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PubMed2005

Sodium butyrate-mediated differentiation of colorectal cancer cells: regulation of PKCbetaII by PI 3-kinase.

The present study focuses on a putative regulation of PKCbetaII by phosphatidylinositol-3 kinase (PI 3-kinase) in colorectal carcinoma cells; little is known about the role and activity of PKCbetaII in these cells. We examined the activity of PI 3-kinase in two adenocarcinoma cell lines, HT29 cells that differentiate only after stimulation with appropriate agents, and Caco-2 cells that can differentiate spontaneously. The activity of PI 3-kinase as well as the activity of PKCbetaII appeared to decrease only in HT29 cells in which differentiation was induced by sodium butyrate. In HT29 cells infected with recombinant adenovirus encoding constitutively active PI 3-kinase, the activity of alkaline phosphatase was almost completely blocked, and this PI 3-kinase significantly potentiated the activity of PKCbetaII in HT29 cells despite the presence of NaBT in the culture medium. On the contrary, in differentiating Caco-2 cells, the activity of PI 3-kinase was not butyrate-sensitive. In agreement with these findings, the alkaline phosphatase activity was not affected by constitutively active PI 3-kinase overexpressed in Caco-2 cells. These observations suggest that PKCbetaII is regulated by PI 3-kinase in HT29 cells and that the mechanisms of spontaneous differentiation versus butyrate-induced differentiation of adenocarcinoma cells may be different.

Adenocarcinoma↗
PubMed1998

Cyclooxygenase-2 up-regulation after FLAP transfection in human adenocarcinoma cell line HT29 cl.19A.

Five-lipoxygenase-activating protein (FLAP) is usually described as an essential protein to activate the leukotriene (LTs) synthesis via the 5-lipoxygenase pathway. In the enterocyte model HT29 cl.19A cell line, 5-lipoxygenase metabolism was found despite the lack of FLAP expression. Therefore HT29 cl.19A represents an original mammalian model to study FLAP-dependent leukotriene synthesis. In FLAP cDNA transfected HT29 cl.19A cells, FLAP expression led to an increase in cyclooxygenase pathway products (mainly PGE2) without an increase in 5-lipoxygenase metabolism. This increase in PGE2 synthesis was associated with a cyclooxygenase-2 upregulation in comparison to untransfected HT29 cl.19A cells. These results suggest a possible interaction between the two major pathways of arachidonic acid metabolism.

5-Lipoxygenase-Activating Proteins↗
PubMed2004

Enhanced DNA-directed effects of FdUMP[10] compared to 5FU.

FdUMP[N] molecules and conjugates are much more effective at inhibiting the proliferation of human tumor cells than is the widely used anticancer drug 5-fluorouracil (5FU). We have evaluated the inhibition of thymidylate synthase (TS), the extent of DNA damage, cell cycle arrest, and the induction of apoptosis by FdUMP[10] and 5FU in the human colorectal cancer cell line HT29. The magnitude and duration of TS inhibition following exposure of HT29 cells to FdUMP[10] at 1 x 10(-8) M was greater than that which occurred following exposure of these cells to 5FU at 1 x 10(-6) M. FdUMP[10] exposure also resulted in much more extensive DNA damage to HT29 cells than occurred following exposure to 100-fold higher concentrations of 5FU. Although exposure of HT29 cells to both drugs resulted in S-phase arrest, more complete accumulation of cells in S-phase was achieved following FdUMP[10] exposure at much lower drug concentrations. FdUMP[10] was also much more effective at inducing apoptosis in HT29 cells than was 5FU. The results are consistent with FdUMP[10] being much more efficient that 5FU at inducing DNA damage that results in apoptotic cell death in colon cancer cells.

Colonic Neoplasms↗
PubMedDate not supplied

A fragment of vasoactive intestinal peptide, VIP(10-28), is an antagonist of VIP in the colon carcinoma cell line, HT29.

The 19 amino acid carboxyl terminus fragment of vasoactive intestinal peptide (VIP), VIP(10-28), inhibits [125I]VIP binding in intact HT29 colonic adenocarcinoma cells and in membranes from these cells. However, VIP(10-28) alone has no effect on adenylate cyclase activity (membranes) or cyclic AMP synthesis (intact cells) in HT29 cells although VIP receptor agonists are markedly stimulatory. The indicated antagonist character of VIP(10-28) was confirmed by rightward parallel shifts of VIP dose response curves in the presence of VIP(10-28) in adenylate cyclase and cyclic AMP synthesis experiments. The equilibrium dissociation constant values for VIP(10-28) from these experiments agree with values from inhibition binding studies. The lack of effect of VIP(10-28) on forskolin dose response curves in HT29 adenylate cyclase assays indicates the specificity of the VIP(10-28) antagonism, thus suggesting that VIP(10-28) may be a useful tool in studying VIP receptor regulation and other aspects of the mechanisms of VIP action. The potential regulatory role of a proteolytically generated fragment of VIP acting antagonistically at VIP receptors is discussed.

Adenocarcinoma↗
PubMed2005

[Effect of exogenous expression of heparanase gene on invasive ability of colorectal cancer cell line HT29].

BACKGROUND & OBJECTIVE: Heparanase (Hpa) is an endoglycosidase that degrades heparin sulfate--the main polysaccharide constituent of extracellular matrix (ECM) and basement. It can enhance the invasive and metastatic potential of malignant tumors and cell lines by destroying ECM and basement. This study was to explore the effects of heparanase gene on the invasive ability of colorectal cancer cell line HT29. METHODS: Heparanase gene was transfected into HT29 cells. Cell growth kinetics was assessed by MTT assay, and in vitro invasive ability was assessed with Boyden chamber. Xenograft and orthotopic implantation of histologically intact tumor in nude mice were constructed to observe the effect of exogenous expression of heparanase gene on invasive ability of HT29 cells. RESULTS: After transfection, the growth rate of heparanase-transfected HT29 (HT29-Hpa) cells was obviously higher than those of untransfected HT29 cells and empty vector-transfected HT29 (HT29-KZ) cells; the invasive cell number was significantly larger in HT29-Hpa cells than in untransfected HT29 cells and HT29-KZ cells (45.5+/-0.5 vs. 29.3+/-0.1 and 30.1+/-0.2, P<0.01). The entity neoplasm formed by HT29-Hpa cells (12 mm x 9 mm x 10 mm) was bigger than that formed by untransfected HT29 cells (6 mm x 8 mm x 6 mm). The prevalence of liver metastasis caused by orthotopic implantation of xenograft was significantly higher in HT29-Hpa group than in control group (71.43% vs. 14.29%, P<0.01). CONCLUSION: Exogenous heparanase gene can facilitate the growth, invasion, and metastasis of HT29 cells.

Animals↗
PubMed2001

Protective activity of butyrate on hydrogen peroxide-induced DNA damage in isolated human colonocytes and HT29 tumour cells.

Epidemiological studies support the involvement of short-chain fatty acids (SCFA) in colon physiology and the protective role of butyrate on colon carcinogenesis. Among the possible mechanisms by which butyrate may exert its anti-carcinogenicity an antioxidant activity has been recently suggested. We investigated the effects of butyrate and mixtures of SCFA (butyrate, propionate and acetate) on DNA damage induced by H(2)O(2) in isolated human colonocytes and in two human colon tumour cell lines (HT29 and HT29 19A). Human colonocytes were isolated from endoscopically obtained samples and the DNA damage was assessed by the comet assay. H(2)O(2) induced DNA damage in normal colonocytes in a dose-dependent manner which was statistically significant at concentrations over 10 microM. At 15 microM H(2)O(2) DNA damage in HT29 and HT29 19A cells was significantly lower than that observed in normal colonocytes (P < 0.01). Pre-incubation of the cells with physiological concentrations of butyrate (6.25 and 12.5 mM) reduced H(2)O(2) (15 microM) induced damage by 33 and 51% in human colonocytes, 45 and 75% in HT29 and 30 and 80% in HT29 19A, respectively. Treatment of cells with a mixture of 25 mM acetate + 10.4 mM propionate + 6.25 mM butyrate did not induce DNA damage, while a mixture of 50 mM acetate + 20.8 mM propionate + 12.5 mM butyrate was weakly genotoxic only towards normal colonocytes. However, both mixtures were able to reduce the H(2)O(2)-induced DNA damage by about 50% in all cell types. The reported protective effect of butyrate might be important in pathogenetic mechanisms mediated by reactive oxygen species, and aids understanding of the apparent protection toward colorectal cancer exerted by dietary fibres, which enhance the butyrate bioavailability in the colonic mucosa.

Biopsy↗
PubMed1990

Effect of VIP on the glycogen metabolism of human colon adenocarcinoma cells studied by 13C nuclear magnetic resonance spectroscopy.

Metabolic pathways of glucose utilization have been investigated in a human colon adenocarcinoma cell line (HT29) using carbon-13 Nuclear Magnetic Resonance spectroscopy. HT29 cells were adapted to grow on a polystyrene beaded microcarrier and were perfused when attached to the beads in a specially designed NMR cell. Abnormalities in carbohydrate metabolism already observed in several cancer cells were studied in HT29 cells fed with (1-13C)-enriched glucose. The cells were first perfused with a glucose-free medium for 2 h in order to deplete the intracellular store of glycogen, and they were subsequently perfused with a medium containing enriched glucose at an initial concentration of 5.5 mM. Sequential 13C-NMR spectra, recorded at 100.5 MHz (5 min accumulation), show that HT29 cells were able to utilize glucose through the glycolytic pathway while storing glucose as glycogen (glucose was utilized at a rate of 3.9 mumol/mg protein/hr). The glycolytic activity determined by the amount of lactic acid produced was 4.6 microns/mg protein/hr, corresponding to the formation of 1.2 lactic acid per glucose molecule. Glycogen accumulation corresponded to 16 micrograms/mg of protein. Treatment of HT29 with 10 nM vasoactive intestinal peptide (VIP) induced a transient decrease in the level of labelled glycogen to 50% of the initial value. Control level was recovered 12 min after VIP loading.

Adenocarcinoma↗
PubMed1990

Respiration of mitochondria isolated from differentiated and undifferentiated HT29 colon cancer cells in the presence of various substrates and ADP generating systems.

1. Oxygen consumption was investigated in two cultured subpopulations of either undifferentiated (Glc+ cells) or differentiated (Glc- cells) HT29 colon cancer cells and in the corresponding isolated mitochondria. In Glc+ cells, a decrease of the respiration is induced by the presence of glucose (Crabtree effect), whereas it is not the case in Glc- cells. 2. The oxidative phosphorylation rate of Glc- mitochondria is found to be much higher than that of Glc+ mitochondria, due to a higher efficiency to oxidize glutamine, glutamate, 2-oxoglutarate, succinate or malate. 3. In both types of mitochondria, respiration can be supported by the ADP formed by adenylate kinase or nucleotide diphosphate kinase, and, although to a lesser extent in Glc- mitochondria, by hexokinase. 4. Glc+ cells are characterized by a low respiration capacity and a high glycolytic flux leading to the Crabtree effect. Glc- cells are characterized by a better correlation between a moderate glycolytic flux and a high respiratory capacity.

Adenosine Diphosphate↗
PubMed1990

Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose.

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.

Cell Division↗
PubMed2001

Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells.

PURPOSE: A new mucus-secreting in vitro drug absorption model based on monolayers of goblet-cell like sub-clones of the human colon carcinoma cell line HT29 obtained by methotrexate (MTX) treatment was investigated. METHODS: Twelve sub-clones were isolated and characterized by light microscopy (LM), transelectron microscopy (TEM), confocal laser scanning microscopy (CLSM), transepithelial electrical resistance (TEER) and the transport of a paracellular marker FITC-Dextran (Mw 4400) (FD-4). RESULTS: Significant differences of microscopical appearance, TEER-values and permeability of FD-4 between the sub-clones were evident. However, two of them, namely MTX-D1 and MTX-E12. formed tight confluent monolayers with a thick mucus-layer on the apical surface. They were used to compare the apparent permeability coefficient (Papp) of a series of lipophilic drugs, which should be affected by the mucus-layer, namely barbiturates (barbituric acid, barbital, phenobarbital, methylphenobarbital and heptabarbital) and testosterone, as a reference, to mucus-free Caco-2 cells. The permeability of drugs with a partition coefficient (log P) > 1 was decreased in the mucus-producing cell lines. Testosterone, the most lipophilic compound, showed a decrease of up to 43%. CONCLUSIONS: We demonstrated that the mucus layer is a significant barrier to drug absorption for lipophilic drugs. In conclusion, our model may serve as a suitable in-vitro cell culture model to study the influence of the mucus layer on drug diffusion.

Algorithms↗
PubMed2003

Developing novel oncolytic adenoviruses through bioselection.

Mutants of human adenovirus 5 (Ad5) with enhanced oncolytic activity were isolated by using a procedure termed bioselection. Two mutants, ONYX-201 and ONYX-203, were plaque purified from a pool of randomly mutagenized Ad5 that was repeatedly passaged in the human colorectal cancer cell line HT29, and they were subsequently characterized. ONYX-201 and ONYX-203 replicated more rapidly in HT29 cells than wild-type Ad5, and they lysed HT29 cells up to 1,000-fold more efficiently. The difference was most profound when cells were infected at a relatively low multiplicity of infection, presumably due to the compounding effects of multiple rounds of infection. This enhanced cytolytic activity was observed not only in HT29 cells but also in many other human cancer cell lines tested. In contrast, the cytotoxicity of the bioselected mutants in a number of normal primary human cells was similar to that of wild-type Ad5, thus enhancing the therapeutic index (cytotoxicity in tumor cells versus that in normal cells) of these oncolytic agents. Both ONYX-201 and -203 contain seven single-base-pair mutations when compared with Ad5, four of which were common between ONYX-201 and -203. The mutation at nucleotide 8350, shared by both mutant viruses, was shown to be essential for the observed phenotypes. This mutation was mapped to the i-leader region of the major late transcription unit, resulting in the truncation of 21 amino acids from the C terminus of the i-leader protein. This work demonstrates that bioselection is a powerful tool for developing novel tumor-selective oncolytic viruses. Other potential applications of this technology are discussed.

Adenoviruses, Human↗
PubMed1999

Short exposure of intestinal epithelial cells to TNF-alpha and histamine induces Mac-1-mediated neutrophil adhesion independent of protein synthesis.

Neutrophils play an important role in intestinal inflammation by interacting with intestinal epithelial cells. In this study, we evaluated neutrophil adhesion to intestinal epithelial cells using intestinal epithelial cell line HT29 stimulated with tumor necrosis factor alpha (TNF-alpha) and histamine for a short time (30 min). The TNF-alpha and histamine stimulation markedly increased neutrophil adhesion. The increased adhesion was inhibited by anti-CD11b and anti-CD18 monoclonal antibodies (mAbs), but not by anti-CD11a and anti-CD54 (ICAM-1) mAbs. It is interesting that flow cytometric analysis revealed that ICAM-1 expression on HT29 cells was not changed by TNF-alpha and histamine stimulation. Moreover, the increased adhesion was inhibited by proteinase K treatment but not cycloheximide treatment of HT29 cells. Together these observations suggest that short exposure of HT29 cells to TNF-alpha and histamine induces CD11b/CD18 (Mac-1)-dependent but CD11a/CD18 (LFA-1)-independent neutrophil adhesion to intestinal epithelial cells, and ICAM-1 is not likely to be involved in the interactions. Furthermore, epithelial cell ligand(s) for neutrophils is likely protein molecule(s) that is expressed on the cell by stimulation independent protein synthesis. However, it is also possible that neutrophil activating factor(s), which stimulates neutrophils to bind with epithelial ligands via Mac-1, is expressed by epithelial cells during stimulation.

Adenocarcinoma↗
PubMed1995

Expression of hormone-sensitive lipase in the human colon adenocarcinoma cell line HT29.

Hormone-sensitive lipase expression was studied in the human colon adenocarcinoma cell line, HT29. Diacylglycerol lipase and cholesterol esterase [corrected] activities in HT29 cells were inhibited by known inhibitors of hormone-sensitive lipase (diethyl-p-nitrophenyl phosphate, NaF and HgCl2) to the same extent as in human adipocytes. A polyclonal antiserum directed against rat hormone-sensitive lipase inhibited 89% of HT29 cell lipase activity. HT29 hormone-sensitive lipase was the same size as the adipocyte enzyme as was its mRNA. Complete homology between mRNA sequences in HT29 and adipocyte was demonstrated using ribonuclease protection assay. These data are consistent with the expression of a protein closely related, if not identical, to the enzyme expressed in human adipose tissue. HT29 is the first human cell line where hormone-sensitive lipase expression has been shown.

Adenocarcinoma↗
PubMed1997

A self-assembled antibody electrode for HT29 tumour cells.

The present paper describes a self-assembled monoclonal-antibody immunosensor for HT29 tumour cells. This immunosensor is composed of two parts. One is a three-electrode system and the other is a layer of anti-(human colonic tumour) monoclonal antibody DF-I, which self-assembled on to the gold electrode. Two kinds of cells, HT29 tumour cells and normal fibroblast cells, were compared using the antibody electrode. The result was the same as that obtained by using immune luminescence. The HT29 cells can be captured specifically by the assembled anti-HT29 monoclonal-antibody electrode and can be measured by an electrochemical method.

Antibodies, Monoclonal↗
PubMed2006

Modulation of key elements of the Wnt pathway by apple polyphenols.

Glycogen synthase kinase-3beta (GSK3beta) is one of the key elements of the Wnt pathway involved in the regulation of beta-catenin homeostasis. The inhibition of GSK3beta kinase activity might lead to the onset of beta-catenin/TCF/LEF-mediated gene transcription, representing a potentially mitogenic stimulus. Apple polyphenols have been shown to mediate several biological effects that might be of interest with respect to chemoprevention. The objective of the study was to elucidate whether apple polyphenols also modulate key elements of the Wnt pathway, an effect that might limit the usefulness of these compounds for the prevention of carcinogenesis. A polyphenol-rich apple juice extract (AE02) was found to effectively inhibit the kinase activity of GSK3beta, immunoprecipitated from HT29 cells. Treatment of HT29 cells with AE02 for 24 h resulted in a concentration-dependent decrease of the cellular GSK3beta kinase activity. The inhibition of the kinase activity in HT29 cells was observed at polyphenol concentrations corresponding to the concentration of the constituents in the original apple juice. The apple characteristic dihydrochalcone glycoside phloridzin was found to be inactive up to 500 muM. The free aglycon phloretin as well as the flavonol quercetin effectively inhibited isolated GSK3beta, but did not affect the respective kinase activity within HT29 cells. In accordance with the inhibition of GSK3beta kinase activity by AE02, treatment of HT29 cells resulted in a significant decrease of phosphorylated beta-catenin. However, the total intracellular beta-catenin level was also found to be diminished, indicating that the interference of the apple constituents with GSK3beta was not associated with a stabilization of beta-catenin in HT29 cells. In line with these results, TCF/LEF-mediated gene transcription remained unaffected by treatment with AE02 as shown in a reporter gene approach. We can assume from the results that at consumer-relevant concentrations apple polyphenols do not mediate growth-stimulating effects in HT29 cells via the Wnt pathway.

Blotting, Western↗
PubMed2001

A heat labile soluble factor from Bacteroides thetaiotaomicron VPI-5482 specifically increases the galactosylation pattern of HT29-MTX cells.

The aim of this work was to set up and validate an in vitro model to study a molecular response of an intestinal host cell line (HT29-MTX), to a non-pathogen microflora component. We found that Bacteroides thetaiotaomicron strain VPI-5482 had the capacity to change a specific glycosylation process in HT29-MTX cells via a mechanism that involved a soluble factor. Differentiated HT29-MTX cells were grown in the presence of 20% of spent culture supernatant from the B. thetaiotaomicron during 10 days. Glycosylation processes were followed using a large panel of lectins and analysed using confocal microscopy, western blotting and flow cytometry techniques. Our results show that a B. thetaiotaomicron soluble factor modified specifically the galactosylation pattern of HT29-MTX cells, whereas other glycosylation steps remained mainly unaffected. Further characterization of this soluble factor indicates that it is a heat labile, low molecular weight compound. Reverse transcript-PCR (RT-PCR) analysis was unable to show any significant change in mRNA expression level of the main galactosyltransferases expressed in HT29-MTX cells. By contrast, galactosyltransferase activities dramatically increased in HT29-MTX cells treated by the soluble extract of B. thetaiotaomicron, suggesting a post-translational regulation of these activities. Our in vitro model allowed us to study the cross-talk between a single bacteria and intestinal cells. The galactosylation process appears to be a target of this communication, thus uncovering a new window to study the functional consequences of co-operative symbiotic bacterial-host interactions.

Bacteroides↗
PubMed1987

Rapid lateral diffusion of lectin-labelled glycoconjugates in the human colonic adenocarcinoma cell line HT29. Comparison with the synthetic lipid analogue diI-C14.

The lateral diffusion of lectin-labelled glycoconjugates was studied in the human colon carcinoma cell line HT29 using fluorescence photobleaching techniques. HT29 cells were grown in either Dulbecco's modified Eagle's medium with glucose (25 mM; DMEM-Glu) or with galactose (25 mM; DMEM-Gal). Cell cultivation in the DMEM-Gal medium was assumed to promote a transformation of the cells to become small-intestinal-like with characteristic microvilli and associated enzymes. The diffusion of glycoconjugates labelled with fluoresceinated Triticum vulgaris agglutinin (Wheat germ agglutinin; WGA), Ricinus communis agglutinin-I (RCA-I), Concanavalia ensiformis agglutinin (ConA), Ulex europaeus agglutinin-I (UEA-I) and Arachis hypogaea agglutinin (PNA) was in all cases rapid, with a diffusion constant (D) ranging between 0.4 and 0.8 X 10(-8) cm2 s-1. As a comparison the diffusion of the fluorescent synthetic lipid analog diI-C14 was characterized by D = 0.8 - 1.0 X 10(-8) cm2 s-1. The diffusion of lectin-labelled surface components could not be related to the presence of microvilli on HT29 cells grown in DMEM-Gal, which ought to yield an apparently lower diffusion rate. The results indicate either that surface glycoconjugates in HT29 cells are dominated by glycolipid, or that the labelled glycoproteins are more or less free to diffuse in the plane of the membrane.

Adenocarcinoma↗