RESUMEN
In this study, we generated transgenic (Tg) mice, which overexpressed transforming growth factor (TGF)-ß stimulated clone-22 (TSC-22), and investigate the functional role of TSC-22 on their development and pathogenesis. We obtained 13 Tg-founders (two mice from C57BL6/J and 11 mice from BDF1). Three of 13 Tg-founders were sterile, and the remaining Tg-founders also could generate only a limited number of the F1 generation. We obtained 32 Tg-F1 mice. Most of the Tg-mice showed marked obesity. Histopathological examination could be performed on 31 Tg-mice; seventeen mice died by some disease in their entire life and 14 mice were killed for examination. Most of the Tg-mice examined showed splenic abnormality, in which marked increase of the megakaryocytes, unclearness of the margin of the red pulp and the white pulp, and the enlargement of the white pulp was observed. B cell lymphoma was developed in 10 (71%) of 14 disease-died F1 mice. These results indicate that constitutive over-expression of TSC-22 might disturb the normal embryogenesis and the normal lipid metabolism, and induce the oncogenic differentiation of hematopoietic cells.
Asunto(s)
Linfoma de Células B/etiología , Obesidad/etiología , Proteínas Represoras/fisiología , Bazo/patología , Animales , Células Cultivadas , Femenino , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/metabolismo , Obesidad/patología , Bazo/metabolismoRESUMEN
A p53 functional analysis system, which can identify the types of abnormality of p53, such as loss of function, dominant negative function, or gain of oncogenic function, is now required. In this study, we examined the functional diversity of several mutations of p53 derived from human head and neck cancer cells. The entire open reading frame of p53 cDNA was subcloned into a mammalian expression vector, pEGFP-C3, and genetic mutations were determined. Then, intracellular localization and transcriptional activity of the tumor-derived p53 proteins were examined in Saos-2 cells. A mutant-p53 (Glu17Lys, His193Leu) or a truncated p53 (Delta121) did not activate the reporters containing p53 responsive elements from p21waf1, BAX, MDM2, p53AIP1, and PUMA genes at all. However, a mutant-p53 (Asn30Ser) showed the transcriptional activity on all of the reporters as wild-type p53 did. On the other hand, a mutant-p53 (Asp281His) activated the p21waf1 promoter strongly and the MDM2 promoter faintly, but did not activate the BAX promoter. Interestingly, this mutant-p53 prevented Saos-2 cells from undergoing apoptosis after treatment with a DNA damaging agent, adriamycin. This mutant-p53 induced cell cycle arrest but not apoptosis. Furthermore, another mutant-p53 (Glu17Lys, His193Leu) also prevented the cells from undergoing apoptosis after DNA damage probably in a transcription-independent manner. These results suggest that some cancer cells may contain the oncogenic mutation of the p53 gene, and the oncogenic p53 protein prevents cancer cells from undergoing apoptosis after DNA damage. Detailed information for mutated p53 gene in cancer cells might provide useful suggestions for the therapeutic strategy.
Asunto(s)
Apoptosis , Daño del ADN , Genes p53 , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Mutación , Secuencia de Bases , Western Blotting , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , ADN Complementario/metabolismo , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteína p53 Supresora de Tumor/químicaRESUMEN
Although tissue-specific apoptosis in the exocrine glands in estrogen-deficient mice may contribute to the development of autoimmune exocrinopathy, the molecular mechanism responsible for tissue-specific apoptosis remains obscure. Here we show that RbAp48 overexpression induces p53-mediated apoptosis in the exocrine glands caused by estrogen deficiency. RbAp48-inducible transfectant results in rapid apoptosis with p53 phosphorylation (Ser9) and alpha-fodrin cleavage. Reducing the expression of RbAp48 through small interfering RNA inhibits the apoptosis. Prominent RbAp48 expression with apoptosis was observed in the exocrine glands of C57BL/6 ovariectomized (OVX) mice but not in OVX estrogen receptor alpha(-/-), p53(-/-), and E2F-1(-/-) mice. Indeed, transgenic expression of the RbAp48 gene induced apoptosis in the exocrine glands but not in other organs. These findings indicate that estrogen deficiency initiates p53-mediated apoptosis in the exocrine gland cells through RbAp48 overexpression and exerts a possible gender-based risk of autoimmune exocrinopathy in postmenopausal women.
Asunto(s)
Apoptosis/genética , Proteínas Portadoras/metabolismo , Receptor alfa de Estrógeno/deficiencia , Receptor alfa de Estrógeno/genética , Proteínas Nucleares/metabolismo , Glándulas Salivales/fisiología , Animales , Anexina A5/metabolismo , Western Blotting , Proteínas Portadoras/genética , Caspasas/análisis , Línea Celular Tumoral , Femenino , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Células HT29 , Células HeLa , Humanos , Inmunohistoquímica , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Proteínas Nucleares/genética , Especificidad de Órganos , Ovariectomía , Fosforilación , ARN Interferente Pequeño/metabolismo , Proteína 4 de Unión a Retinoblastoma , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Células U937RESUMEN
The alpha-fodrin N-terminal portion (AFN) autoantigen mediates in vivo immunoregulation of autoimmune responses in primary Sjögren's syndrome (SS). We further examined this process and found that cleavage products of AFN were frequently detected in the salivary gland duct cells of SS patients. In in vitro studies using human salivary gland HSY cells, anti-Fas-induced apoptosis resulted in specific cleavage of alpha-fodrin into the 120-kd fragment, in association of alpha-fodrin with mu-calpain, and activation of caspase 3. Significant proliferative responses against AlphaFN autoantigen were observed in the peripheral blood mononuclear cells (PBMCs) from SS patients with higher pathological score (grade 4) and with short duration from onset (within 5 years). In vivo roles of AFN peptides were investigated using PBMCs from patients with SS, systemic lupus erythematosus, and rheumatoid arthritis. Significant proliferative T-cell responses of PBMCs to AFN peptide were detected in SS but not in systemic lupus erythematosus or rheumatoid arthritis. AFN peptide induced Th1-immune responses and accelerated down-regulation of Fas-mediated T-cell apoptosis in SS. Our data further elucidate the in vivo role of AFN autoantigen on the development of SS and suggest that the AFN autoantigen is a novel participant in peripheral tolerance.
Asunto(s)
Autoantígenos/inmunología , Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/metabolismo , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/patología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Autoantígenos/química , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Calpaína/metabolismo , Proteínas Portadoras/síntesis química , Proteínas Portadoras/química , Proteínas Portadoras/genética , Estudios de Casos y Controles , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Activación Enzimática , Femenino , Furanos/farmacología , Glutatión Transferasa/metabolismo , Humanos , Inmunohistoquímica , Japón/epidemiología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/efectos de la radiación , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Proteínas de Microfilamentos/síntesis química , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Peso Molecular , Glándula Parótida/citología , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Timidina/metabolismo , Receptor fas/metabolismoRESUMEN
A firm, ulcerated tumor formed on the left side of the tongue of an elderly woman. Histopathological analysis showed that this unusual neoplasm was composed of monomorphic polygonal cells that exhibited a clear cytoplasm containing large amounts of periodic acid Schiff (PAS)-positive, diastase-digestive material. Most of the tumor cells stained immunohistochemically for Cytokeratin, high-molecular, CAM5.2, and epithelial membrane antigen (EMA), but were negative for alpha-smooth muscle actin, vimentin, glial fibrillary acid protein (GFAP), and S-100 protein. These findings supported a diagnosis of clear cell adenocarcinoma. Although patients with this type of tumor generally have a favorable prognosis, the tumor in our patient was fast-growing and contained a large number of Ki-67 positive cells, which are known to be highly proliferative. Thus, this case highlights the fact that even clear cell adenocarcinomas that are usually slow-growing should be investigated by conventional morphological techniques and their proliferative activity quantified in order to select the most appropriate treatment strategy.
Asunto(s)
Adenocarcinoma de Células Claras/patología , Neoplasias de la Lengua/patología , Anciano , Anciano de 80 o más Años , Biomarcadores , Biomarcadores de Tumor/análisis , Proliferación Celular , Citoplasma/ultraestructura , Resultado Fatal , Femenino , Humanos , Inmunohistoquímica , Queratinas/análisis , Antígeno Ki-67/análisis , Mucina-1/análisisRESUMEN
Mutations of p53 tumor suppressor gene increase with tumor progression in colorectal cancers. In this study, we examined the expressions of p33ING1, p14ARF, MDM2 and p21WAF1 mRNA in 25 advanced colorectal cancers by quantitative RT-PCR method, and compared the expression levels of p33ING1, p14ARF, p21WAF1 and MDM2 in relation to p53 status in the tumors. Fifteen of 25 colorectal cancers (60%) showed abnormal accumulation of p53 protein in the nucleus, and the remaining 10 colorectal cancers (40%) were negative for p53 immunostaining. We found a G --> T transition (nonsense mutation) at the first nucleotide of codon 298 (exon 8) in one p53-negative case, and a frame shift mutation on exon 7 in another p53-negative case. In remaining eight p53-negative cases, there was no mutation in the entire open reading frame of p53 cDNA. Interestingly, in eight cases with p53 wild-type gene, 6 cases (75%) showed a marked down-regulation of p14ARF mRNA, and three cases (37.5%) over-expressed MDM2 mRNA. Only one case with wild-type p53 gene showed normal level expression of p53 regulatory-factors (p33ING1, p14ARF, and MDM2). Thus, p53 tumor suppressor pathway was disrupted in 24 of 25 colorectal cancers (96%).
Asunto(s)
Proteínas de Ciclo Celular/genética , Neoplasias Colorrectales/genética , Genes p53/fisiología , Mutación , Proteínas Nucleares/genética , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteína p14ARF Supresora de Tumor/genética , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN , Femenino , Genes Supresores de Tumor , Humanos , Proteína Inhibidora del Crecimiento 1 , Péptidos y Proteínas de Señalización Intracelular , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-mdm2 , ARN Mensajero/análisis , Proteínas Supresoras de TumorAsunto(s)
Adenocarcinoma/genética , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/genética , Mutación/genética , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/patología , Núcleo Celular/patología , Transformación Celular Neoplásica/patología , Colon/patología , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN , Regulación Neoplásica de la Expresión Génica/fisiología , Silenciador del Gen/fisiología , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Antígeno Ki-67/genética , Pérdida de Heterocigocidad/genética , Mutación Missense , Mutación Puntual , Valor Predictivo de las Pruebas , Proteína p53 Supresora de Tumor/análisisRESUMEN
We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Neoplasias de la Boca/genética , Proteínas Nucleares , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genética , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , Proteínas de Unión al ADN , Formaldehído , Genes Supresores de Tumor , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Inmunohistoquímica , Proteína Inhibidora del Crecimiento 1 , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Adhesión en Parafina , Biosíntesis de Proteínas , Proteínas/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , ARN Mensajero/biosíntesis , Fijación del Tejido , Proteína p14ARF Supresora de Tumor/biosíntesis , Proteína p14ARF Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas Supresoras de TumorRESUMEN
We isolated a metastasizing human esophageal squamous cell carcinoma (SCC) cell line, T.Tn-AT1, from a parental non-metastasizing cell line, T.Tn, by in vitro selection and by use of a nude mouse orthotopic inoculation model. Then, we compared the expression profiles of 9206 genes in T.Tn-AT1 and T.Tn by cDNA microarray analysis. The gene expression profiles of T.Tn and T.Tn-AT1 were very similar, and only 34 genes showed more than 3-fold differential expression. Among the 34 genes, 29 genes were down-regulated and only 5 genes were up-regulated in T.Tn-AT1 cells. Subsequently, we confirmed the expression levels of 14 of the 34 genes in T.Tn and T.Tn-AT1 cells by means of reverse transcription-polymerase chain reaction. The expression of 8 genes (KAL1, HPGD, NDN, REG1A, CXCR4, SPOCK, DIAPH2 and AIF1) was down-regulated and that of one gene (VNN2) was up-regulated in T.Tn-AT1 cells. These 9 genes encoded proteins associated with metastatic processes, such as adhesion, migration, inflammation, proliferation, and differentiation. Thus, these genes might regulate the metastasis of esophageal SCC, and could be predictive markers for lymph node metastasis of esophageal SCC.
Asunto(s)
Neoplasias Esofágicas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Células Clonales , Cartilla de ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , Neoplasias Esofágicas/patología , Perfilación de la Expresión Génica/métodos , Humanos , Metástasis de la Neoplasia/genética , Células Tumorales CultivadasRESUMEN
Vesnarinone has been shown to be a unique anti-proliferating, differentiation-inducing and apoptosis inducing drug against several human malignancies, including leukemia and several solid tumors. Furthermore, vesnarinone potentiates the effect of conventional cytotoxic chemotherapy or radiation therapy. Combination of differentiation-inducing therapy by vesnarinone with conventional chemotherapy or radiation therapy might be second- or third-line therapy in patients with advanced cancer. Analysis of the molecular mechanisms of the tumor differentiation therapy by vesnarinone might provide selective and targeted molecules for novel tumor dormancy therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Quinolinas/uso terapéutico , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Humanos , Ratones , Neoplasias/radioterapia , Pirazinas , Quinolinas/química , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificaciónRESUMEN
TSC-22 gene was composed of three exons and its length was approximately 5.5 kb including 2.9 kb promoter region. The transcription starting site was located at 7 and 29 bp downstream from TATA box. Promoter analysis revealed that 2146 bp of TSC-22 promoter was activated by several differentiation inducing drugs. Although originally TSC-22 was isolated as a TGF-beta-inducible gene, TSC-22 promoter was not activated by the enhanced TGF-beta signaling. We found 3 copies of the Shaw-Kamens sequence (AUUUA) in the human TSC-22 mRNA 3'-UTR and identified three proteins (40, 20, and 15 kDa) which bound to this. Only the 40 kDa protein-RNA complex was decreased by treatment with TGF-beta 1. Moreover, the TSC-22 mRNA 3'-UTR destabilized the heterologous luciferase mRNA, but the destabilization was recovered with TGF-beta 1. These observations suggest that up-regulation of TSC-22 mRNA by TGF-beta 1 is achieved by mRNA stabilization, but not by transcriptional activation.
Asunto(s)
Estabilidad del ARN , Proteínas Represoras/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/farmacología , Regiones no Traducidas 3' , Secuencia de Bases , Línea Celular , Células HeLa , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Elementos de Respuesta , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Activación Transcripcional , Factor de Crecimiento Transformador beta1RESUMEN
Tubular gland structures of colorectal cancer (CRC) have been demonstrated to undergo dedifferentiation at the primary site, and then the gland structures are re-formed in the liver metastases. In this study, we examined the degree of differentiation of the gland structure of 48 cases of CRCs (24 cases with synchronous liver metastasis, 24 cases without metastasis) by the modified Gleason grading system. We also investigated the role of ZO-1, one of the tight junction proteins, in the morphological changes, i.e., dedifferentiation and redifferentiation, of CRCs at the primary site and liver metastases. Liver-metastasized CRCs (2.47+/-0.37) showed a lower score in the modified Gleason grading system than the corresponding primary tumors (3.28+/-0.36) did, i.e., the tumor cells had undergone redifferentiation at liver metastases. ZO-1 was expressed at the apical cell borders of normal colorectal epithelium, the luminal side of which has tubular gland structures. In comparison with this normal epithelium, the ZO-1 expression level was frequently reduced in primary CRC with liver metastasis (20.8%) and ZO-1 was reexpressed in liver metastasized cancers (79.2%). Furthermore, it was demonstrated by an immunoprecipitation-western blotting analysis on 5 cases of CRC with liver metastasis that ZO-1 bound to epidermal growth factor receptor (EGFR) irrespective of the phosphorylation status of EGFR, and that EGFR associated ZO-1 was highly tyrosine-phosphorylated only in the primary CRC, but was dephosphorylated in the liver-metastasized cancers. Our observations suggest that tyrosine phosphorylation of ZO-1 leads to down-regulation of the function of ZO-1 and dedifferentiation of the glands in CRCs, and these phenomena contribute to liver metastases, and redifferentiation of the glands occurs in the liver metastases.
Asunto(s)
Adenocarcinoma/secundario , Neoplasias Colorrectales/patología , Receptores ErbB/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/secundario , Proteínas de la Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , Fosfoproteínas/biosíntesis , Procesamiento Proteico-Postraduccional , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Especificidad de Órganos , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Fosforilación , Proteína de la Zonula Occludens-1RESUMEN
Pre-therapeutic evaluation of p53 gene is very important for treating patients with head and neck cancer. However, the analysis for p53 gene has generally been done by immunohistochemistry, polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and direct sequencing. Functional analysis system for p53 transcriptional activity in mammalian cells is now required. We developed a functional analysis system for p53 transcriptional activity in cancer cells. We used two human head and neck cancer cell lines harboring mutated p53 gene, HSG (Asn30Ser) and TYS (Asp281His), and a human osteosarcoma cell line, Saos-2 as a control. We transfected these cells with luciferase reporter plasmids containing promoter sequence of p53 target genes (p21waf1, BAX, MDM2, p53AIP1 or PUMA). After treating the cells with chemotherapeutic drugs, alteration of the luciferase activity was measured. In HSG cells, none of the target gene promoters was activated by treatment with chemotherapeutic drugs. In TYS cells, p21waf1 promoter was markedly activated by treatment with chemotherapeutic drugs, but Bax and p53AIP1 promoter was not activated. This type of mutated-p53 in TYS cells prevents cell death from DNA damage, and probably accumulates genetic alterations and accelerates the malignant progression of the cells by DNA damaging therapy. Thus, analysis for the diverse function of mutated-p53 may help to determine the therapeutic strategy, especially for chemotherapy and radiation in the individual patients with head and neck cancer.
Asunto(s)
Sustitución de Aminoácidos , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/genética , Transformación Celular Neoplásica/genética , ADN de Neoplasias/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Genes p53 , Neoplasias de Cabeza y Cuello/genética , Mutación Missense , Proteínas de Neoplasias/fisiología , Proteínas Nucleares , Mutación Puntual , Proteínas Proto-Oncogénicas c-bcl-2 , Proteína p53 Supresora de Tumor/fisiología , Antineoplásicos/efectos adversos , Apoptosis , Proteínas Reguladoras de la Apoptosis , Carcinoma de Células Escamosas/patología , Codón/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Daño del ADN , ADN de Neoplasias/genética , Progresión de la Enfermedad , Genes Reporteros , Neoplasias de Cabeza y Cuello/patología , Humanos , Luciferasas/análisis , Luciferasas/biosíntesis , Proteínas de Neoplasias/genética , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , Células Tumorales Cultivadas/patología , Proteína p53 Supresora de Tumor/genética , Proteína X Asociada a bcl-2RESUMEN
Lymph node metastasis is commonly found in esophageal squamous cell carcinoma (SCC). In this study, we examined the molecular and genetic characteristics of a human esophageal SCC cell line, T.Tn. T.Tn cells formed tumors at s.c. tissue in nude mice when inoculated with Matrigel, but did not metastasize to any organs. T.Tn cells expressed low level of proMMP2 and a trace level of proMMP9. However, T.Tn cells expressed high level of TIMP1 and TIMP2, and beta-catenin and E-cadherin. We found a point mutation of p53 gene at codon 213 (CAT-->CGT) in T.Tn cells. The mutated-p53 protein did not show transcriptional activity on p21(waf1), MDM2 and Bax promoters. Thus, T.Tn cells are low tumorigenic and weakly invasive but not metastasizing in nude mice, and T.Tn cells are suitable parental cells for establishing a model system to study invasion and metastasis of esophageal SCC.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Proteína p53 Supresora de Tumor/genética , Animales , Cadherinas/biosíntesis , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundario , División Celular/fisiología , Colagenasas/biosíntesis , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , Precursores Enzimáticos/biosíntesis , Neoplasias Esofágicas/metabolismo , Gelatinasas/biosíntesis , Humanos , Inmunohistoquímica , Metaloproteinasa 9 de la Matriz , Metaloendopeptidasas/biosíntesis , Ratones , Ratones Desnudos , Mutación , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Transactivadores/biosíntesis , Transactivadores/genética , Activación Transcripcional , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/fisiología , beta CateninaRESUMEN
It is important to examine the abnormality of the entire p53 tumor suppressor pathway in head and neck cancer. We examined the mRNA expressions of p53 regulatory factors, p33ING1 and p14ARF, and a p53-target gene, p21WAF1 in head and neck cancer. Nine of 14 benign pleomorphic adenomas (PAs) and 7 of 8 malignant salivary gland tumors (MSGTs) expressed p33ING1 mRNA. Thirteen of 14 PAs expressed p14ARF mRNA, however, only 1 of 8 MSGTs expressed p14ARF mRNA. Eight of 14 PAs and 7 of 8 MSGTs expressed p21WAF1 mRNA. In salivary gland tumors, there was clear correlation between the expression of p33ING1 and p21WAF1 (p<0.0001, r2=0.53). However, there was no correlation between the expression of p14ARF and p21WAF1 (p=0.6543, r2=0.009). Twenty-six of 28 oral squamous cell carcinomas (SCCs) expressed p33ING1 mRNA. Nineteen of 28 oral SCCs expressed p14ARF mRNA. All of the oral SCCs expressed p21WAF1 mRNA. In oral SCCs, the expressions of both p33ING1 (p=0.009, r2=0.181) and p14ARF (p=0.0009, r2=0.271) correlated with the expression of p21WAF1. Interestingly, 24 of 26 oral SCCs (92%) showed either abnormality of p53 itself or loss of expression of p53 regulatory factors, p33ING or p14ARF. These results suggest that head and neck cancer often involve the dysfunction of p53 tumor suppressor pathway.
Asunto(s)
Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Southern Blotting , Proteínas de Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , Cartilla de ADN/química , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor , Neoplasias de Cabeza y Cuello/etiología , Humanos , Técnicas para Inmunoenzimas , Proteína Inhibidora del Crecimiento 1 , Péptidos y Proteínas de Señalización Intracelular , Proteínas Nucleares , Pronóstico , Proteínas/genética , Proteínas/metabolismo , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteína p14ARF Supresora de Tumor/genética , Proteína p14ARF Supresora de Tumor/metabolismo , Proteínas Supresoras de TumorRESUMEN
We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.
Asunto(s)
Tolerancia a Radiación/efectos de los fármacos , Proteínas Represoras/farmacología , Neoplasias de las Glándulas Salivales/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Transporte Activo de Núcleo Celular/efectos de la radiación , Secuencias de Aminoácidos/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ciclo Celular/efectos de la radiación , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Citoplasma/metabolismo , Citoplasma/efectos de la radiación , Proteínas Fluorescentes Verdes , Humanos , Leucina Zippers/fisiología , Proteínas Luminiscentes/genética , Señales de Localización Nuclear/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Neoplasias de las Glándulas Salivales/tratamiento farmacológico , Eliminación de Secuencia , Transfección , Células Tumorales CultivadasRESUMEN
We analyzed the K-ras gene mutation and the polymorphism of peroxisome proliferator activated receptor gamma (PPARgamma) gene, which is known as a master gene for adipocyte differentiation and a tumor suppressor gene, in patients with colorectal cancer by polymerase chain reaction-restriction fragment length polymorphism. We detected a mutation of K-ras gene at codon 12 in 8 of 39 colorectal cancers. We also detected a polymorphism of PPARgamma gene at codon 12 in 10 of 39 patients with colorectal cancer. Homozygous polymorphism (Ala12Ala) in PPARgamma gene was found only in one patient, but heterozygous polymorphism (Pro12Ala) was found in 9 of 29 patients. Surprisingly, all 10 colorectal cancers developed in the patients with polymorphism of PPARgamma gene carrying the wild-type K-ras gene. These findings suggest that PPARgamma gene polymorphism may be implicated with the development of colorectal cancers, in which K-ras gene is not mutated.
Asunto(s)
Neoplasias Colorrectales/genética , Genes ras/genética , Polimorfismo Genético/genética , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Adulto , Neoplasias Colorrectales/patología , Femenino , Frecuencia de los Genes , Humanos , Masculino , Persona de Mediana Edad , Mutación/genéticaRESUMEN
Several investigators have demonstrated that TGF-beta stimulated clone-22 (TSC-22) regulates cell growth and differentiation, and cell death. TSC-22 is a putative transcriptional regulator containing a leucine zipper-like structure and a nuclear export signal. We previously showed the cytoplasmic localization of TSC-22 and the nuclear translocation of TSC-22 concomitant with induction of apoptosis in salivary gland cancer cells. In the present study, we attempted to identify the active domain of TSC-22 protein that regulated the biological functions of TSC-22. We constructed three mammalian expression vectors, which could produce full length TSC-22 only in cytoplasm, the leucine zipper structure of TSC-22 in both cytoplasm and nucleus, and the leucine zipper structure only in nucleus. Then, we transfected a salivary gland cancer cell line, HSG with these expression vectors, and investigated the growth profile of the transfectants. None of the TSC-22 constructs inhibited the monolayer growth and the anchorage-dependent colony formation of HSG cells. However, the leucine zipper structure of TSC-22 markedly inhibited the anchorage-independent colony formation of HSG cells (p<0.001; one way ANOVA). Full length TSC-22 also suppressed the anchorage-independent colony formation of HSG cells, although the effect of full length TSC-22 was much lower than those of the leucine zipper constructs. These observations suggest that the leucine zipper structure in TSC-22 protein is an active domain that negatively regulates the growth of salivary gland cancer cells.