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PubMed2002

Benzo(a)pyrene activates extracellular signal-related and p38 mitogen-activated protein kinases in HT29 colon adenocarcinoma cells: involvement in NAD(P)H:quinone reductase activity and cell proliferation.

Benzo(a)pyrene (BP) is a polyaromatic hydrocarbon generated from the combustion of fossil fuel. Human exposure results primarily through dietary sources and smoking. Little is known about the effect of BP on mitogen-activated protein (MAP) kinases, which include extracellular signal-related kinase (ERK), Jun N-terminal kinase (JNK), and p38. We investigated the participation of BP-induced MAP kinase activation in cell growth and increases in activity of the detoxification enzyme NAD(P)H:quinone reductase (QR). In HT29 human colon carcinoma cells, 10 nM BP activated ERK and p38 but not JNK after 24 h. Treatment with 10 nM BP increased QR activity within 24 h and tritiated thymidine ([(3)H]thyd) incorporation after 48 h and reduced cell viability after 72 h. Using the MAP kinase inhibitors PD98059 and SB203580, we investigated the relative contributions of ERK and p38 to QR activity and [(3)H]thyd cell incorporation. Inhibition of ERK eliminated BP-induced QR activity, whereas inhibition of p38 had no effect on QR activity. Treatment of cells with 10 nM BP increased [(3)H]thyd incorporation by 50% after 48 h. This increase was eliminated by ERK but not p38 inhibition. In conclusion, 10 nM BP activates ERK and p38, but only ERK contributes to BP-induced QR activity. ERK, but not p38 activation participated in [(3)H]thyd incorporation. In summary, BP influences cellular signaling pathways at concentrations present in routine human exposures.

Benzo(a)pyrene↗

PubMed1998

Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate.

O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant post-translational modification found on nuclear and cytoplasmic proteins and is thought to be a dynamically regulated modification much like phosphorylation. In this study we have demonstrated that O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbama te (PUGNAc), a potent in vitro inhibitor of the enzyme responsible for the removal of O-GlcNAc from proteins (peptide O-GlcNAc-beta-N-acetylglucosaminidase), can be used to increase O-GlcNAc levels on nuclear and cytoplasmic proteins in vivo. Overall, PUGNAc caused approximately a 2-fold increase in O-GlcNAc levels in the human colon cancer cells, HT29, although the effects on individual proteins varied. The increase appeared to be the result of the direct inhibition of the peptide O-GlcNAc-beta-N-acetylglucosaminidase since neither the O-GlcNAc transferase nor UDP-GlcNAc levels were affected by the treatment. O-GlcNAc levels in other cell lines tested (NIH 3T3, CV-1, and HeLa) were also affected by PUGNAc, although the effects on HeLa cells were minimal. At the concentrations tested, PUGNAc was non-toxic and had no affect on the growth rate of any of the cell lines examined. Interestingly, we demonstrated that an increase in O-GlcNAc levels on the transcription factor Sp1 resulted in a reciprocal decrease in its level of phosphorylation, supporting the hypothesis that O-GlcNAc competes with phosphate on some proteins. These studies demonstrate that PUGNAc is an effective inhibitor of O-GlcNAc turnover within cells and can be used to selectively alter the extent of O-GlcNAc on cellular proteins.

Acetylglucosamine↗

PubMed2003

Angiopoietin-1 inhibits vascular permeability, angiogenesis, and growth of hepatic colon cancer tumors.

Angiopoietin (Ang)-1 and -2 are critical regulators of embryonic and postnatal neovascularization. Ang-1 activates the endothelial cell-specific tyrosine kinase receptor Tie-2, which in turn leads to enhanced endothelial cell survival and stabilization. The effects of Ang-1 on tumor angiogenesis remain controversial; although we have previously demonstrated that Ang-1 overexpression in colon cancer cells leads to a decrease in s.c. tumor growth, others have shown that Ang-1 may be proangiogenic. Few studies have addressed the role of the Angs in tumors growing in the organ of metastatic growth. We hypothesized that overexpression of Ang-1 may inhibit the growth of colon cancers growing in the liver by inhibition of angiogenesis. We also wanted to investigate the mechanisms by which Ang-1 affects angiogenesis in vivo. Human colon cancer cells (HT29) were stably transfected with an Ang-1 construct or an empty vector (pcDNA) and injected directly into the livers of nude mice. After 37 days, livers were harvested and weighed, and tumor sizes were measured. In an additional experiment, to validate the paracrine effect of Ang-1, various mixtures of control cells and Ang-1-transfected cells were injected into livers, and tumor growth was assessed. Direct effects of recombinant Ang-1 on angiogenesis were studied with an in vivo Gelfoam angiogenesis assay. The impact of Ang-1 on vascular permeability was investigated using an intradermal Miles assay with conditioned media from transfected cells. Liver weights (P < 0.05), tumor volumes (P < 0.05), vessel counts (P < 0.01), and tumor cell proliferation (P < 0.01) in the Ang-1 group were significantly lower than those in the control (pcDNA) group. Tumor vessels in the Ang-1 group developed a significantly higher degree of pericyte coverage (P < 0.02) than vessels in pcDNA tumors. In the cell mixture experiment, even as few as a 1:10 mixture of Ang-1-transfected cells/control cells resulted in a significant reduction of hepatic tumor volumes (P < 0.04). In the angiogenesis assay, vessel counts in Gelfoam implants were significantly decreased by the addition of Ang-1 (P < 0.01). Finally, conditioned medium from Ang-1-transfected cells decreased vascular permeability more than that from control cells (P < 0.05). Our results suggest that Ang-1 is an important regulator of angiogenesis and vascular permeability and that this effect may be secondary to increasing periendothelial support and vessel stabilization. Thus, Ang-1 could potentially serve as an antineoplastic or anti-permeability agent for patients with metastatic colorectal cancer.

Angiogenesis Inducing Agents↗

PubMed1997

The alpha-galactosyl specific lectin from Artocarpus integrifolia distinguishes between two lymphoma lines with different metastatic potential.

A lectin purified from the seeds of the Vietnamese Artocarpus integrifolia distinguishes between the mouse T-cell lymphoma cell lines Eb and ESb, with low and high metastatic potential, respectively. It agglutinates Eb cells as well as human erythrocytes, but not ESb cells or the human colon carcinomas cells HT29. The haemagglutinin is specific for alpha-galactosyl residues and has a molecular mass of 62 kDa.

Agglutination Tests↗

PubMed1999

Glutathione redox potential in response to differentiation and enzyme inducers.

The reduced glutathione (GSH)/oxidized glutathione (GSSG) redox state is thought to function in signaling of detoxification gene expression, but also appears to be tightly regulated in cells under normal conditions. Thus it is not clear that the magnitude of change in response to physiologic stimuli is sufficient for a role in redox signaling under nontoxicologic conditions. The purpose of this study was to determine the change in 2GSH/GSSG redox during signaling of differentiation and increased detoxification enzyme activity in HT29 cells. We measured GSH, GSSG, cell volume, and cell pH, and we used the Nernst equation to determine the changes in redox potential Eh of the 2GSH/GSSG pool in response to the differentiating agent, sodium butyrate, and the detoxification enzyme inducer, benzyl isothiocyanate. Sodium butyrate caused a 60-mV oxidation (from -260 to -200 mV), an oxidation sufficient for a 100-fold change in protein dithiols:disulfide ratio. Benzyl isothiocyanate caused a 16-mV oxidation in control cells but a 40-mV oxidation (to -160 mV) in differentiated cells. Changes in GSH and mRNA for glutamate:cysteine ligase did not correlate with Eh; however, correlations were seen between Eh and glutathione S-transferase (GST) and nicotinamide adenine dinucleotide phosphate (NADPH):quinone reductase activities (N:QR). These results show that 2GSH/GSSG redox changes in response to physiologic stimuli such as differentiation and enzyme inducers are of a sufficient magnitude to control the activity of redox-sensitive proteins. This suggests that physiologic modulation of the 2GSH/GSSG redox poise could provide a fundamental parameter for the control of cell phenotype.

Adenocarcinoma↗

PubMed1995

Effects of the excretory/secretory products of six nematode species, parasites of the digestive tract, on the proliferation of HT29-D4 and HGT-1 cell lines.

The excretory/secretory (ES) products of the nematode parasite Trichostrongylus colubriformis have been found to increase the in vitro proliferation of the epithelial cell line HT29-D4. To assess the specificity of this effect, ES products from other trichostrongyle species were tested on colonic (HT29-D4) and gastric (HGT-1) tumour cell lines. Adult worms of six different nematode species, parasites of the stomach or the small intestine of ruminants, were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h. The conditioned media were then added at different concentrations to the culture medium of the two cell lines. A stimulation of the HT29-D4 cell growth occurred with the ES products of two parasite species of the small intestine, at the concentrations of 0.1 microgram protein/ml (Trichostrongylus vitrinus) and 1.0-5.0 micrograms/ml (Cooperia curticei). Inversely, a decrease in cell number was observed with the ES products of another intestinal species, Nematodirus battus at concentrations of 1.0-5.0 micrograms/ml. With the ES products of the abomasal nematodes, a proliferation of HT29-D4 cells was obtained at 0.25-5.0 micrograms/ml with ES products of Teladorsagia circumcincta but no significant effect was observed for Haemonchus contortus. On the tumoral gastric cell line HGT-1, the ES products from the 6 nematode species gave a similar stimulative effect. These in vitro results suggest that nematode parasite species secrete or excrete component(s) which could affect the epithelial regeneration of the host digestive tract.

Animals↗

PubMed2000

Monoclonal antibodies specific for human tumor-associated antigen 90K/Mac-2 binding protein: tools to examine protein conformation and function.

As part of our effort to identify glycoproteins that contribute to colon cancer progression, we have previously described a family of structurally related glycoproteins expressing beta1-6 branched asparagine(Asn)-linked oligosaccharides defined by monoclonal antibody (MAb 1H9), which are differentially expressed, processed, and glycosylated by human colon carcinoma cell lines (Laferté and Loh [1992]; Biochem J; 283:193-201). MAb 1H9 immunoprecipitates three glycoproteins having apparent sizes of 92-100, 66-70, and 25 kDa, the size heterogeneity attributable to cell-type specific glycosylation differences. We report on the basis of partial protein and cDNA sequence information, that the 100-kDa glycoprotein detected by MAb 1H9 is identical to the 90-kDa glycoprotein variably known as tumor-associated antigen 90K (TAA90K), Mac-2 binding protein, and cyclophilin C-associated protein. Using a PCR-based cloning strategy, the complete cDNA encoding TAA90K was cloned into the eukaryotic expression vector pCDNA-3 (pCD-TAA90K(wt)) and the protein expressed in COS-1 cells. A [(35)S]methionine-labeled 60-kDa polypeptide, processed to an endoglycosidase H-sensitive 74-kDa glycoprotein in the presence of dog pancreas microsomes, was detected in a coupled transcription/translation in vitro reaction. The in vitro-translated 60-kDa polypeptide and N-glycanase-treated TAA90K (60-kDa species) immunoprecipitated from HT29 cells were shown to be structurally identical by limited proteolytic peptide mapping. Using a new panel of 11 TAA90K-specific monoclonal antibodies, including five specific for human TAA90K and six cross-reactive with a 90-kDa species expressed by COS-1 cells, we have detected conformational differences between recombinant wild-type TAA90K, in vitro-synthesized TAA90K, and mutant forms of TAA90K containing point mutations at residues 189, 223, and 259. Furthermore, we have shown that these mutant forms of TAA90K, as well as a truncated form of TAA90K containing amino acid residues 1-383, are defective in secretion. These studies demonstrate the potential usefulness of TAA90K-specific monoclonal antibodies for examining the structure and function of TAA90K, and highlight the contribution of specific amino acid residues to its normal processing and secretion.

3T3 Cells↗

PubMed2005

Characterizing lipopolysaccharide and core lipid A mutant O1 and O139 Vibrio cholerae strains for adherence properties on mucus-producing cell line HT29-Rev MTX and virulence in mice.

Components of lipopolysaccharide (LPS), i.e. capsule, O antigen, core oligosaccharide, as well as the toxin-coregulated pili are among the factors which significantly contribute to intestinal colonization by Vibrio cholerae O1 and O139. To further address the contribution of LPS to V. cholerae virulence, we performed in vivo colonization experiments and mucus layer attachment studies with defined LPS and capsule mutants of O1 and O139. We investigated the interaction of V. cholerae strains with the differentiated human intestinal cell line HT29-Rev MTX, and found 3-5-fold reduced efficiencies for attachment by defined LPS and capsule mutants of O1 and O139 in comparison with the wild-type strains. In addition, two O1/O139-specific core oligosaccharide biosynthetic gene products, WavJ and WavD, were characterized and tested for colonization. We demonstrate that single and double knockout mutants in wavJ and wavD have an effect on core oligosaccharide biosynthesis, and that these mutants show an attenuated growth in the presence of novobiocin. Curiously, in the mouse intestinal colonization model, only the O139 wavJ,D mutants demonstrated reduced colonization.

Animals↗

PubMed2005

GRIM-19 interacts with nucleotide oligomerization domain 2 and serves as downstream effector of anti-bacterial function in intestinal epithelial cells.

Nucleotide oligomerization domain 2 (NOD2) functions as a mammalian cytosolic pathogen recognition molecule, and variants have been associated with risk for Crohn disease. We recently demonstrated that NOD2 functions as an anti-bacterial factor limiting survival of intracellular invasive bacteria. To gain further insight into the mechanism of NOD2 activation and signal transduction, we performed yeast two-hybrid screening. We demonstrate that GRIM-19, a protein with homology to the NADPH dehydrogenase complex, interacts with endogenous NOD2 in HT29 cells. GRIM-19 is required for NF-kappaB activation following NOD2-mediated recognition of bacterial muramyl dipeptide. GRIM-19 also controls pathogen invasion of intestinal epithelial cells. GRIM-19 expression is decreased in inflamed mucosa of patients with inflammatory bowel diseases. GRIM-19 may be a key component in NOD2-mediated innate mucosal responses and serve to regulate intestinal epithelial cell responses to microbes.

Animals↗

PubMed1999

Enhancement of antibody dependent cellular cytotoxicity (ADCC) by combination of cytokines.

Monoclonal antibodies (MAb) specific for tumor-associated antigens (TAA) can induce an immunological cellular attack of tumor cells by a process termed antibody dependent cellular cytotoxicity (ADCC). Cytokines may augment ADCC by direct activation of immune cells or by enhancement of TAA on tumor cells. Thus, we investigated whether ADCC by MAb 17-1A and BR55-2, which recognize TAA on colorectal tumor cells, can be augmented by 3-day incubation with different concentrations of IL-2, IL-4, IL-6, IL-12, IFN-alpha, IFN-gamma, GM-CSF, M-CSF, and TNF-alpha. ADCC was assessed by a new flowcytometric cytotoxicity assay (Flieger et al. Immunol Methods 1995; 180:1-13) using PKH-2 labeled HT29 cells as targets and PKH-26 labeled peripheral blood mononuclear cells from three healthy volunteers as effector cells. We found three reaction patterns with the cytokines tested: (a) cytokines, which increase ADCC (IL-2, IL-12, IFN-alpha, and IFN-gamma, which represent Thl cytokines); (b) cytokines with no effect (GM-CSF, M-CSF, and TNF-alpha); and (c) cytokines, which decrease ADCC (IL-4 and IL-6, which represent Th2 cytokines). Then, we tested cytokines that increase ADCC in combination with the other cytokines. We found that the combinations IL-2/IFN-alpha, IL-2/IFN-gamma, IL-2/IL-12, and IL-12/IFN-alpha potentiated ADCC. By contrast, IL-4 reduced the IL-2, IL-12, and IFN-alpha-induced ADCC. Since the Thl response, cooperation of monocytes and CD4 cells is involved, we plan to elucidate by magnetic cell sorting (MACS) separation techniques, which cells are involved in cytokine-induced ADCC. Our results may be useful for finding combinations of cytokines and MAb for the locoregional treatment of colorectal cancer.

Antibodies, Monoclonal↗

PubMed1988

Alpha-2A and alpha-2B adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype.

The affinities of 34 adrenergic antagonists for alpha-2 adrenergic receptors were determined from homogenate radioligand binding studies using [3H]yohimbine and [3H]rauwolscine. It has been suggested that alpha-2 adrenergic receptors can be subdivided into alpha-2A and alpha-2B subtypes. Oxymetazoline is selective for alpha-2A receptors, whereas prazosin is alpha-2B selective. Five different tissues were used, each of which has only one of the two subtypes: human platelet (alpha-2A), HT29 cell line (alpha-2A), human cerebral cortex (alpha-2A), neonatal rat lung (alpha-2B), and NG108-15 cell line (alpha-2B). The drug affinities were highly correlated when alpha-2A tissues were compared with alpha-2A tissues (r = 0.97 to 0.98) or when the two alpha-2B tissues were compared (r = 0.99). By contrast, comparison of an alpha-2A tissue with an alpha-2B tissue resulted in poor correlations (r = 0.77 to -0.87). Three new subtype selective drugs were identified among these drugs on the basis of at least a 10-fold greater affinity for one subtype. All three were selective for the alpha-2B subtype: ARC-239 (100-fold selective), chlorpromazine (18-fold selective), and 7-hydroxychlorpromazine (17-fold selective). These studies, by demonstrating distinct pharmacological profiles for the two alpha-2 adrenergic receptor subtypes in several different tissues, further support the existence and definition of these subtypes. The identification of a cell line for each subtype should be useful in the further study of alpha-2 adrenergic receptor subtypes.

Animals↗

PubMed1993

HT29-18-C1 intestinal cells: a new model for studying the epithelial transport of drugs.

A polarized differentiated subclone of HT29, a human colon carcinoma cell line, was used to measure the passage of drugs across the intestinal epithelium. These cells, HT29-18-C1, when grown on permeable filters, formed tight monolayers of high electrical resistance (> 400 omega cm2). Electron micrographs revealed the presence of numerous apical microvilli and well developed junctional complexes. The transepithelial passage of various drugs was studied: the transepithelial permeability coefficients ranged from 0.8 x 10(-6) cm/s for fluorescein, to 4.8 x 10(-5) cm/s for the most lipophilic molecule, testosterone. For the five compounds tested, there was a coarse correlation between the apparent transepithelial permeability coefficient and the octanol/buffer distribution coefficient. The HT29-18-C1 subclone thus represents a new in-vitro model for studying the intestinal absorption of drugs.

1-Octanol↗

PubMed2002

[Effect of Macrovipera lebetina and Cerastes cerastes venoms on adherence to integrins of cancerous cells (IGR39, HT29-D4 and IGROV1)].

In this work, we provide experimental arguments in favor of the fact that components from Macrovipera lebetina and Cerastes cerastes venoms bind to IGR39 melanoma cells but not to HT29D4 cells that derive from carcinoma adenome. Furthermore, Macrovipera lebetina and Cerastes cerastes venoms inhibit the adherence of IGR39 and HT 29-D4 to various extracellular matrix proteins. Macrovipera lebetina and Cerastes cerastes venoms did not inhibit the non specific adherence of IGR 39 cells to polylysine. In addition, binding of components from Cerastes cerastes venom to IGR39 cells is inhibited by GRGDS peptide and by monoclonal antibidy anti-av, while these two components have no effect on the adherence of IGR39 to Macrovipera lebetina venom.

Animals↗

PubMed1996

Sphingosine and its methylated derivative N,N-dimethylsphingosine (DMS) induce apoptosis in a variety of human cancer cell lines.

In the study of apoptosis initiated by various signals including ligands binding to cell membrane receptors such as Fas and TNFRI, the sphingomyelin pathway and its resulting metabolites, the sphingolipids, have been suggested to be involved in the signaling pathway. In earlier studies we presented data which indicated that sphingosine (Sph) itself was increased during apoptosis induced by phorbol myristate acetate (PMA) in HL60 cells and tumor necrosis factor (TNF) in neutrophils, and when added exogenously was able to induce apoptosis. We report here that Sph and its methylated derivative N,N,-dimethylsphingosine (DMS) are able to induce apoptosis in cancer cells of both hematopoietic and carcinoma origin. In human leukemic cell lines CMK-7, HL60 and U937, treatment with 20 microM Sph for 6 hr caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRT18, MKN74 and COLO205 were shown to be more susceptible to apoptosis upon addition of DMS (>50%) than of Sph (<50%), yet were weakly or not sensitive to N,N,N-trimethylsphingosine (TMS). Under the same conditions, in the presence of serum, neither Sph-1-phosphate nor ceramide analogues C2-, C6- or C8-ceramide were able to induce apoptosis in any cell lines. However, in the absence of serum, ceramide analogues induced apoptosis in leukemia cell lines after 18 hr, yet much less so than Sph or DMS. Furthermore, apoptosis induced by Sph or DMS could not be inhibited by the ceramide synthase inhibitor fumonisin B1. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by Sph, DMS or C2Cer was inhibited by protease inhibitors. Our data further support the evidence that the catabolic pathway of sphingomyelin involving Sph and other metabolites is an integral part of the apoptosis pathway.

Animals↗

PubMed1996

A redox-based mechanism for induction of interleukin-1 production by nitric oxide in a human colonic epithelial cell line (HT29-Cl.16E).

We evaluated the effects of two NO donors, sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1), characterized by alternative redox states, i.e. nitrosonium ion (NO+) and nitric oxide (NO.) respectively, on intracellular interleukin-1 (IL-1) production, by a human colonic epithelial cell line (HT29-Cl.16E). SNP was able to induce intracellular IL-1 alpha production up to 10 h incubation, in a dose-dependent manner. Several experiments provide evidence that the NO+ redox form, and not the free radical NO., is implicated in the IL-1 alpha production: (i) SIN-1, devoid of any NO+ character, led to a very weak IL-1 production as compared with SNP; (ii) the reductive action of a thiol such as cysteine on NO+ led to a dose-dependent increase in NO, concentration, measured as NO2-/NO3- accumulation, and to large decrease in IL-1 production. Dibutyryl cGMP had no effect on IL-1 production, this finding supporting the concept that a cGMP-independent pathway is involved in the intracellular signalling of NO+. Together these results point out that NO, depending on its redox form, is able to modulate IL-1 production in cultured colonic epithelial cells.

Cell Line↗

PubMed2004

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

Animals↗

PubMed1993

Differential regulation of carcinoembryonic antigen and biliary glycoprotein by gamma-interferon.

Carcinoembryonic antigen (CEA), biliary glycoprotein (BGP), and non-specific cross-reacting antigen (NCA) are three closely related cell surface glycoproteins induced by gamma-interferon (IFN-gamma) in colonic epithelial cells. Maximal induction of CEA by IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the colon carcinoma cell line HT-29 occurs at 5-6 days with maximal secreted levels at 14 ng/ml for IFN-gamma and 20 ng/ml for TNF-alpha. Cell viability was reduced to 67% of controls for TNF-alpha and to 36% for IFN-gamma. Dose-response curves showed maximal induction of CEA at 500 units/ml for TNF-alpha and at 200 units/ml for IFN-gamma. Combinations of the two lymphokines revealed that the CEA induction effects were additive and the cytotoxicity effects were synergistic. Northern blot analysis of HT-29 cells treated with IFN-gamma and probed with specific probes for BGP, CEA, and NCA showed a 2-fold increase in mRNA level for BGP, and a greater than 10-fold induction for CEA and NCA. Similar results were obtained for the SW403 cell line, but in the case of the LS174T cell line, CEA mRNA levels remained constant before and after IFN-gamma treatment, while BGP and NCA mRNA levels increased by 2-5-fold. Polymerase chain reaction analysis of the four alternatively spliced transcripts of BGP revealed no differential induction of one transcript over another by IFN-gamma. A comparison of the kinetics of induction of the mRNA levels for BGP and CEA by IFN-gamma in the HT29 cell line revealed a half-time of < 6 h for BGP and 48 h for CEA. The induction of CEA mRNA was completely inhibited with either cycloheximide (protein synthesis inhibitor) or actinomycin D (RNA synthesis inhibitor), but the induction of BGP mRNA was superinduced by cycloheximide. The difference in the kinetics of induction and effect of cycloheximide on CEA and BGP mRNAs suggest that the two genes are regulated differently in the same cell line. We conclude that the regulation occurs mainly at the posttranscriptional level for CEA and involves mRNA stability. BGP regulation may be more complex, involving transcriptional and posttranscriptional regulation, and more closely resembles the regulation of MHC class II mRNA by IFN-gamma in epithelial cells. The mRNA stability effects may be mediated by the dramatically different sequences present in the 3'-untranslated regions of CEA and BGP.

Antigens, CD↗

PubMed2005

Casein kinase II (CK2) enhances death-inducing signaling complex (DISC) activity in TRAIL-induced apoptosis in human colon carcinoma cell lines.

Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli, as well as being elevated in many human cancers examined. We have demonstrated that CK2 is a key survival factor that protects human colon carcinoma cells from TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. We determined that inhibition of CK2 phosphorylation events by DRB (5,6-dichlorobenzimidazole) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis, in the absence of effects in normal cells. Sensitization was caspase dependent, and independent of regulation via NF-kappaB. Further, inhibition of phosphorylation by CK2 did not modify the expression level of antiapoptotic proteins. Analysis of TRAIL-induced death-inducing signaling complex (DISC) formation demonstrated enhanced formation of the DISC, enhanced cleavage of caspase-8 and cleavage of Bid in the presence of DRB, thereby facilitating the release of proapoptotic factors from the mitochondria with subsequent downregulation of the expression of XIAP and c-IAP1. Further, silencing of CK2alpha in HT29 cells following transfection of CK2alpha shRNA abrogated CK2 kinase activity while simultaneously increasing TRAIL sensitivity. These findings demonstrate that CK2 plays a critical antiapoptotic role by conferring resistance to TRAIL at the level of the DISC.

Adenocarcinoma↗