RESUMO
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.
Assuntos
Carcinoma Ductal Pancreático/imunologia , Macrófagos/fisiologia , Pâncreas/patologia , Neoplasias Pancreáticas/imunologia , Células Estreladas do Pâncreas/fisiologia , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Metaplasia , Camundongos , Neoplasias Pancreáticas/patologia , Receptor Cross-TalkRESUMO
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.
Assuntos
Celulas Principais Gástricas/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Peptídeos/metabolismo , Estômago/patologia , Animais , Biomarcadores/metabolismo , Células Cultivadas , Mucosa Gástrica/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Metaplasia/diagnóstico , Camundongos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pepsinogênio C/genética , Pepsinogênio C/metabolismo , Peptídeos/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Complexos Ubiquitina-Proteína Ligase , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Transformação Celular Neoplásica , Colo/patologia , Neoplasias do Colo/genética , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante/genética , Animais , Colo/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismoRESUMO
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.
Assuntos
Claudina-2/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/fisiologia , Miofibroblastos/fisiologia , Junções Íntimas/fisiologia , Anfirregulina , Células CACO-2 , Comunicação Celular , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proliferação de Células , Meios de Cultivo Condicionados , Família de Proteínas EGF , Impedância Elétrica , Receptores ErbB/metabolismo , Humanos , Mucosa Intestinal/metabolismo , LigantesRESUMO
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.
Assuntos
Biomarcadores Tumorais/análise , Lasers , Microdissecção/métodos , Proteínas de Neoplasias/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adenoma/metabolismo , Adenoma/patologia , Animais , Calgranulina A/metabolismo , Estudos de Casos e Controles , Humanos , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Camundongos , Proteoma/análise , Proteômica , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Antígenos Virais de Tumores/fisiologia , Transformação Celular Viral/genética , Fatores de Transcrição E2F/metabolismo , Enterócitos/metabolismo , Regulação da Expressão Gênica , Proteína do Retinoblastoma/metabolismo , Vírus 40 dos Símios/fisiologia , Animais , Enterócitos/virologia , Perfilação da Expressão Gênica , Hiperplasia/etiologia , Intestino Delgado/patologia , Camundongos , Camundongos Transgênicos , Proteína Supressora de Tumor p53/genéticaRESUMO
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.
Assuntos
Proliferação de Células , Neoplasias Colorretais/patologia , Genes APC , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Sequência de Bases , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Fosforilação , Reação em Cadeia da Polimerase , Transdução de SinaisRESUMO
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.
Assuntos
Receptores ErbB/metabolismo , Pâncreas/metabolismo , Pancreatopatias/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proliferação de Células , Quimiotaxia , Modelos Animais de Doenças , Receptores ErbB/genética , Fibrose , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Pâncreas/patologia , Pancreatopatias/genética , Pancreatopatias/patologia , Pancreatopatias/prevenção & controle , Proteínas Recombinantes/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
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.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Fator de Transcrição E2F2/metabolismo , Gastroenteropatias/virologia , Hiperplasia/virologia , Vírus 40 dos Símios/patogenicidade , Animais , Fator de Transcrição E2F2/deficiência , Fator de Transcrição E2F4/metabolismo , Enterócitos/química , Enterócitos/virologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína p130 Retinoblastoma-Like/metabolismoRESUMO
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.
Assuntos
Linhagem da Célula/fisiologia , Intestino Delgado/citologia , Fosfoproteínas/biossíntese , Proteínas Repressoras/biossíntese , Transcrição Gênica , Animais , Linhagem Celular , Chlorocebus aethiops , Células Enteroendócrinas/citologia , Células Caliciformes/citologia , Histona Desacetilases/metabolismo , Humanos , Intestino Delgado/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Correpressor 1 de Receptor Nuclear , Celulas de Paneth/citologia , Fosfoproteínas/genética , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
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.
Assuntos
Técnicas de Cultura de Células , Neoplasias Colorretais , Receptores Frizzled/metabolismo , Organoides/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Neoplasias Colorretais/patologia , Neoplasias Colorretais/fisiopatologia , Receptores Frizzled/genética , Técnicas de Transferência de Genes , Humanos , Morfogênese , Organoides/citologia , Retroviridae/genética , Retroviridae/metabolismo , Proteínas Wnt/genéticaRESUMO
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.
Assuntos
Diferenciação Celular , Polaridade Celular , Colo/enzimologia , Células Epiteliais/enzimologia , Mucosa Intestinal/patologia , Quinases da Família src/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Proliferação de Células , Forma Celular , Cromogranina A/metabolismo , Colo/citologia , Impedância Elétrica , Mucosa Intestinal/citologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Mucinas/metabolismo , Ocludina , Organoides , Fenótipo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases , Junções Íntimas/metabolismo , Fatores de Tempo , Proteína da Zônula de Oclusão-1 , Quinases da Família src/deficiência , Quinases da Família src/genéticaRESUMO
The mechanism whereby cyclooxygenase-2 and its prostaglandin (PG) products are involved in colonic carcinogenesis is not fully understood. Prostacyclin (PGI(2)) is a major PG with antiapoptotic activity and is produced in the gastrointestinal tract. We reported previously that a human colorectal cancer (CRC) cell line, HCA-7, produces significant levels of PGE(2), PGD(2), thromboxane, and PGF(2alpha), but not PGI(2). We now report that human colonic fibroblast cell lines produce significant amounts of PGI(2) and that fibroblast lines derived from normal-appearing colonic mucosa of hereditary nonpolyposis CRC individuals produce 50-fold more PGI(2) than normal fibroblast lines derived from individuals with nonhereditary CRC. Coculture of HCA-7 cells with hereditary nonpolyposis CRC fibroblasts, but not normal fibroblasts, markedly reduced butyrate-induced apoptosis of HCA-7 cells. This antiapoptotic effect was inhibited by the cyclooxygenase-2 inhibitor rofecoxib and was restored by the stable PGI(2) analogue carbaprostacyclin. PGI(2) binds either G protein-coupled cell surface PGI(2) receptor or the nuclear peroxisome proliferator-activated receptor (PPAR) delta. PPAR delta likely mediates this antiapoptotic effect because HCA-7 cells express this receptor, and another PPAR delta agonist, docosahexaenoic acid, mimics the effect. We propose a novel mechanism by which stromal production of PGI(2) promotes survival of colonocytes through PPAR delta activation. This mechanism may have relevance to maintenance of cells in the normal crypt and to clonal expansion of mutant colonocytes during tumorigenesis.
Assuntos
Apoptose/fisiologia , Colo/citologia , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Epoprostenol/biossíntese , Técnicas de Cocultura , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Células Epiteliais/metabolismo , Epoprostenol/análogos & derivados , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Lactonas/farmacologia , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/metabolismo , Células Estromais/metabolismo , Sulfonas , Células Tumorais CultivadasRESUMO
The activin signaling pathway parallels the transforming growth factor (TGF)-beta pathway. Both use extracellular ligands and cell surface receptors that are structurally and functionally related, as well as the same intracellular mediators (SMADs 2-4) to transmit these signals. Members of both pathways have been characterized previously as tumor suppressor genes on the demonstration of inactivating mutations in human neoplasms, e.g., genetic inactivation of the activin type I receptor was reported recently in pancreatic cancer. Here, we present evidence of selection for mutations of the activin A type II receptor (ACVR2) gene during human gastrointestinal carcinogenesis. Two 8-bp polyadenine tracts of the ACVR2 gene are targets for inactivating frameshift mutations in gastrointestinal neoplasms having microsatellite instability (MSI). These mutations are similar to those of the 10-bp polyadenine tract within the TGF-beta type II receptor (TGFBR2), a well-characterized target of frameshift mutations in the same neoplasms. We identified biallelic mutations of ACVR2 in 25 of 28 MSI colorectal and pancreatic cancers. In addition, a mutation in the ACVR2 gene combined with loss of the wild-type allele was found in a non-MSI pancreatic cancer. This evidence is compatible with a high degree of selection for inactivation of the ACVR2 gene in tumorigenesis, supporting ACVR2 as a candidate tumor suppressor gene in gastrointestinal cancers.
Assuntos
Receptores de Activinas Tipo II/genética , Neoplasias Colorretais/genética , Neoplasias Duodenais/genética , Neoplasias Pancreáticas/genética , Receptores de Activinas Tipo II/biossíntese , Neoplasias Colorretais/metabolismo , Neoplasias Duodenais/metabolismo , Mutação da Fase de Leitura , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/genética , Células Tumorais CultivadasRESUMO
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.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Mucosa Intestinal/citologia , Animais , Linhagem Celular Transformada , Camundongos , Camundongos TransgênicosRESUMO
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.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Vírus 40 dos Símios/fisiologia , Animais , Células Cultivadas , Enterócitos/virologia , Fibroblastos/virologia , Camundongos , Camundongos TransgênicosRESUMO
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.
Assuntos
Apoptose/fisiologia , Esôfago de Barrett/patologia , Refluxo Gastroesofágico/patologia , NF-kappa B/metabolismo , Apoptose/efeitos da radiação , Esôfago de Barrett/metabolismo , Linhagem Celular , Dano ao DNA , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Refluxo Gastroesofágico/metabolismo , Humanos , NF-kappa B/antagonistas & inibidores , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Raios UltravioletaRESUMO
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.
Assuntos
Movimento Celular , Colo/metabolismo , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Neuropeptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Cicatrização , Proteínas rac de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/enzimologia , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/enzimologia , Receptores ErbB/deficiência , Receptores ErbB/genética , Camundongos , Camundongos Knockout , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/genética , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Pseudópodes/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transfecção , Cicatrização/efeitos dos fármacos , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP , Quinases da Família src/antagonistas & inibidoresRESUMO
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.
Assuntos
Colo/crescimento & desenvolvimento , Desenvolvimento Embrionário , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Animais , Animais não Endogâmicos , Análise por Conglomerados , Colo/embriologia , Colo/ultraestrutura , DNA Complementar , Epitélio/metabolismo , Feminino , Imuno-Histoquímica , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
BACKGROUND & AIMS: The surface epithelium of the colon is being replaced constantly with cells derived from the stem cells of the crypt. Although the location of the stem cells is known, there are no markers for these cells. This study tested the hypothesis that colonic stem cells might be isolated and cultured on the basis of specific integrin expression patterns in normal human colonic epithelium. METHODS: Integrin expression in normal human colonic mucosa was determined by using indirect immunofluorescence. Crypt cells were then isolated as single cells from normal colon tissues and the expression pattern of integrins was analyzed by flow cytometry. Based on the specific expression of integrin beta1 in colonic crypts, the cells were sorted by using a flow cytometer, and colony assays in soft agar were performed to evaluate the clonogenicity of the sorted cells. RESULTS: By immunofluorescence, the cells located in the lower one third of crypts expressed higher levels of beta1-integrin than the cells in the remainder of the crypt. When isolated crypt cells were stained with the beta1-integrin antibody and examined in a flow cytometer, there were 2 peaks of fluorescence. Sorting of crypt cells based on staining with anti-beta1 integrin antibody produced a cell population with a significantly enhanced ability to form colonies. CONCLUSIONS: beta1-integrin is a candidate surface marker for the proliferative zone of the human colonic crypt. Our in vitro culture system for the clonal growth of a single colonic crypt cell suspension could facilitate the identification of other candidate stem cell markers.