cytochalasin-d and Carcinoma--Squamous-Cell

Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1β precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells.. HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay.. Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental calculus was significantly inhibited by cytochalasin D, z-YVAD-fmk and glyburide, indicating NLRP3 inflammasome involvement. In permeability assays, dental calculus attenuated the barrier function of HSC-2 cell monolayers.. Dental calculus induces pyroptotic cell death in human oral epithelial cells and the crystalline structure plays a major role in this process. Oral epithelial cell death induced by dental calculus might be important for the etiology of periodontitis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Carcinoma, Squamous Cell; Caspase 1; Cell Death; Cell Line, Tumor; Cell Membrane Permeability; Cytochalasin D; Dental Calculus; Epithelial Cells; Humans; Inflammasomes; Interleukin-1beta; L-Lactate Dehydrogenase; Macrophages; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Tumor Necrosis Factor-alpha

The fate of GAS and epithelial cells following internalization was determined in this study. HEp-2 cells harbouring intracellular bacteria were treated with antibiotics to kill extracellular adherent bacteria, washed, and the fate of bacteria and epithelial cells was assessed up to 24 h post-infection. In the absence of antibiotics, massive bacterial growth was apparent in the cell medium, accompanied by extensive cell death, suggesting that intracellular bacteria had multiplied and damaged the monolayer. Addition of the internalization inhibitor, cytochalasin D, either pre- or post-internalization prevented bacterial growth and cell injury; post-internalization treatment with chloramphenicol had the same effect. Analysis of three apoptotic markers in HEp-2 cells - chromatin condensation, DNA laddering and translocation of phosphatidylserine onto the cell-surface membrane - indicated that HEp-2 cells underwent apoptosis. Taken together, the data presented here support a model in which internalized bacteria can induce their own externalization into the medium by a process that requires both an intact host-cell cytoskeleton and de novo synthesis of bacterial proteins. Concomitantly, intracellular and, apparently, extracellular free bacteria induce apoptosis through their cytotoxic activity, and release essential nutrients required for their growth.

Topics: Anti-Bacterial Agents; Apoptosis; Bacterial Adhesion; Bacterial Proteins; Carcinoma, Squamous Cell; Chloramphenicol; Culture Media; Cytochalasin D; Epithelial Cells; Humans; Nucleic Acid Synthesis Inhibitors; Phosphatidylserines; Protein Synthesis Inhibitors; Streptococcus pyogenes; Tumor Cells, Cultured

Using patch clamp and ion-selective fluorescence dye techniques, we investigated the influence of actin cytoskeleton rearrangements on the activity of calcium entry channels in plasma membrane of human carcinoma A431 cells. It is shown that disruption of actin microfilaments by cytohalasin D has no significant effect on calcium release from the stores and its entry from the extracellular space. It also does not interfere with the activation of inositol 1,4,5-trisphosphate (IP3)-dependent high-selective low-conductance calcium channels Imin. The treatment of cells with calyculin A induces the formation of actin filament layer beneath plasma membrane and also inhibits Imin activation and calcium entry through the plasma membrane, though calcium efflux from the stores was nearly unchanged. Thus, it is concluded that calcium signalling in A431 cells can be modulated by actin cytoskeleton rearrangements, and may be well described in terms of "conformational coupling" model.

Topics: Actins; Calcium; Calcium Channel Agonists; Calcium Channels; Calcium Signaling; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cytochalasin D; Cytoskeleton; Electric Conductivity; Extracellular Space; Humans; Inositol 1,4,5-Trisphosphate; Ion Channel Gating; Marine Toxins; Oxazoles; Patch-Clamp Techniques; Thapsigargin; Tumor Cells, Cultured

Previous studies have shown that EGF can induce the tyrosine phosphorylation of caveolin-1 in murine fibroblasts following ErbB1 (EGF receptor) mutation or overexpression, but the cell signaling events linking EGF action with caveolin phosphorylation are not fully established. In this regard, we examined multiple human carcinoma cell lines that express various ErbB family members, including A431 epidermoid carcinoma cells and several squamous carcinoma cell lines. In all cases, EGF treatment induced the tyrosine phosphorylation of caveolin-1 in a time- and EGF dose-dependent manner, and immunoblotting analysis revealed that this phosphorylation occurred at tyrosine-14. The EGF-dependent phosphorylation of caveolin-1 was observed at low temperatures (4 degrees C) and was enhanced by caveolae-disrupting agents (cyclodextrin), suggesting that this EGF-dependent system is in a low temperature-stable arrangement that allows for their interaction under conditions where mobility in the membrane is altered. To further assess the events linking EGF action with caveolin phosphorylation, we evaluated the ligand specificity of these responses and their dependence on known effectors of EGF receptor function. We observed that EGF and HB-EGF, but not heregulin, promoted caveolin-1 phosphorylation in A431 cells, suggesting that these responses are linked to EGF receptor activation and not solely occurring via the activation of other endogenous ErbB family members. In addition, the EGF-induced phosphorylation of caveolin-1 in A431 cells was blocked by the Src kinase antagonists PP1 and PP2, but not by the MEK inhibitor PD98059, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin, or cytoskeleton-disrupting agents, such as cytochalasin D, colchicine, and nocadazole. Altogether, these data indicate that multiple human carcinoma cells exhibit an EGF receptor-dependent tyrosine phosphorylation of caveolin-1 and that this process is sensitive to Src family kinase inhibitors. These observations support a role for caveolin tyrosine phosphorylation in the profile of cellular responses by which Src potentiates cancer progression following EGF receptor overexpression.

Topics: Androstadienes; Carcinoma, Squamous Cell; Caveolae; Caveolin 1; Caveolins; Cell Line; Chromones; Colchicine; Cyclodextrins; Cytochalasin D; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Humans; Kinetics; Morpholines; Nucleic Acid Synthesis Inhibitors; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; src-Family Kinases; Tumor Cells, Cultured; Tyrosine; Wortmannin

Addition of epidermal growth factor (EGF) to A431 human epidermal carcinoma cells results in actin reorganization and phosphorylation of several cytoskeletal proteins. In the present study, we found that EGF treatment of this cell line also results in the redistribution and tyrosine phosphorylation of ZO-1. In normal polarized epithelial cells, ZO-1 is restricted to the cytoplasmic surface of the most apical of the intercellular junctions, the tight junction. In contrast, ZO-1 in the majority of unstimulated A431 cells in small subconfluent islands colocalizes with actin along the lateral cell membranes and in rare microspikes and membrane ruffles. Exposure to EGF results in a transient redistribution of actin into an apically positioned ring. ZO-1 becomes highly focused at apical sites of cell contact and co-localizes with the newly formed band of perijunctional actin. Coincidently, ZO-1 and another tight junction protein, ZO-2, become transiently phosphorylated on tyrosine residues, as determined by anti-phosphotyrosine immunoblotting. Pre-treatment of A431 cells with cytochalasin D, which disrupts normal microfilament organization, does not affect EGF-dependent phosphorylation of the EGF receptor. However, cytochalasin D pretreatment blocks both the EGF-induced ZO-1 rearrangement and tyrosine phosphorylation, suggesting that these responses are dependent on an intact actin microfilament system. We speculate that the transient tyrosine phosphorylation of ZO-1 in response to EGF treatment may be involved in remodeling of intercellular junctions in A431 cells.

Topics: Actins; Blotting, Western; Carcinoma, Squamous Cell; Cell Line; Cytochalasin D; Epidermal Growth Factor; Humans; Intercellular Junctions; Membrane Proteins; Phosphoproteins; Phosphorylation; Phosphotyrosine; Tumor Cells, Cultured; Tyrosine; Zonula Occludens-1 Protein

Previous studies have identified two bacterial factors involved in enteropathogenic Escherichia coli (EPEC) infection. A plasmid-mediated EPEC adherence factor (EAF) is responsible for initial and localized adherence. A chromosomally encoded E. coli attachment and effacement factor (eae) is involved in effacement of the eukaryotic cell surface and characteristic "pedestal" formation. By using isogenic strains deficient in either EAF, eae, or both, the process of EPEC adherence and entry in vitro was examined. While EAF proved necessary and sufficient for efficient bacterial association with HEp-2 cells, both EAF and eae were required for efficient effacement of and entry into these cells and other cultured cell lines. Invasion mediated by eae was markedly inhibited by cytochalasin D and colchicine. Afimbrial adhesion or type I pili from uropathogenic strains of E. coli substituted for EAF in EAF-Eae+ strains to provide initial adherence to HEp-2 cells and to facilitate actin condensation.

Topics: Adhesins, Escherichia coli; Bacterial Adhesion; Bacterial Outer Membrane Proteins; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Colchicine; Cytochalasin D; Cytoskeleton; Escherichia coli; Humans; Laryngeal Neoplasms; Microscopy, Electron; Microvilli; Plasmids; Tumor Cells, Cultured

Topics: Actins; Carcinoma, Squamous Cell; Cell Line; Cycloheximide; Cytochalasin D; Cytochalasins; Humans; Kinetics

In HeLa and HEp2, cell lines derived from human carcinoma, application of cytochalasin D (CD) is followed in minutes by generalized cell contraction and zeiosis. Simultaneously, actin, myosin and tropomyosin, mostly from cables, become relocated in condensed masses. Most of these occupy the bases of the zeiotic knobs protruding at the cell surface. In contrast to most nontransformed cell types, in these cells both the protrusions and the contractile proteins are concomitantly translocated centripetally to the cell apex to form an aggregate of zeiotic knobs and a subjacent, membrane-associated, actin-based microfilamentous cap containing tropomyosin and myosin. The redistribution of these contractile proteins is antagonized by pretreatment with inhibitors of energy metabolism, or with cyclic adenine nucleotide. The centripetal movement appears to be guided by microtubules, which tend a radiate toward apical aggregates. Under the influence of CD 10 nm filaments associate in bundles. These processes are rapidly reversed after withdrawal of CD. The changes in distribution of actin, myosin and tropomyosin in relation to cell surface structures are considered in terms of the hypothesis that CD induces contraction of the microfilament apparatus of the cortex which, at least in part, remains tethered to plasma membrane.

Topics: Carcinoma, Squamous Cell; Cell Line; Contractile Proteins; Cytochalasin D; Cytochalasins; Cytoskeleton; Fluorescent Antibody Technique; HeLa Cells; Humans; Microtubules; Neoplasms, Experimental