valinomycin and Colonic-Neoplasms

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; beta Catenin; Butyrates; Colonic Neoplasms; Curcumin; Cytoskeletal Proteins; DNA-Binding Proteins; Enzyme Inhibitors; G2 Phase; Humans; Hydroxamic Acids; Lymphoid Enhancer-Binding Factor 1; Mice; Sulindac; Trans-Activators; Transcription Factors; Up-Regulation; Valinomycin

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C1 cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C1 was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10 microM) increased furosemide-insensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanide-insensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloride-depleted cells. When cells with normal chloride content were depolarized (135 mM medium potassium + 10 microM valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl- approximately Br- > NO3- > I-. The electrogenic transport pathway was inhibited in chloride-depleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.

Topics: Anions; Biological Transport; Bumetanide; Carrier Proteins; Cations; Cell Membrane Permeability; Cell Polarity; Cell Size; Chloride Channels; Chlorides; Colforsin; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Electrophysiology; Furosemide; Gluconates; Humans; Intestinal Mucosa; Intracellular Fluid; Ionomycin; Nitrates; Organ Specificity; Quinolinium Compounds; Sodium-Potassium-Chloride Symporters; Tumor Cells, Cultured; Valinomycin

The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.

Topics: Animals; Cell Membrane; Chloride Channels; Chlorides; Colonic Neoplasms; Epithelium; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Intestinal Mucosa; Ion Channels; Kinetics; Membrane Proteins; Potassium Channels; Rats; Tumor Cells, Cultured; Valinomycin

N-(4-Methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (MPCU) is a new agent that exhibits high therapeutic activity against human and rodent tumor models. Initial studies indicated that in vitro [3H]MPCU was concentrated 4- to 6-fold in GC3/c1 human colon adenocarcinoma cells in an azide-sensitive manner. In this study the dependence of uptake and concentrative accumulation of MPCU upon temperature, plasma membrane potential, and the electrochemical potential of mitochondria has been examined. Accumulation and efflux of MPCU were temperature dependent. At 3.6 microM MPCU, initial rates of uptake (15 s) were 1.4, 38.0, and 84.2 pmol/min/10(6) cells at 2 degrees C, 23 degrees C, and 37 degrees C, respectively. The rate of uptake and concentrative accumulation within GC3/c1 cells was not altered in high K+ buffer or by 1 mM ouabain, indicating that plasma membrane potential was not significant in these processes. Concentrative accumulation, but not initial uptake, was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol, and sodium azide. Glucose partially antagonized the inhibition of these agents which uncouple oxidative phosphorylation. Oligomycin, an inhibitor of mitochondrial ATP synthase, did not inhibit uptake or concentrative accumulation of MPCU. However, oligomycin in the presence of 2-deoxyglucose significantly inhibited concentrative accumulation of MPCU. These results suggested that concentrative accumulation of MPCU was dependent upon the mitochondrial transmembrane gradient rather than ATP, although direct implication of ATP could not be excluded. To examine which component of this gradient was predominant in causing MPCU sequestration, the ionophores valinomycin and nigericin were used. Valinomycin, which collapses the charge gradient across the mitochondrial matrix membrane, caused only slight inhibition of MPCU accumulation, and the effect was similar at 2 or 10 mumol. In contrast, nigericin (which collapses the pH gradient and increases mitochondrial membrane potential) inhibited by approximately 90% concentrative accumulation of MPCU. These data suggested that MPCU was being concentrated in mitochondria and that this was dependent upon the pH gradient across mitochondrial membrane. In cells exposed to MPCU or the analogue N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea, enlargement of mitochondria was observed within 24 h and appeared to be the initial morphological change associated with drug treatment. These res

Topics: Adenocarcinoma; Antineoplastic Agents; Biological Transport; Cell Compartmentation; Colonic Neoplasms; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Microscopy, Electron; Mitochondria; Nigericin; Oligomycins; Ouabain; Sulfonylurea Compounds; Uncoupling Agents; Valinomycin

The tumor lysis syndrome, consisting of severe hyperkalaemia, hyperphosphatemia and hypocalcemia, occurs after the effective induction chemotherapy of rapidly growing responsive tumors. The metabolic abnormalities are thought to be secondary to the release of intracellular products. For the purpose to examine quantitative relation between cellular potassium release and drug sensitivity, we compare the inhibition of valinomycin (K-ionophore)-induced-hyperpolarization (MPR Test) with that of succinate dehydrogenase activity (SDI test). Our present research revealed a high correlation of MPR test and SDI test, and suggested the significant association of drug sensitivity with potassium release from cancer cells. Therefore, it seems appropriate to monitor potassium levels when therapy of a responsive tumor is initiated.

Topics: Animals; Cell Line; Cell Membrane Permeability; Cell Survival; Colonic Neoplasms; Drug Screening Assays, Antitumor; Female; Humans; Membrane Potentials; Potassium; Urinary Bladder Neoplasms; Uterine Neoplasms; Valinomycin