cytochalasin-b and Colonic-Neoplasms

Membrane androgen receptors (mAR) have been implicated in the regulation of cell growth, motility and apoptosis in prostate and breast cancer. Here we analyzed mAR expression and function in colon cancer.. Using fluorescent mAR ligands we showed specific membrane staining in colon cell lines and mouse xenograft tumor tissues, while membrane staining was undetectable in healthy mouse colon tissues and non-transformed intestinal cells. Saturation/displacement assays revealed time- and concentration-dependent specific binding for testosterone with a KD of 2.9 nM. Stimulation of colon mAR by testosterone albumin conjugates induced rapid cytoskeleton reorganization and apoptotic responses, even in the presence of anti-androgens. The actin cytoskeleton drug cytochalasin B effectively inhibited the pro-apoptotic responses and caspase-3 activation. Interestingly, in vivo studies revealed that mAR activation resulted in a 65% reduction of tumor incidence in chemically induced Balb/c mice colon tumors.. Our results demonstrate for the first time that functional mARs are predominantly expressed in colon tumors and that their activation results in induction of anti-tumor responses in vitro and extensive reduction of tumor incidence in vivo.

Topics: Animals; Apoptosis; Caspase 3; Cell Line, Tumor; Colonic Neoplasms; Cytochalasin B; Fluorescent Antibody Technique; Humans; In Situ Nick-End Labeling; Membrane Proteins; Mice; Mice, Inbred BALB C; Receptors, Androgen

Tumor necrosis factor-alpha (TNF-alpha) is an important inflammation cytokine without known direct effect on DNA. In this study, we found that TNF-alpha can cause DNA damages through reactive oxygen species. The mutagenic effect of TNF-alpha is comparable with that of ionizing radiation. TNF-alpha treatment in cultured cells resulted in increased gene mutations, gene amplification, micronuclei formation, and chromosomal instability. Antioxidants significantly reduced TNF-alpha-induced genetic damage. TNF-alpha also induced oxidative stress and nucleotide damages in mouse tissues in vivo. Moreover, TNF-alpha treatment alone led to increased malignant transformation of mouse embryo fibroblasts, which could be partially suppressed by antioxidants. As TNF-alpha is involved in chronic inflammatory diseases, such as chronic hepatitis, ulcerative colitis, and chronic skin ulcers, and these diseases predispose the patients to cancer development, our results suggest a novel pathway through which TNF-alpha promotes cancer development through induction of gene mutations, in addition to the previously reported mechanisms, in which nuclear factor-kappaB activation was implicated.

Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Colonic Neoplasms; Cytochalasin B; DNA Damage; Fluoresceins; Gene Amplification; Humans; Mice; Mutagens; Mutation; Oxidative Stress; Plasmids; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Although infection of the intestinal and biliary tracts by Cryptosporidium parvum is a major problem in patients with the acquired immunodeficiency syndrome, the specific microbial and host molecules involved in C. parvum infection are unknown. We tested the hypothesis that lectin-carbohydrate interactions and cytoskeleton reorganization are involved in the infection of biliary and intestinal epithelia by C. parvum.. In vitro models of cryptosporidial infection using human biliary and intestinal epithelial cell lines were used to assay C. parvum attachment and invasion.. Exposure of C. parvum sporozoites to the sugar, galactose-N-acetylgalactosamine (Gal/GalNAc), and to bovine mucin reduced C. parvum attachment to biliary and intestinal epithelia up to 70%. Preincubation of cell monolayers with either lectins specific to Gal/GalNAc, or glycosidases that specifically release Gal/GalNAc oligosaccharides from glycoproteins, decreased attachment up to 80%. Cytochalasin B and cytochalasin D, but not nocodazole, decreased invasion of cells by C. parvum up to 70% without affecting attachment. During cell invasion (but not attachment), confocal microscopy showed recruitment of actin (but not tubulin) in biliary and intestinal epithelia directly adjacent to C. parvum.. Gal/GalNAc epitopes of glycoproteins on the epithelial apical membrane and Gal/GalNAc-specific sporozoite surface lectins are involved in the mechanism(s) of C. parvum attachment to intestinal and biliary epithelial cells, and actin remodeling in host cells is required for C. parvum invasion.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Bile Ducts; Cattle; Cell Adhesion; Cell Line; Colonic Neoplasms; Cryptosporidiosis; Cryptosporidium parvum; Cytochalasin B; Cytochalasin D; Epithelial Cells; Glycoside Hydrolases; Humans; Intestinal Mucosa; Lectins; Mucins; Nocodazole; Tumor Cells, Cultured

The role of the polymorphonuclear leukocyte (PMN) cytoskeleton during the transmigration across colonic epithelial cells is not very well understood. In order to study the role of different components of the PMN cytoskeleton during transepithelial migration across a colonic epithelial cell monolayer (T84), PMN were preincubated with drugs affecting either the actin cytoskeleton (cytochalasin B, iota toxin of Clostridium perfringens, and phalloidin) or the microtubules (colchicine and taxol). The role of PMN myosin during transepithelial migration was investigated using the inhibitor 2,3-butanedione monoxime (BDM) and DC3B toxin. PMN intracellular Ca2+, during neutrophil adhesion and translocation across the epithelium, was assessed by the Ca2+ chelator 1, 2bis-(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM). Transmigration of PMN was initiated by applying either interleukin-8 or formyl-met-leu-phe (fMLP). While colchicine and taxol preexposure did not influence PMN transepithelial migration, treatment with cytochalasin B, iota toxin, phalloidin, BDM, DC3B toxin and BAPTA-AM greatly diminished migration of PMN across T84 monolayers. Similarly, cell-cell contacts established between PMN and epithelial cells during the transmigration were diminished after treatment of PMN with iota toxin or cytochalasin B. These data show that the neutrophil actin cytokeleton and myosin, but not the microtubules, evoke a Ca2+ -dependent motility that facilitates migration across the colonic epithelial barrier.

Topics: Actins; ADP Ribose Transferases; Bacterial Toxins; Calcium; Cell Adhesion; Cells, Cultured; Chelating Agents; Chemotaxis, Leukocyte; Colchicine; Colonic Neoplasms; Cytochalasin B; Cytoskeleton; Diacetyl; Egtazic Acid; Epithelial Cells; Humans; Interleukin-8; Intestinal Mucosa; Microtubules; Myosins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Paclitaxel; Phalloidine; Tumor Cells, Cultured

Patients with cancer cachexia exhibit increased glucose flux and lactate production in skeletal muscle. The aim of this study was to examine the direct effect of cancer cell-conditioned media on glucose metabolism in L6 myoblasts. Media from PANC-1 and Colo 320 cells caused a marked time-dependent and concentration-dependent increase of 2-deoxyglucose uptake in GLUT-4 transfected L6 myoblasts. This effect was greater than maximal acute stimulation by insulin and the effect of insulin was additive. Glucose utilization and lactate production increased in parallel to glucose uptake. The effect was inhibited by the protein synthesis inhibitor, cycloheximide and the glucose transport inhibitor, cytochalasin B. The bioactive factor had a molecular weight of approximately 5,000 and the biological activity was destroyed by proteinase K digestion. Radioimmunoassay and immunoneutralization studies indicated the major factor involved is not TNFalpha, IL-1beta, insulin, IGF-I or IGF-II. Further purification and characterization are needed to reveal the identity of this novel factor or factors which may have other metabolic effects that contribute to the cancer cachexia and insulin resistance.

Topics: Animals; Biological Factors; Cell Line; Colonic Neoplasms; Culture Media, Conditioned; Cycloheximide; Cytochalasin B; Deoxyglucose; Dose-Response Relationship, Drug; Endopeptidase K; Glucose; Glucose Transporter Type 4; Humans; Insulin; Lactic Acid; Molecular Weight; Monokines; Monosaccharide Transport Proteins; Muscle Proteins; Muscles; Pancreatic Neoplasms; Rats; Somatomedins; Tumor Cells, Cultured

Hemidesmosomes (HDs) are cellular junctions that anchor epithelial cells to the extracellular matrix (ECM) and are associated morphologically with the cytoskeleton. Hemidesmosomal molecular components include two proteins involved in linking intermediate filaments, HD1/plectin and BP230, and two transmembrane proteins, BP180 and the alpha6beta4 integrin, a laminin receptor. In cells lacking BP230 and BP180, HD1/plectin still associates with alpha6beta4 integrin, forming HD-like structures, called type II HDs. In the present study, we used an intestinal epithelial cell line that expresses HD1/plectin and the alpha6beta4 integrin to investigate the regulation of assembly of these proteins in type II HDs. These compounds were found to be clustered at sites of cell-ECM contact and their polarized localization was influenced by either cell confluency or extracellular matrix deposition. Conventional and immunoelectron microscopy showed that HD1/plectin and the beta4 integrin subunit are colocalized in an adhesion structure. Using cytoskeleton-disrupting drugs and confocal microscopy, we demonstrated that type II HDs are made up of numerous individual plaques whose assembly into a cluster requires actin filaments, but not microtubules.

Topics: Actin Cytoskeleton; Actins; Antigens, Surface; Cell Adhesion; Cell Count; Cell Size; Colonic Neoplasms; Cytochalasin B; Cytoskeleton; Desmosomes; Epithelial Cells; Extracellular Matrix; Humans; Integrin alpha6beta4; Integrins; Intermediate Filament Proteins; Intestinal Mucosa; Keratins; Microscopy, Confocal; Microscopy, Electron; Microtubules; Plectin; Pseudopodia; Tumor Cells, Cultured; Vinblastine; Vinculin

Hemidesmosomes (HDs) mediate adhesion of epithelial cells to the extracellular matrix and have morphological associations with intermediate-size filaments (IFs). Hemidesmosomal molecular components including HD1, the two bullous pemphigoid antigens, and the integrin alpha 6 beta 4 have been identified in HDs of stratified and complex epithelium. In this study, we report that HT29-Fu cells, a human colonic tumor cell line, express two hemidesmosomal components (HD1, alpha 6 beta 4) associated in an adhesion structure termed type II HDs. Immunofluorescence studies showed a colocalization of HD1 and alpha 6 beta 4 in basal patches between actin stress fibers. Using cytochalasin B or vinblastine, two drugs which disrupt the cytoskeleton, we demonstrate that the redistribution of HD1 was probably induced by the reorganization of the basal cytokeratin network. We also show that in vitro HD1 binds to polymerized cytokeratin intermediate filaments; this suggests that HD1 in intestinal epithelial cells functions as a linker protein connecting cytokeratin filaments to the basal plasma membrane, probably through the beta 4 subunit of the integrin alpha 6 beta 4.

Topics: Actins; Adenocarcinoma; Cell Adhesion; Cell Differentiation; Cell Polarity; Colonic Neoplasms; Cytochalasin B; Cytoskeleton; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Neoplasm Proteins; Organelles; Tumor Cells, Cultured; Vinblastine

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.

Topics: Apoptosis; Carcinoma; Cell Adhesion; Colonic Neoplasms; Cycloheximide; Cytochalasin B; DNA, Neoplasm; Epithelial Cells; Epithelium; fas Receptor; HT29 Cells; Humans; Hyaluronan Receptors; Hyaluronic Acid; Kinetics; Molecular Weight; Protease Inhibitors; Protein Synthesis Inhibitors; Solubility; Tumor Cells, Cultured

The liver is frequently colonized by metastatic tumor cells despite its dense population of macrophages (Kupffer cells). We have studied the interactions between metastatic colon carcinoma cells (DHD) and syngeneic Kupffer cells under different experimental conditions in vitro. In an adhesion assay the binding of DHD cells to Kupffer cell monolayers was shown to be time and temperature dependent, reaching a maximum level after about 90 min of incubation at 37 degrees C. In contrast, only a low level of binding could be observed at 4 degrees C. The level of binding could be increased by pretreatment of the Kupffer cells with phorbol 12-myristate 13-acetate. A firm interaction between the two cell types was shown to be dependent on the presence of calcium- and trypsin-sensitive structures on the surface of the Kupffer cells. Pretreatment of the macrophages with the cytoskeletal inhibitors colchicine and cytochalasin B was also found to reduce significantly the binding of tumor cells. This binding was also inhibited to a large extent by D-mannose and N-acetyl-D-galactosamine. The Kupffer cells were not cytotoxic against the colon carcinoma cells.

Topics: Animals; Carcinoma; Cell Adhesion; Cell Line; Colchicine; Colonic Neoplasms; Cytochalasin B; Cytotoxicity, Immunologic; Egtazic Acid; Kupffer Cells; Liver Neoplasms; Macrophages; Male; Monosaccharides; Peritoneal Cavity; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Trypsin

The effects of insulin on glucose transport and metabolism were examined in cultured HT29 human colonic adenocarcinoma cells. The presence of glucose transporters was verified by D-glucose displaceable [3H]cytochalasin B binding. The Kd and Bmax values from cytochalasin B binding studies were 190 +/- 30 nM and 8.4 +/- 1.4 pmol/mg protein, respectively. Glucose transport determined with 3-O-methylglucose showed saturable kinetics with a Km of 5.8 +/- 0.4 mM and a Vmax of 0.047 +/- 0.003 mumol/mg protein per min at 25 degrees C. Moreover, in HT29 cells, two classes of insulin binding sites were detected in radioligand binding experiments. Although insulin failed to stimulate glucose transport, it was found to activate glycolysis in HT29 cells. Glucose consumption increased from 0.33 +/- 0.03 mumol/mg protein per h to 0.49 +/- 0.05 mumol/mg protein per h and lactate production was augmented from 0.67 +/- 0.04 mumol/mg protein per h to 0.87 +/- 0.06 mumol/mg protein per h in response to 10(-7) to 10(-5) M insulin. Insulin also enhanced mannose metabolism. Apart from these two hexoses, HT29 cells exhibited a surprisingly narrow substrate specificity. With the possible exception of glyceraldehyde, little lactate was produced from alternative substrates, including adenosine, inosine, ribose, deoxyribose, dihydroxyacetone, galactose and fructose either with or without insulin. Despite its limited utilization by the glycolytic pathway, adenosine was readily salvaged for de novo synthesis of adenine nucleotides. These findings suggest that insulin directly influences substrate utilization through the glycolytic pathway in HT29 cells without activating the glucose transport pathway.

Topics: 3-O-Methylglucose; Adenocarcinoma; Adenosine; Adenosine Triphosphate; Biological Transport; Colonic Neoplasms; Cytochalasin B; Epithelium; Glucose; Glycolysis; Hexoses; Insulin; Insulin-Like Growth Factor I; Methylglucosides; Monosaccharide Transport Proteins; Tumor Cells, Cultured

We measured the permeability of normal, adenomatous, colitic and malignant large bowel epithelial cells by immersing fragments of large bowel mucosa in radiolabelled inulin and comparing autoradiograph grain density inside and outside cells after incubation. All the carcinomas studied showed extensive uptake of inulin within 5 min, while normal, adenomatous and colitic epithelial cells completely excluded inulin for 30 min. We found no difference in the proportion of epithelial cells incorporating uridine into RNA in carcinomatous and normal mucosa, and this suggests that the increased inulin permeability of carcinoma cell membranes was not due to leakage into non-viable cells. Experiments with cytochalasin B also showed that increased pinocytosis by carcinoma cells could not account for the difference. The relative impermeability of adenomatous and colitic cells suggests that increased permeability is not caused by increased proliferation. The consistent finding of increased permeability in the plasma membranes of carcinoma cells suggests that this may be more than an epiphenomenon of malignancy. It also suggests that measurement of cell permeability may have a role in distinguishing malignant from benign epithelial neoplasms.

Topics: Adenoma; Cell Membrane Permeability; Colitis, Ulcerative; Colon; Colonic Neoplasms; Cytochalasin B; Epithelium; Humans; In Vitro Techniques; Intestinal Absorption; Intestinal Mucosa; Inulin; Pinocytosis; Rectal Neoplasms

We studied the translocation of dimeric IgA across epithelium, using neoplastic human colon cells in culture as a source of epithelial cells, and immunoelectronmicroscopy with peroxidase-labeled antigens and antibodies. The cells had some of the ultrastructural characteristics of normal, mature epithelial cells, i.e., polarity, desmosomal junctions, and secretory component on their basal and lateral plasma membranes. Horseradish peroxidase-labeled dimeric IgA, exposed to the cells at 0 degrees C, bound selectively to secretory component on the cell surfaces. At 37 degrees C, the bound dimeric IgA was taken into the cells by endocytosis and transported apically through the cytoplasm in vesicles. After 30 min, IgA was discharged across the apical surface. Neither colchicine (10(-4) M) nor cytochalasin B (10(-5) M) interfered with binding or endocytosis of dimeric IgA, but colchicine inhibited intracellular transport of the IgA-containing vesicles. These experiments demonstrated that dimeric IgA can be transported through living intestinal epithelial cells in vitro. The transport includes 1) specific binding of IgA dimers to secretory component on plasma membranes, 2) endocytosis of IgA in vesicles, 3) transcytoplasmic transport of the IgA-containing vesicles by a process involving microtubules, and 4) discharge of IgA at the apical surfaces.

Topics: Binding Sites, Antibody; Biological Transport; Cell Transformation, Neoplastic; Colchicine; Colonic Neoplasms; Cytochalasin B; Horseradish Peroxidase; Humans; Immunoglobulin A; Secretory Component; Trypsin