Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HT29 Cells”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 955 records · Page 53Linked to original sources
PubMed2005

In vitro and ex vivo intestinal tissue models to measure mucoadhesion of poly (methacrylate) and N-trimethylated chitosan polymers.

PURPOSE: The adhesion of a range of polymers based on poly(2-(dimethylamino-ethyl) methacrylate (pDMAEMA) was assessed using human mucus-secreting and non mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12) and HT29 monolayers, as well as excised non-everted intestinal sacs from rats. Differentiation of mucoadhesion from bioadhesion was achieved by pre-treatment with the mucolytic agent, N-acetyl cysteine (NAC). Adherence of pDMAEMA polymers was compared to that obtained with the mucoadhesive, N-trimethylated chitosan (TMC). METHODS: The quantity of adherent coumarin 343-conjugated polymers to HT29, E12, and intestinal sacs was measured by fluorescence. Confocal laser scanning microscopy (CLSM), light microscopy, and fluorescent microscopy were used to provide direct evidence. Measurements of transepithelial electrical resistance (TEER), permeability to FITC-dextran 4000 (FD-4), and the release of lactate dehydrogenase (LDH) were used to assess potential cytotoxicity of polymers. RESULTS: Adherence of unquaternized and of 10%, 24%, and 32% methyl iodide-quaternized pDMAEMA polymers was measured in E12, HT29, and sacs. All pDMAEMA polymers showed significantly higher levels of adhesion to mucus (mucoadhesion) than to epithelium (bioadhesion). Colocalization of pDMAEMA with mucus was confirmed in E12 by microscopy. TMC showed equally high levels of mucoadhesion as unquaternized and 24% quaternized pDMAEMA, but displayed higher levels of bioadhesion. pDMAEMA-based polymers demonstrated lower levels of adherence to E12 and rat sacs in the presence of NAC, whereas adherence of TMC was unchanged. pDMAEMA significantly decreased the permeability of FD-4 across E12 monolayers and sacs and was less cytotoxic in E12 than in HT29. In contrast, TMC increased the permeability of FD-4 across E12 and sacs and was less cytotoxic in E12 than in HT29. CONCLUSIONS: Human mucus-producing E12 monolayers can be used to assess polymer mucoadhesion and give similar data to isolated rat intestinal sacs. pDMAEMA displayed similar levels of mucoadhesion and lower levels of bioadhesion than a chitosan derivative and it was not cytotoxic. pDMAEMA decreased FD-4 flux in the presence of mucus, whereas TMC increased it. The combination of mucus and methacrylate polymers appears to increase barrier function of the apical membrane.

Animals↗
PubMed2004

Butyrate regulation of glycosylation-related gene expression: evidence for galectin-1 upregulation in human intestinal epithelial goblet cells.

Glycosylation of mucins produced by human intestinal goblet cells plays a crucial role in their functions: mucus gel physico-chemical protective properties, host-bacteria interactions, cell-cell adhesion, cell migration, and cell signaling. Colonic mucin glycosylation can be modified by luminal metabolites of fiber fermentation like butyrate. Our aim was to assess the effect of butyrate on the expression of a large panel of glycosylation-related genes in human intestinal epithelial goblet cells HT29-Cl.16E. We found that only a very scarce group of genes: 9 out of 252 were evidenced by microarray screening, and only three had their modulation significantly confirmed by real time PCR quantification. The most striking effect of butyrate was its 8- to 18-fold increase of galectin-1 gene expression, which was confirmed at the protein level, specifically with a central and apical intracellular localization. Significant butyrate effects will be discussed in regard to their possible link with mucins expressed by HT29-Cl.16E cells.

Butyrates↗
PubMed1998

System for dynamic measurements of membrane capacitance in intact epithelial monolayers.

Dynamic measurements of exocytosis have been difficult to perform in intact epithelial monolayers. We have designed a system that estimates with +/-1% accuracy (99% confidence) the total membrane capacitance of monolayers represented by a lumped model. This impedance measurement and analysis system operates through a conventional transepithelial electrophysiology clamp, performing all signal measurements as frequently as every 5 s. Total membrane capacitance (the series combination of apical and basolateral membranes) is the inverse of one of three unique coefficients that describe the monolayer impedance. These coefficients are estimated using a weighted, nonlinear, least-squares algorithm. Using the estimated coefficients, solution ranges for individual membrane parameters are calculated, frequently providing results within +/-20% of true values without additional electrophysiological measurements. We determined the measurement system specifications and statistical significance of estimated parameters using 1) analytical testing with circuit simulation software and equation-generated data; 2) a system noise analysis combined with Monte Carlo simulations; and 3) analog model circuits for calibration of the electronic system and to check equation-generated results. Finally, the time course of capacitance changes associated with purinergically stimulated mucin exocytosis are quantified in monolayers of the colonic goblet cell-like cell line HT29-CI.16E.

Algorithms↗
PubMed2001

Detection of repair activity during the DNA damage-induced G2 delay in human cancer cells.

All eukaryotic cells manifest cell cycle delay after exposure to DNA damaging agents. It has been proposed that such cell cycle checkpoints may allow DNA repair but direct evidence of such activity during the radiation-induced G2 delay has been lacking. We report here that cells arrested in G2 by radiation (2-3 Gy) and etoposide incorporate bromodeoxyuridine (BrdU) at discrete foci in the nucleus. We detected G2 cells with CENP-F, a nuclear protein maximally expressed in G2. Caffeine and okadaic acid, both established radiosensitizers, inhibit the incorporation of BrdU in G2 cells. Radioresistant HT29 and OVCAR cells demonstrate BrdU foci formation more frequently during the G2 delay when compared to the more radiosensitive A2780 cell line. The repair foci formed during G2 may be followed through mitosis and observed in daughter cells in G1. Taken together, these observations are consistent with the detection of DNA repair activity during the radiation-induced G2 delay after relatively low doses of radiation.

Bromodeoxyuridine↗
PubMed1997

Characterization of the promoter of human adipocyte hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5' untranslated region (5'-UTR). A single 5'-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5'-UTR is mutually exclusive. The short and long 5'-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5'-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

Adipocytes↗
PubMed1999

Biogenesis of alpha6beta4 integrin in a human colonic adenocarcinoma cell line involvement of calnexin.

The heterodimer alpha6beta4 is a major integrin and the main laminin receptor in epithelia. The alpha6 integrin subunit is proteolytically cleaved, probably by furin, and glycosylated during its biosynthesis. In the present work, we have investigated the kinetics of the assembly process of alpha6beta4 heterodimers in the colonic adenocarcinoma cell line HT29-D4. We demonstrate that the association of alpha6 and beta4 precursors occurs within the ER, while the endoproteolytic cleavage of pro-alpha6 occurs later, probably in the trans-Golgi network. When pro-alpha6 was blocked within the ER by treatment with brefeldin A, its maturation processing was completely prevented without any consequence on its association with beta4 subunit. Low temperature (20 degrees C) also blocked pro-alpha6 maturation, like brefeldin A, but in addition impaired the integrin assembly. Calnexin, an ER resident protein chaperone, was found to be associated with both the alpha6 and beta4 subunit precursors. Our data suggest that calnexin might be responsible for the prolonged retention of pro-alpha6 within the ER compartment and for the defect of integrin subunit association observed at low temperature.

Adenocarcinoma↗
PubMed1997

Lymphotoxin-beta receptor signaling complex: role of tumor necrosis factor receptor-associated factor 3 recruitment in cell death and activation of nuclear factor kappaB.

The binding of heterotrimeric lymphotoxin, LT alpha1 beta2, to the LTbeta receptor (LTbeta R), a member of the tumor necrosis factor receptor (TNFR) superfamily, induces nuclear factor kappaB (NF-kappaB) activation and cell death in HT29 adenocarcinoma cells. We now show that treatment with LT alpha1 beta2 or agonistic LTbeta R antibodies causes rapid recruitment of TNFR-associated factor 3 (TRAF3) to the LTbeta R cytoplasmic domain. Further, stable overexpression of a TRAF3 mutant that lacks the RING and zinc finger domains inhibits LTbeta R-mediated cell death. The inhibition is specific for LTbeta R cell death signaling, since NF-kappaB activation by LT alpha1 beta2 and Fas-mediated apoptosis are not inhibited in the same cells. The mutant and endogenous TRAF3s are both recruited at equimolar amounts to the LTbeta R, suggesting that the mutant disrupts the function of the signaling complex. These results implicate TRAF3 as a critical component of the LTbeta R death signaling complex and indicate that at least two independent signaling pathways are initiated by LTbeta R ligation.

Adenocarcinoma↗
PubMed1997

Dominant negative mutants of TRAF3 reveal an important role for the coiled coil domains in cell death signaling by the lymphotoxin-beta receptor.

Ligation of the lymphotoxin-beta receptor (LTbetaR) recruits tumor necrosis factor receptor-associated factor-3 (TRAF3) and initiates cell death in HT29 adenocarcinoma cells. The minimal receptor binding domain (TRAF-C) defined by two hybrid analyses is not sufficient for direct recruitment to the ligated receptor. A series of TRAF3 deletion mutants reveal that a subregion of the coiled coil motif is required for efficient recruitment to the LTbetaR. Furthermore, the ability of TRAF3 to self-associate maps to an adjacent subregion. A TRAF3 deletion mutant that lacks the N-terminal zinc RING and zinc finger motifs, but retains the coiled coil and TRAF-C motifs, competitively displaces endogenous TRAF3 from the LTbetaR. A second TRAF3 mutant that lacks the receptor binding domain, yet contains the TRAF3 self-association domain, prevents TRAF3 homodimers from being recruited to the LTbetaR. Both of these mutants have a dominant negative effect on cell death and demonstrate that the recruitment of TRAF3 oligomers is necessary to initiate signal transduction that activates the cell death pathway.

Apoptosis↗
PubMed1991

The E1b oncogene of adenovirus confers cellular resistance to cytotoxicity of tumor necrosis factor and monoclonal anti-Fas antibody.

The cell lines KB8, 16, and 18 are KB cells which constitutively express adenovirus type 2 (Ad2) E1a, E1a plus E1b, and E1b genes, respectively. We show here that KB18 cells are completely resistant to cytolysis by tumor necrosis factor (TNF) or anti-Fas, although KB8 and KB or KB16 are highly and moderately sensitive, respectively. The levels of receptors for TNF and anti-Fas of KB18 were almost the same as compared with those of KB or other KB-cell lines. Expression of manganous superoxide dismutase (MnSOD) mRNA in KB18 was about 20-fold higher than that in KB or KB8 cells. KB, HT29, and A673 cells infected with dl337 (an Ad5 mutant defective in E1b function) are highly sensitive to TNF or anti-Fas, although wild-type Ad2-infected cells are resistant. Our results indicate that the E1b oncogene can confer cellular resistance to cytolysis by either TNF or anti-Fas in both KB cells and adenovirus-infected human cell lines through influencing intracellular events including regulation of MnSOD genes. Furthermore, we describe how anti-Fas mimics only the cytolytic activity of TNF, whereas TNF also has many other biological activities.

Adenoviruses, Human↗
PubMed2001

Involvement of nuclear factor kappaB in c-Myc induction by tubulin polymerization inhibitors.

We showed previously that microtubule disassembly by vinblastine induces the proto-oncogene c-myc in epithelial mammary HBL100 cells. In this study, we demonstrate that vinblastine treatment in these cells, in contrast to what was observed with the colon adenocarcinoma cell line HT29-D4, activated the transcription factor NFkappaB, which has been involved in c-myc regulation. The microtubule disassembly also induced IkappaB degradation. Using transient transfection analysis, we show that the trans-activation of c-myc by vinblastine was decreased when NFkappaB binding sites on c-myc promoter were mutated. Additionally, we demonstrate that microtubule dissolution trans-activated a thymidine kinase-CAT construct containing an NFkappaB binding site at -1180 to -1080 bp relative to the P1 promoter. Thus, vinblastine up-regulates the enhancer activity of the NFkappaB binding site. These results suggest that microtubule disassembly induced by vinblastine can trans-activate the c-myc oncogene through NFkappaB. Taking into consideration the paradoxical roles of both c-myc and NFkappaB in proliferation or apoptosis, this data reveals the complex action mechanism of this microtubule interfering agent.

Antineoplastic Agents, Phytogenic↗
PubMed1991

Effects of fatty acids on expression of genes encoding subunits of cytochrome c oxidase and cytochrome c oxidase activity in HT29 human colonic adenocarcinoma cells.

As the primary and preferred energy source of normal colonic epithelial cells, fatty acids may play a unique role in the differentiation and physiology of these cells. We have shown that expression levels of COXIII, a mitochondrial gene encoding one of the 13 subunits of cytochrome c oxidase, are abnormally low in colon tumors and colonic tissue at genetic risk for developing tumors but increase following in vitro treatment of HT29 human colonic adenocarcinoma cells with the fatty acid butyrate. The present studies investigate the specificity of fatty acids in effecting cytochrome c oxidase subunit expression and enzymatic activity in HT29 cells. The data demonstrate that, depending upon their chain length, metabolizable unbranched fatty acids increase expression of two subunits encoded by mitochondrial genes (I and III) and enhance cytochrome c oxidase activity. However, none of the fatty acids had an effect on expression of two subunits encoded by nuclear genes (IV and Va). These findings suggest that the low levels of COXIII expression exhibited in colonic tumors may represent a limiting factor in the assembly of functional cytochrome c oxidase and contribute to the depressed enzyme activity reported in these tumors. By elevating expression of subunits I and III and enzymatic activity, fatty acids may enhance the potential for cellular respiration. The more differentiated phenotype which is reported in colorectal carcinoma cell lines treated with fatty acids in vitro may be, therefore, associated with correction of metabolic abnormalities in transformed cells.

Adenocarcinoma↗
PubMed1995

Heat stress or rotavirus infection of human epithelial cells generates a distinct hyperphosphorylated form of keratin 8.

The two major intermediate filament (IF) phosphoglycoproteins of human simple epithelia are keratins 8 and 18 (K8/18). Previous studies showed that heat stress and, in some cases, viral infection of cultured cells can be associated with alteration in IF organization. Here we show that heat stress of the human colonic cell line HT29 increased K8 and K18 phosphorylation and glycosylation and generated a distinct hyperphosphorylated form of K8 (HK8) that was previously noted upon G2/M arrest of epithelial cells. In contrast, rotavirus infection of HT29 cells resulted in phosphorylation changes similar to those induced by heat stress but did not alter K8/18 glycosylation. The identity of HK8 was determined using phosphatase treatment, tryptic phosphopeptide mapping, and K8-specific antibodies. A time course of heat stress showed that the increased phosphorylation and glycosylation of K8/18 occurred several hours after induction of heat shock protein 70 expression. Rotavirus altered the organization of the K8/18 network in most infected cells, whereas the effect of heat stress on K8/18 organization was less prominent. However, in vitro filament assembly of purified K8/18 was not significantly altered after isolation from heat-stressed or rotavirus-infected cells, despite having increased solubility and hyperphosphorylation. Our results indicate that increased glycosylation and/or phosphorylation of K8 and K18, in association with heat stress or rotavirus infection, does not significantly alter in vitro keratin filament assembly, whereas the association of these modifications with filament reorganization in cells is more dramatic. Generation of HK8 appears to be a late event and to occur in common with several forms of stress including heat, viral infection, and arrest of cells in G2/M.

Blotting, Western↗
PubMed2003

Apoptosis induced by cryo-injury in human colorectal cancer cells is associated with mitochondrial dysfunction.

Cryotherapy, a method of in situ ablation, is used in the treatment of colorectal liver metastases with variable results. During the treatment, the central area of treated tumor undergoes necrotic destruction by lethal cryo-injury; however, the cellular response of tumor exposed to sublethal cryo-injury at the peripheral zone is unclear. In our study, we have identified the induction of apoptosis by cryo-injury at -10 degrees C in 4 colorectal cancer cell lines (HT29, HCT116, KM12C and KM12SM). The apoptosis was characterized by chromatin condensation, transferase-mediated dUTP nick end-labeling (TUNEL) staining, proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and cytokeratin 18, and activation of caspase-3. The occurrence and intensity of cryo-induced apoptosis did not correlate with the functional status of p53 in the cell lines studied. The expression of anti-apoptotic proteins (Bcl-2, Bcl-X(L)) and pro-apoptotic proteins (Bax, Bcl-X(S), Bad, and Bak) in response to cryo-injury varied in this cell line panel. The basal level of Bcl-2/Bax protein ratio correlated inversely to the apoptotic rate. We further demonstrated that Bax level decreased in cytosol and increased in mitochondria, followed by a loss of mitochondrial membrane potential after cryo-injury in HT29 cells. These findings indicate that cryo-injury induces apoptosis in colorectal cancer cells via disruption of mitochondrial integrity. The cryo-induced apoptosis was also identified in a nude mouse tumor xenograft model. Our elucidation of the apoptosis pathway induced by cryo-injury implies that synergistic combination of cryosurgery with pharmacological agents that augment of apoptosis induction may have clinical relevance in treating colorectal liver metastasis.

Animals↗
PubMed1977

Use of a permanent cell line extracts to study the tumor associated immune reactions in colorectal cancer patients by leucocyte migration inhibition test.

Crude extracts of colorectal surgical tumors, either individual or pooled, and HT29 line cells were compared in leucocyte migration inhibition test. HT29 cell extract gave more positive reactions with leucocytes of colorectal cancer patients than the other ones, and less with control leucocytes. It was thus used for all the subsequent experiments. As a whole, we obtained 44% (30/68) of positive inhibitions in patients afflicted with a colorectal carcinoma, against 9% (3/31e in normal controls and 20% (8/40) in patients bearing a carcinoma from another organ. Among the latest patients, those having a gynecological carcinoma were rarely positive, contrasting with those suffering from a carcinoma from the aerodigestive organs. It seemed that the antigen(s) involved in the reaction had no organ specificity, but rather a broad tissue specificity.

Adenocarcinoma↗
PubMed1994

Saccharomyces boulardii inhibits secretagogue-mediated adenosine 3',5'-cyclic monophosphate induction in intestinal cells.

BACKGROUND/AIMS: The yeast Saccharomyces boulardii inhibits the secretion induced by cholera toxin (CT) in rat jejunum. The present study was aimed at unraveling the mechanism by which S. boulardii protects intestinal cells against CT. METHODS: CT-induced adenosine 3',5'-cyclic monophosphate (cAMP) levels were measured by radioimmunoassay in intestinal epithelial cells IEC-6 or HT29-D4 cells exposed to whole yeast or to culture medium conditioned by S. boulardii (Sb-conditioned medium). RESULTS: Sb-conditioned medium significantly reduced CT-induced cAMP levels in IEC-6 cells. This effect was eliminated by heat treatment, trypsin hydrolysis, and trichloroacetic acid precipitation of Sb-conditioned medium. When conditioned medium was fractionated on polyacrylamide gel under nondenaturing conditions, neutralizing activity was shown to be associated with a 120-kilodalton protein. The neutralizing activity was not attributable to proteolytic activity against CT. Sb-conditioned medium reduced the amount of cAMP induced by CT as well as Escherichia coli thermolabile toxin or forskolin in HT29-D4 cells. The modulation of secretagogue-induced cAMP by Sb-conditioned medium did not occur in the presence of pertussis toxin. CONCLUSIONS: These results suggest that the neutralization of CT by S. boulardii is mediated by a specific yeast protein and involves a receptor that is negatively coupled to adenylate cyclase.

Animals↗
PubMed2002

Modulation of HMG-N2 binding to chromatin by butyrate-induced acetylation in human colon adenocarcinoma cells.

Butyrate, a short chain fatty acid (SCFA), is generated by anaerobic fermentation of undigested carbohydrates within the colon. Butyrate enhances acetylation of core histones, a process directly linked to the formation of active chromatin and gene expression. However, additional chromatin components also contribute to the formation of transcriptionally active chromatin. The high mobility group protein N2 (HMG-N2), a nonhistone protein, is involved in chromatin structure modulation. We examined the effects of butyrate on HMG-N2 expression, hyperacetylation and chromatin binding. HT29 human adenocarcinoma cells were incubated with butyrate. Levels of HMG-N2 mRNA and of total or acetylated HMG-N2 protein were analyzed. Protein dynamics were investigated with transfected cells expressing HMG-N2-EGFP fusion proteins. Treatment of HT29 cells with butyrate led to significant hyperacetylation of HMG-N2. Levels of HMG-N2 protein remained unchanged. Northern blot analysis revealed a significant reduction in HMG-N2 mRNA levels after treatment with butyrate. Analysis of HMG-N2-EGFP transfected HT29 cells demonstrated that butyrate treatment changes the binding properties of HMG-N2-EGFP to chromatin. In addition, butyrate treatment resulted in solubilization of endogenous acetylated HMG-N2 into the supernatant of permeabilized cells. We demonstrate that butyrate treatment is associated with hyperacetylation of HMG-N2 protein in HT29 cells. The modulation of this nonhistone chromatin protein resulted in altered binding properties to chromatin. This may represent an additional step in changing chromatin structure and composition with subsequent consequences for transcription and gene expression. Modulation of nonhistone chromatin proteins, like the ubiquitous HMG-N2 proteins, may be partly responsible for the wide range of butyrate-associated effects.

Acetylation↗
PubMed1998

Regulation of stimulated cyclic AMP synthesis by urocanic acid.

Urocanic acid (UCA) has been shown to mediate the UVB radiation-induced immunosuppression initiated in the skin by UV-induced isomerization from the trans to the cis isomer. However, the mechanism by which cis-UCA acts is still unclear. Therefore, the present study was undertaken to determine the effect of trans- and cis-UCA on cyclic adenosine 3',5'-monophosphate (cAMP) synthesis in human dermal fibroblasts, Golden Syrian hamster hepatocytes and in the human adenocarcinoma cell line, HT29. Neither trans- nor cis-UCA was able to stimulate cAMP synthesis directly in any of the models tested. In human dermal fibroblasts, cis-UCA, in contrast to trans-UCA, specifically inhibited cAMP synthesis induced by either prostaglandin (PG) E1 or PGE2 with a maximum inhibitory effect of 25-30% at cis-UCA concentrations greater than 1 microM and half-maximum inhibitory effect (EC50) observed at 35 nM. The effect of cis-UCA was not to stimulate phosphodiesterase and cAMP breakdown. The inhibitory effect of cis-UCA (an imidazole derivative) was not mediated through stimulation of the alpha 2-adrenergic receptor. The inhibitory effect of cis-UCA on stimulated cAMP synthesis was a function of the cell density and was only significant when the fibroblasts were confluent or postconfluent. In contrast to the studies with human dermal fibroblasts, an inhibitory effect of cis-UCA was not observed in either isolated hamster hepatocytes or HT29 cells, in which cAMP synthesis was stimulated by glucagon and vasoactive intestinal peptide, respectively. These results point to a possible regulation of cAMP synthesis in fibroblasts as one mechanism by which cis-UCA exerts its biological effect in the skin.

Adrenergic alpha-Agonists↗