RESUMO
Pancreatic adenocarcinoma, with an incidence/death ratio of 0.99, has the worst prognosis of all cancers. Risk factors associated with the sporadic form of pancreatic adenocarcinoma are unknown and less than 10% of patients receive curative treatment (surgery associated with radiation therapy or chemotherapy) with a low 5-year survival rate (10 to 20%). In more than 90% of patients, the tumor discovered at diagnosis is not resectable or has already metastasized. Thus, a better understanding of the etiology of pancreatic cancer is essential to identify new prognostic markers and new therapeutic targets. There is a wealth of data on the identification of genetic alterations associated with pancreatic cancer and their role in its development. This review will focus on the current knowledge of genetic alterations associated with two pancreatic lesions that can potentially evolve into pancreatic adenocarcinoma, Pancreatic Intraepithelial Neoplasia (PanIN) and Intraductal Papillary Mucinous Neoplasm (IPMN). These two lesions share a large panel of typical genetic alterations which are close to those found in pancreatic adenocarcinoma. A better understanding of these alterations may lead to therapeutic targets that could help prevent the progression of PanIN and IPMN to cancer.
Assuntos
Neoplasias Pancreáticas/genética , Lesões Pré-Cancerosas/genética , Adenocarcinoma Mucinoso/genética , Carcinoma in Situ/genética , Carcinoma Papilar/genética , Humanos , Proteínas Proto-Oncogênicas/genética , Telômero/ultraestrutura , Proteínas Supressoras de Tumor/genéticaRESUMO
Pancreatitis-associated protein 1 (PAP1) belongs to the Reg family of secretory proteins. Several important biological roles have been attributed to PAP1 but the signaling pathways activated by this protein remain only partially understood. Here, we describe the intracellular pathways triggered by PAP1 in a pancreatic acinar cell line. Taking advantage of the fact that PAP1 induces its own transcription, we performed ChIP assays to analyze the recruitment of transcriptional factors on its promoter. Our results show that PAP1 increased the transactivation activity of pap1 and the binding on its promoter of the nuclear factors C/EBPbeta, P-CREB, P-ELK1, EGR1, STAT3, and ETS2, which are downstream targets of MAPK signaling. p44/42, p38, and JNK MAPKs activity increased after PAP1 treatment. In addition, pharmacological inhibition of these kinases markedly inhibited the induction of pap1 mRNA. Taken together, these results indicated that the mechanism of PAP1 action involves the activation of the MAPK superfamily.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Lectinas Tipo C/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Humanos , Lectinas Tipo C/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Pâncreas/citologia , Proteínas Associadas a Pancreatite , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ativação TranscricionalRESUMO
A panel of kinases whose inhibition increased apoptosis of pancreatic adenocarcinoma cells in vitro was recently established. The aim of this work was to observe in a mouse xenograft model whether inhibition of these kinases would alter pancreatic tumor growth. Rate of apoptosis, caspase-3 activity and cell viability were assessed in two pancreatic cancer cell lines, MiaPaCa2 and BxPC3, after inhibiting selected kinases by transfection of specific siRNAs. For in vivo experiments, MiaPaCa2 cells were injected into the pancreas of nude mice, where they formed tumors. Inhibition of kinases was obtained by repeated intraperitoneal (i.p.) injections of modified O-Methyl (OMe) siRNAs specific for the selected kinases. Tumor volumes were assessed after 21 days. Among selected kinases, PAK7, MAP3K7 and CK2alpha were those whose inhibition increased apoptosis the most in vitro. Simultaneous inhibition of two of them increased apoptosis up to five times. Moreover, inhibiting these kinases had little effect on 10 non-pancreatic cell lines, suggesting pancreatic specificity. In vivo, OMe-siRNAs induced significant but incomplete inhibition of kinase expression (45-75%). Nevertheless, such inhibition resulted in a twofold increase in caspase-3 activity in tumors and a strong reduction in tumor volume (about 75%). In vivo inhibition by OMe-siRNAs of three survival kinases apparently specific for pancreatic cancer cells, PAK7, MAP3K7 and CK2alpha, decreases significantly the growth of xenografted MiaPaCa2 cells. This strategy is therefore of potential clinical interest.
Assuntos
Caseína Quinase II/genética , Terapia Genética , MAP Quinase Quinase Quinases/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , RNA Interferente Pequeno/genética , Quinases Ativadas por p21/genética , Animais , Apoptose , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/metabolismoRESUMO
OBJECTIVE: Yersinia pseudotuberculosis causes ileitis and mesenteric lymphadenitis by mainly invading the Peyer's patches that are positioned in the terminal ileum. Whereas toll-like-receptor 2 (TLR2) controls mucosal inflammation by detecting certain microbiota-derived signals, its exact role in protecting Peyer's patches against bacterial invasion has not been defined. DESIGN: Wild-type, Tlr2-, Nod2- and MyD88-deficient animals were challenged by Y pseudotuberculosis via the oral or systemic route. The role of microbiota in conditioning Peyer's patches against Yersinia through TLR2 was assessed by delivering, ad libitum, exogenous TLR2 agonists in drinking water to germ-free and streptomycin-treated animals. Bacterial eradication from Peyer's patches was measured by using a colony-forming unit assay. Expression of cryptdins and the c-type lectin Reg3 beta was quantified by quantitative reverse transcriptase polymerase chain reaction analysis. RESULTS: Our data demonstrated that Tlr2-deficient mice failed to limit Yersinia dissemination from the Peyer's patches and succumbed to sepsis independently of nucleotide-binding and oligomerisation domain 2 (NOD2). Recognition of both microbiota-derived and myeloid differentiation factor 88 (MyD88)-mediated elicitors was found to be critically involved in gut protection against Yersinia-induced lethality, while TLR2 was dispensable to systemic Yersinia infection. Gene expression analyses revealed that optimal epithelial transcript level of the anti-infective Reg3 beta requires TLR2 activation. Consistently, Yersinia infection triggered TLR2-dependent Reg3 beta expression in Peyer's patches. Importantly, oral treatment with exogenous TLR2 agonists in germ-free animals was able to further enhance Yersinia-induced expression of Reg3 beta and to restore intestinal resistance to Yersinia. Lastly, genetic ablation of Reg3 beta resulted in impaired clearance of the bacterial load in Peyer's patches. CONCLUSIONS: TLR2/REG3 beta is thus an essential component in conditioning epithelial defence signalling pathways against bacterial invasion.
Assuntos
Nódulos Linfáticos Agregados/microbiologia , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Receptor 2 Toll-Like/metabolismo , Infecções por Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Deleção de Genes , Perfilação da Expressão Gênica/métodos , Vida Livre de Germes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteínas Associadas a Pancreatite , Nódulos Linfáticos Agregados/metabolismo , Nódulos Linfáticos Agregados/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Receptor 2 Toll-Like/genéticaRESUMO
We have used a quantitative fluorescent cDNA microarray hybridization approach to identify pancreatic genes induced by the cellular stress promoted by acute pancreatitis in the mouse. We report the cloning and characterization of one of them that encodes the stress-induced proteins (SIP). The mouse SIP gene is organized into five exons and expands over approximately 20 kilobase pairs. Exon 4 (38 base pairs) is alternatively spliced to generate two transcripts. Northern blot and in situ hybridization showed that both SIP mRNAs are rapidly and strongly induced in acinar cells of the pancreas with acute pancreatitis. They are also constitutively expressed in several other tissues, although with different ratios. They encode proteins of 18 and 27 kDa (SIP(18) and SIP(27)). SIP(27) is identical to the thymus-expressed acidic protein (TEAP) protein, formerly described as a thymus-specific protein. Expression of the SIP(18) and SIP(27)/EGFP or V5 fusion proteins showed that both are nuclear factors. We monitored SIP expression in NIH3T3 cells submitted to various stress agents. UV stress, base damaging, mutagenic stress, ethanol, heat shock, and oxidative stress induced the concomitant expression of SIP(18) and SIP(27) mRNAs. Finally, transient transfection of SIP(18) and SIP(27) expression plasmids induced death by apoptosis in COS7 cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. In conclusion, the SIP gene is an important element of cellular stress response. It is expressed in many tissues and induced by a variety of stress agents affecting many cellular pathways. SIP generates, by alternative splicing, two nuclear proteins that can promote cell death by apoptosis.
Assuntos
Processamento Alternativo , Proteínas de Transporte/fisiologia , Proteínas de Choque Térmico , Células 3T3 , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Sequência de Bases , Proteínas de Transporte/genética , Clonagem Molecular , Hibridização in Situ Fluorescente/métodos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
We report that exposure of mouse embryonic fibroblasts to transforming growth factor beta-1 (TGFbeta-1) (5 ng/ml) results in a strong activation of p8 mRNA expression that precedes the induction of cell growth. Involvement of the p8 promoter in the regulation was demonstrated by using a p8-chloramphenicol acetyltransferase construct. We therefore speculated that p8 might be a mediator of TGFbeta-1 in these cells. The incorporation of [(3)H]thymidine on treatment with TGFbeta-1 was indeed significantly higher in p8(+/+) fibroblasts than in p8(-/-) fibroblasts. Smad transcriptional activity was used as marker of the TGFbeta-1 signalling pathway, to probe the lower p8(-/-) response to TGFbeta-1. Two Smad-binding elements (SBEs)-luciferase constructs were transfected into p8(-/-) and p8(+/+) embryonic fibroblasts before treatment with TGFbeta-1. A lower level of Smad transactivation was observed in p8(-/-) embryonic fibroblasts, under basal conditions and after stimulation with TGFbeta-1. To test whether Smad underexpression in p8(-/-) cells was actually due to p8 depletion, p8(-/-) embryonic fibroblasts were transfected with a human p8 expression plasmid together with an SBE-luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFbeta-1-treated cells to the level found in p8(+/+) cells. We concluded that TGFbeta-1 activates p8 expression, which in turn enhances the Smad-transactivating function responsible for TGFbeta-1 activity.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Substâncias de Crescimento/genética , Proteínas de Neoplasias , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Células 3T3 , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , DNA/biossíntese , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Substâncias de Crescimento/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Homozigoto , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Timidina/metabolismo , Transativadores/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional , TransfecçãoRESUMO
Expression of the Cdx1 homeobox gene in epithelial intestinal cells promotes cellular growth and differentiation. Cdx1and the Pancreatitis Associated Protein I (PAP I) are concomitantly expressed in the epithelial cells of the lower part of the intestinal crypts. Because Cdx1 is a transcription factor and PAP I, in other tissues, is a proliferative factor, we looked for a relationship between these two proteins in the intestinal-derived IEC-6 cells. After stable transfection with a Cdx1 expression vector, they produce high levels of the PAP I transcript and protein indicating a functional link between the two genes. Demonstration of Cdx1 binding to the PAP I promoter region and suppression of PAP I induction after deletion of the corresponding sequence indicated that Cdx1 is a transcription factor controlling PAP I gene expression in intestinal cells. By infecting IEC-6 cells with adenoviruses expressing PAP I, we demonstrated that PAP I induces mitosis in these cells. On the other hand, inhibition of the PAP I expression in the IEC-6 Cdxl-expressing cells using an antisense strategy confirmed the requirement of this protein for the effect of Cdx1 on cell growth. Finally, addition of the immunopurified PAP I to the culture medium promotes cell growth of the IEC-6 cells in a dose-dependent manner. Maximal effect was obtained at 1 ng/ml. Taken together these results demonstrate that PAP I is a target of the Cdx1 homeobox gene in intestinal cells which participates in the regulation of intestinal cell growth via an autocrine and/or paracrine mechanism.
Assuntos
Proteínas de Fase Aguda/metabolismo , Antígenos de Neoplasias , Biomarcadores Tumorais , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/citologia , Lectinas Tipo C , Fatores de Transcrição/metabolismo , Proteínas de Fase Aguda/genética , Animais , Divisão Celular , Linhagem Celular , Células Epiteliais/citologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Mitose , Proteínas Associadas a Pancreatite , Regiões Promotoras Genéticas , Ratos , Elementos de Resposta , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Pancreatitis associated protein I is a secretory stress protein first characterized in pancreas during pancreatitis but also expressed in several tissues including hepatic, gastric, and colon cancer. Its concentration in serum can be significant. The relationship of pancreatitis associated protein I to skin cancers was investigated in normal melanocytes, melanoma tumors, and melanoma cell lines. None of them expressed pancreatitis associated protein I, even after stress induction. Adenovirus-mediated pancreatitis associated protein I expression, however, reduced cell adhesion to laminin-1 and fibronectin with a loss of integrin participation. Pancreatitis associated protein I expression stimulated haptotactic and directed migrations of some melanoma cells, but only directed migration was activated in normal melanocytes. Importantly, directed migration and spreading on fibronectin of the responsive melanoma cells were also enhanced when purified rat pancreatitis associated protein I was added to the culture medium of noninfected cells. This indicates that effects in infected cells were elicited by pancreatitis associated protein I after its secretion. Exogenous pancreatitis associated protein I can therefore modify the adhesion and motility of normal and transformed melanocytes, suggesting a potential interaction with melanoma invasivity.
Assuntos
Proteínas de Fase Aguda/farmacologia , Antígenos de Neoplasias , Biomarcadores Tumorais , Movimento Celular/efeitos dos fármacos , Lectinas Tipo C , Melanócitos/efeitos dos fármacos , Melanócitos/fisiologia , Melanoma/fisiopatologia , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Adenoviridae/genética , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Transferência de Genes , Humanos , Melanócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Proteínas Associadas a PancreatiteRESUMO
OBJECTIVES: The increasing evidence of the benefits of neonatal screening for cystic fibrosis (CF) indicates that this procedure could soon be implemented throughout France. The screening strategy currently used involves the detection of infants with elevated levels of immunoreactive trypsinogen (IRT) (approximately 1% of the population), followed by the detection of CFTR gene mutations. However, genetic analysis has certain drawbacks, the most important of which being the management of heterozygotes, and in France the requirement by law of previous informed consent. In cases of CF, pancreatic alterations are already present in utero. A previous study has demonstrated the value of pancreatitis-associated protein (PAP) as a screening test for CF, and has indicated that a feasible two-stage strategy could involve the following: 1) selection of infants with elevated PAP levels; 2) in this group of infants, subsequent detection of those with elevated IRT levels for direct CF diagnosis by the sweat test thereby avoiding the use of genetic analysis. The study aim was to evaluate this strategy in a large number of neonates. METHODS AND RESULTS: The aforementioned strategy was evaluated in a prospective study involving 47,213 infants in the Provence region of France. In infants with a PAP > 7.5 ng/mL (1.28%), 176 had an elevated IRT level > 700 ng/mL (0.37%). In this limited population sample (0.37% of the total), the sweat test diagnosed five cases of CF. A sixth case involving the monozygous twin of an infant with diagnosed CF remained undetected, probably because of a registration error. Genetic analysis confirmed the diagnosis, and also detected another case in an infant with two CFTR mutations but with a normal phenotype at 20 months of age. As the observed incidence was similar to that which had previously been reported, and as no further case was subsequently detected two years after the end of the study, this indicated that the sensitivity of this screening strategy was satisfactory. Its specificity makes the direct diagnosis of CF cases by the sweat test feasible, without further selection by genetic analysis. CONCLUSION: The PAP/IRT technique for CF detection seems to be suitable for mass screening, without the drawbacks of genetic testing.
Assuntos
Proteínas de Fase Aguda/metabolismo , Antígenos de Neoplasias , Biomarcadores Tumorais , Fibrose Cística/sangue , Fibrose Cística/diagnóstico , Lectinas Tipo C , Triagem Neonatal/métodos , Tripsinogênio/sangue , Fibrose Cística/epidemiologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Ensaio de Imunoadsorção Enzimática/normas , Estudos de Viabilidade , França/epidemiologia , Testes Genéticos/métodos , Humanos , Recém-Nascido , Mutação/genética , Triagem Neonatal/normas , Proteínas Associadas a Pancreatite , Seleção de Pacientes , Avaliação de Programas e Projetos de Saúde , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
We report here that the mere fact of changing culture medium for fresh medium induced in several cell lines the expression of stress-activated genes including protein kinases p38, JNK and ERK1/2 and the transcription factor C/EBPbeta. As a consequence, p8, a gene induced by stress in several tissues, was strongly up-regulated. Induction did not occur after change for cell-conditioned medium. Induction was however transient, with a peak at 60 min for p38, at 15-30 min for JNK and at 15 min for ERK1/2, at 2-3 hours for C/EBPbeta and at 4-6 hours for p8. Repression of the induction was due to the secretion of thermolabile molecule(s) that progressively conditioned the medium. As low as 25% of conditioned medium added to fresh culture medium was sufficient to abolish the stress response. Taken together, our data indicate that the renewal of culture medium induces a transient cellular stress that may be a source of artifacts in experiments performed shortly after a change of culture medium.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/genética , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas de Neoplasias , Células 3T3 , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas Estimuladoras de Ligação a CCAAT , Meios de Cultivo Condicionados/química , Expressão Gênica/fisiologia , Células HT29 , Células HeLa , Humanos , MAP Quinase Quinase 4 , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA Mensageiro/análise , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Most attacks of acute pancreatitis are self-limiting, suggesting that the pancreatic cells adapt their phenotype to prevent progression of the disease. Such phenotypic change must involve a coordinated modification in the expression of numerous genes. To identify differentially expressed genes, high-density mouse cDNA microarrays were hybridized with cDNA probes from both healthy pancreas and pancreas affected by acute pancreatitis. From the 7981 mouse genes analyzed, 239 showed significant changes in their expression during the acute phase of pancreatitis. Among them, 107 genes were up-regulated whereas 132 were down-regulated. They include genes whose function was not previously related to pancreatitis, suggesting that they are involved in some way into the acute pancreatic response. Finally, 40% of differentially expressed genes corresponded to ESTs. Demonstration that a large quantity of unexpected or yet uncharacterized genes showed altered expression during acute pancreatitis underscores the interest of a genome-based investigation. Some of these genes are certainly involved in the cellular defense against pancreatitis and, as such, deserve being studied further.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Pancreatite/genética , Reação de Fase Aguda , Animais , Regulação para Baixo , Camundongos , Camundongos Endogâmicos BALB C , Regulação para CimaRESUMO
BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha contributes to the development of acute pancreatitis. Because TNF-alpha is involved in the control of apoptosis, we studied its interaction with the pancreatic apoptotic pathway. METHODS: Pancreatic acinar AR4-2J cells were used. Apoptosis was monitored by morphologic and biochemical criteria. RESULTS: TNF-alpha induced apoptosis in AR4-2J cells. Induction was strongly enhanced in cells treated with actinomycin D, suggesting that TNF-alpha activated concomitantly an antiapoptotic mechanism through newly synthesized proteins. This mechanism involved activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases because their inhibition worsened TNF-alpha-induced apoptosis. The antiapoptotic pancreatitis-associated protein (PAP) I is a candidate for mediating TNF-alpha activity. Its expression is induced by TNF-alpha, and cells overexpressing PAP I show significantly less apoptosis on exposure to TNF-alpha. We examined whether TNF-alpha induction of PAP I expression was mediated by NF-kappaB or MAP kinases by using specific inhibitors of both pathways. Inhibition of NF-kappaB had no effect. However, inhibitors of MEK1 eliminated PAP I induction. CONCLUSIONS: TNF-alpha induces concomitantly proapoptotic and antiapoptotic mechanisms in pancreatic AR4-2J cells. Antiapoptotic mechanisms are mediated by NF-kappaB and MAP kinases, and PAP I is one of the effectors of apoptosis inhibition.
Assuntos
Proteínas de Fase Aguda/fisiologia , Antígenos de Neoplasias , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Lectinas Tipo C , NF-kappa B/antagonistas & inibidores , Pâncreas/efeitos dos fármacos , Pâncreas/fisiopatologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas de Fase Aguda/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Dactinomicina/farmacologia , Sinergismo Farmacológico , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Pâncreas/patologia , Proteínas Associadas a Pancreatite , Proteínas Serina-Treonina Quinases/fisiologia , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
We have previously reported that down-regulation of Cdx1 and Cdx2 mRNA expression is associated with colon carcinogenesis, and that coordinated reexpression of these genes in the HT29 colon cancer-derived cell line leads to a reduced malignant phenotype. Here we show that restoring Cdx1 and Cdx2 expression in HT29 cells enhanced the antigen presentation system, as reflected by a strong induction of the concentration of HLA-I molecules at the cell surface, resulting from increased expression of the HLA-I mRNA. Expression of the LMP2 proteasomal protein was also strongly induced by Cdx1 and Cdx2 at the transcriptional level, whereas TAP1 expression which is under the control of the same bidirectional promoter as LMP2 remained unchanged. Furthermore, expression of the adhesion molecule ICAM-1, which works in concert with HLA-I, and of the cell death promoter Fas was also increased upon Cdx1 and Cdx2 expression. Taken together, these results suggest that loss of Cdx1 and Cdx2 expression during colorectal carcinogenesis could favor the escape of tumor cells from the immune system. In conclusion, restoration of Cdx1 and Cdx2 expression should be considered in immunotherapeutic strategies for colorectal cancer.
Assuntos
Proteínas Aviárias , Neoplasias do Colo/genética , Cisteína Endopeptidases , Genes Homeobox/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Homeodomínio/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Antígenos CD58/genética , Antígenos CD58/metabolismo , Fator de Transcrição CDX2 , Proteína Ligante Fas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes MHC Classe I , Células HT29 , Proteínas de Homeodomínio/metabolismo , Humanos , Imunidade Celular , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interferon gama/farmacologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas/genética , Proteínas/metabolismo , TransativadoresRESUMO
We have used a microarray-based strategy to characterize, at the molecular level, the pancreatic emergency program set up by the pancreatic cells in response to pancreatitis. In this strategy, the phenotype of the pancreatitis-affected pancreas is established by characterization of a large number of its transcripts using a high-density mouse cDNA microarray. This method allows identification of transcripts differentially expressed during pancreatitis. We describe here the cloning, sequencing, and expression analysis of a new gene, named PIP49 (Pancreatitis Induced Protein 49). Its very strong expression is specific of acinar cells and occurs rapidly after initiation of the acute phase of pancreatitis. Analysis of its primary and secondary structures strongly suggests that PIP49 encodes a putative transmembrane protein.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Pâncreas/metabolismo , Pancreatite/metabolismo , Doença Aguda , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Hibridização In Situ , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Pâncreas/patologia , Pancreatite/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de ProteínaRESUMO
BACKGROUND & AIMS: Homeobox genes are involved in establishing and maintaining differentiated patterns in adult tissues. Cdx1 might carry out that function in the intestinal epithelium because its expression is specific to that tissue and increases during development. METHODS: Cdx1 expression was induced in IEC-6 intestinal epithelial cells by stable transfection, and subsequent changes in cell growth, resistance to apoptosis, migration, and differentiation were monitored. RESULTS: Compared with control, IEC-6/Cdx1 cells proliferated more rapidly, were more resistant to apoptosis, and migrated 3-4 times faster, as shown by an in vitro wound assay. IEC-6/Cdx1 cells in culture formed multilayers. Morphology of the top layer was similar to that of columnar epithelium, with cells showing typical features of differentiated enterocytes, including complex junctions and well-developed microvilli with glycocalix. Expression of 2 markers of enterocyte differentiation, aminopeptidase N and villin, was induced in IEC-6/Cdx1 cells. Aminopeptidase N was targeted to the basolateral membrane, and villin was localized to the cytoplasm. Actin filaments, which were mostly present in transcytoplasmic stress fibers in control cells, were redistributed to the cortex in Cdx1-transfected cells. CONCLUSIONS: Cdx1 expression in IEC-6 cells induces phenotypic changes characteristic of differentiating enterocytes, suggesting an important role for Cdx1 in the transition from stem cells to proliferating/transit cells.
Assuntos
Proteínas Aviárias , Proteínas de Homeodomínio/fisiologia , Mucosa Intestinal/citologia , Actinas/metabolismo , Animais , Apoptose/fisiologia , Antígenos CD13/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Proteínas dos Microfilamentos/metabolismo , Fenótipo , Ratos , Células-Tronco/citologia , Transfecção , Fator de Crescimento Transformador beta/farmacologiaRESUMO
BACKGROUND: Peritoneal colonization is a crucial event in the pathogenesis of peritonitis and its local complications. Adherence to the serosal mesothelium is mediated in a number of microorganisms derived from the digestive tract (especially E. coli) by type-1 fimbriae which have an oligosaccharide specificity. PURPOSE: To evaluate the effect of repeated peritoneal washes with saline solution and oligosaccharides on E. coli peritoneal adherence in a rat peritonitis model. METHODS: Sixty rats were randomized in 3 groups of 20. E. coli was inoculated at a constant concentration of 10(8)/mL per 100 g of weight. Then, peritoneal washes were achieved daily during three consecutive days (D1, D2, D3), with saline solution in Group I (control group), Methyl alpha-D-Mannoside (MADM) in Group II, and p-Nitro-phenyl alpha-D-Mannoside (pNADM) in Group III. Peritoneal samples were obtained before and after lavage at D1, D2, and D3. Microbial recovery was expressed as cfu/mg of tissue, and converted into a percentage of the initial value. A 10% threshold defined efficiency of the wash (inhibition of adherence for 90% of bacteries). RESULTS: Compared with data from Group I, E. coli peritoneal adherence was significantly lower after washes in Group III (D1: p = 0.03; D2: p = 0.009; D3: p = 0.003). Repeated washes were more efficient in Group III than in Group II (D1: p = 0.1; D2: p = 0.5; D3: p = 0.001). CONCLUSION: These results suggest that the addition of oligosaccharides, especially of pNADM, reduces the peritoneal adherence of E. coli when a peritoneal wash is performed for peritonitis.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Manosídeos/uso terapêutico , Peritonite/microbiologia , Tensoativos/uso terapêutico , Animais , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/fisiologia , Metilmanosídeos , Lavagem Peritoneal , Peritônio/microbiologia , Peritonite/tratamento farmacológico , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Cloreto de SódioRESUMO
AIM: To determine whether pancreatitis associated protein (PAP) is a marker for cystic fibrosis which could be used in neonatal screening for the disease. METHODS: PAP was assayed on screening cards from 202,807 neonates. Babies with PAP > or = 15 ng/ml, or > or = 11.5 ng/ml and immunoreactive trypsinogen (IRT) > or = 700 ng/ml were recalled for clinical examination, sweat testing, and cystic fibrosis transmembrane regulator (CFTR) gene analysis. RESULTS: Median PAP value was 2.8 ng/ml. Forty four cases of cystic fibrosis were recorded. Recalled neonates (n = 398) included only 11 carriers. A receiver operating characteristic curve analysis showed that PAP above 8.0 ng/ml would select 0.76% of babies, including all those with cystic fibrosis, except for one with meconium ileus and two with mild CFTR mutations. Screening 27,146 babies with both PAP and IRT showed that only 0.12% had PAP > 8.0 ng/ml and IRT > 700 ng/ml, including all cases of cystic fibrosis. CONCLUSION: PAP is increased in most neonates with cystic fibrosis and could be used for CF screening. Its combination with IRT looks promising.
Assuntos
Proteínas de Fase Aguda/análise , Antígenos de Neoplasias , Biomarcadores Tumorais , Fibrose Cística/diagnóstico , Lectinas Tipo C , Triagem Neonatal/métodos , Biomarcadores/sangue , Fibrose Cística/sangue , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Recém-Nascido , Proteínas Associadas a Pancreatite , Valor Preditivo dos Testes , Estudos Prospectivos , Tripsinogênio/sangueRESUMO
BACKGROUND: Urine is supersaturated in calcium oxalate, which means that it will contain calcium oxalate crystals that form spontaneously. Their size must be controlled to prevent retention in ducts and the eventual development of a lithiasis. This is achieved, in part, by specific inhibitors of crystal growth. We investigated whether promoters of crystal nucleation could also participate in that control, because for the same amount of salt that will precipitate from a supersaturated solution, increasing the number of crystals will decrease their average size and facilitate their elimination. METHODS: Albumin was purified from commercial sources and from the urine of healthy subjects or idiopathic calcium stone formers. Its aggregation properties were characterized by biophysical and biochemical techniques. Albumin was then either attached to several supports or left free in solution and incubated in a metastable solution of calcium oxalate. Kinetics of calcium oxalate crystallization were determined by turbidimetry. The nature and efficiency of nucleation were measured by examining the type and number of neoformed crystals. RESULTS: Albumin, one of the most abundant proteins in urine, was a powerful nucleator of calcium oxalate crystals in vitro, with the polymers being more active than monomers. In addition, nucleation by albumin apparently led exclusively to the formation of calcium oxalate dihydrate crystals, whereas calcium oxalate monohydrate crystals were formed in the absence of albumin. An analysis of calcium oxalate crystals in urine showed that the dihydrate form was present in healthy subjects and stone formers, whereas the monohydrate, which is thermodynamically more stable and constitutes the core of most calcium oxalate stones, was present in stone formers only. Finally, urinary albumin purified from healthy subjects contained significantly more polymers and was a stronger promoter of calcium oxalate nucleation than albumin from idiopathic calcium stone formers. CONCLUSIONS: Promotion by albumin of calcium oxalate crystallization with specific formation of the dihydrate form might be protective, because with rapid nucleation of small crystals, the saturation levels fall; thus, larger crystal formation and aggregation with subsequent stone formation may be prevented. We believe that albumin may be an important factor of urine stability.