RESUMEN
Oxyntic atrophy in the stomach leads to chief cell transdifferentiation into spasmolytic polypeptide expressing metaplasia (SPEM). Investigations of preneoplastic metaplasias in the stomach are limited by the sole reliance on in vivo mouse models, owing to the lack of in vitro models for distinct normal mucosal lineages and metaplasias. Utilizing the Immortomouse, in vitro cell models of chief cells and SPEM were developed to study the characteristics of normal chief cells and metaplasia. Chief cells and SPEM cells isolated from Immortomice were cultured and characterized at both the permissive (33°C) and the nonpermissive temperature (39°C). Clones were selected on the basis of their transcriptional expression of specific stomach lineage markers (named ImChief and ImSPEM) and protein expression and growth were analyzed. The transcriptional expression profiles of ImChief and ImSPEM cells were compared further by using gene microarrays. ImChief cells transcriptionally express most chief cell markers and contain pepsinogen C and RAB3D-immunostaining vesicles. ImSPEM cells express the SPEM markers TFF2 and HE4 and constitutively secrete HE4. Whereas ImChief cells cease proliferation at the nonpermissive temperature, ImSPEM cells continue to proliferate at 39°C. Gene expression profiling of ImChief and ImSPEM revealed myelin and lymphocyte protein 2 (MAL2) as a novel marker of SPEM lineages. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cell lines resemble in vivo chief and SPEM cell lineages. These cell culture lines provide the first in vitro systems for studying the molecular mechanisms of the metaplastic transition in the stomach.
Asunto(s)
Células Principales Gástricas/metabolismo , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/metabolismo , Péptidos/metabolismo , Estómago/patología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Mucosa Gástrica/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Metaplasia/diagnóstico , Ratones , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pepsinógeno C/genética , Pepsinógeno C/metabolismo , Péptidos/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP , Proteínas de Unión al GTP rab3/genética , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.
Asunto(s)
Carcinoma Ductal Pancreático/inmunología , Macrófagos/fisiología , Páncreas/patología , Neoplasias Pancreáticas/inmunología , Células Estrelladas Pancreáticas/fisiología , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibrosis , Metaplasia , Ratones , Neoplasias Pancreáticas/patología , Receptor Cross-TalkRESUMEN
Recent progress has been made in the identification of protein-coding genes and miRNAs that are expressed in and alter the behavior of colonic epithelia. However, the role of long non-coding RNAs (lncRNAs) in colonic homeostasis is just beginning to be explored. By gene expression profiling of post-mitotic, differentiated tops and proliferative, progenitor-compartment bottoms of microdissected adult mouse colonic crypts, we identified several lncRNAs more highly expressed in crypt bottoms. One identified lncRNA, designated non-coding Nras functional RNA (ncNRFR), resides within the Nras locus but appears to be independent of the Nras coding transcript. Stable overexpression of ncNRFR in non-transformed, conditionally immortalized mouse colonocytes results in malignant transformation, as determined by growth in soft agar and formation of highly invasive tumors in nude mice. Moreover, ncNRFR appears to inhibit the function of the tumor suppressor let-7. These results suggest precise regulation of ncNRFR is necessary for proper cell growth in the colonic crypt, and its misregulation results in neoplastic transformation.
Asunto(s)
Transformación Celular Neoplásica , Colon/patología , Neoplasias del Colon/genética , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/genética , Animales , Colon/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Ratones , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismoRESUMEN
The colonic epithelium is composed of a polarized monolayer sheathed by a layer of pericryptal myofibroblasts (PCMFs). We mimicked these cellular compartments in vitro to assess the effects of paracrine-acting PCMF-derived factors on tight junction (TJ) integrity, as measured by transepithelial electrical resistance (TER). Coculture with 18Co PCMFs, or basolateral administration of 18Co conditioned medium, significantly reduced TER of polarized Caco-2 cells. Among candidate paracrine factors, only keratinocyte growth factor (KGF) reduced Caco-2 TER; basolateral KGF treatment led to time- and concentration-dependent increases in claudin-2 levels. We also demonstrate that amphiregulin (AREG), produced largely by Caco-2 cells, increased claudin-2 levels, leading to epidermal growth factor receptor-mediated TER reduction. We propose that colonic epithelial TJ integrity can be modulated by paracrine KGF and autocrine AREG through increased claudin-2 levels. KGF-regulated claudin-2 induction may have implications for inflammatory bowel disease, where both KGF and claudin-2 are upregulated.
Asunto(s)
Claudina-2/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/fisiología , Miofibroblastos/fisiología , Uniones Estrechas/fisiología , Anfirregulina , Células CACO-2 , Comunicación Celular , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Proliferación Celular , Medios de Cultivo Condicionados , Familia de Proteínas EGF , Impedancia Eléctrica , Receptores ErbB/metabolismo , Humanos , Mucosa Intestinal/metabolismo , LigandosRESUMEN
The development of pancreatic fibrosis has been shown to be a major component in several diseases of the pancreas including pancreatic cancer, chronic pancreatitis, and type 2 diabetes mellitus, but its actual role in the progression of these disorders is still unknown. This fibrosis is characterized by stromal expansion and the excessive deposition of extracellular matrix (ECM) that replaces pancreatic tissue. This eventually leads to dysregulation of ECM turnover, production of cytokines, restriction of blood flow, and often exocrine and endocrine insufficiencies. Activated pancreatic stellate cells (PSCs) have been identified as key mediators in the progression of pancreatic fibrosis, serving as the predominant source of excess ECM proteins. Previously, we found that overexpression of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pancreatic islets led to intraislet fibrosis. HB-EGF binds to and activates two receptors, epidermal growth factor receptor (EGFR) and ErbB4, as well as heparin moieties and CD9/DRAP27. To understand the mechanism underlying the induction of fibrogenesis by HB-EGF, we utilized a hypomorphic allele of Egfr, the Waved-2 allele, to demonstrate that EGFR signaling regulates fibrogenesis in vivo. Using an in vitro cell migration assay, we show that HB-EGF regulates both chemoattraction and stimulation of proliferation of PSCs via EGFR activation.
Asunto(s)
Receptores ErbB/metabolismo , Páncreas/metabolismo , Enfermedades Pancreáticas/metabolismo , Transducción de Señal , Animales , Línea Celular , Proliferación Celular , Quimiotaxis , Modelos Animales de Enfermedad , Receptores ErbB/genética , Fibrosis , Factor de Crecimiento Similar a EGF de Unión a Heparina , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Páncreas/patología , Enfermedades Pancreáticas/genética , Enfermedades Pancreáticas/patología , Enfermedades Pancreáticas/prevención & control , Proteínas Recombinantes/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Simian virus 40 large T antigen (TAg) contributes to cell transformation, in part, by targeting two well-characterized tumor suppressors, pRb and p53. TAg expression affects the transcriptional circuits controlled by Rb and by p53. We have performed a microarray analysis to examine the global change in gene expression induced by wild-type TAg (TAg(wt)) and TAg mutants, in an effort to link changes in gene expression to specific transforming functions. For this analysis we have used enterocytes from the mouse small intestine expressing TAg. Expression of TAg(wt) in the mouse intestine results in hyperplasia and dysplasia. Our analysis indicates that practically all gene expression regulated by TAg in enterocytes is dependent upon its binding and inactivation of the Rb family proteins. To further dissect the role of the Rb family in the induction of intestinal hyperplasia, we have screened several lines of transgenic mice expressing a truncated TAg (TAg(N136)), which is able to interfere with the Rb pathway but lacks the functions associated with the carboxy terminus of the protein. This analysis confirmed the pivotal association between the Rb pathway and the induction of intestinal hyperplasia and revealed that upregulation of p53 target genes is not associated with the tumorigenic phenotype. Furthermore, we found that TAg(N136) was sufficient to induce intestinal hyperplasia, although the appearance of dysplasia was significantly delayed.
Asunto(s)
Antígenos Virales de Tumores/fisiología , Transformación Celular Viral/genética , Factores de Transcripción E2F/metabolismo , Enterocitos/metabolismo , Regulación de la Expresión Génica , Proteína de Retinoblastoma/metabolismo , Virus 40 de los Simios/fisiología , Animales , Enterocitos/virología , Perfilación de la Expresión Génica , Hiperplasia/etiología , Intestino Delgado/patología , Ratones , Ratones Transgénicos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Tumor-derived cell lines are indispensable tools for understanding the contribution of activated signaling pathways to the cancer phenotype and for the design and testing of targeted signal therapies. In our study, we characterize 10 colorectal carcinoma cell lines for the presence of mutations in the wnt, Ras/MAPK, PI3K and p53 pathways. The mutational spectrum found in this panel of cell lines is similar to that detected in primary CRC, albeit with higher frequency of mutation in the beta-catenin and B-Raf genes. We have monitored activation of the wnt and Ras/MAPK pathways in these cells and analyzed their sensitivity to selective signaling inhibitors. Using beta-catenin subcellular distribution as a marker, we show that cells harboring APC mutations have low-level activated wnt signaling, which can be blocked by the extracellular wnt inhibitor DKK-1, suggesting autocrine activation of this pathway; proliferation of these cells is also blocked by DKK-1. In contrast, cells with beta-catenin mutations are unresponsive to extracellular wnt inhibition. Constitutive phosphorylation of MAPK is present in the majority of the cell lines and correlates with B-Raf but not K-Ras mutations; correspondingly, the proliferation of cells harboring mutations in B-Raf, but not K-Ras, is exquisitely sensitive inhibition of the MAPK pathway. We find no correlation between PI3K mutation or loss of PTEN expression and increased sensitivity to PI3K inhibitors. Our study discloses clear-cut differences in responsiveness to signaling inhibitors between individual mutations within an activated signaling pathway and suggests likely targets for signal-directed therapy of colorectal carcinomas.
Asunto(s)
Proliferación Celular , Neoplasias Colorrectales/patología , Genes APC , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Secuencia de Bases , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Humanos , Fosforilación , Reacción en Cadena de la Polimerasa , Transducción de SeñalRESUMEN
SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Virus 40 de los Simios/fisiología , Animales , Células Cultivadas , Enterocitos/virología , Fibroblastos/virología , Ratones , Ratones TransgénicosRESUMEN
Apoptosis is an important mechanism for maintaining tissue homeostasis and for preventing the proliferation of cells with mutations that could result in malignancy. Barrett's epithelium has been reported to be more resistant to apoptosis than normal esophageal squamous epithelium. We have explored the contribution of the nuclear factor-kappaB (NF-kappaB) pathway to apoptotic resistance in non-neoplastic, telomerase-immortalized esophageal squamous (NES) and Barrett's (BAR-T) epithelial cell lines. We exposed these cells to UV-B irradiation in doses known to cause DNA damage and to induce apoptosis in normal cells, and studied apoptosis as well as the expression of phospho-H2AX, NF-kappaB, Bcl-2, XIAP, cIAP-1, and survivin proteins. We also used Bay 11-7085 and siRNAs to NF-kappaB and Bcl-2 to assess the effects of NF-kappaB and Bcl2 inhibition on apoptosis. UV-B irradiation at low doses (50 and 100 J/m(2)) caused DNA damage in both NES and BAR-T cells but significantly increased apoptosis only in NES cells. UV-B irradiation caused a decrease in the levels of NF-kappaB, Bcl-2, cIAP-1, XIAP, and survivin in NES cells but increased the levels of those proteins in BAR-T cells. The resistance of BAR-T cells to apoptosis induced by low-dose UV-B irradiation was abolished by Bay 11-7085 and by siRNA for NF-kappaB and was decreased significantly by siRNA for Bcl-2. We conclude that the ability of Barrett's epithelial cells to activate the NF-kappaB pathway when they have sustained DNA damage allows them to resist apoptosis. This capacity to avoid apoptosis despite genotoxic damage may underlie the persistence and malignant predisposition of Barrett's metaplasia.
Asunto(s)
Apoptosis/fisiología , Esófago de Barrett/patología , Reflujo Gastroesofágico/patología , FN-kappa B/metabolismo , Apoptosis/efectos de la radiación , Esófago de Barrett/metabolismo , Línea Celular , Daño del ADN , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Epiteliales/efectos de la radiación , Reflujo Gastroesofágico/metabolismo , Humanos , FN-kappa B/antagonistas & inhibidores , Fenotipo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Rayos UltravioletaRESUMEN
Obtaining protein profiles from a homogeneous cell population in tissues can significantly improve our capability in protein biomarker discovery. In this study, unique protein profiles from the top and bottom sections of mouse crypts and Apc(Min+/-) adenomas were obtained using laser capture microdissection (LCM) combined with MALDI MS. Statistically significant protein peaks with differential expression were selected, and a set of novel protein biomarkers were identified. Immunohistochemistry was performed to confirm the differentially expressed protein biomarkers found by LCM combined with MALDI MS. To validate the relevance of the findings in human colorectal cancer (CRC), S100A8 was further confirmed in human CRC using immunohistochemistry. In addition, S100A8 was found to have an increased expression at different human CRC stages (Duke's A-D) compared with controls at both protein (n = 168 cases) and RNA (n = 215 cases) levels. Overall LCM combined with MALDI MS is a promising method to identify intestinal protein biomarkers from minute amounts of tissue. The novel protein biomarkers identified from the top and bottom crypts will increase our knowledge of the specific protein changes taking place during cell migration from the crypt bottom to top. In addition, the identified cancer protein biomarkers will aid in the exploration of colorectal tumorigenesis mechanisms as well as in the advancement of molecularly based diagnosis of colorectal cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Rayos Láser , Microdisección/métodos , Proteínas de Neoplasias/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adenoma/metabolismo , Adenoma/patología , Animales , Calgranulina A/metabolismo , Estudios de Casos y Controles , Humanos , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Ratones , Proteoma/análisis , Proteómica , Reproducibilidad de los ResultadosRESUMEN
It has proved to be impossible to culture epithelial cells from the gastrointestinal tract of adult animals. Researchers have had to use either cell lines derived from newborn rat small intestine or colon carcinoma cell lines that have retained some of the properties of the gastrointestinal mucosa. We have described a method for establishing conditionally immortalized cell lines from the stomach, small intestine, colon, pancreas, and liver from tissue obtained from a transgenic mouse strain carrying a temperature-sensitive mutant of the SV40 large T gene (the "Immortomouse"). This immortalizing gene has proved to be useful for establishing cell lines from a number of transgenic mice following crossbreeding of the Immortomouse with the transgenic mouse of interest. These cell lines are being used in numerous studies. In this review we describe the methods for developing such lines and list the range of cell lines that have been developed from colon, small intestine, stomach, liver, and pancreas of a number of transgenic mice.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Técnicas de Cultivo de Célula/métodos , Células Epiteliales/citología , Mucosa Intestinal/citología , Animales , Línea Celular Transformada , Ratones , Ratones TransgénicosRESUMEN
Cells in tissues do not exist as isolated entities but are part of the three-dimensional tissue architecture. Consequently, some aspects of cell behaviour cannot be mimicked by simple in vitro monolayer culture systems. Moreover, cell shape and behaviour is not rigid but is dynamic and can be regulated by intrinsic and extrinsic factors. For example, tumour cells in epithelium-derived cancer such as colorectal cancer often retain significant features of the colonic mucosa. However, as the tumour progresses, the morphology of the tumour cells often undergoes a transition from an epithelial morphology to a mesenchymal morphology. This transition is important as it signifies a change in the tumour phenotype to a more aggressive, invasive, and eventually metastatic phenotype. In vitro models that allow the study of this transition are needed. One such model is the LIM1863 colon carcinoma cells that normally grow as organoids but can be adapted to efficiently undergo an epithelial to mesenchymal transition that can be reversed. This system has allowed the study of the genes such as Frizzled 7 that are involved in this dynamic and reversible epithelial to mesenchymal transition.
Asunto(s)
Técnicas de Cultivo de Célula , Neoplasias Colorrectales , Receptores Frizzled/metabolismo , Organoides/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Técnicas de Cultivo de Célula/instrumentación , Línea Celular , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/fisiopatología , Receptores Frizzled/genética , Técnicas de Transferencia de Gen , Humanos , Morfogénesis , Organoides/citología , Retroviridae/genética , Retroviridae/metabolismo , Proteínas Wnt/genéticaRESUMEN
BACKGROUND AND AIMS: PTK6 is an intracellular src-related tyrosine kinase that regulates differentiation in the intestine, where knockout animals have increased proliferative activity and growth characteristics. To explore the phenotype further we attempted to establish epithelial cell lines from the intestinal mucosa. METHOD: We mated Ptk6 null mice with a tsSV40 large T transgenic mouse (Immortomouse) to obtain null mice carrying the SV40 gene. Intestinal tissues from these mice were cultured. RESULTS: We established a Ptk6 null epithelial cell line from the colonic mucosa. Consistent with a role of Ptk6 in cell differentiation, these cells have the characteristics of a stable progenitor cell. In monolayer culture, the cells form domes in the monolayer when confluent. When cultured on Transwell filters, the cells polarize and form an electrically resistant barrier. Formation of tight junctions was confirmed by demonstrating expression of ZO1 and occludin at the apical junctions, whereas E-cadherin localized to the basolateral membrane. When cultured in collagen gel, the Ptk6 null cells form complex organoids, some of which resemble cups of cells. These organoids contain cells with differentiated phenotypes. Using immunohistochemistry and confocal microscopy we have been able to identify villin-positive (absorptive cells) and a small percentage of mucin-containing cells (goblet cells) and chromogranin A-positive cells (endocrine cells). CONCLUSION: This conditionally immortalized cell line represents an excellent cell culture model system for exploring the mechanisms of cell function and epithelial differentiation in the colonic mucosa.
Asunto(s)
Diferenciación Celular , Polaridad Celular , Colon/enzimología , Células Epiteliales/enzimología , Mucosa Intestinal/patología , Familia-src Quinasas/metabolismo , Animales , Cadherinas/metabolismo , Línea Celular , Proliferación Celular , Forma de la Célula , Cromogranina A/metabolismo , Colon/citología , Impedancia Eléctrica , Mucosa Intestinal/citología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Mucinas/metabolismo , Ocludina , Organoides , Fenotipo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas , Uniones Estrechas/metabolismo , Factores de Tiempo , Proteína de la Zonula Occludens-1 , Familia-src Quinasas/deficiencia , Familia-src Quinasas/genéticaRESUMEN
Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.
Asunto(s)
Movimiento Celular , Colon/metabolismo , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Neuropéptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Cicatrización de Heridas , Proteínas de Unión al GTP rac/metabolismo , Familia-src Quinasas/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Colon/efectos de los fármacos , Colon/enzimología , Activación Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/enzimología , Receptores ErbB/deficiencia , Receptores ErbB/genética , Ratones , Ratones Noqueados , Neuropéptidos/antagonistas & inhibidores , Neuropéptidos/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas , Seudópodos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Transfección , Cicatrización de Heridas/efectos de los fármacos , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1 , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Factor de Transcripción E2F2/metabolismo , Enfermedades Gastrointestinales/virología , Hiperplasia/virología , Virus 40 de los Simios/patogenicidad , Animales , Factor de Transcripción E2F2/deficiencia , Factor de Transcripción E2F4/metabolismo , Enterocitos/química , Enterocitos/virología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína p130 Similar a la del Retinoblastoma/metabolismoRESUMEN
Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.
Asunto(s)
Linaje de la Célula/fisiología , Intestino Delgado/citología , Fosfoproteínas/biosíntesis , Proteínas Represoras/biosíntesis , Transcripción Genética , Animales , Línea Celular , Chlorocebus aethiops , Células Enteroendocrinas/citología , Células Caliciformes/citología , Histona Desacetilasas/metabolismo , Humanos , Intestino Delgado/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear , Células de Paneth/citología , Fosfoproteínas/genética , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3RESUMEN
During late embryogenesis, the mouse colon develops from a pseudostratified, undifferentiated endoderm to a single-layered columnar epithelium with accompanying mesenchymal maturation. To identify regulatory genetic programs underlying these morphological changes, we profiled gene expression of the developing mouse colon by microarray from embryonic day (E)13.5 to E18.5. Unbiased cluster analysis of 13,484 cDNA elements revealed two distinct groups of genes whose expression changes reflect the dynamic morphological events of the epithelium and mesenchyme during this period. Additional analyses revealed two subsets of genes whose expression is either upregulated or downregulated over the same developmental period. Of those genes whose expression increases from E13.5 to E18.5 (n = 158), known functions include acquisition and/or maintenance of colonic differentiation. Genes whose transcription is downregulated over this period (n = 49) have demonstrated roles in nuclear organization, transcriptional regulation, and cell proliferation. These results provide the basis for a molecular portrait of colonic development during late embryogenesis and should be a valuable resource for investigators interested in colonic development and neoplasia, as well as comparative organogenesis.
Asunto(s)
Colon/crecimiento & desarrollo , Desarrollo Embrionario , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Animales , Animales no Consanguíneos , Análisis por Conglomerados , Colon/embriología , Colon/ultraestructura , ADN Complementario , Epitelio/metabolismo , Femenino , Inmunohistoquímica , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
BACKGROUND: Smad proteins play a key role in TGF-beta signaling that regulates cell proliferation, differentiation, and apoptosis. Mice deficient in Smad3 develop colonic adenocarcinoma. MATERIALS AND METHODS: We developed a Smad3-deficient colonocyte cell line that was used to study TGF-beta-mediated growth inhibition and induction of apoptosis was compared to young adult mouse colonocyte (YAMC) control cells. Growth inhibition was assessed by cell count and ((3)H)-thymidine incorporation assay. Transcriptional response to TGF-beta was measured by transfecting the reporters p3TP-Lux and p(CAGA)(9)-MLP-luc. TGF-beta-induced apoptosis was assessed using ELISA and Hoechst staining. Mediators of cell-cycle arrest and apoptosis were assayed by Western blot. RESULTS: Smad3-/- cells were resistant to TGF-beta-mediated growth inhibition compared to control cells. Ninety-eight percent of cell count growth inhibition observed in YAMC cells, while 34% inhibition was observed in Smad3-/- cells after TGF-beta treatment. ((3)H)-thymidine incorporation was inhibited by 61% in YAMC cells, while Smad3-/- cells showed 25% inhibition after TGF-beta treatment. Smad3-/- cells were deficient in luciferase reporter induction by TGF-beta. TGF-beta induced apoptosis 8-fold in YAMC cells, but had no effect on apoptosis in Smad3-/- cells. p21(Cip11) and PAI-1 are induced in YAMC cells by TGF-beta, but unchanged in Smad3-/- cells. TGF-beta decreases cyclin D1 levels in YAMC cells but does not affect levels in Smad3-/- cells. CONCLUSIONS: Our findings suggest that the loss of Smad3 contributes to resistance of TGF-beta growth inhibition and apoptosis in colonic epithelium. This may represent a mechanism by which cells are able to escape antiproliferative controls and embark on a pathway toward neoplasia.
Asunto(s)
Apoptosis/fisiología , Colon/fisiología , Proteínas de Unión al ADN/fisiología , Inhibidores de Crecimiento/fisiología , Mucosa Intestinal/fisiología , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , División Celular/fisiología , Línea Celular , Proteínas de Unión al ADN/deficiencia , Ratones , Ratones Transgénicos , Mutación , Regiones Promotoras Genéticas/fisiología , Proteína smad3 , Transactivadores/deficienciaRESUMEN
A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.
Asunto(s)
Metaloendopeptidasas/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animales , Línea Celular , Femenino , Metaloproteasas/fisiología , Ratones , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in both normal pancreatic islets and in pancreatic cancers, but its role in pancreatic physiology and disease is not known. This report examines the effects of HB-EGF overexpression in mouse pancreas. METHODS: Transgenic mice were established using a tissue-specific promoter to express an HB-EGF complementary DNA in pancreatic beta cells, effectively elevating HB-EGF protein 3-fold over endogenous levels. RESULTS: Mice overexpressing HB-EGF in pancreatic islets showed both endocrine and exocrine pancreatic defects. Initially, islets from transgenic mice failed to segregate alpha, beta, delta, and PP cells appropriately within islets, and had impaired separation from ducts and acini. Increased stroma was detected within transgenic islets, expanding with age to cause fibrosis of both endocrine and exocrine compartments. In addition to these structural abnormalities, subsets of transgenic mice developed profound hyperglycemia and/or proliferation of metaplastic ductal epithelium. Both conditions were associated with severe stromal expansion, suggesting a role for islet/stromal interaction in the onset of the pancreatic disease initiated by HB-EGF. Supporting this conclusion, primary mouse fibroblasts adhered to transgenic islets when the 2 tissues were cocultured in vitro, but did not interact with nontransgenic islets. CONCLUSIONS: An elevation in HB-EGF protein in pancreatic islets led to altered interactions among islet cells and among islets, stromal tissues, and ductal epithelium. Many of the observed phenotypes appeared to involve altered cell adhesion. These data support a role for islet factors in the development of both endocrine and exocrine disease.