HT29 Cl Research Articles

T1700 Protein Expression Profiling Using LC ESI/QTOF Mass Spectrometry in a Colon Carcinoma Cell Line (HT29 Cl.16E) and in Inflammatory Bowel Disease Colonic Epithelial Cells

T1700 Protein Expression Profiling Using LC ESI/QTOF Mass Spectrometry in a Colon Carcinoma Cell Line (HT29 Cl.16E) and in Inflammatory Bowel Disease Colonic Epithelial Cells

Differential proteomic analysis of HT29 Cl.16E and intestinal epithelial cells by LC ESI/QTOF mass spectrometry

Intestinal epithelial cells (IECs) play a key role in Crohn's disease, a chronic inflammatory bowel disease which requires invasive examinations to be diagnosed. The comparison of the cellular protein expression profiles of Crohn's disease patients and healthy subjects is fundamental for the identification of proteins clinically relevant as new biomarkers or as drug targets. For this purpose a differential label-free nano-LC ESI/QTOF mass spectrometry (MS) approach combined with targeted MS/MS analysis has been developed and applied to isolated IECs. We report here a study of the protein variations in IECs from healthy subjects (H) and Crohn's disease patients (CD). The method was previously validated using HT29 Cl.16E cell line, normal or treated with interferon-gamma as a model of inflammation. Subcellular fractions proteins were extracted from HT29 and IECs and for each fraction monodimensional gel-electrophoresis was performed and the proteins subjected to tryptic digestion. The resulting peptides were analysed by LC ESI/QTOF MS and the obtained chromatographic runs were aligned with msInspect software. The peptides differently expressed were statistically evaluated using the Proteios Software Environment (ProSE) and identified by LC ESI/QTOF MS/MS analysis and database search. The preliminary results obtained allowed the identification of many proteins involved in the inflammation processes.

Chronic PKC-β activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation

We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl- current (ISC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane ISC generation. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC alpha-, beta1-, gamma-, and epsilon-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-alpha membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-beta1, decreased PKC-beta2 membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta1 and a significant reduction in PKC-epsilon mass occurred. Thus activated PKC-beta1 and/or PKC-epsilon prevented, whereas activated PKC-alpha facilitated, apical membrane ISC-cAMP. PKC-beta-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC beta-isoform.

Chronic PKC-β2activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane CFTR targeting

We investigated the effects of chronically applied PKC-stimulating phorbol esters on subcellular CFTR expression and localization in polarized HT-29 Cl.19A monolayers. Modulation of PKC activity with the PKC-beta-specific agonist 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) or nonisoform-selective PMA altered monolayer CFTR immunofluorescence. A decrease in the CFTR signal within the luminal cellular pole was noted with both phorbol esters. Volumetric analysis of the intracellular CFTR signal revealed that both compounds promoted CFTR accumulation into punctate vesicle-like structures found adjacent to the cellular tight junction [labeled with zona occludens (ZO)-1 antibody], extending basally (DOPPA) into the cell. Puncta were more frequent with DOPPA and larger in size with PMA. DOPPA also promoted ZO-1 accumulation at tricellular corners associated with enhanced CFTR puncta number. The observed loss of CFTR immunofluorescence signal induced by low-dose PMA was related to CFTR sequestration into fewer cytoplasmic puncta and correlated with larger increases in PKC substrate phosphorylation. Both phorbol esters downregulated steady-state cellular CFTR mRNA levels by 70%. However, the effects of DOPPA and PMA were largely independent of CFTR biosynthesis: expression levels were 80-85% of control, and the glycosylation status of immunoprecipitated protein remained largely unchanged. Thus changes in cellular CFTR localization correlated with our companion study showing that PMA-induced inhibition of transcellular cAMP-dependent short-circuit current (ISC) was accompanied by cytoplasmic PKC-beta2 accumulation and modest activation of PKC-beta1 and PKC-epsilon. The inhibitory effect of DOPPA on ISC was related solely to increased cytoplasmic PKC-beta2 levels. Thus PKC-beta2 is hypothesized to participate in the regulation of CFTR apical plasma membrane targeting within the constitutive cellular biosynthetic pathway.

Antisense Inhibition of Entamoeba histolytica Cysteine Proteases Inhibits Colonic Mucus Degradation

The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. Cysteine protease-deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [(35)S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. Trophozoites with reduced cysteine protease activity were ineffective at degrading [(35)S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease-deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis.

Eotaxin-3/CCL26 gene expression in intestinal epithelial cells is up-regulated by interleukin-4 and interleukin-13 via the signal transducer and activator of transcription 6

Several inflammatory processes of the bowel are characterized by an accumulation of eosinophils at sites of inflammation. The mechanisms that govern mucosal infiltration with eosinophils are not fully understood. Eotaxin-3/CCL-26 belongs to a family of CC chemokines, which are potent chemoattractants for eosinophils. In this study, we hypothesized that intestinal epithelial cells could release eotaxin-3. We demonstrate that the T helper 2 type cytokines interleukin-4 or interleukin-13 increase eotaxin-3 mRNA levels and eotaxin-3 protein expression in the human intestinal epithelial cell lines HT-29 CL.19A and T84 in a dose-dependent manner. Addition of actinomycin-D prior to interleukin-4/-13 stimulation led to decreases in eotaxin-3 mRNA levels similar to those observed in controls without interleukin-4/-13. Interleukin-4 and interleukin-13 activated signal transducer and activator of transcription 6 which was found to bind the two canonical signal transducer and activator of transcription 6 binding sites located in the eotaxin-3 promoter. Experiments with the eotaxin-3 promoter luciferase constructs revealed that the most proximal signal transducer and activator of transcription 6 binding site located between positions −62 and −71 relative to the transcriptional start was necessary for full eotaxin-3 promoter activity. Importantly, we present evidence that the signal transducer and activator of transcription 6 is necessary and sufficient for interleukin-4 or interleukin-13 mediated eotaxin-3 gene up-regulation using HT-29 CL.19A cells expressing a dominant-negative signal transducer and activator of transcription 6. Overall, these results demonstrate that epithelial eotaxin-3 is up-regulated in the context of a T helper 2 mediated inflammatory bowel disease via the signal transducer and activator of transcription 6, thus suggesting that the intestinal epithelium actively participates in the recruitment of eosinophils at the site of inflammation.

Expression of human TFF3 in relation to growth of HT-29 cell subpopulations: involvement of PI3-K but not STAT6

The trefoil factor family (TFF) peptides 1 and 2 (TFF1 and 2) are expressed in mucus cells of the stomach, whereas TFF3 is localized in goblet cells of the intestine. In the present study, we aimed to determine whether phosphatidylinositol 3-kinase (PI3-K) or signal transducer and activator of transcription protein 6 (STAT6) is involved in the expression of goblet cell specific markers. TFF3 expression was analyzed by RT-PCR, Northern blot, and radioimmunoassay (RIA) in relation to cell growth in subclones of HT-29 cells including the CL.16E and methotrexate (MTX) cell lines, which both exhibit a phenotype of mucus-secreting intestinal cells. A 30-fold increase in TFF3 mRNA levels and a 10-fold increase in TFF3-cell content were observed between the early proliferative and the late confluency states. The levels of MUC2 and MUC3 mRNA were also increased in the course of the differentiation process. A three to fourfold increase in PI3-K and Akt activities was observed in early post-confluent cells as compared with pre-confluent cells. Exposure of pre- and post-confluent cells to LY294002, a specific PI3-K inhibitor, for 1–4 days profoundly reduced TFF3 and MUC2 expression. A marked reduction in mucin granules content was also observed in LY-treated cells. Inhibition of the mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 did not modify the course of differentiation of the goblet cell lines. Moreover, stable transfection of HT-29 CL.16E cells with a dominant negative form of STAT6 had no effect on TFF3 induction. Together, these data indicate that PI3-K promotes the expression of TFF3 and MUC2 and that the PI3-K/Akt pathway may play a pivotal role in intestinal goblet cell differentiation.

IL-4 and IL-13 up-regulate intestinal trefoil factor expression: requirement for STAT6 and de novo protein synthesis.

The development of intestinal goblet cell hyperplasia/hypertrophy during nematode infection involves the Th2 cytokines IL-4 and IL-13 via STAT6 activation. This is thought to play an important role in host protective immunity against the infection. In this study we demonstrate that IL-4 and IL-13 up-regulate the specific goblet cell product trefoil factor-3 (TFF3) from the mucus-producing HT-29 CL.16E and HT-29 cells selected by adaptation to methotrexate. Up-regulation of TFF3 mRNA and protein levels occurred in a time- and dose-dependent fashion and was accompanied by up-regulation of the goblet cell product mucin 2 (MUC2). Addition of actinomycin D before IL-4/IL-13 stimulation led to decreases in TFF3 mRNA levels similar to those observed in controls without IL-4/IL-13. Furthermore, IL-4-mediated increased TFF3 transcription required de novo protein synthesis. Stable transfection of HT-29 CL.16E cells with a truncated dominant-negative form of STAT6 produced a cell line that was unresponsive to IL-4/IL-13. Although only one consensus STAT6 binding site is contained in the TFF3 gene, located in the intron 1, it did not operate as an enhancer in the context of an SV40 promoter/luciferase construct. Thus, STAT6 activation mediates a transcriptional enhancement of TFF3 by induction of de novo synthesized protein in goblet cells.

Immunolocalization of a new intestinal antiproliferative factor in human intestinal epithelial cells.

A new intestinal antiproliferative factor (IAF) with an approximate molecular weight of 120 kDa has been purified from the human small intestine. This factor blocks the progression of human colon adenocarcinoma cells HT-29 from the G1 to the S phase. IAF, specific of the lower part of the digestive tract, was detected rather late in mouse embryonic development. For determination of the specific intestinal cell producing IAF, long-term differentiated mucus-secreting HT-29 Cl 16E and enterocytic HT-29 Cl 19A cell lines were used. IAF is synthesized exclusively in the intestinal goblet cells; it is processed in the RER and Golgi complex before being excreted in secretory vesicles independently of mucin secretion. IAF can be considered a growth inhibitor of intestinal proliferation for the same reason as TGF-beta. However, two features differentiate it from TGF-beta: (1) the intestinal cell type synthesizing it, and (2) the delay in its expression in embryonic development. Particular interest was paid to IAF expression in pathological conditions using human colon biopsies. IAF was consistently recovered in biopsies from patients with inflammatory bowel diseases and benign tumors, but it was never detected in malignant tumors. IAF could represent a marker of colon cancer owing to its absence from malignant tumors.

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.19 A 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.19 A cells, FLAP expression led to an increase in cyclooxygenase pathway products (mainly PGE 2) without an increase in 5-lipoxygenase metabolism. This increase in PGE 2 synthesis was associated with a cyclooxygenase-2 up-regulation in comparison to untransfected HT29 cl.19A cells. These results suggest a possible interaction between the two major pathways of arachidonic acid metabolism.

Identification of a human enterocyte lipoxin A4 receptor that is regulated by interleukin (IL)-13 and interferon gamma and inhibits tumor necrosis factor alpha-induced IL-8 release.

Epithelial cells of the alimentary tract play a central role in mucosal immunophysiology. Pathogens and/or agonists that interact with mucosal surfaces often elicit epithelial responses that upregulate inflammation. Therefore, it was of interest to explore potential epithelial targeted antiinflammatory signals. Here we identified and sequenced a human enterocyte lipoxin (LX) A4 [5(S), 6(R),15(S)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic acid] receptor, and demonstrate that transcription of this receptor was controlled by cytokines, of which lymphocyte-derived interleukin (IL)-13 and interferon gamma were the most potent. When lipoxins and LXA4 stable analogs were evaluated for enterocyte functional as well as immune responses, lipoxins sharply inhibited TNF-alpha-induced IL-8 release but did not alter either barrier function or agonist-stimulated chloride secretion. 15R/S-methyl-LXA4 and 16-phenoxy-LXA4 each attenuated (IC50 approximately 10 nM) IL-8 release. Cyclooxygenase (COX) II is emerging as an important component in wound healing and proliferation in intestinal epithelia and when acetylated by acetylsalicylic acid (aspirin) initiates the biosynthesis of a LXA4 receptor ligand. We therefore determined whether colonic cell lines (HT-29 Cl.19A, Caco-2, or T84) express the COX II isozyme. Results for RT-PCR and Western blot analysis showed that COX I as well as an IL-1beta- and TNF-alpha-inducible COX II are expressed in HT-29 Cl.19A. In addition, aspirin-treated enterocytes generated 15R-HETE, a precursor of 15-epi-LXA4 biosynthesis, whose potent bioactions were mimicked by the stable analog 15R/S-methyl-LXA4. Taken together, these results identify an endogenous pathway for downregulating mucosal inflammatory events and suggest a potential therapeutic benefit for LXA4 stable analogs.

Production of arachidonic acid metabolites by the colon adenocarcinoma cell line HT29 cl.19A and their effect on chloride secretion.

Eicosanoids were found in large amounts in the colonic mucosa of patients suffering from inflammatory bowel diseases and colonic adenocarcinoma. The aim of this study was to evaluate the role of the intestinal epithelial cells in the arachidonic acid metabolism and their functional response to certain eicosanoids. We used the human adenocarcinoma epithelial cell line HT29 cl.19A cell, which is an in vitro model of colon carcinoma and ion transport. These cells were found to express 5- and 15-lipoxygenase, leukotriene A4 hydrolase and cyclooxygenase-1 and -2 mRNAs. We observed an arachidonic acid metabolism via 5-lipoxygenase pathway despite the lack of FLAP mRNA expression and that certain eicosanoids such as hydroperoxy- and hydroxyeicosatetraenoic acids stimulate chloride secretion.

PRODUCTION OF ARACHIDONIC ACID LIPOXYGENASE METABOLITES BY THE INTESTINAL EPITHELIAL CELL LINE HT29 CL. 19A AND THEIR EFFECT ON CHLORIDE SECRETION.

Arachidonic acid (AA) lipoxygenase products may be important in the pathogenesis of diarrhoea associated with intestinal inflammation, because they may stimulate electrogenic chloride secretion in the epithelium. However, the role of the epithelial cells as producers or as targets for lipoxygenase products remains controversial. To clarify this role, we measured the following on the same clone of a chloride-secreting intestinal cell line of human origin (HT29 cl. 19A): 1) the expression of the mRNAs of 5-lipoxygenase, platelet and leukocyte type 12-lipoxygenase, 15-lipoxygenase, LTA4 hydrolase, Five-Lipoxygenase-Activating-Protein (FLAP), prostaglandin H (PGH) synthase-1 and PGH synthase-2 after total RNA extraction, reverse transcription and cDNA amplification by PCR, using specific primers; 2) the formation of AA metabolites by high pressure liquid chromatography (HPLC) and 3) the effect of such metabolites on the stimulation of short-circuit current (Isc). an index of chloride secretion, in filter-grown HT29 cl. 19A cells mounted in Ussing chambers. Results: The mRNAs of 5-lipoxygenase, 15-lipoxygenase, LTA4 hydrolase, PGH synthase-1 and PGH synthase-2 were equally expressed in HT29 cl. 19A cells at different stages of culture, but FLAP, platelet and leukocyte type 12-lipoxygenase mRNAs were not expressed. When dispersed HT29 cl. 19A cells were incubated with [1-14C] arachidonic acid, calcium ionophore A23187, Mg2+ and Ca2+, they produced 5-hydroxyeicosatetraenoic acid (5-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), and a peak containing leukotriene B4 (LTB4) unresolved from its 6-trans-stereoisomers, and 5(S)-12(S)-di-HETEs. No peptidoleukotriene were detected. Unlabelled AA metabolites, analyzed by HPLC and enzyme immunoassay, were mainly 6-trans-stereoisomers of LTB4 and unidentified di-HETEs, but only a small amount of LTB4 was detected. In addition to prostaglandin E2 (PGE2), which was the most potent compound, several hydroperoxyeicosatetraenoic acids (HPETEs) produced by autoxidation of AA (5-, 9-, 11-, 12- and 15-HPETEs) induced bumetanide-sensitive chloride secretion by HT29 cl. 19A cells (Δ Isc: 3.2 ± 0.4 to 12.1 ± 3.7 μA cm-2). The 9-, 12- and 15-HETEs obtained by triphenylphosphine reduction of the corresponding HPETEs, displayed the same activity as the parent compound, but the 5- and 11-HETEs lose their potencies. LTB4 had no effect on chloride secretion. The present results suggest that the enterocyte may participate in the production of AA metabolites through the lipoxygenase and prostaglandin synthase pathways. In addition, the enterocytes are a target for the lipoxygenase pathway metabolites they and other cells produce.

Tumour Necrosis factor-α induces morphological and functional alterations of intestinal HT29 cl.19A cell monolayers

TNF-alpha is a widely distributed proinflammatory cytokine, involved in many disease states. Although it has widely distributed effects, a precise mechanism of action has never been described, in particular at the epithelial level. Morpho-functional changes of the intestinal epithelial monolayer HT29 cl.19A exposed to TNF-alpha were therefore assessed, using electron microscopy (including freeze-fracture replica analysis), as well as measurement of mannitol, Na+ and horseradish peroxidase fluxes across intestinal HT29 cl.19A cell monolayers using Ussing chambers. TNF-alpha receptors were induced on HT29 cl.19A cells by a small non-toxic dose of IFN-gamma (5 U/ml). After 4 h of the combined presence of TNF-alpha (10 ng/ml) and IFN-gamma (5 U/ml), the tight junction structure was altered as shown by a significant decrease in the average strand number measured in the apico-basal direction (5.50 +/- 2.70 vs 3.73 +/- 1.39 in control and treated cells respectively, P < 0.0001) and by a significant decrease in junctional depth (0.27 +/- 0.14 and 0.17 +/- 0.10 microns in control and treated cells respectively, P < 0.0001). These results are in agreement with a decrease in number of 'kiss' sites between contiguous membranes of TNF-alpha treated cells observed in ultrathin sections.(ABSTRACT TRUNCATED AT 250 WORDS)

DISTINCT SIGNALING MEDIATES CHLORIDE SECRETION INDUCED BY TUMOR PROMOTER BILE-SALTS AND PHORBOL ESTERS IN HUMAN COLONIC CELLS

Tumor promoter phorbol esters directly activate protein kinase C (PKC). Similarly tumor promoter bile salts activate PKC in the human colon epithelial cell line HT-29 cl.19A, as well as in platelets. Bile salts were also able to directly activate the purified brain enzyme. We designed experiments to investigate the effects of bile salts on Cl- secretion in HT-29cl.19A cell monolayers and the involvement of PKC in this process. Sodium deoxycholate (DOG) gradually evoked a slow increase of the short-circuit current (I-sc) in the Ussing chamber-mounted cell monolayers. By contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited a large increase in I-sc which rapidly plateaued. The I-sc response was representative of enhanced Cl- secretion, since it was abrogated in Cl--free medium, and was prevented by Cl- channel blocker diphenylamine-2-carbonylate as well as by bumetanide, a Na+/K+/Cl- transporter inhibitor. Sequential applications of TPA and DOC at suboptimal doses elicited additive effects. Neither DOC nor TPA increased the levels of cAMP in these cells. However DOG, at variance with TPA, increased the intracellular content of inositol trisphosphate, presumably due to triggering of the phosphoinositide pathway. From our studies and others, it is suggested that DOG-induced unspecific activation of some membrane-associated proteins, elicits a rise in Ca2+ from extracellular and internal sources which combines its effects with those due to PKC activation.

Mononuclear cells from infants allergic to cow's milk secrete tumor necrosis factor α, altering intestinal function

Background/Aims: Intestinal dysfunction observed during cow's milk allergy (CMA) is incompletely understood, and neither the effector cells nor the mediators responsible have been clearly identified. This study was undertaken to better characterize the implication of mononuclear cells in food allergy. Methods: Peripheral blood mononuclear cells (PBMC) from infants with CMA were cultured in the presence of cow's milk proteins (CMP), and the release of tumor necrosis factor α (TNF-α), Interferon gamma (IFN-γ), and interleukin 4 and 6 was measured. The effect of culture supernatants was tested on HT29 cl.19A intestinal cell monolayers mounted in Ussing chambers. Results: When stimulated by CMP, PBMC from infants with CMA released more TNF-α than those from control infants (429 ± 92 vs. 205 ± 34 pg/mL). Culture supernatants did not directly stimulate electrogenic chloride secretion by HT29 cl.19A cells, but epithelial barrier capacity was altered as shown by the significant decrease in electrical resistance (85 ± 17 vs. 135 ± 14 Ω·cm2 in controls) and the increases in intact horseradish peroxidase, [14C]mannitol, and 22Na+ fluxes. These effects were reversed when culture supernatants were neutralized with anti-TNF-α antibodies. Recombinant human-TNF-α altered the HT29 cl.19A epithelial barrier capacity, and its effect was highly potentiated by IFN-γ. Conclusions: These results indicate that during CMA, the high level of TNF-α released by mononuclear cells after milk protein challenge acts synergistically with IFN-γ to increase the intestinal permeability.

Relation between chloride secretion and intracellular cyclic adenosine monophosphate in a cloned human intestinal cell line HT-29 cl 19A.

The relation between the intracellular cyclic adenosine monophosphate (cAMP) content and the electrogenic chloride secretion induced by cholera toxin was studied in secretory HT-29 cl 19A cell monolayers. Cells were treated by the mucosal addition of cholera toxin (5 micrograms/ml) for 10, 45, or 90 minutes in Ussing chambers. After 10 minutes, the mean (SEM) intracellular cAMP content (3.2 (0.2) pmol/mg protein) and short circuit current (Isc) (1.9 (0.3) microA.cm-2) did not differ significantly from the corresponding basal values. At 45 minutes, a significant increase in the Isc (22.2 (5.7) microA.cm-2) was accompanied by a significant elevation in cAMP (10(1.7) pmol/mgh protein). At 90 minutes, when the stimulated Isc plateaued (35.2 (5.2) microA.cm-2), the cAMP value (99.2 (23.8) pmol/mg protein) increased further. The protein kinase C (PKC) activity of the cells was not affected by cholera toxin. Treatment of cell monolayers by different concentrations of DbcAMP (10(5), 5 x 10(-5), 10(-3) M) showed that the minimal concentration of DbcAMP (serosal) which significantly increased the Isc (delta 4.5 microA.cm-2) was 10(-4) M, and that this was accompanied by an increase in cAMP of delta 6.7 pmol/mg protein: Compared with DbcAMP, cholera toxin stimulated the Isc (at 45 minutes) to a much higher degree with a comparable elevation of cAMP. It is concluded that in cl 19A cells there is a threshold value of increase in intracellular cAMP that induces chloride secretion. Cholera toxin stimulated chloride secretion can be explained predominantly by an increase in intracellular cAMP that is unrelated to PKC activity.

Basolateral mechanisms of intracellular pH regulation in the colonic epithelial cell line HT29 clone 19A.

The intracellular pH (pHi) of the colonic tumour cell line HT29 cl.19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer, grown in a polarized manner on permeable supports, were examined. An amiloride-sensitive Na+/H+ exchange and a stilbene-insensitive Cl-/HCO3- exchange mechanism have been identified in the basolateral membrane. Removal of Na+ from the basolateral solution caused a decrease of pHi by 0.50 +/- 0.09 unit (n = 4). Amiloride or Na(+)-free solution at the apical side had no effect on pHi. Cl- removal at the basolateral side led to an increase of pHi by 0.20 +/- 0.03 unit (n = 4) whereas apical removal had no influence on pHi. This effect was independent of Na+ and was insensitive to 0.2 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid. A basolateral Cl-/HCO3- exchanger is the most likely explanation for this observation. The Na+/H+ exchange mechanism in the basolateral membrane is an acid extruder, whereas the Cl-/HCO3- exchanger is an acid loader. Both of these mechanisms are important for the maintenance of intracellular pH in HT29 cl.19A cells.

Effect of cytokines on the epithelial function of the human colon carcinoma cell line HT29 cl 19A.

In various intestinal diseases, lymphoid cell infiltration of the lamina propria might be an important factor causing intestinal dysfunction through cytokine release. The effects of such cytokines were therefore measured on chloride secretion and macromolecular transport in intestinal HT29 cl 19A cells. Cells were grown on transwell filters and serosally exposed for two to 48 hours to 1% or 5% concentrations of phytohaemagglutinin (PHA) activated or non-activated healthy human mononuclear cell culture supernatants. These supernatants were tested for their interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), and interleukin 6 (IL6) concentration. The mean (SE) were 4.72 (1.63), 3.17 (0.14), and 4.47 (1.12) ng/ml in activated supernatants. Intestinal function was studied in Ussing chambers: chloride secretion was assessed from the variations in short circuit current, and epithelial barrier capacity was monitored by the electrical resistance of the HT29 cl 19A cell monolayers and by measuring intact or degraded fluxes of the macromolecular tracer horseradish peroxidase. Release of lactate dehydrogenase (LDH) in the presence of various concentrations of cytokines was assessed as a test for cellular injury. The results indicate that serosal cytokines do not directly stimulate short circuit current. Long term (48 hours) exposure, however, significantly reduced both short circuit current (6.79 (0.65) v 2.90 (1.16) microA/cm2 in control v treated cells) and barrier capacity as shown by the decrease in resistance of the HT29 cl 19A cell monolayers from 158 (10) to 36 (9) ohms.cm2, and the increase in intact fluxes from 167 (33) in controls to 874 (206) ng/h.cm2, in cytokine rich supernatant treated cells and in degraded fluxes from 1113 (114) to 2327 (234) ng/h.cm2. This decreased barrier capacity was associated with an increase in release of LDH F(21.7 (1.2) and 43.6 (2.0) UI/h.cm2 in 1% and 5% cytokine treated monolayers v 10.8 (2.1) UI/h.cm2 in control monolayers). The results indicate that cytokines do not directly stimulate intestinal chloride secretion but that prolonged exposure to these agents reduced both electrolyte transport and barrier capacity to macromolecules through cellular and paracellular pathways.

Bile acids, non-phorbol-ester-type tumor promoters, stimulate the phosphorylation of protein kinase C substrates in human platelets and colon cell line HT29.

Protein kinase C (PKC) is the target for a number of tumor promoters. The mechanism underlying the promoting effects of bile acids in colorectal cancer is not understood. We report that sodium deoxycholate (DOC) triggered activation of PKC in physiological conditions. The biphasic effects of DOC upon PKC activation were Ca(2+)-stimulated and did not require phosphatidylserine (PtdSer) as phospholipid co-factor. The optimal rate of activation was obtained at 0.4 mM DOC and reached approximately half the maximal rate of activation obtained in the presence of PtdSer. Similarly to PtdSer, DOC supported diacylglycerol- as well as phorbol-ester-mediated PKC activation. The reciprocal effects of PtdSer and DOC upon PKC in either 0.5 mM CaCl2 or 0.5 mM EGTA suggest that DOC interacts with the phospholipid-binding domain to elicit PKC activation. DOC-supported enzyme activation exhibited substrate specificity different from that of PtdSer-supported enzyme activation. All tested primary and secondary bile acids activated PKC to various extents, with DOC being the most potent. We suggest that amphipathic bile acids acting in a PtdSer-like manner provide the hydrophobic environment required for PKC activation. Treatment of 32P-labeled platelets and colonic cells HT29 Cl.19A with DOC enhanced the phosphorylation of endogenous substrates for PKC. Colonic cells responsive at 50 microM DOC, appeared to be 10-fold more sensitive than platelets. We suggest that direct or indirect activation of PKC by bile acids may account for the promoting effects of these non-phorbol-ester-type tumor promoters.