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1.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934845

RESUMO

Commensal microbiota contribute to gut homeostasis by inducing transcription of mucosal genes. Analysis of the impact of various microbiota on intestinal tissue provides an important insight into the function of this organ. We used cDNA microarrays to determine the gene expression signature of mucosa isolated from the small intestine and colon of germ-free (GF) mice and animals monoassociated with two E. coli strains. The results were compared to the expression data obtained in conventionally reared (CR) mice. In addition, we analyzed gene expression in colon organoids derived from CR, GF, and monoassociated animals. The analysis revealed that the complete absence of intestinal microbiota mainly affected the mucosal immune system, which was not restored upon monoassociation. The most important expression changes observed in the colon mucosa indicated alterations in adipose tissue and lipid metabolism. In the comparison of differentially expressed genes in the mucosa or organoids obtained from GF and CR mice, only six genes were common for both types of samples. The results show that the increased expression of the angiopoietin-like 4 (Angptl4) gene encoding a secreted regulator of lipid metabolism indicates the GF status.


Assuntos
Perfilação da Expressão Gênica , Vida Livre de Germes/genética , Mucosa Intestinal/metabolismo , Organoides/metabolismo , Animais , Biomarcadores/metabolismo , Colo/metabolismo , Escherichia coli/fisiologia , Regulação da Expressão Gênica , Sistema Imunitário/metabolismo , Imunidade nas Mucosas , Camundongos Endogâmicos BALB C , Microbiota
2.
Genesis ; 54(3): 101-14, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26864984

RESUMO

The Wnt pathway plays a crucial role in self-renewal and differentiation of cells in the adult gut. In the present study, we revealed the functional consequences of inhibition of canonical Wnt signaling in the intestinal epithelium. The study was based on generation of a novel transgenic mouse strain enabling inducible expression of an N-terminally truncated variant of nuclear Wnt effector T cell factor 4 (TCF4). The TCF4 variant acting as a dominant negative (dn) version of wild-type (wt) TCF4 protein decreased transcription of ß-catenin-TCF4-responsive genes. Interestingly, suppression of Wnt/ß-catenin signaling affected asymmetric division of intestinal stem cells (ISCs) rather than proliferation. ISCs expressing the transgene underwent several rounds of division but lost their clonogenic potential and migrated out of the crypt. Expression profiling of crypt cells revealed that besides ISC-specific markers, the dnTCF4 production downregulated expression levels of epithelial genes produced in other crypt cells including markers of Paneth cells. Additionally, in Apc conditional knockout mice, dnTCF activation efficiently suppressed growth of Apc-deficient tumors. In summary, the generated mouse strain represents a convenient tool to study cell-autonomous inhibition of ß-catenin-Tcf-mediated transcription.


Assuntos
Mucosa Intestinal/citologia , Intestino Delgado/citologia , Células-Tronco/citologia , Via de Sinalização Wnt , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diferenciação Celular , Divisão Celular , Proliferação de Células , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco/metabolismo , Fator de Transcrição 4 , Transcrição Gênica , beta Catenina/metabolismo
3.
Gastroenterology ; 144(2): 381-391, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23142137

RESUMO

BACKGROUND & AIMS: The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice. METHODS: We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc(+/Min) and Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization. RESULTS: We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer-derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist. CONCLUSIONS: TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.


Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Cancers (Basel) ; 14(17)2022 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-36077674

RESUMO

Trophoblastic cell surface antigen 2 (TROP2) is a membrane glycoprotein overexpressed in many solid tumors with a poor prognosis, including intestinal neoplasms. In our study, we show that TROP2 is expressed in preneoplastic lesions, and its expression is maintained in most colorectal cancers (CRC). High TROP2 positivity correlated with lymph node metastases and poor tumor differentiation and was a negative prognostic factor. To investigate the role of TROP2 in intestinal tumors, we analyzed two mouse models with conditional disruption of the adenomatous polyposis coli (Apc) tumor-suppressor gene, human adenocarcinoma samples, patient-derived organoids, and TROP2-deficient tumor cells. We found that Trop2 is produced early after Apc inactivation and its expression is associated with the transcription of genes involved in epithelial-mesenchymal transition, the regulation of migration, invasiveness, and extracellular matrix remodeling. A functionally similar group of genes was also enriched in TROP2-positive cells from human CRC samples. To decipher the driving mechanism of TROP2 expression, we analyzed its promoter. In human cells, this promoter was activated by ß-catenin and additionally by the Yes1-associated transcriptional regulator (YAP). The regulation of TROP2 expression by active YAP was verified by YAP knockdown in CRC cells. Our results suggest a possible link between aberrantly activated Wnt/ß-catenin signaling, YAP, and TROP2 expression.

5.
Genesis ; 49(3): 142-51, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21309068

RESUMO

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located on chromosome 17p13.3, a region frequently hypermethylated or deleted in human neoplasias. In mouse, Hic1 is essential for embryonic development and exerts an antitumor role in adult animals. Since Hic1-deficient mice die perinatally, we generated a conditional Hic1 null allele by flanking the Hic1-coding region by loxP sites. When crossed to animals expressing Cre recombinase in a cell-specific manner, the Hic1 conditional mice will provide new insights into the function of Hic1 in developing and mature tissues. Additionally, we used gene targeting to replace sequence-encoding amino acids 186-893 of Hic1 by citrine fluorescent protein cDNA. We demonstrate that the distribution of Hic1-citrine fusion polypeptide corresponds to the expression pattern of wild-type Hic1. Consequently, Hic1-citrine "reporter" mice can be used to monitor the activity of the Hic1 locus using citrine fluorescence.


Assuntos
Alelos , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Fatores de Transcrição Kruppel-Like/genética , Animais , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/metabolismo , Feminino , Deleção de Genes , Marcação de Genes , Genes Reporter , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/genética
6.
Sci Rep ; 9(1): 1629, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733598

RESUMO

The first step in the development of human colorectal cancer is aberrant activation of the Wnt signaling pathway. Wnt signaling hyperactivation is predominantly caused by loss-of-function mutations in the adenomatous polyposis coli (APC) gene that encodes the pathway negative regulator. In order to identify genes affected by the Apc loss, we performed expression profiling of intestinal epithelium isolated from mice harboring a conditional Apc allele. The gene encoding transcriptional factor msh homeobox 1 (Msx1) displayed robust upregulation upon Apc inactivation. Histological analysis of the Apc-deficient epithelium revealed that in the small intestine, the Msx1 protein was localized exclusively in ectopic crypts, i.e., in pockets of proliferating cells abnormally positioned on the villi. Ablation of the Msx1 gene leads to the disappearance of ectopic crypts and loss of differentiated cells. Moreover, tumors arising from Msx1-deficient cells display altered morphology reminiscent of villous adenomas. In human tumor specimens, MSX1 displayed significantly increased expression in colonic neoplasia with a descending tendency during the lesion progression towards colorectal carcinoma. In summary, the results indicate that Msx1 represents a novel marker of intestinal tumorigenesis. In addition, we described the previously unknown relationship between the Msx1-dependent formation of ectopic crypts and cell differentiation.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Diferenciação Celular , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Camundongos Knockout , Via de Sinalização Wnt , beta Catenina/metabolismo
7.
Sci Rep ; 9(1): 20188, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31874996

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

8.
Genes (Basel) ; 9(9)2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30200414

RESUMO

T-cell factor 4 (TCF4), together with ß-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

9.
Eur J Pharmacol ; 559(2-3): 109-14, 2007 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-17292884

RESUMO

CART (cocaine- and amphetamine-regulated transcript) peptides have been studied for ten years. We report specific binding of 125I-CART(61-102) to the rat adrenal pheochromocytoma PC12 cell line, both intact cells and cell membranes. Saturation binding to intact plated cells resulted in Kd of 0.48+/-0.16 nM and Bmax of 2228+/-529 binding sites/cell. 125I-CART(61-102) was also bound to PC12 cells differentiated using nerve growth factor to the neuronal phenotype with non-specific binding below 20%, and Kd of 1.90+/-0.27 nM and Bmax of 11,194+/-261 binding sites/cell. In competitive binding experiments, CART(61-102), CART(55-102) and di-iodinated CART(61-102) were bound to PC12 cell membranes with Ki in low nM range; their affinity to intact non-differentiated and differentiated cells was in low 10(-8) M range. In order to prove that iodination did not eliminate the pharmacological properties of CART, we tested the biological activity of di-iodinated CART(61-102). It decreased food intake in in vivo feeding experiment on fasted mice in a dose of 1 microg/mouse to the same extent as CART(61-102) in a dose of 0.5 microg/mouse.


Assuntos
Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neoplasias das Glândulas Suprarrenais , Animais , Ligação Competitiva , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Isótopos de Iodo/metabolismo , Radioisótopos do Iodo/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neurônios/patologia , Células PC12 , Fragmentos de Peptídeos/metabolismo , Fenótipo , Feocromocitoma , Ligação Proteica , Ratos , Fatores de Tempo
10.
Int J Oncol ; 28(2): 559-65, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16391813

RESUMO

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.


Assuntos
Butiratos/farmacologia , Células HT29/patologia , Adenocarcinoma , Fosfatase Alcalina/metabolismo , Antígenos CD/metabolismo , Apoptose , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Clonais , Neoplasias Colorretais , Células HT29/efeitos dos fármacos , Células HT29/ultraestrutura , Humanos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Int J Mol Med ; 17(1): 69-75, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16328013

RESUMO

beta-catenin has a dual function; it is implicated in intercellular junctions and transcriptional co-activation. Here we examined the regulation of the expression and localization of beta-catenin in HT29 colorectal adenocarcinoma cells. Our results showed that inhibition of PI-3 kinase with wortmannin was accompanied by a considerably reduced expression of beta-catenin. This effect was overcome by butyrate and occurred at the protein level, not at the level of mRNA. Moreover, NaBT significantly increased the phosphorylation of the ribosomal protein, S6, known to participate in the translational control of gene expression. This was accompanied by the increased phosphorylation of p70 S6K and MAPKs, the effector proteins that are upstream of protein S6 in the distinct signaling pathways. These facts indicate that different signaling pathways may be involved in the regulation of beta-catenin synthesis. Modulation of beta-catenin expression induced by NaBT appeared to occur at the level of protein translation, suggesting that NaBT may act as a translational regulator.


Assuntos
Adenocarcinoma/metabolismo , Butiratos/metabolismo , Neoplasias Colorretais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , beta Catenina/metabolismo , Fosfatase Alcalina/metabolismo , Androstadienos/metabolismo , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina/metabolismo , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Wortmanina , beta Catenina/genética
12.
Transl Oncol ; 9(2): 99-107, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27084425

RESUMO

Neoplastic growth is frequently associated with genomic DNA methylation that causes transcriptional silencing of tumor suppressor genes. We used a collection of colorectal polyps and carcinomas in combination with bioinformatics analysis of large datasets to study the expression and methylation of Hypermethylated in cancer 1 (HIC1), a tumor suppressor gene inactivated in many neoplasms. In premalignant stages, HIC1 expression was decreased, and the decrease was linked to methylation of a specific region in the HIC1 locus. However, in carcinomas, the HIC1 expression was variable and, in some specimens, comparable to healthy tissue. Importantly, high HIC1 production distinguished a specific type of chemotherapy-responsive tumors.

13.
Int J Oncol ; 26(3): 793-9, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15703838

RESUMO

Sodium butyrate or glucose deprivation induce a more differentiated phenotype in many cancer cells. The aim of this study was to determine whether the induction effect of butyrate and/or glucose deprivation is dependent, in some way, on the differentiation state of individual cell lines. Sodium butyrate enhanced alkaline phosphatase activity and induced formation of an ultrastructurally more differentiated phenotype in both HT29 and HT115 cell lines. Interestingly, the more invasive HT115 cells responded more strongly to butyrate treatment. On the other hand, the differentiation effect of glucose deprivation was much less prominent in the HT115 cell line in comparison with HT29 cells. Our data confirm the influence of the malignant potential of the cells on their response to treatment with differentiation and apoptosis-inducing agents. Butyrate treatment also enhanced the adhesiveness of HT115 cells. Since E-cadherin was not found in these cells, while the level of CEACAM1 was increased, it is obvious that the CEACAM1 molecules are involved in HT115 cell-cell adhesion.


Assuntos
Adenocarcinoma/patologia , Butiratos/farmacologia , Neoplasias Colorretais/patologia , Glucose/metabolismo , Invasividade Neoplásica , Antígenos CD/farmacologia , Antígenos de Diferenciação/farmacologia , Adesão Celular , Moléculas de Adesão Celular , Diferenciação Celular , Humanos , Isobutiratos , Células Tumorais Cultivadas
14.
Mol Cancer Res ; 13(7): 1139-48, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25934696

RESUMO

UNLABELLED: Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses "danger" signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells. IMPLICATIONS: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells.


Assuntos
Carcinogênese/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Azoximetano , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais , Técnicas de Silenciamento de Genes , Humanos , Intestinos/citologia , Camundongos , Camundongos Transgênicos , Regulação para Cima
15.
FEBS Lett ; 543(1-3): 81-6, 2003 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-12753910

RESUMO

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3'-kinase (PI-3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI-3K by wortmannin, phosphorylation of 70-kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non-transformed cells but not in RSV-transformed cells. Thus PI-3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v-src oncogene, suggesting that changes in the PI-3K signalling pathway upstream of p70 S6k are induced by RSV transformation.


Assuntos
Fator de Iniciação 2B em Eucariotos/fisiologia , Proteína Oncogênica pp60(v-src)/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Quinases S6 Ribossômicas 70-kDa/fisiologia , Transdução de Sinais , Androstadienos/farmacologia , Animais , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/patogenicidade , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Viral , Cricetinae , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Wortmanina
16.
Int J Oncol ; 25(5): 1459-64, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15492839

RESUMO

In epithelial cells, the cell surface glycoprotein E-cadherin is a key molecule in the establishment of cell-cell adhesion. In addition to its contribution to cell adhesion, E-cadherin was found to induce ligand-independent activation of the EGF receptor (EGFR), likely as a result of their co-clustering. As it has also been reported that ligand activation of the overexpressed EGFRs disturb E-cadherin-mediated cell-cell adhesion, we analyzed E-cadherin-EGFR interactions and their consequences in A431 cells and in two colorectal cancer cell lines using immunoblotting and analyzes of several protein kinase activities. Activation of the PI3-K/Akt/GSK-3 signaling pathway upon EGF treatment that we observed in the analyzed cells indicates that EGFRs are functional even in the colorectal cancer cells containing a low density of EGFRs. The transactivation of EGFR by E-cadherin did not occur either in the colorectal cancer cells tested or in A431 cells containing a high density of both EGFRs and E-cadherin on their surface. This observation suggests that high amounts of both molecules on the surface of tumour cells did not predetermine ligand-independent activation of EGFRs.


Assuntos
Caderinas/biossíntese , Carcinoma/genética , Carcinoma/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptores ErbB/biossíntese , Caderinas/farmacologia , Humanos , Immunoblotting , Ligantes , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas
17.
Int J Oncol ; 23(6): 1755-60, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14612951

RESUMO

Morphological and biochemical studies of HT29 cells treated with sodium butyrate and/or glucose-deprived revealed both apoptotic and differentiation response. The main apoptotic response was accompanied with an increase of floating cells. However, the ultrastructural analysis of adherent cells showed the typical apoptotic character of the nucleus in some of them. In addition, remarkable changes of mitochondria, assumed as early stages starting the apoptotic cascade, were observed. These changes were represented not only by alterations of mitochondrial morphology, but also by the number of mitochondria and their localization.


Assuntos
Apoptose , Mitocôndrias/fisiologia , Anestésicos Intravenosos/farmacologia , Western Blotting , Diferenciação Celular , Citoplasma/ultraestrutura , Glucose/metabolismo , Células HT29 , Humanos , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Oxibato de Sódio/farmacologia , Frações Subcelulares
18.
Int J Mol Med ; 13(4): 601-5, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15010863

RESUMO

Growth factors and hormones activate global and selective protein translation by phosphorylation and therefore activation of p70 S6 kinase through a wortmannin-sensitive phosphoinositide-3 kinase (PI-3K) antiapoptotic pathway and a rapamycin-sensitive signalling pathway of mTOR. Here we demonstrate that the phosphorylation of 40S ribosomal protein S6, a physiological substrate p70 S6 kinase, was highly increased by growth-stimulation of the cytolytic T cells (CTLL2) with interleukin 2 (IL2), which was accompanied with the increased phosphorylation of p70 S6K. The activity of p70 S6K and phosphorylation of the S6 protein was completely blocked by rapamycin and significantly decreased upon treatment of the cells with wortmannin, indicating an involvement of the PI-3K pathway in concert with the signalling pathway of mTOR in IL2-dependent phos-phorylation of ribosomal protein S6. The phosphorylation and activity of PKB/Akt in IL2-stimulated CTLL2 cells were rapamycin-insensitive and reduced upon wortmannin treatment of the cells, confirming a requirement for PI-3K for Akt activity. The data support the hypothesis that Akt may act downstream to PI-3K and upstream to mTOR in an IL2-mediated signal transduction pathway that controls phosphorylation of the regulatory protein S6 in CTLL2 cells.


Assuntos
Interleucina-2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Proteína S6 Ribossômica/química , Androstadienos/farmacologia , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Immunoblotting , Camundongos , Proteínas Proto-Oncogênicas c-akt , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Wortmanina
19.
Int J Mol Med ; 10(6): 779-84, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12430007

RESUMO

The high level of alkaline phosphatase activity in HT29 cells induced after 2 or 5 days of butyrate treatment was decreased during their prolonged exposure (about 30 days) to this agent together with a decrease of sensitivity to apoptosis. However, an enormous additive effect on alkaline phosphatase activity was found after butyrate treatment of glucose-starved cells. In concert with this finding, the substructural analysis revealed a dense brush border, tendency to polarization and morphologically normal mitochondria. It can be concluded that prolonged butyrate treatment of HT29 cells attenuated their response to this agent. On the other hand, glucose deprivation, as another inductor of differentiation, was found to increase the sensitivity of HT29 cells to butyrate.


Assuntos
Butiratos/metabolismo , Diferenciação Celular/fisiologia , Glucose/metabolismo , Células HT29/metabolismo , Células HT29/ultraestrutura , Humanos , Microscopia Eletrônica
20.
Mol Cancer Ther ; 13(4): 812-22, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24552772

RESUMO

The Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/ß-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with ß-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or ß-catenin-induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of ß-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Monensin/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Monensin/uso terapêutico , Neoplasias Experimentais , Ensaios Antitumorais Modelo de Xenoenxerto , Xenopus , Peixe-Zebra , beta Catenina/metabolismo
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