of upregulation in A549 cells and enhanced their migratory and invasive activity, confirming the involvement of TGF-beta signaling. However, HOXD3 reduced the expression of TGF-beta-independent genes coding for desmosomal components such as desmoglein, desmoplakin and plakoglobin which are known to suppress tumor invasion and metastasis. These results suggest that HOXD3 enhances the invasive and metastatic potential of cancer cells through the TGF-beta-dependent and -independent pathways.
Assuntos
Movimento Celular , Proteínas de Ligação a DNA , Proteínas de Homeodomínio/fisiologia , Neoplasias/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Regulação para Baixo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Neoplásico/biossíntese , Fatores de Transcrição , Transfecção , Fator de Crescimento Transformador beta/biossíntese , Células Tumorais Cultivadas , Regulação para CimaRESUMO
HOX genes are known not only as master genes that control the morphogenesis, but also as regulator genes that maintain tissue or organ specificity in the adult body. We hypothesized that dysregulated expression of HOX genes was associated with tumor development and malignant progression such as invasion and metastasis. In this study, we analyzed the expression patterns of 39 HOX genes in human invasive ductal breast cancer tissues and normal tissues by the real-time RT-PCR method. We found 11 HOX genes (HOXA1, A2, A3, A5, A9, C11, D3, D4, D8, D9 and D10) expression levels of which were significantly different between cancerous and normal tissues. All 10 genes except HOXC11 were expressed at lower levels in cancerous tissues than normal tissues. Comparing expression levels of each HOX gene among the different types of cancer tissues, the expression level of HOXB7 was lower in lymph node metastasis-positive cancer tissues than negative cancer tissues; those of HOXD12 and D13 were higher in progesterone receptor-positive cancer tissues than negative cancer tissues; and the expression level of HOXC5 was lower in cancerous tissues with mutated-type p53 than in normal and cancerous tissues with wild-type p53. These results suggest that the aberrant expression of HOX genes is related to the development of breast cancer and malignant behavior of cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Homeobox/genética , Adulto , Idoso , Mama/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Análise por Conglomerados , Progressão da Doença , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Mutação , Invasividade Neoplásica , Metástase Neoplásica , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: The purpose of this research was to identify molecular clues to tumor progression and lymph node metastasis in esophageal cancer and to test their value as predictive markers. EXPERIMENTAL DESIGN: We explored the gene expression profiles in cDNA array data of a 36-tissue training set of esophageal squamous cell carcinoma (ESCC) by using generalized linear model-based regression analysis and a feature subset selection algorithm. By applying the identified optimal feature sets (predictive gene sets), we trained and developed ensemble classifiers consisting of multiple probabilistic neural networks combined with AdaBoosting to predict tumor stages and lymph node metastasis. We validated the classifier abilities with 18 independent cases of ESCC. RESULTS: We identified 71 genes of 1289 cancer-related genes of which the expression correlated with tumor stages. Of the 71 genes, 47 significantly differed between the Tumor-Node-Metastasis pT1/2 and pT3/4 stages. Cell cycle regulators and transcriptional factors possibly promoting the growth of tumor cells were highly expressed in the early stages of ESCC, whereas adhesion molecules and extracellular matrix-related molecules possibly promoting invasiveness increased in the later stages. For lymph node metastasis, we identified 44 genes with predictive values, which included cell adhesion molecules and cell membrane receptors showing higher expression in node-positive cases and cell cycle regulators and intracellular signaling molecules showing higher expression in node-negative cases. The ensemble classifiers trained with the selected features predicted tumor stage and lymph node metastasis in the 18 validation cases with respective accuracies of 94.4% and 88.9%. This demonstrated the reproducibility and predictive value of the identified features. CONCLUSION: We suggest that these characteristic genes will provide useful information for understanding the malignant nature of ESCC as well as information useful for personalizing the treatments.
Assuntos
Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Algoritmos , Biomarcadores Tumorais , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Proliferação de Células , DNA Complementar/metabolismo , Progressão da Doença , Feminino , Humanos , Ligantes , Modelos Lineares , Linfonodos/patologia , Metástase Linfática , Masculino , Metástase Neoplásica , Análise de Sequência com Séries de OligonucleotídeosRESUMO
"Seven in absentia homolog" (SIAH) is E3 ubiquitin ligase gene. It is reported that mice have three SIAH homolog genes, Siah1a, Siah1b and Siah2, while men have SIAH1 and SIAH2 genes. It has recently been reported that, among these genes, mouse Siah1b gene is a direct target of p53. However, the nature of human "SIAH1b" gene remains unknown. In this study, we identified it on chromosome X by using a computed searching methods (in silico cloning). Using cDNA from human tissues, we attempted to amplify this region by PCR, but failed to amplify it. By analyzing the structure of human "SIAH1b" gene, we found that Alu was inserted immediately before its coding region, and that this gene had a number of base substitutions in its coding region, compared to its homolog genes in mouse or man. From these findings, we suggest that human "SIAH1b" gene had been inactivated by insertion of Alu, and estimate that the inactivation occurred about 30-58 million years ago from the substitution rates of base during evolution.
Assuntos
Inativação Gênica , Proteínas/genética , Animais , Processamento Eletrônico de Dados , Evolução Molecular , Humanos , Camundongos , Proteínas Nucleares , Análise de Sequência , Cromossomos Sexuais , Ubiquitina-Proteína LigasesRESUMO
Homeobox genes regulate sets of genes that determine cellular fates in embryonic morphogenesis and maintenance of adult tissue architecture by regulating cellular motility and cell-cell interactions. Our previous studies showed that a specific member, HOXD3, when overexpressed, enhanced cell motility and invasiveness of human lung cancer A549 cells (Hamada et al. Int. J. Cancer 2001; 93: 516-25 [19]). In the present study, we investigated the roles of HOXD3 in motile and invasive behavior of human malignant melanoma cells. Of seven melanoma cell lines examined here, six cell lines expressed the HOXD3 gene, whereas normal melanocytes did not. We transduced the HOXD3-antisense gene expression vector into two cell lines (A375M and MMIV). The cell transduced with the HOXD3-antisense gene showed reduced in vitro invasion of Matrigel. The transduction of the HOXD3-antisense gene also decreased cell spreading, haptotactic activity to vitronectin and laminin-1, and phagokinetic activity. To find the difference of gene expression between the HOXD3-antisense-transduced A375M cells and the control A375MNeo2 cells, we carried out cDNA microarray analysis. The results of the microarray analysis indicated that the increased expression of cdc42-interacting protein 4, KIAA0554 and tropomyosin 1, which are all associated with the cytoskeletal system, may be involved in the reduction of motile and invasive activity by the HOXD3-antisense gene transduction.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Homeodomínio/genética , Melanoma/patologia , Transdução Genética/métodos , Animais , Sequência de Bases , Linhagem Celular , Movimento Celular , Quimiotaxia , Primers do DNA , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Homeobox , Humanos , Melanoma/fisiopatologia , Camundongos , Invasividade Neoplásica , Reação em Cadeia da Polimerase , Fatores de Transcrição , Transfecção , Células Tumorais CultivadasRESUMO
We investigated the effects of transforming growth factor-beta (TGF-beta) on biological behavior of a weakly malignant rat mammary carcinoma ER-1 cell line. TGF-beta enhanced the tumorigenic and metastatic capacity of ER-1 cells and their in vitro invasiveness to rat mesothelial and endothelial cell. Further cell biological analysis indicated that the increased invasive and metastatic capacity of ER-1 cells by TGF-beta was due to the increase in cell motility and adhesion to the mesothelial and endothelial cell monolayers. Thus, it is suggested that TGF-beta acts on ER-1 cells as a progression-enhancing factor which stimulates their adhesive and motile activities.
Assuntos
Neoplasias Mamárias Experimentais/patologia , Metástase Neoplásica/patologia , Fator de Crescimento Transformador beta/farmacologia , Proteínas de Xenopus , Animais , Anticorpos/farmacologia , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Feminino , Receptores de Hialuronatos/imunologia , Ácido Hialurônico/farmacologia , Hialuronoglucosaminidase/farmacologia , Proteínas Imediatamente Precoces/genética , Cinética , Camundongos , Invasividade Neoplásica , Proteínas Nucleares/genética , Transativadores/genéticaRESUMO
AIE-75 has been known as a 75-kDa autoantigen detected in the serum of autoimmune enteropathy (AIE) and as a colon cancer-related antigen, and now designated as a gene causative of Usher syndrome type 1C hereditary syndromic hearing loss. It binds to a novel putative tumor suppressor MCC2 that is homologous to MCC (mutated in colon cancer) through a PSD-95/Dlg/ZO-1 (PDZ) domain. To clarify the functional role in colon cancer cells, we transfected AIE-75 gene into SW480 colon cancer cells which do not express AIE-75. Expression of AIE-75 suppressed growth of SW480 cells in vitro in correlation with the expression levels. It was due mainly to G2/M phase cell cycle arrest associated with mitotic slippage, resulting in emergence of hyperploid giant-nucleated or multi-nucleated cells. Screening of proteins that bound to PDZ domains of AIE-75 by a yeast two hybrid system showed that three serine/threonine phosphatase catalytic subunits (PP2AC-alpha, PP2AC-beta, and PPP6C) could bind to AIE-75. Since PP2AC is known to regulate G2/M checkpoint, we suggest that AIE-75 interacts with PP2AC and prevent cells to transit mitotic phase.
Assuntos
Adenocarcinoma/genética , Proteínas de Transporte/genética , Ciclo Celular , Neoplasias do Colo/genética , Perfilação da Expressão Gênica , Proteínas Adaptadoras de Transdução de Sinal , Adenocarcinoma/patologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Neoplasias do Colo/patologia , Proteínas do Citoesqueleto , Humanos , Fosfoproteínas Fosfatases/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais CultivadasRESUMO
The degree of E- and P-cadherin expressions inversely correlate with the progression stage of human melanoma. In the present study, we analyzed mechanisms of down-regulation of E- and P-cadherin gene expressions in 8 human melanoma cell lines. In 5 of the 8 melanoma cell lines, E-cadherin expression was lost or markedly decreased compared to that in normal melanocytes, and 4 of the 5 melanoma cell lines lost P-cadherin expression. All of the melanoma cell lines expressed snail, which is known to encode a transcription repressor for E-cadherin, at a higher level than melanocytes whereas expression levels of the snail varied among cell lines. Transduction of snail gene into MMAc cells which expressed a high level of E-cadherin and an extremely low level of snail decreased expression of E-cadherin but not P-cadherin. In contrast, transduction of antisense-snail gene into A375M cells which expressed no E-cadherin and a high level of snail restored expression of E-cadherin but not P-cadherin. Methylation-specific PCR analysis revealed CpG methylation in the promoter region of E-cadherin of MeWo and AKI cells. Further, the treatment with a demethylating agent, 5-azacytidine led AKI and A375M cells to re-express both E- and P-cadherin. The results show E-cadherin gene is silenced by at least two distinct mechanisms (methylation and transrepression by Snail) in human melanoma cell lines whereas P-cadherin gene seems to be silenced by methylation but not by snail.
Assuntos
Caderinas/biossíntese , Perfilação da Expressão Gênica , Inativação Gênica , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Metilação de DNA , Proteínas de Ligação a DNA/farmacologia , Regulação para Baixo , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição da Família Snail , Fatores de Transcrição/farmacologia , Células Tumorais Cultivadas , Dedos de ZincoRESUMO
The APC (adenomatous polyposis coli) gene status in a familial adenomatous polyposis (FAP) patient who developed hepatoblastoma was analyzed by the yeast color assay. Although a single base insertion at codon 462 resulting in truncation of its product was documented in hepatoblastoma cells, no additional somatic mutation was detectable in the whole coding sequence of the APC gene. The nuclear accumulation of beta-catenin without mutation in the exons 2-4 of the beta-catenin gene, however, was observed in the tumor cells by immunohistochemistry. The similar nuclear accumulation of beta-catenin without an additional somatic mutation in its gene, in the absence of somatic mutation in cluster region of the APC gene, has been previously reported in the single FAP case. Moreover, review in the hepatoblastoma cases in the FAP families showed a relatively later onset of the disease when compared with the sporadic cases. These observations suggest that accumulation of beta-catenin without an additional somatic mutation in the APC gene might be a possible mechanism for tumorigenesis of hepatoblastoma in the FAP families.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/complicações , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Hepatoblastoma/complicações , Neoplasias Hepáticas/complicações , Transativadores/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA/métodos , Saúde da Família , Hepatoblastoma/genética , Hepatoblastoma/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Mutação , Transativadores/genética , Proteína Supressora de Tumor p53/genética , beta CateninaRESUMO
BACKGROUND: Tissue factor (TF), expressed in endothelial cells (ECs) and enriched in human atherosclerotic lesions, acts as a critical initiator of blood coagulation in acute coronary syndrome. Basic fibroblast growth factor (bFGF) induces the proliferation and migration of ECs and plays a role in angiogenesis and restoration of endothelial integrity. As TF is implicated in angiogenesis, we studied the effect of bFGF on TF gene and protein expression. Methods: Human umbilical vein ECs (HUVECs) were exposed to bFGF. TF mRNA was assessed by Northern blot and TF protein was assessed by Western blot. TF promoter activity was assessed by transient transfection assay and transcription factor was identified by electro mobility shift assay. RESULTS: bFGF increased TF mRNA and protein expression in HUVECs. Increased TF mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. Transient transfection assays of the human TF promoter-luciferase construct (-786/+121 bp) demonstrated that bFGF induced transcription was dependent on the elements within the -197 to -176 bp relative to the transcription start site of the human TF gene. This region contains NF-kappaB like binding site. Electro mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of NF-kappaB transcription factor to TF promoter. Nucleotide substitution to disrupt NF-kappaB like site reduced bFGF stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator activated receptor-alpha, reduced basal and bFGF stimulated TF expression. CONCLUSIONS: These results indicate that bFGF may increase TF production in ECs through activation of transcription at NF-kappaB binding site, and control coagulation in vessel walls. Fibrate can inhibit TF expression and therefore reduce the thrombogenecity of human atherosclerotic lesions.
RESUMO
Prostate cancer is one of the most common tumors in males and its incidence is steadily increasing worldwide. Serotonin or 5-hydroxytryptamine (5-HT) is a well-known neurotransmitter that mediates a wide variety of physiological effects. An increase in the number of 5-HT-releasing neuroendocrine (NE) cells has been correlated with tumor progression. However, it is particularly unclear whether released 5-HT or the release of 5-HT has a role in tumor cell growth. We hypothesized that 5-HT synthesis and metabolism in NE cells regulate the growth of prostate cancer cells. In the present study, 5-HT was found to play a role as a cell growth factor in prostate cancer cells. Moreover, the pharmacological inhibition of 5-HT synthesis and metabolism interrupted the growth of prostate cancer cells. To confirm the existence of 5-HT in prostate cancer cells, we performed ELISA, HPLC, RT-PCR and immunohistochemical analyses. A high expression of tryptophan hydroxylase (TPH-1), dopa decarboxylase (DDC) and monoamine oxidase A (MAO-A) was noted in the prostate cancer cells when compared with normal prostate cells. Previous studies showed that 5-HT stimulated the proliferation of prostate cancer cells mediated by 5-HT receptors 5-HTR1A and R1B. However, cell proliferation was significantly inhibited when siRNA for both DDC and TPH-1 was transfected to the cells. Consequently, we propose that the secretion system of prostate NE cells capable of 5-HT synthesis and metabolism plays a significant role in prostate tumor generation and progression. These findings provide crucial clues for the development of potential pharmacotherapeutics to slow prostate tumor progression.