RESUMEN
BACKGROUND: Expression of acinar cell-specific genes requires the pancreas transcription factor 1alpha (Ptf1alpha). p48 is the only component of Ptf1alpha that is involved in both acinar gene regulation and pancreatic ontogenesis. MATERIALS AND METHODS: To determine whether Ptf1alpha/p48 expression is regulated during pancreatitis, acute pancreatitis was induced in rats by repeated caerulein injections; early chronic pancreatitis by the combined administration of caerulein and cyclosporin A; and focal pancreas fibrosis by trinitrobenzene sulfonic acid infusion into the pancreatic duct. AR42J cells were used to examine caerulein effects on acinar cells. Ptf1alpha/p48 expression was examined using immunohistochemistry, Western blotting, and qRT-PCR methods. RESULTS: In acute pancreatitis, Ptf1alpha/p48 decreased markedly within 6 h as determined by Western blotting and immunohistochemistry. After 24 h, Ptf1alpha/p48 increased continuously and normalized at day six. In contrast, pancreas amylase reached a nadir at 48 h, when Ptf1alpha/p48 had largely recovered. In the early chronic pancreatitis model Ptf1alpha/p48 levels did not completely recover even at day 14, and this was associated with a failure to restore normal histology and amylase content. qRT-PCR showed that p48 mRNA were reduced after pancreatitis induction and were followed by a decrease in elastase mRNA. In the focal pancreas fibrosis model, Ptf1alpha/p48 expression was undetectable in areas with substantial acinar cell loss and tubular complexes. Caerulein did not affect Ptf1alpha/p48 expression in AR42J cells. CONCLUSIONS: Ptf1alpha/p48 protein and mRNA levels are regulated in acute and chronic experimental pancreatitis. Inability to re-express Ptf1alpha/p48 after injury may preclude acinar cell differentiation and appropriate pancreatic regeneration.
Asunto(s)
Páncreas/fisiología , Pancreatitis/inducido químicamente , Factores de Transcripción/fisiología , Enfermedad Aguda , Animales , Western Blotting , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Enfermedad Crónica , Ensayos Clínicos Controlados como Asunto , Regulación de la Expresión Génica , Pancreatitis/genética , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Regeneración/genética , Regeneración/fisiología , Factores de Transcripción/genéticaRESUMEN
AIMS/HYPOTHESIS: We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. METHODS: Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice. RESULTS: The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols. CONCLUSIONS/INTERPRETATION: These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.
Asunto(s)
Diferenciación Celular/fisiología , Insulina/metabolismo , Células Madre/citología , Animales , Células Cultivadas , Embrión de Mamíferos , Proteínas de Homeodominio/genética , Secreción de Insulina , Islotes Pancreáticos/embriología , Islotes Pancreáticos/metabolismo , Ratones , Regiones Promotoras Genéticas , TransfecciónRESUMEN
Islet transplantation as a potential treatment for diabetes has been investigated extensively over the past 10 years. Such an approach, however, will always be limited mainly because it is difficult to obtain sufficiently large numbers of purified islets from cadaveric donors. One alternative to organ or tissue transplantation is to use a renewable source of cells. Stem cells are clonogenic cells capable of both self-renewal and multilineage differentiation. These cells have the potential to proliferate and differentiate into any type of cell and to be genetically modified in vitro, thus providing cells which can be isolated and used for transplantation. Recent studies have given well-defined differentiation protocols, which can be used to guide stem cells into specific cell lineages as neurons, cardiomyocytes and insulin-secreting cells. Moreover, these derived cells have been useful in different animal models. In this regard, insulin-secreting cells derived from R1 mouse embryonic stem cells restore blood glucose concentrations to normal when they are transplanted into streptozotocin-induced diabetic animals. These results show that diabetes could be among the first applications of stem cell therapy.
Asunto(s)
Trasplante de Células , Diabetes Mellitus/terapia , Trasplante de Células Madre , Animales , Diferenciación Celular , Embrión de Mamíferos/citología , Humanos , Insulina/genética , Trasplante de Islotes Pancreáticos , TransfecciónRESUMEN
The majority of human pancreatic adenocarcinomas display a ductal phenotype; experimental studies indicate that tumors with this phenotype can arise from both acinar and ductal cells. In normal pancreas acinar cells, the pancreas transcription factor 1 transcriptional complex is required for gene expression. Pancreas transcription factor 1 is a heterooligomer of pancreas-specific (p48) and ubiquitous (p75/E2A and p64/HEB) basic helix-loop-helix proteins. We have examined the role of p48 in the phenotype of azaserine-induced rat DSL6 tumors and cancers of the human exocrine pancreas. Serially transplanted acinar DSL6 tumors express p48 whereas DSL6-derived cell lines, and the tumors induced by them, display a ductal phenotype and lack p48. In human pancreas cancer cell lines and tissues, p48 is present in acinar tumors but not in ductal tumors. Transfection of ductal pancreas cancers with p48 cDNA did not activate the expression of amylase nor a reporter gene under the control of the rat elastase promoter. In some cell lines, p48 was detected in the nucleus whereas in others it was cytoplasmic, as in one human acinar tumor. Together with prior work, our findings indicate that p48 is associated with the acinar phenotype of exocrine pancreas cancers and it is necessary, but not sufficient, for the expression of the acinar phenotype.
Asunto(s)
Adenocarcinoma/genética , Diferenciación Celular/genética , Secuencias Hélice-Asa-Hélice/genética , Páncreas/patología , Neoplasias Pancreáticas/genética , Factores de Transcripción/genética , Adenocarcinoma/patología , Secuencia de Aminoácidos , Animales , Antimetabolitos Antineoplásicos/farmacología , Azaserina/farmacología , Modelos Animales de Enfermedad , Humanos , Datos de Secuencia Molecular , Neoplasias Pancreáticas/patología , Fenotipo , ARN Mensajero/análisis , Ratas , Células Tumorales CultivadasRESUMEN
Shigella entry into epithelial cells is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment in a macropinocytic process. Using affinity chromatography on HeLa cell extracts, we show here that the hyaluronan receptor CD44 associates with IpaB, a Shigella protein that is secreted upon cell contact. Overlay and solid-phase assays indicated that IpaB binds directly to the extracellular domain of CD44; binding is saturable and inhibitable, with a half-maximal inhibitory concentration of 175 nM. Immunoprecipitation experiments showed that IpaB associates with CD44 during Shigella entry. CD44 is recruited at bacterial entry sites and localizes at the plasma membrane of cellular extensions induced by Shigella. Pretreatment of cells with an anti-CD44 monoclonal antibody resulted in inhibition of Shigella-induced cytoskeletal reorganization, as well as inhibition of bacterial entry, whereas transfection of CD44 in cells that are deficient for CD44 results in increased bacterial binding to cells and internalization. The IpaB-CD44 interaction appears to be required for Shigella invasion by initiating the early steps of the entry process.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Células Epiteliales/microbiología , Receptores de Hialuranos/fisiología , Shigella flexneri/patogenicidad , Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Estructura Terciaria de Proteína , TransfecciónRESUMEN
Shigella flexneri is an enteroinvasive bacterium responsible for bacillary dysentery in humans. Bacterial entry into epithelial cells is a crucial step for the establishment of the infection. It is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment by a macropinocytic process. We show in this study that the membrane-cytoskeleton linker, ezrin, a member of the ERM (ezrin, radixin, moesin) family, plays an active role in the process of Shigella uptake. Ezrin is highly enriched in cellular protrusions induced by the bacterium and is found in close association with the plasma membrane. In addition, Shigella entry is significantly reduced in cells transfected with a dominant negative allele of ezrin with entry foci showing much shorter cellular protrusions. These results indicate that ezrin not only acts as a membrane-cytoskeleton linker, but may also mediate extension of cellular projections in the presence of signals such as those elicited by invading microorganisms.
Asunto(s)
Fosfoproteínas/fisiología , Shigella flexneri/patogenicidad , Actinas/metabolismo , Animales , Membrana Celular/microbiología , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Proteínas del Citoesqueleto , Citoesqueleto/microbiología , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Disentería Bacilar/etiología , Disentería Bacilar/microbiología , Células HeLa , Humanos , Células LLC-PK1 , Microscopía Confocal , Microscopía Inmunoelectrónica , Modelos Biológicos , Fosfoproteínas/genética , Transducción de Señal , Porcinos , Transfección , VirulenciaRESUMEN
Recent data suggest that p120-catenin plays a role in the regulation of functionality of E-cadherin, a protein essential for the establishment and maintenance of cell-cell contacts. Since dysfunction of intercellular adhesiveness is an alteration frequently observed in colon cancer we have studied the expression and distribution of p120-catenin in human colorectal tumors. In normal colon, p120-catenin was observed in the crypt and surface epithelium; the cells showed reactivity both in the membrane and in the cytosol. Thirteen primary tumors were examined for p120-catenin expression: they were graded as uniformly positives (+) (4); heterogeneous (+/-) (6), with a diminished expression, detected mainly in the cytosol; and negatives (-) (3). Although the number of tumors was low, the reduction in p120-catenin correlated with a larger size of the tumors (p = 0.038). Association of p120-catenin to the cytoskeleton was also determined in 5 tumors by detergent extraction and Western blot; this analysis shows that lack of reactivity in the membrane was accompanied by absence of p120-catenin in the cytoskeleton-associated fraction. Analysis of E-cadherin was performed in order to compare the distribution of this protein and p120-catenin. Although no complete correlation was found between the expression of both proteins (p = 0.077), our results showed that alterations in the level or distribution of p120-catenin were accompanied by lack of E-cadherin reactivity in the membrane, whereas absence of p120-catenin in the cytoskeleton fraction was associated with important decreases in the amount of E-cadherin in this same fraction. These results show that alterations in p120-catenin levels are a common event in colorectal tumors, and suggest that the distribution of this protein and E-cadherin is coordinately regulated.
Asunto(s)
Moléculas de Adhesión Celular/análisis , Neoplasias Colorrectales/química , Fosfoproteínas/análisis , Anticuerpos Monoclonales , Western Blotting , Cadherinas/análisis , Cateninas , Membrana Celular/química , Neoplasias Colorrectales/ultraestructura , Citoesqueleto/química , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Catenina deltaRESUMEN
1. HT-29 M6 cells are a subpopulation of HT-29 cells that, contrarily to the parental cells, establish tight cell contacts and differentiate. Cell-to-cell contacts in HT-29 M6 cells are also regulated by protein kinase C; addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) decreases the homotypic contacts of these cells. We show here that HT-29 cells or HT-29 M6 cells treated with PMA contain lower levels of functional E-cadherin, determined by analysing the association of this protein with the cytoskeleton. No significant differences in the localization of alpha-, beta-, or p120-catenins were detected under the three different conditions. 2. Dysfunction of E-cadherin can be reversed by incubation of HT-29 cells with the tyrosine kinase inhibitor herbimycin A. On the other hand an augmentation of c-src activity in HT-29 cells or HT-29 M6 cells treated with PMA was observed with respect to control HT-29 M6 cells. The phosphorylation status of catenins was also investigated; in HT-29 or in HT-29 M6 cells treated with PMA, dysfunction of E-cadherin was accompanied by an increased phosphorylation of p120-catenin and by an elevated association of this protein to E-cadherin. These results suggest a role for pp60src and the pp60src substrate p120-catenin in the control of E-cadherin function in HT-29 cells.
Asunto(s)
Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Adhesión Celular/fisiología , Mucosa Intestinal/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transactivadores , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales , Epitelio/metabolismo , Humanos , Intestinos/citología , Fosforilación , Unión Proteica , beta CateninaRESUMEN
A combination of immunohistochemical, biochemical, and recombinant DNA techniques were used to investigate class I expression in 26 pancreatic adenocarcinomas and 6 autologous tumor-derived cells. The prevalence of HLA losses was found to be comparable to that observed in other tumor types (> 35%), using monomorphic and locus-specific antibodies. In one patient, the original tumor tissue, a tumor derived cell line (IMIM-PC-2), and EBV-transformed lymphocytes were available for study. The patient's phenotype was A25, A30, B18, B18. However, A30 allele product could not be detected in the original tumor not in the cultured tumor cells. In addition, A30 allele could not be isolated from cDNA or genomic clones from the cultured tumor cells whereas it was isolated from the autologous lymphoblastoid cell line. Using isoelectric focusing analysis a significant reduction in the B18 heavy chain product was also observed in the tumor cell line, IMIM-PC-2, suggesting the absence of expression of one allele. Further studies revealed loss of heterozygosity at DR and other loci of chromosome 6 and cytogenetic data strongly suggested deletion of a full chromosome 6. This work indicates for the first time that loss of a full HLA haplotype occurs in tumor tissue and suggests that this mechanism may contribute to the progression of human cancer.
Asunto(s)
Adenocarcinoma/genética , Antígenos de Neoplasias/genética , Genes MHC Clase I , Antígenos HLA/genética , Haplotipos/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Alelos , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/inmunología , Cromosomas Humanos Par 6/genética , Cromosomas Humanos Par 6/ultraestructura , ADN Complementario/genética , Repeticiones de Dinucleótido , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Antígenos HLA/biosíntesis , Antígenos HLA/inmunología , Haplotipos/inmunología , Heterocigoto , Humanos , Técnicas para Inmunoenzimas , Inmunofenotipificación , Focalización Isoeléctrica , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Células Tumorales CultivadasRESUMEN
Incubation of HT-29 M6 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) induces cell scattering, loss of cellular contacts and inactivation of E-cadherin. We have investigated the involvement of different protein kinase C (PK-C) isoforms in these processes using specific activators. Thymeleatoxin, a derivative of mezerein that activates conventional PK-Cs (cPK-Cs) but not novel PK-Cs (nPK-Cs), promoted effects that were similar to those of PMA, i.e. at concentrations of 200 nM it induced scattering of HT-29 M6 colonies, loss of homotypic contacts and dissociation of E-cadherin from the cytoskeleton. Among the isoforms activated by this compound, only cPK-C alpha was detected in HT-29 M6 cells by Western blot. The specificity of this compound with respect to the rest of the PK-C isoforms present in these cells was determined; thymeleatoxin induced, as did PMA, the translocation of cPK-C alpha from the cytosol to the membrane and the cytoskeleton, and its partial down-regulation. On the other hand, thymeleatoxin did not modify the cellular levels or localization of nPK-C epsilon or atypical PK-C zeta. "In vitro' assays also showed that thymeleatoxin did not activate nPK-C epsilon at the concentrations added to the cell cultures. These results indicate that thymeleatoxin is selective for cPK-C alpha over nPK-C epsilon and show a role for the former enzyme in the regulation of cell-cell contacts and the inactivation of E-cadherin in HT-29 M6 cells.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Isoenzimas/fisiología , Proteína Quinasa C/fisiología , Secuencia de Aminoácidos , Anticuerpos/farmacología , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Ésteres del Forbol/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteína Quinasa C-alfa , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The importance of E-cadherin protein in the establishment and maintenance of homotypic contacts in epithelial cells has already been determined. We report here that the association of E-cadherin to cytoskeleton, required for the functionality of this protein, increases progressively after seeding; in HT-29 M6 cells 4-5 days were required for detecting most of E-cadherin in the Triton X-100-insoluble (cytoskeleton-associated) fraction. The phorbol ester TPA differently affected E-cadherin levels in HT-29 M6 cells; at day 2-3, when most E-cadherin was found not-associated to the cytoskeleton, very important decreases (90%) in the total levels of this protein were detected as soon as 6 h after the addition of this compound. However, on later days (day 5), the predominant effect by 6 h was a translocation from the Triton-insoluble to the soluble fraction. The E-cadherin-associated proteins alpha-catenin and beta-catenin were not significantly affected by treatment with TPA.
Asunto(s)
Cadherinas/metabolismo , Mucosa Intestinal/metabolismo , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transactivadores , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Activación Enzimática/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Humanos , Intestinos/efectos de los fármacos , Intestinos/ultraestructura , Factores de Tiempo , alfa Catenina , beta CateninaRESUMEN
Cytokeratin (CK) expression in tumors generally reflects the CK pattern of the corresponding normal epithelium. Pancreas cancers express CK of simple epithelia 7, 8, 18 and 19, as normal ductal cells. To analyze whether CK of complex or stratified epithelia are abnormally expressed in pancreas cancers, we have used polypeptide-specific mouse monoclonal antibodies (MAbs) detecting CK 5, CK 10, CK 13, CK 14 and CK 17, and an antibody detecting CK 13, CK 15 and CK 16. The streptavidin-peroxidase technique was applied on sections of fresh-frozen specimens of normal pancreas and of pancreas cancer. None of these polypeptides were expressed by normal acinar and centro-acinar cells. CK 5, CK 14 and CK 17 were expressed by less than 5% of cells in normal ducts, whereas CK 10, CK 13, CK 15 and CK 16 were not expressed at all. In tumors, CK 14, CK 15/16 and CK 17 were detected in the majority of cases studied; CK 5, CK 10 and CK 13 were present in a sub-population of pancreas cancers. CK of complex/stratified epithelia were detected in areas of glandular differentiation, but expression was more intense in areas of squamous differentiation. In pancreatitis adjacent to cancer, CK of complex/stratified epithelia were weakly detected or undetectable. These results suggest that up-regulation of these CK takes place in pancreas cancer. The CK phenotype may be of help in the differential diagnosis of this tumor.
Asunto(s)
Queratinas/análisis , Páncreas/química , Neoplasias Pancreáticas/química , Anticuerpos Monoclonales , Humanos , Neoplasias Pancreáticas/patología , Fenotipo , Regulación hacia ArribaRESUMEN
Expression of intermediate filaments (IF) is regulated during development and differentiation. The authors have studied the expression of vimentin and cytokeratins (CK) 4, 7, 8, 13, 18, 19 in normal pancreas, chronic pancreatitis, and pancreas cancer using monoclonal antibodies. Immunohistochemical assays were performed on fresh frozen tissue sections and on cultured pancreas cancer cells using the streptavidin-peroxidase method. In normal pancreas, acinar cells expressed CK 8 and 18, whereas ductal cells expressed CK 7, 8, 18, and 19. CK 4 was expressed by 5-10% of pancreas duct cells in all specimens of normal pancreas. CK 13 was not detected in any epithelial cells of normal pancreas or pancreatitis. CK 7, 8, 18, and 19 were homogeneously expressed in all pancreas cancers, whereas CK 4 was expressed only in 5-50% of cells in 10/16 tumors. Foci of squamous metaplasia expressed CK 13 but showed partial loss of expression of CK 7, 8, 18, and 19. Thirteen pancreas cancer cell lines examined showed homogeneous expression of CK 7, 8, 18, and 19; 2/11 lines expressed CK 4 weakly, and 6/11 expressed vimentin. CK 13 was not detected in any of the lines. These results indicate that pancreas cancer cells consistently express cytokeratin polypeptides characteristic of ductal epithelial cells and that this phenotype is retained in pancreas cancer cell lines. In addition, squamous metaplasia is associated with a coordinate change in the expression of CK polypeptides.