thapsigargin and Colonic-Neoplasms

In the present study, we investigated whether celecoxib could induce the expression of NKG2D ligands in clonogenic colon cancer cells, and increase their susceptibility to NK cell-mediated cell death. Celecoxib and its non-coxib analog, 2,5-dimethyl celecoxib, induced ULBP-1 and DR5 in both COX-2 negative HCT-15 cells and COX-2 positive HT-29 cells. Celecoxib increased their susceptibility to NK92 cells in both DELFIA assay and soft agar colony forming assay. The inducibility of ULBP-1 and DR5 by celecoxib was not different between CD44- and CD44+ HCT-15 cells, and CD133- and CD133+ HT-29 cells. Celecoxib increased the susceptibility of highly clonogenic CD44+ HCT-15 and CD133+ HT-29 cells to NK92 cells, at least comparable to less clonogenic CD44- HCT-15 and CD133- HT-29 cells, respectively. In addition, celecoxib induced CHOP, and thapsigargin, an inducer of ER (endoplasmic reticulum) stress, induced DR5 but not ULBP1 in HCT-15. Taken together, these findings suggest that celecoxib induces the expression of ULBP-1 as well as DR5 in clonogenic colon cancer cells via COX-2 and ER stress-independent pathways, and increases their susceptibility to NK cells.

Topics: AC133 Antigen; Antigens, CD; Celecoxib; Colonic Neoplasms; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cytotoxicity, Immunologic; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Glycoproteins; GPI-Linked Proteins; HT29 Cells; Humans; Hyaluronan Receptors; Intracellular Signaling Peptides and Proteins; Killer Cells, Natural; Peptides; Pyrazoles; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sulfonamides; Thapsigargin; Transcription Factor CHOP

GRP78, a major endoplasmic reticulum chaperone and signaling regulator, is commonly overexpressed in cancer. Moreover, induction of GRP78 by a variety of anti-cancer drugs, including histone deacetylase inhibitors, confers chemoresistance to cancer, thereby contributing to tumorigenesis. Thus, therapies aimed at decreasing GRP78 levels, which results in the inhibition of tumor cell proliferation and resensitization of tumor cells to chemotherapeutic drugs may hold promise for cancer treatment. Despite advances in our understanding of GRP78 actions, little is known about endogenous inhibitors controlling its expression. As endogenous regulators, microRNAs (miRNAs) play important roles in modulating gene expression; therefore, we sought to identify miRNA(s) that target GRP78, under the hypothesis that these miRNAs may serve as therapeutic agents. Here, we report that three miRNAs (miR-30d, miR-181a, miR-199a-5p) predicted to target GRP78 are down-regulated in prostate, colon and bladder tumors, and human cancer cell lines. We show that in C42B prostate cancer cells, these miRNAs down-regulate GRP78 and induce apoptosis by directly targeting its 3' untranslated region. Importantly, we demonstrate that the three miRNAs act cooperatively to decrease GRP78 levels, suggesting that multiple miRNAs may be required to efficiently control the expression of some genes. In addition, delivery of multiple miRNAs by either transient transfection or lentivirus transduction increased the sensitivity of cancer cells to the histone deacetylase inhibitor, trichostatin A, in C42B, HCT116 and HL-60 cells. Together, our results indicate that the delivery of co-transcribed miRNAs can efficiently suppress GRP78 levels and GRP78-mediated chemoresistance, and suggest that this strategy holds therapeutic potential.

Topics: 3' Untranslated Regions; Adenocarcinoma; Animals; Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Heat-Shock Proteins; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Lentivirus; Male; Mice; Mice, Nude; MicroRNAs; Neovascularization, Pathologic; Prostatic Neoplasms; RNA; RNA, Messenger; Thapsigargin; Transcription, Genetic; Transfection; Tumor Stem Cell Assay; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.. Cl(-) secretion was measured as changes in short-circuit current across voltage-clamped T(84) colonic epithelial cells. Protein expression was measured by western blotting and intracellular Ca(2+) levels by Fura-2 fluorescence.. While acute (15 min) treatment of T(84) cells with a cholinergic G(q) PCR agonist, carbachol (CCh), rapidly stimulated Cl(-) secretion, subsequent CCh-induced responses were attenuated in a biphasic manner. The first phase was transient and resolved within 6 h but this was followed by a chronic phase of attenuated responsiveness that was sustained up to 48 h. CCh-pretreatment did not chronically alter responses to another G(q)PCR agonist, histamine, or to thapsigargin or forskolin which elevate intracellular Ca(2+) and cAMP, respectively. This chronically acting antisecretory mechanism is not shared by neurotransmitters that activate G(s)PCRs. Conditioned medium from CCh-pretreated cells mimicked its chronic antisecretory actions, suggesting involvement of an epithelial-derived soluble factor but further experimentation ruled out the involvement of epidermal growth factor receptor ligands. Acute CCh exposure did not chronically alter surface expression of muscarinic M(3) receptors but inhibited intracellular Ca(2+) mobilization upon subsequent agonist challenge.. These data reveal a novel, chronically acting, antisecretory mechanism that downregulates epithelial secretory capacity upon repeated G(q)PCR agonist exposure. This mechanism involves release of a soluble factor that uncouples receptor activation from downstream prosecretory signals.

Topics: Adenocarcinoma; Calcium; Carbachol; Cell Line, Tumor; Chlorides; Colforsin; Colonic Neoplasms; Epithelial Cells; Histamine; Humans; Patch-Clamp Techniques; Protein Kinase C; Receptor, Muscarinic M3; Receptors, G-Protein-Coupled; Thapsigargin

p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity.

Topics: Active Transport, Cell Nucleus; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Drug Evaluation, Preclinical; Ellipticines; Green Fluorescent Proteins; Humans; Leupeptins; Osteosarcoma; Recombinant Fusion Proteins; Thapsigargin; Tumor Suppressor Protein p53

N,N-dimethyl-D-erythro-sphingosine (DMS), an N-methyl derivative of sphingosine, is an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In previous reports, DMS-induced intracellular Ca2+ increase concentration ([Ca2+]i) was studied in T lymphocytes, monocytes, astrocytes and neuronal cells. In the present study, we studied DMS-induced increase of [Ca2+]i in HCT116 human colon cancer cells. We found that the DMS-induced increase of [Ca2+]i in colon cancer cells is composed of Ca2+ release from intracellular Ca2+ stores and subsequent Ca2+ influx. The Ca2+ release is not related to modulation of inositol 1,4,5-trisphosphate (IP3) receptor or ryanodine receptor. On the other hand, the Ca2+ influx is mediated largely through Ca2+ channels sensitive to verapamil, nifedipine, Ga3+, and La3+. Furthermore, we found that the response is inhibited by bepridil and Ni2+, specific inhibitors of Na+-Ca2+-exchanger, suggesting involvement of Na+-Ca2+ exchanger in the DMS-induced [Ca2+]i increase in colon cancer cells. This inhibition was also observed in U937 monocytes, but not in 1321N1 astrocytes.

Topics: Calcium; Calcium Channel Blockers; Chelating Agents; Colonic Neoplasms; Egtazic Acid; Enzyme Inhibitors; Flow Cytometry; HCT116 Cells; Humans; Inositol 1,4,5-Trisphosphate Receptors; Ryanodine Receptor Calcium Release Channel; Sodium-Calcium Exchanger; Sphingosine; Thapsigargin

Glucose regulated protein-78, GRP78 has been implicated in the protection of tumor cells from cytotoxic damage and apoptosis. When protein profiles of colon cell lines were investigated we found remarkably high GRP78 expression in two cell lines. These cell lines express elevated levels of the transcription factor c-Myb due to genomic amplification of the c-myb locus and we hypothesized that c-Myb regulates GRP78 expression in colon cancer cells. The promoters of human and murine GRP78 and the related family member GRP94 were examined and potential c-Myb binding sites were identified and characterized. DNA binding studies with recombinant c-Myb and nuclear extracts together with ChIP assays on colon cell lines validated these sites. Endogenous GRP78 expression was further induced in these colon cells in response to Thapsigargin treatment, a potent inducer of the unfolded protein response. Transactivation studies with the human GRP78 promoter in colon cell lines showed reporter activity was dependent upon the presence of a conserved c-Myb binding site independent of sequences associated with the unfolded protein response. Finally, over-expression of c-Myb induced the endogenous GRP78 gene. These data suggest that amplification of c-myb in tumor cells may lead to robust GRP78 gene induction, which may in turn assist cells in survival under conditions of oxygen deprivation and nutrient stress.

Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Cell Line, Tumor; Colonic Neoplasms; Electrophoresis, Gel, Two-Dimensional; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Genes, myb; Heat-Shock Proteins; Humans; Molecular Chaperones; Molecular Sequence Data; Promoter Regions, Genetic; Thapsigargin; Transcriptional Activation

Bax is a crucial mediator of the mitochondrial pathway for apoptosis, and loss of this proapoptotic Bcl-2 family protein contributes to drug resistance in human cancers. We report here that the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (THG) induces apoptosis of human colon cancer HCT116 cells through a Bax-dependent signaling pathway controlling the cytosolic release of mitochondrial apoptogenic molecules. Treating HCT116 cells with THG results in caspase-8 activation; Bid cleavage; Bax conformational change and mitochondrial translocation; the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 into the cytosol; caspase-3 activation; and apoptosis. In contrast, knockout of Bax completely abrogates the full processing/activation of caspase-3 but has no effect on the processing of caspase-8 and the initial cleavage of caspase-3 to p24 fragment after THG treatment. The caspase-8-specific inhibitor z-IETD-fmk, as well as pan-caspase inhibitor z-VAD-fmk, but not the calpain inhibitor E-64d, prevents Bid cleavage, Bax conformational change, and subsequent caspase-3 processing and apoptosis. Caspase-8 processing is dependent on de novo protein synthesis; DR5 expression is strongly up-regulated by THG treatment. Moreover, the absence of Bax blocks THG-induced Omi and Smac release from mitochondria, and expression of cytosolic Omi (GFP-IETD-Omi) or Smac (GFP-IETD-Smac) restores the sensitivity of Bax-knockout HCT116 cells to apoptosis in response to THG treatment. Taken together, our results indicate that Bax-dependent Smac and Omi release plays an essential role in caspase-3 activation and apoptosis induced by THG in human colon cancer HCT116 cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Calpain; Carrier Proteins; Caspase 8; Caspase 9; Caspases; Colonic Neoplasms; Enzyme Activation; Enzyme Inhibitors; High-Temperature Requirement A Serine Peptidase 2; Humans; Intracellular Signaling Peptides and Proteins; Mitochondria; Mitochondrial Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Serine Endopeptidases; Signal Transduction; Thapsigargin; Transfection; Tumor Cells, Cultured

Thapsigargin (TG), by inducing perturbations in cellular Ca(2+) homeostasis, can induce apoptosis, but the molecular mechanisms remain to be fully elucidated. We have recently reported that TG-induced apoptosis appears to involve the DR5-dependent apoptotic pathway that cross talks with the mitochondrial pathway via TG-induced Bid cleavage. In this study, we have utilized Bax-proficient and -deficient HCT116 human colon cancer cells to investigate the effect of Bax deficiency on TG-induced apoptosis and TG regulation of the DR5 and mitochondrial pathways. Our results indicate that Bax-deficient cells are less sensitive to undergo apoptosis following TG treatment. Our results further demonstrate that TG-induced apoptosis is coupled with DR5 upregulation and caspases 8 and 3 activation, as well as Bid cleavage in both Bax-proficient and -deficient cells, although caspase 3 activation was reduced in Bax-deficient cells. TG also promoted the release of cytochrome c into cytosol and caspase 9 activation in Bax-proficient cells but not in Bax-deficient cells. These findings suggest that although Bax is not absolutely required for death receptor (DR)-dependent signals, it appears to be a key molecule in TG-regulated mitochondrial events. Bax-deficient cells were relatively more resistant to Apo2L/TRAIL than the Bax-proficient counterparts. However, the combination of Apo2L/TRAIL and TG was more effective in mediating apoptosis in both Bax-proficient and -deficient cells and that was coupled with activation of caspases 8 and 3. Although both agents in combination also induced cytochrome c release into cytosol and caspase 9 activation in Bax-proficient cells, these events were abrogated in Bax-deficient cells. Our results thus suggest that the combination of Apo2L/TRAIL and TG appears to bypass the Bax deficiency-induced defects in the mitochondrial (intrinsic) pathway by engaging the DR5-dependent apoptotic signals (extrinsic pathway).

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Calcium; Carcinogens; Colonic Neoplasms; Endoplasmic Reticulum; Humans; Membrane Glycoproteins; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Thapsigargin; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Interleukin 1 beta (IL-1 beta) levels are elevated in the colonic mucosa of patients with ulcerative colitis (UC). We propose that IL-1 beta may also be elevated in the circular muscle layer of the colon and may be partially responsible for the motility dysfunction observed in patients with UC.. Cohort analytic study.. Research laboratory in a tertiary academic medical center.. Normal smooth muscle was obtained from the disease-free margins of human sigmoid colon specimens resected from patients with cancer and compared with specimens from patients with UC.. An enzyme-linked immunosorbent assay was used to measure IL-l beta. Standard muscle chambers were used to measure force changes. Single muscle cells were isolated by enzymatic digestion, and cell shortening in response to neurokinin A (NKA) and thapsigargin was measured under a microscope. Cytosolic Ca(2+) (calcium) concentrations were measured by standard techniques.. Effects of IL-1 beta on smooth muscle function in normal and UC colons.. In patients with UC, IL-1 beta was elevated in the muscularis propria, and sigmoid circular smooth muscle contractions in response to NKA and thapsigargin were significantly reduced. In fura-2-loaded cells from patients with UC, the NKA-induced Ca(2+) signal was also significantly reduced in Ca(2+)-free medium, indicating the reduced intracellular Ca(2+) stores after UC. Exposure of normal cells to IL-1 beta mimicked the changes observed in patients with UC. An IL-1 beta-induced reduction in contraction and release of intracellular Ca(2+) in response to NKA was partially restored by the hydrogen peroxide scavenger catalase.. In patients with UC, IL-1 beta was increased in colonic circular muscles and may contribute to motor dysfunction after UC through production of hydrogen peroxide.

Topics: Adenosine Triphosphate; Calcium; Colitis, Ulcerative; Colon; Colon, Sigmoid; Colonic Neoplasms; Cytosol; Enzyme-Linked Immunosorbent Assay; Gastrointestinal Motility; Humans; Hydrogen Peroxide; In Vitro Techniques; Interleukin-1; Muscle Contraction; Muscle, Smooth; Neurokinin A; Thapsigargin

Studies with mouse embryo fibroblasts deficient for the BCL-2 family multidomain proapoptotic proteins BAX and BAK have revealed that both of these proteins are essential for apoptosis induced by multiple stimuli, suggesting that these proapoptotic proteins are functionally overlapping in these cells [M. C. Wei et al., Science (Wash. DC), 292: 727-730, 2001; W. X. Zong et al., Genes Dev., 15: 1481-1486, 2001]. We have determined the effect of several different apoptotic stimuli in a Bax-deficient human epithelial cancer cell line (HCT116BaxKO). We show that this cell line expresses functional BAK protein and is defective in manifestation of apoptosis induced by the BH3-only proteins BIK and BID as well as extrinsic stimuli that engage the death receptors, tumor necrosis factor receptor, tumor necrosis factor-related apoptosis-inducing ligand receptor, and Fas. In addition, this cell line is deficient for apoptosis induced by cytotoxic agents such as UV, staurosporine, and thapsigargin that induce either mitochondrial or endoplasmic reticulum stress. Our results suggest that BAX plays a critical role in the manifestation of apoptosis paradigms induced by multiple stimuli in human epithelial cancer cells. Our results also suggest that the integrity of BAX may have important consequences in the progression of epithelial tumors and in determining the outcome of chemotherapeutic regimens of such tumors.

Topics: Antibodies; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Carcinoma; Colonic Neoplasms; Epithelial Cells; fas Receptor; Humans; Membrane Glycoproteins; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Staurosporine; Thapsigargin; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation and angiogenesis in colorectal cancer. We examined a possible involvement of store-operated calcium (SOC) entry in human colon carcinoma cells (HRT-18), which require calcium for proliferation. Acetyl-salicylic-acid (ASA), mefenamic acid (MEF) and sulindac sulfide (SUS) inhibited cell proliferation with the following order of potency: SUS > MEF >> ASA. SUS but not MEF and ASA induced apoptosis following low-dose treatment. Furthermore, SUS and MEF significantly altered the cell cycle distribution. The ability of NSAIDs to inhibit SOC entry was assessed by measuring the intracellular calcium concentration ([Ca2+]i) in response to calcium store depletion using the endoplasmic calcium ATPase inhibitor thapsigargin. SUS and MEF, but not ASA significantly inhibited SOC entry. A causal link between SOC entry inhibition and anti-proliferative activity was tested using the inorganic SOC entry inhibitor La3+ and the specific organic inhibitor N-1-n-octyl-3,5-bis-(4-pyridyl)triazole (DPT). Both La3+ and DPT inhibited cell proliferation and SOC entry. Analogous to MEF, the anti-proliferative effect of DPT was mediated by cell cycle arrest and not by induction of apoptosis. These data indicate a role of SOC entry for cell proliferation in cancer cells and suggest a novel anti-proliferative NSAID mechanism in addition to its known influence on lipid metabolism.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Calcium; Cell Cycle; Cell Division; Cell Separation; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Flow Cytometry; Humans; Inhibitory Concentration 50; Lanthanum; Lipid Metabolism; Mefenamic Acid; Pyridines; Sulindac; Thapsigargin; Time Factors; Triazoles; Tumor Cells, Cultured

IL-8 is a chemokine that activates and recruits neutrophils and plays a major role in intestinal inflammation. Signal transduction pathways mediated by protein kinases are central in regulating IL-8 gene expression, however, little is known about the role of Ca2+ in this event. In this study, we characterize the effect of intracellular Ca2+ on interleukin-8 gene expression in T84 human colonic epithelial cells.. Cells were stimulated with Ca2+ ionophore, A23187 or thapsigargin, a Ca2+-ATPase inhibitor. Semi-quantitative RT-PCR was used to examine IL-8 mRNA and ELISA for protein quantification. Reporter gene techniques were used to determine transcription rate.. A23187 and thapsigargin caused a dose- and time-dependent accumulation of IL-8 mRNA and protein production which was dependent on the release of Ca2+ from intracellular stores. FK506, a specific inhibitor of calcineurin, inhibited A23187- and thapsigargin-induced IL-8 mRNA expression in a dose dependent manner. Reporter gene studies and actinomycin D chase experiments showed that A23187 and thapsigargin enhanced IL-8 gene transcription and stabilized IL-8 mRNA transcripts, respectively.. Intracellular Ca2+ plays an important role in regulating IL-8 transcriptionally and posttranscriptionally through calcium/calmodulin-dependent calcineurin.

Topics: Adenocarcinoma; Calcimycin; Calcineurin; Calcium; Colon; Colonic Neoplasms; Dose-Response Relationship, Drug; Epithelial Cells; Gene Expression; Humans; Interleukin-8; Ionophores; Kinetics; Protein Kinase C; RNA, Messenger; Tetradecanoylphorbol Acetate; Thapsigargin; Transcription, Genetic; Tumor Cells, Cultured

Regulation of capacitative Ca(2+) entry was studied in two different multidrug resistance (MDR) protein (MRP1) overexpressing cell lines, HT29(col) and GLC4/ADR. MRP1 overexpression was accompanied by a decreased response to thapsigargin. Moreover, inhibition of capacitative Ca(2+) entry by D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was abolished in MRP1 overexpressing cells. Both PDMP and the MRP1 inhibitor MK571 greatly reduced InsP(3)-mediated (45)Ca(2+) release from intracellular stores in HT29 cells. Again, these effects were virtually abolished in HT29(col) cells. Our results point to a modulatory role of MRP1 on intracellular calcium concentration ([Ca(2+)](i)) homeostasis which may contribute to the MDR phenotype.

Topics: Adenocarcinoma; ATP-Binding Cassette Transporters; Calcium; Calcium Radioisotopes; Colonic Neoplasms; Gene Expression; Homeostasis; Humans; Inositol 1,4,5-Trisphosphate; Morpholines; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; Thapsigargin; Tumor Cells, Cultured

N(omega)-nitro-L-arginine (LNNA) inhibits the synthesis of heat shock proteins in animals and cultured cells exposed to heat stress. Heat shock protein synthesis is known to be Ca2+-dependent. In this study, we have characterized the effect of LNNA on [Ca2+]i before and after heat stress in human colon carcinoma T84 cells. In untreated cells incubated in the presence of external Ca2+, the resting [Ca2+]i was 201+/-3 nM. If these cells were exposed to 45 degrees C for 10 min, [Ca2+]i increased by 50+/-2%. Preincubation with LNNA (100 microM) without subsequent heating led to a decrease in [Ca2+]i in a LNNA concentration-dependent manner. Preincubation with LNNA followed by heating increased [Ca2+]i to levels 88+/-5% greater than cells heated without LNNA pretreatment. Incubating cells in medium without external Ca2+ (no heating, no LNNA treatment) lowered resting [Ca2+]i to 115+/-2 nM and greatly reduced the increase in [Ca2+]i observed if cells were heated in the presence of Ca2+, indicating that external Ca2+ plays an important role in the maintenance of [Ca2+]i in T84 cells. With external Ca2+ absent, LNNA pretreatment further reduced [Ca2+]i in unheated cells, and heating failed to enhance [Ca2+]i. We determined (with external Ca2+ present) that the heat-stress induced increase in [Ca2+]i in T84 cells was blocked by dichlorobenzamil, a Na+/Ca2+ exchanger inhibitor, suggesting that the exchanger mediates Ca2+ entry. The median inhibitory concentration (IC50) in cells not treated with LNNA was 0.970+/-0.028 microM. With LNNA pretreatment, the IC50 was 5.099+/-0.107 microM. Heat stress of T84 cells did not affect the binding affinity of the Na+/Ca2+ exchanger for external Ca2+, but it increased the maximal velocity of the exchanger. In unheated cells, preincubation with LNNA decreased the binding affinity of the exchanger for Ca2+, but after heat treatment, both the binding affinity and maximal velocity of the exchanger increased. Our data are consistent with the idea that LNNA affects the activity of the Na+/Ca2+ exchanger. We also determined there are intracellular Ca2+ pools in T84 cells sensitive to thapsigargin, monensin, and ionomycin. Treatment with TMB-8, a blocker of Ca2+ sequestration and mobilization, or ionomycin inhibited the LNNA-induced decrease in [Ca2+]i observed in the absence of external Ca2+, suggesting that LNNA promotes Ca2+ sequestration.

Topics: Amiloride; Calcium; Cell Compartmentation; Cell Survival; Colonic Neoplasms; Cytosol; Enzyme Inhibitors; Heat-Shock Response; Humans; Ionophores; Monensin; Nitric Oxide Synthase; Nitroarginine; Sodium-Calcium Exchanger; Thapsigargin; Tumor Cells, Cultured

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl- secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl- conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I- > Cl-; for forskolin, Cl- > I-). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I- > Cl-). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anions; Bumetanide; Calcium; Cell Membrane; Cell Polarity; Charybdotoxin; Chloride Channels; Colforsin; Colonic Neoplasms; Cyclic AMP; Cytosol; Humans; Kinetics; Membrane Potentials; Models, Biological; Thapsigargin; Tumor Cells, Cultured

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Topics: Calcimycin; Calcium; Carcinoma; Cations, Divalent; Colonic Neoplasms; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Half-Life; Humans; Membrane Proteins; RNA, Messenger; Terpenes; Thapsigargin; Transcription, Genetic; Tumor Cells, Cultured

1. Thapsigargin, a sesquiterpene lactone, was shown to cause electrogenic anion secretion in monolayers of human colonic epithelial cells, an effect which was crucially dependent upon calcium and did not involve eicosanoid formation. 2. To measure the secretory effect calcium needed to be present in the external bathing solution. By means of Fura-2 fluorescence measurements thapsigargin was shown to raise Cai by around 250 nM when the bathing solution contained calcium. In the nominal absence of external calcium thapsigargin raised Cai by only 60 nM, but from a lower basal value. This was insufficient to cause secretion. 3. Effects of other calcium-dependent secretagogues (e.g. lysylbradykinin) were inhibited in the presence of thapsigargin, whereas kinin responses were potentiated if the peptide was added following a stimulus which increases cyclic AMP. 4. From the data given here and the known behaviour of colonic epithelia it is concluded that thapsigargin increases Cai by a non-ionophoric mechanism by release from internal stores. Calcium-stimulated calcium influx then follows resulting in the opening of basolateral K channels, increasing the electrochemical gradient for chloride efflux, or alternatively by activating anion channels in the apical membrane. It is concluded that thapsigargin is a potentially important tool for examining epithelial mechanisms.

Topics: Adenocarcinoma; Anions; Calcimycin; Calcium; Colforsin; Colonic Neoplasms; Electrophysiology; Epithelium; Humans; Plant Extracts; Thapsigargin; Tumor Cells, Cultured