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1.
Am J Physiol Cell Physiol ; 279(4): C1100-6, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11003590

RESUMO

Uncoupling protein-2 (UCP-2) is a mitochondrial protein expressed in adipocytes and has recently been involved in the control of energy dissipation. Because obesity is characterized by an imbalance between energy intake and expenditure and by an enhanced adipocyte-derived secretion of tumor necrosis factor-alpha (TNF-alpha), we asked whether TNF-alpha could directly influence UCP-2 expression in adipocytes. Experiments performed in differentiated 3T3F442A preadipocytes showed that TNF-alpha (10 ng/ml) induced a reduction of UCP-2 trancripts, assessed by Northern blot analysis. A significant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF-alpha stimulation of the cells. The characterization of the mechanisms responsible for the TNF-alpha effect on UCP-2 expression demonstrates an involvement of the TNF-alpha-induced inducible (i) nitric oxide synthase (NOS) expression. Cell treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mmol/l) significantly diminished the TNF-alpha-mediated sustained downregulation of UCP-2 expression, whereas cell treatment with a nitric oxide (NO) donor (10(-3) mol/l S-nitroso-L-glutathione) mimicked the TNF-alpha effect on UCP-2 expression. Moreover, Western blot analysis clearly showed that TNF-alpha alone induces the expression of iNOS after 12-24 h treatment of differentiated 3T3F442A cells. These experiments demonstrate that TNF-alpha directly downregulates UCP-2 expression via NO-dependent pathways that involve the induction of iNOS expression.


Assuntos
Adipócitos/metabolismo , Regulação para Baixo/fisiologia , Glutationa/análogos & derivados , Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Óxido Nítrico/metabolismo , Proteínas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células 3T3 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Northern Blotting , Western Blotting , Diferenciação Celular , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glutationa/farmacologia , Canais Iônicos , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Compostos Nitrosos/farmacologia , Proteínas/genética , RNA Mensageiro/biossíntese , S-Nitrosoglutationa , Fator de Necrose Tumoral alfa/farmacologia , Proteína Desacopladora 2
2.
Anal Bioanal Chem ; 354(5-6): 615-9, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15067456

RESUMO

Human cell lines cultured under standardized conditions are found to react after a wide variety of aggressive but sub-lethal treatments by expressing stress proteins, mainly of the HSP68, HSP70 and HSP90 families. The stress reaction is a repair mechanism which rapidly takes place after minute damages caused to cell structures. It was therefore proposed to develop a sensitive biomarker to monitor environmental pollution, especially related to metals, based on the evaluation of stress protein production. This was achieved by computer-assisted densitometric analysis of autoradiographies of electrophoretically separated (35)S-labelled proteins. The results indicate that our biological models consisting in HT29 and HepG2 cell-lines react to low concentrations of cadmium or nickel by a clear-cut increase of stress proteins expression. In most cases, this effect is much more significant and much more rapid to observe than changes in growth curves. It may constitute a reliable index of cell susceptibility to environmental aggressions.

3.
Int J Biochem Cell Biol ; 27(4): 385-91, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7788560

RESUMO

The current study was undertaken to investigate the expression of stress proteins (HSP) in cultured human HT29 cells submitted to stressing events under in vitro conditions. Heat shocks (45 degrees C, for 15-60 min) or cold shocks (+ 1 degree C for 4 hr) were found to modify cell growth (growth curves) and to enhance HSP expression. In most cases, changes in HSP expression are much more pronounced than changes in cell growth. Exposure to 8% ethanol for 15 min resulted in both growth inhibition and HSP overexpression. Propanol-1 was found to be more toxic since 5% concentration given for 15 min stops cell growth. 2.5% propanol-1 for 15 min induces a slight reduction of cell growth but a clear-cut overexpression of stress proteins. We conclude that expression of stress proteins, especially those of the HSP68/70 family, constitutes a more sensitive response than changes in growth rate in case of external aggression. This could make our model an interesting biological sensor to environmental physical or chemical pollutants.


Assuntos
Proteínas de Choque Térmico/biossíntese , 1-Propanol/farmacologia , Temperatura Baixa , Meio Ambiente , Etanol/farmacologia , Temperatura Alta , Humanos , Modelos Biológicos , Células Tumorais Cultivadas
4.
Int J Biochem ; 24(8): 1315-21, 1992 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1644212

RESUMO

1. The current study was undertaken to test the existence of functionally distinct intracellular pools of cholesterol depending on the origin: neosynthesis or exogenous. 2. This was performed on two subpopulations, either differentiated or undifferentiated, of the HT29 cell line. 3. A parallel study was also carried out on Caco-2 cells. 4. First we checked the ability of differentiated HT29 cells to secrete lipids into the medium and found that lipid production was efficient but less so than in Caco-2 cells. 5. In contrast, undifferentiated HT29 cells were unable to secrete lipids into the medium. 6. Then we studied the fate of [14C]cholesterol incorporated into micellar preparations and of [14C]mevalonate in the different models. 7. The data obtained with labelled exogenous cholesterol show that it enters the membrane cholesterol pool as well as, for the differentiated models, the cholesteryl ester pool. 8. Similarly, labelled newly synthesized cholesterol could be used for membrane formation as well as for incorporation into cholesteryl esters. 9. Thus, in HT29 subpopulations as well as in Caco-2 cells, the results suggest the existence of a common pool of cholesterol whatever its origin.


Assuntos
Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Diferenciação Celular , Colesterol/biossíntese , Ésteres do Colesterol/biossíntese , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Intestinos/efeitos dos fármacos , Cinética , Micelas , Nocodazol/farmacologia , Triglicerídeos/biossíntese , Células Tumorais Cultivadas
5.
Biochim Biophys Acta ; 1085(2): 265-71, 1991 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-1892896

RESUMO

The ACAT activity was studied on different subpopulations deriving from HT29 cells, a human colon carcinoma cell line. Grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G + cells). From this heterogeneous population, differentiated cells were selected by glucose deprivation and grown either on medium without glucose (G - cells) or in standard medium containing 25 mM glucose (G-Rev cells). The G- and G-Rev cells have the features of differentiated small intestine cells. The two types of differentiated cells (G- and G-Rev) exhibited similar ACAT activities and the kinetic characteristics of the enzyme were also similar. A time-course study showed increasing activity during the exponential phase and a decrease just after confluency. It was possible to stimulate the enzyme by micellar or lipoprotein cholesterol. In contrast, the ACAT activity was hardly detectable in undifferentiated G + cells. In addition, all the experimental conditions known to stimulate ACAT activity, and confirmed in the differentiated HT29 cells, were inefficient in the undifferentiated G + cells. Therefore, the different models derived from HT29 cells provide the opportunity to study cholesterol esterification as well as the consequences of its aberrances in intestinal cells.


Assuntos
Colesterol/metabolismo , Colo/metabolismo , Esterol O-Aciltransferase/metabolismo , Adenocarcinoma , Diferenciação Celular , Membrana Celular/metabolismo , Ésteres do Colesterol/metabolismo , Colo/citologia , Humanos , Cinética , Metabolismo dos Lipídeos , Lipoproteínas LDL/metabolismo , Células Tumorais Cultivadas
6.
Int J Cancer ; 47(4): 633-8, 1991 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-1847359

RESUMO

Involvement of ornithine decarboxylase (ODC) in proliferation of the HT29 cell line and its control by either fetal calf serum (FCS) or vasoactive intestinal peptide (VIP) as an external signal increasing cAMP level were investigated. Activation of the polyamine-producing system appears to be a necessary step in the proliferative response of HT29 cells since cell growth is arrested by addition of difluoromethylornithine (DFMO, an inhibitor of ODC), then restored by further addition of putrescine into the culture medium. FCS deprivation results in decreased activity of ODC and arrest of cell growth. Addition of FCS induces reactivation of ODC peaking at 9 hr and re-initiates proliferation but does not affect cAMP level. VIP strongly and rapidly stimulated cAMP accumulation, which resulted in significant activation of ODC. When VIP-induced cAMP formation was hindered by the alpha 2-adrenergic agonist UK14304, activation of ODC was no longer observed. The dose-response curve for ODC activation by VIP indicates an EC50 value of 0.078 nM which falls within the range of physiological concentrations for this peptide. However, VIP fails to stimulate proliferation when cells are cultured either in an FCS-free medium or in the presence of a growth-limiting concentration of FCS. We conclude that the mechanisms of ODC activation by either FCS or VIP are different, the latter involving cAMP formation. Activation of ODC to produce polyamines is necessary to support the proliferative process in our model but the VIP-induced activation of the enzyme alone is not sufficient to promote cell growth.


Assuntos
Neoplasias do Colo/patologia , Ornitina Descarboxilase/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/enzimologia , AMP Cíclico/análise , Eflornitina/farmacologia , Sangue Fetal/fisiologia , Humanos , Ornitina Descarboxilase/análise , Células Tumorais Cultivadas
8.
Int J Cancer ; 46(2): 320-5, 1990 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-2384278

RESUMO

The metabolism of human low-density lipoproteins was studied in 2 subpopulations deriving from cells of HT29, a human colon carcinoma cell line. When grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G+ cells). From this heterogeneous population, a subpopulation with features of differentiated small-intestinal cells was selected by glucose deprivation (G- cells). The characteristics of the LDL receptor were first investigated. The results showed that the binding of 125I-LDL to G+ and G- cells performed at 4 degrees C was saturable and specific. The Kd values were not statistically different in the 2 cell subpopulations. The Bmax of G+ cells was 55 +/- 6 ng 125I-LDL/mg cell protein and showed no changes whatever the phase of culture. In G- cells, the Bmax was higher during the exponential phase of culture and decreased in the post-confluent phase (82 +/- 5 versus 15 +/- 6.8 ng 125I-LDL/mg cell protein). Cellular degradation of 125I-LDL was effective in both cell subpopulations but time-course studies showed that, in post-confluent G- cells, degradation was slowed as compared to G+ cells (4 hr vs. 2 hr to reach maximal degradation). The rate of LDL processing at 37 degrees C was enhanced by pre-incubation with FCS-supplemented medium, suggesting the existence of a serum component which stimulates the total degradation of 125I-LDL. Concerning regulation of the LDL receptor activity, we demonstrated that pre-incubation of G+ cells with LDL induced 80% down-regulation of receptor number in both phases of culture. This was also observed in G- cells during the exponential phase while only a 20% decrease of the receptor number was observed in post-confluent G- cells. The LDL degradation of G+ cells resulted in an inhibition of the cholesterogenic activity by 30% and 60% depending on the phase of culture. In G- cells, LDL pre-incubation inhibited cholesterol synthesis to the same extent (45%) in the exponential phase but did not affect the rate of cholesterol synthesis when cells were confluent. The defective regulatory role of LDL on receptor number and cholesterol synthesis suggests that, in the post-confluent differentiated cells, cholesterol derived from LDL does not reach the regulatory pool. Taken together, our findings indicate the existence of functional LDL receptors in the HT29 cell line, either in the differentiated or in the undifferentiated form.


Assuntos
Adenocarcinoma/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/metabolismo , Lipoproteínas LDL/metabolismo , Adenocarcinoma/análise , Linhagem Celular , Transformação Celular Neoplásica/análise , Colesterol/análise , Colesterol/biossíntese , Neoplasias do Colo/análise , Humanos , Radioisótopos do Iodo , Lipoproteínas LDL/análise , Ligação Proteica , Ensaio Radioligante , Receptores de LDL/análise , Receptores de LDL/metabolismo , Células Tumorais Cultivadas/análise , Células Tumorais Cultivadas/metabolismo
9.
Biochem J ; 261(1): 175-9, 1989 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-2505753

RESUMO

Effects of insulin on key steps of carbohydrate metabolism were investigated in cultured HT29 colon cancer cells by two different approaches, i.e. incubation of the cells either in the absence or in the presence of glucose in the medium. In glucose-deprived cells, insulin decreased glycogen breakdown, but did not affect polysaccharide levels when glucose was present. Glycogen synthase became activated after insulin treatment in both conditions, even though the activation was more evident when glucose was omitted. No effect on glycogen phosphorylase activity was evident under our experimental conditions. In cells incubated with glucose, the hormone stimulated in a dose-dependent manner the rates of glucose uptake and lactate release. Concomitantly with the increase in glycolytic rate, insulin caused a strong increase in fructose 2,6-bisphosphate. This effect was not observed in the absence of glucose. It is concluded that the carbohydrate metabolism of cultured HT29 cells responds to insulin, making this biological model suitable for investigations in vitro on the mechanism of insulin action.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Glicogênio/metabolismo , Glicólise , Insulina/farmacologia , Frutosedifosfatos/metabolismo , Glucose/metabolismo , Glicogênio Sintase/metabolismo , Humanos , Lactatos/metabolismo , Fosforilases/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo
10.
Carcinogenesis ; 8(1): 155-61, 1987 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2879649

RESUMO

The sequential histochemical changes during colon carcinogenesis were studied in male Sprague-Dawley rats given 16 weekly subcutaneous injections of 15 mg 1,2-dimethylhydrazine per kg body wt and serially killed at regular intervals. Cryostat sections were used to study the mucus content of the colonic mucosa with the periodic acid Schiff's reaction, and enzyme histochemical methods were applied to investigate the activity of some key enzymes of carbohydrate metabolism at different stages of carcinogenesis. Enlarged mucus-rich crypts with a marked hypercellularity (149% of control as determined morphometrically) appearing very early during carcinogenic treatment revealed almost normal activities of glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase (G6PDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Hyperbasophilic crypts lacking mucus production were observed later and showed a loss of G6Pase, but marked increase of G6PDH and GAPDH activity. Mucus-rich signet ring cell carcinomas showed the same enzymatic pattern as the mucus-rich crypts, whereas mucus-free adenocarcinomas and undifferentiated carcinomas revealed a loss of G6Pase and highly increased G6PDH and GAPDH activities. The results showed that focal changes in polysaccharide content and in the activity of some enzymes of carbohydrate metabolism, as observed in various organs, also accompany the carcinogenic process in the colon. This supports the concept that aberrations in carbohydrate metabolism play an important role during the process of carcinogenesis.


Assuntos
Neoplasias do Colo/induzido quimicamente , Dimetilidrazinas , Metilidrazinas , Lesões Pré-Cancerosas/induzido quimicamente , 1,2-Dimetilidrazina , Fosfatase Ácida/metabolismo , Animais , Neoplasias do Colo/enzimologia , Gluconeogênese , Glucose-6-Fosfatase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicogênio Sintase/metabolismo , Glicólise , Histocitoquímica , Masculino , Via de Pentose Fosfato , Fosforilases/metabolismo , Lesões Pré-Cancerosas/enzimologia , Ratos , Ratos Endogâmicos , gama-Glutamiltransferase/metabolismo
11.
Int J Biochem ; 18(3): 263-9, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-2869987

RESUMO

Effect of glutamine deprivation (GLN- medium) and of its replacement by 4mM ammonium chloride (GLN-/NH4+ medium) or by 4mM glutamate (GLN-/Gt+ medium) was studied on growth rate, morphology and metabolism of HT29 human colon cancer cells. Growth rates were modified as follows: at the first passage, growth of GLN- cells was strongly decreased (doubling time: 192 hr vs 32 hr in control cells grown in GLN+ medium); GLN-/NH4+ cells and GLN-/Gt+ cells were found to have doubling times of 72 and 70 hr, respectively. At the 8th passage, doubling times were decreased in all cases, being: 144 hr for GLN- cells, 60 hr for GLN-/NH4+ cells and 24 hr for GLN-/Gt+ cells, which indicates a capacity of adaptation of the cell-line to new culture conditions. GLN- cells and GLN-/NH4+ cells were found to exhibit an enterocytic type of differentiation (polarization of the cell layer with apical and cystic brush border and tight junctions); GLN-/Gt+ cells remained undifferentiated and comparable to control GLN+ cells. Glycogen level varied according to the phases of the culture, with a trend to lower level in glutamine deprived cells; glucose uptake and lactate production varied as a function of the medium composition and of the phases of the culture. At the 8th passage, all the glutamine deprived cells produced less lactate than control; GLN-/Gt+ cells were found to utilize less glucose than others.


Assuntos
Cloreto de Amônio/farmacologia , Glutamatos/farmacologia , Glutamina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/ultraestrutura , Glucose/metabolismo , Ácido Glutâmico , Glicogênio/metabolismo , Humanos , Lactatos/metabolismo , Microscopia Eletrônica
12.
Int J Biochem ; 18(12): 1123-8, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-3817273

RESUMO

Under the standard conditions of culture, Fru-2,6-P2 level in HT29 cells is transitorily increased as a consequence of medium change; the peak value occurs after 2 hr, followed by a gradual return to a basal value (40 pmol/mg protein) which is maintained as long as medium glucose concentration stands above 2 mM. A 20 hr glucose deprivation lowers Fru-2,6-P2 level to trace value, but, when glucose is reintroduced, the peak value is much higher; large Fru-2,6-P2 accumulation is correlated with higher rates of glucose uptake and lactate release, which suggests an activation of glycolysis at the level of phosphofructokinase-1. Fru-2,6-P2 level depends on the glucose concentration within the range of 0 to 5 mM. At this concentration and above, maximal effect is reached. Previous glucose deprivation renders the Fru-2,6-P2 forming system more sensitive to glucose. When given instead of glucose, fructose enters the glycolytic pathway and produces same effect as glucose on the Fru-2,6-P2 level. Galactose turns it to almost zero which coincides with low glycolytic rate. Acidity of the culture medium favorishes the Fru-2,6-P2 formation; however, change in pH cannot explain the variations of Fru-2,6-P2 level observed under the standard culture conditions. Lactate concentrations over 10 mM in the medium are found to significantly inhibit the Fru-2,6-P2 producing system. Therefore, lactate accumulation in the medium could be an important factor controlling Fru-2,6-P2 level during standard cell culture.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Frutosedifosfatos/metabolismo , Glucose/metabolismo , Hexosedifosfatos/metabolismo , Lactatos/metabolismo , Linhagem Celular , Humanos , Cinética
13.
Int J Biochem ; 17(2): 247-51, 1985.
Artigo em Inglês | MEDLINE | ID: mdl-2989018

RESUMO

Some aspects of carbohydrate metabolism were investigated in three non-malignant, glycogen storing, cell lines derived from a primary culture of rat hepatocytes, and in the Morris hepatoma 3924 cells. The three cell lines show biochemical alterations which are, to a large extent, similar to those found in the hepatoma cells: increased activity of glycolytic enzymes and decreased activity of gluconeogenetic enzymes. An increase of glucose-6-phosphate dehydrogenase activity is also found. The three cell lines, as the Morris hepatoma cells, actively convert glucose into lactate under the in vitro conditions of culture. Fructose is not taken up as quickly as glucose and galactose is not metabolized. As compared with normal hepatocytes, the three cell lines have altered metabolism and growth behaviour. They largely resemble the preneoplastic cells appearing in rat liver at the early stages of experimental carcinogenesis.


Assuntos
Metabolismo dos Carboidratos , Glicogênio Hepático/metabolismo , Fígado/metabolismo , Animais , Linhagem Celular , Células Clonais , Frutose-Bifosfatase/metabolismo , Glucose-6-Fosfatase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glicogênio Sintase/metabolismo , Glicólise , Hexoquinase/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactatos/metabolismo , Masculino , Fosfofrutoquinase-1/metabolismo , Fosforilases/metabolismo , Piruvato Quinase/metabolismo , Ratos , Ratos Endogâmicos
14.
Biochem Biophys Res Commun ; 111(3): 958-63, 1983 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-6301493

RESUMO

It is known that the human exocrine pancreas responds to secretin stimulation more than does VIP, a structurally related peptide. We looked for the receptors for those polypeptides in a human pancreatic cancer cell line grown in culture and in nude mice. By analysing the cAMP responses and the 125I-VIP binding we found VIP receptors with a KD of 1.5 10(-9) M. Secretin stimulates the adenylate cyclase through the VIP receptor sites with a KD of 1.7. 10(-6) M. We noted also that during cell proliferation in culture there was about a 5 fold increase of the cAMP response to VIP.


Assuntos
Adenocarcinoma/metabolismo , AMP Cíclico/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Superfície Celular/isolamento & purificação , Animais , Divisão Celular , Linhagem Celular , Humanos , Camundongos , Camundongos Nus , Receptores de Peptídeo Intestinal Vasoativo , Secretina/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia
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