Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line

Summary Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheat-germ agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(1–3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosac-charide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Characterization of a human colonic adenocarcinoma cell line,LS123

Summary An epithelial cell line, LS123, was established in 1974 from the second in a series of three primary colonic tumors resected from a Caucasian female. The cell line is aneuploid, releases low concentrations of carcinoembryonic antigen (CEA), fails to grow progressively in nude mice, and forms colonies only in enriched semisolid medium developed for tumor stem cells. LS123 cells grow on confluent cell monolayers and in either low serum or serum-free medium. In the chick embryonic skin assay, LS123 cells grew as a well-differentiated abnormal colonic epithelium with little mitotic activity but with some indication of invasion. On floating collagen gels LS123 cells formed a one to three-cell-layer-thick undifferentiated epithelial sheet. The apparent low invasiveness of the cells of this line is supported by the patient's history of three primary colon tumors without systemic metastases during the past 30 yr. Therefore, although LS123 cells possess several properties associated with neoplasia, they have little invasive potential. Thus, LS123 cells may represent an important model for the study of human colon cancer. Presented in part at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, CA, June 6–10, 1982. The work has been partially supported by U.S. Public Health Service Grants CA23871 (L. P. R.), CA24024 and RCDAK04-CA00579 (B. H. T.), and CA27124 and CA22370 (B. D. K.); the latter was awarded through the National Large Bowel Cancer Project.     

The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues.

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.     

Inhibition of plasminogen activator secretion by cyclic AMP in a macrophage-like cell line.

The continuous cell line, J774.2, exhibits many macrophage-like functions such as latex and Fc-mediated phagocytosis, antibody mediated phagocytosis, antibody mediated cytotoxicity, chemotaxis, and lysozyme secretion. Cyclic AMP stimulates Fc-mediated phagocytosis and inhibits the growth of J774.2. To further evaluate the relationship between cyclic AMP and the specialized functions exhibited by these cells. Variants deficient in phagocytosis, adenylate cyclase and cyclic AMP-dependent protein kinase were derived. We have now shown that J774.2 also secretes plasminogen activator and that this secretion is rapidly and specifically inhibited by 8-bromoadenosine 3':5'-cyclic monophosphoric acid (8 Br--cAMP) or cholera toxin under conditions where lysozyme secretion is unaltered. Utilizing protein kinase-deficient variants, the ability of cyclic AMP to inhibit plasminogen activator secretion was shown to be mediated by a cyclic AMP-dependent protein kinase. We conclude that cyclic AMP has diametrically opposing effects on two macrophage-like functions: Fc-mediated phagocytosis and plasminogen activator secretion.     

G protein control of potassium channel activity in a mast cell line

Using the patch-clamp technique, we studied regulation of potassium channels by G protein activators in the histamine-secreting rat basophilic leukemia (RBL-2H3) cell line. These cells normally express inward rectifier K+ channels, with a macroscopic whole-cell conductance in normal Ringer ranging from 1 to 16 nS/cell. This conductance is stabilized by including ATP or GTP in the pipette solution. Intracellular dialysis with any of three different activators of G proteins (GTP gamma S, GppNHp, or AlF-4) completely inhibited the inward rectifier K+ conductance with a half-time for decline averaging approximately 300 s after "break-in" to achieve whole-cell recording. In addition, with a half-time averaging approximately 200 s, G protein activators induced the appearance of a novel time-independent outwardly rectifying K+ conductance, which reached a maximum of 1-14 nS. The induced K+ channels are distinct from inward rectifier channels, having a smaller single-channel conductance of approximately 8 pS in symmetrical 160 mM K+, and being more sensitive to block by quinidine, but less sensitive to block by Ba2+. The induced K+ channels were also highly permeable to Rb+ but not to Na+ or Cs+. The current was not activated by the second messengers Ca2+, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, or by cyclic AMP-dependent phosphorylation. Pretreatment of cells with pertussis toxin (0.1 microgram/ml for 12-13 h) prevented this current's induction both by guanine nucleotides and aluminum fluoride, but had no effect on the decrease in inward rectifier conductance. Since GTP gamma S is known to stimulate secretion from patch-clamped rat peritoneal mast cells, it is conceivable that K+ channels become inserted into the plasma membrane from secretory granules. However, total membrane capacitance remained nearly constant during appearance of the K+ channels, suggesting that secretion induced by GTP gamma S was minimal. Furthermore, pertussis toxin had no effect on secretion triggered by antigen, and triggering of secretion before electrical recording failed to induce the outward K+ current. Finally, GTP gamma S activated the K+ channel in excised inside-out patches of membrane. We conclude that two different GTP-binding proteins differentially regulate two subsets of K+ channels, causing the inward rectifier to close and a novel K+ channel to open when activated.     

Inhibition of macrophage activation by calcium channel blockers and calmodulin antagonists

The biochemical mechanisms by which macrophages become activated to the tumoricidal state are poorly understood. To investigate the role of calcium in this process, the effect of calcium channel blockers and calmodulin antagonists on the acquisition of tumoricidal properties by macrophages activated by a number of different agents was examined. Activation of thioglycollate-stimulated C57BL/6 mouse peritoneal macrophages by macrophage activation factor (MAF) plus LPS, IFN-gamma plus LPS or the calcium ionophore, A23187, was inhibited in a dose-dependent fashion by the calcium channel blockers nifedipine and verapamil. These agents blocked the influx of 45Ca into macrophages activated by MAF plus LPS. Macrophage activation was also inhibited by chlorpromazine, W-7, and calmidazolium at concentrations known to perturb calmodulin function. The data suggest that activation of macrophages to the tumoricidal state is a calcium-dependent process involving the participation of calcium-regulated biochemical reactions whose activities can be modulated by pharmacological agents that frustrate transmembrane calcium fluxes and/or inhibit calmodulin function.     

The role of intracellular lumina in the repolarization process of a colonic adenocarcinoma cell line

When glucose is added to the culture medium, some cells of the undifferentiated HT-29 line derived from a human colonic adenocarcinoma develop spherical structures, demonstrated to be intracellular by the ruthenium red staining method, which are bordered with microvilli, contain osmiophilic substances and resemble intracellular lumina. When glucose is replaced by galactose in the culture medium, the cells differentiate apical membranes bordered with microvilli. Our observations suggest that these new apical membranes correspond to the membranes of intracellular lumina which have opened outside the cells. We suggest that intracellular lumina may represent "compensation" for loss of polarity of epithelial cells and may be an important step in the repolarizing process of the cells.     

Inhibition of enterotoxin-induced porcine colonic secretion by diarylsulfonylureas in vitro

Muscle-stripped piglet colon was used to evaluate changes in short-circuit current (I(sc)) as an indicator of anion secretion. Mucosal exposure to Escherichia coli heat-stable (STa) or heat-labile enterotoxins (LT) stimulated I(sc) by 32 +/- 5 and 42 +/- 7 microA/cm(2), respectively. Enterotoxin-stimulated I(sc) was not significantly affected by either 4,4'-diaminostilbene-2, 2'-disulfonic acid or CdCl(2), inhibitors of Ca(2+)-activated Cl(-) channels and ClC-2 channels, respectively. Alternatively, N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), a compound that inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion, reduced I(sc) by 29 +/- 7 and 34 +/- 11 microA/cm(2), respectively. Two additional diarylsulfonylurea (DASU)-based compounds were evaluated for their effects on enterotoxin-stimulated secretion. The rank order of potency for inhibition by these three closely related DASU structures was identical to that observed for human CFTR. The degree of inhibition by each of these compounds was similar for both STa and LT. The structure- and concentration-dependent inhibition shown indicates that CFTR mediates both STa- and LT-stimulated colonic secretion. Similar structure-dependent inhibitory effects were observed in forskolin-stimulated rat colonic epithelium. Thus DASUs compose a family of inhibitors that may be of therapeutic value for the symptomatic treatment of diarrhea resulting from a broad spectrum of causative agents across species.     

Activation of phosphatidylinositol turnover by neurotensin receptors in the human colonic adenocarcinoma cell line HT29

Association of neurotensin to its receptor in HT29 cells increases the intracellular concentration of inositol phosphates. A rapid (20–30 s), transient stimulation of inositol trisphosphate (275% of the basal level) and inositol bisphosphate (420%) is first observed, followed by a slower, stable increase in inositol monophosphate (170%). Half-maximal stimulation of the three inositol phosphates was obtained with 50–100 nM neurotensin. These results indicate that neurotensin is able to regulate intracellular Ca²⁺ levels in HT29 cells by using inositol trisphosphate as a second messenger.     

Protein kinase-A-mediated secretion of mucin from human colonic epithelial cells

Both Ca(2+)- and cAMP-mediated second messenger cascades acutely regulate mucin secretion from human colonic epithelial cells. To better understand the cAMP-dependent regulation of mucin secretion we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms in mucus-secreting T84 cells, and determined which of these isoforms is responsible for agonist-stimulated mucin secretion. Our results show the presence of both type I and type II PKA in cells that also contain large mucin granules. Forskolin caused a rapid and sustained increase in PKA activity that reached a maximum 5-10 min following its addition. Secretion of mucin was detected 15 min following exposure to forskolin, and continued to increase for a further 15 min before reaching a plateau. Mucin secretion was also measured in the presence of combinations of site-selective cAMP analog pairs, which preferentially activate either type I or type II PKA. Similar levels of mucin secretion were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase mucin secretion. Thus, activation of either type I or type II PKA is able to maximally stimulate secretion of mucins from T84 human colonic epithelial cells.     

Basal lamina and metastatic potential of two tumorigenic clones from a human colonic adenocarcinoma cell line

We isolated from a human colonic adenocarcinoma cell line two clones with highly different metastatic abilities. One of them, which spreads rapidly in culture, produces, when injected in immunosuppressed newborn rats, well differentiated epithelial like tumors limited by a continuous basal lamina and never produces lung metastasis. The other clone, which spreads slowly in culture, produces undifferentiated tumors of irregular shape and with usually no basal lamina; tumor cells are often dispersed in the stroma and metastases are observed in the lungs. These two clones may hence constitute a model for the study of the link between the presence or absence of a basal lamina in human tumors and their ability to metastasize.     

Iron depletion decreases proliferation and induces apoptosis in a human colonic adenocarcinoma cell line, Caco2

Iron is essential for maintaining cellular metabolism of most organisms. Iron chelators such as desferrioxamine have been used clinically in the treatment of iron overload diseases. In the present study, we used human colon adenocarcinoma cells as a proliferating cell model to validate that desferrioxamine inhibits cell proliferation and induces apoptosis. Proteomic analysis revealed that proteins involved in cell proliferation, signal transduction, metabolism and protein synthesis were significantly regulated by the availability of iron, rendering a close correlation between cell apoptosis and the disturbance of mitochondrial, signaling and metabolic pathways. These results provide new insights into the mechanisms of cell proliferation inhibition attributed to iron depletion.     

Inhibition of TASK-1 potassium channel by phospholipase C

Thetwo-pore-domain K⁺ channel, TASK-1, was recently shown tobe a target of receptor-mediated regulation in neurons and in adrenalglomerulosa cells. Here, we demonstrate that TASK-1 expressed inXenopus laevis oocytes is inhibited by differentCa²⁺-mobilizing agonists. Lysophosphatidic acid, via itsendogenous receptor, and ANG II and carbachol, via their heterologouslyexpressed ANG II type 1a and M₁ muscarinic receptors,respectively, inhibit TASK-1. This effect can be mimicked by guanosine5'-O-(3-thiotriphosphate), indicating the involvementof GTP-binding protein(s). The phospholipase C inhibitor U-73122reduced the receptor-mediated inhibition of TASK-1. Downstream signalsof phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmicCa²⁺ concentration, and diacylglycerol) do not mediate theinhibition. Unlike the G_q-coupled receptors, stimulation ofthe G_i-activating M₂ muscarinic receptorcoexpressed with TASK-1 results in an only minimal decrease of theTASK-1 current. However, additional coexpression of phospholipaseC-₂ (which is responsive also to G_i-subunits) renders M₂ receptor activation effective.This indicates the significance of phospholipase C activity in thereceptor-mediated inhibition of TASK-1.

     

Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549

Active oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves-tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K⁺ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K⁺) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K⁺ channels are activated following reoxygenation but not during the hypoxia phase. The K⁺ currents decayed with time following reoxygenation. The decay characteristics of the K⁺ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K⁺ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K⁺ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K⁺ currents. © 1993 Wiley-Liss, Inc.     

Effects of calcium channel blockers on potassium homeostasis.

The known effects of calcium channel blockers on various aspects of potassium homeostasis are reviewed. Regulation of potassium homeostasis requires both renal and external handling mechanisms. Signaling by calcium appears to mediate both of these. Calcium channels have been identified in adrenal glomerulosa cells, and cellular calcium entry has been demonstrated in vitro to be necessary for the synthesis and secretion of aldosterone. Calcium channel antagonists such as verapamil and nifedipine, at pharmacologic doses, can block aldosterone production. In vivo, however, only chronic administration of verapamil appears to attenuate aldosterone responsiveness to angiotensin II. Chronic administration of nifedipine does not have a dramatic effect on aldosterone production following potassium loading. Other studies have demonstrated improved extrarenal potassium disposal following treatment with calcium channel blocking agents. Clinically, there are no reports of either hyperkalemia or hypokalemia with the routine therapeutic use of these agents given alone. This review was prompted by the development of hyperkalemia in a patient with chronic renal failure following the initiation of therapy with the calcium channel blocker diltiazem: however, numerous other etiologies may also have contributed to the development of hyperkalemia in this case. Review of the data indicates that current evidence implicating this class of drugs in the pathogenesis of disordered potassium regulation remains equivocal.     

Effects of anion channel blockers NPPB and DIDS on tobacco pollen tube growth and its mitochondria state

The influence of anion channel blockers NPPB and DIDS on pollen tube growth and its mitochondria functioning was studied by means of fluorescence microscopy and flow cytometry. NPPB (40 μM) blocked pollen tube growth completely, but didn’t change its diameter. DIDS (20–80 μM) caused pollen tube swelling and bursting, suggesting that DIDS-sensitive channels take part in the regulation of pollen tube osmotic balance. The osmotic effect of low DIDS concentration (20 μM) wasn’t accompanied by changes in the tube growth rate. The mapping of plasma membrane potential of pollen tubes using Di-4-ANEPPS revealed the involvement of NPPB-sensitive but not DIDS-sensitive anion channels in the maintenance of the longitudinal membrane potential gradient along the tube surface. The study of isolated pollen mitochondria showed that DIDS increased their capacity to take up potential-dependent dye DiOC₅(3), i.e. caused hyperpolarization of mitochondrial membranes. At the same time DIDS influenced on intramitochondrial ROS content and ROS release from mitochondria. Thus, NPPB and DIDS in different ways influenced on plasma membrane potential distribution along pollen tube, on its osmotic balance, and on mitochondria functioning. This set of data suggests that pollen tube growth is dependent on activity of anion channels that differ in localization and functions.     

Histochemical alterations of mucin in normal colon, inflammatory bowel disease and colonic adenocarcinoma

Summary Loss of sialic acid o-acyl substitutions in colonic mucus was studied using specific histochemical techniques in individuals with a variety of large-bowel diseases and in a control population. Changes found included a focal or field (diffuse) loss of side-chain substitutions which were qualitatively similar in all groups studied. The results were tested statistically using a variety of assumptions that field and/or focal loss of o-acyl substitution may be either abnormal or a normal variant. No statistically significant differences in the prevalence of substitutions were detected between normal males and females or between normal individuals aged 0–29 years and 30–80 years. Significant differences were found between ascending and descending colon in both normal individuals and in the non-neoplastic mucosa of patients with cancer. There were also significant differences between the normal descending colon and cases with cancer of the descending colon. These differences seem unlikely to be due to non-specific factors, since for most assumptions there were also differences between colons containing cancer and those from patients with inflammatory bowel disease. In agreement with the work of other investigators, it seems likely that focal loss of o-acetylation results from an acquired gene mutation. It is not clear whether or not this plays a role in carcinogenesis. Deceased 21 May, 1994.