cytochalasin-d and Urinary-Bladder-Neoplasms

Bacille Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that is used widely as a vaccine for tuberculosis and is used as an effective treatment for superficial bladder carcinoma. Despite being the most successful cancer biotherapy, its mechanism of action and response determinants remain obscure. Here, we establish a model system to analyze BCG interaction with bladder cancer cells, using it to show that these cells vary dramatically in their susceptibility to BCG infection. Unexpectedly, the uptake of BCG by bladder cancer cells occurs by macropinocytosis rather than phagocytosis. BCG entry into bladder cancer cells relied upon Rac1, Cdc42, and their effector kinase Pak1. The difference in susceptibility between BCG-permissive and -resistant bladder cancer cells was due to oncogenic activation of signaling pathways that activate macropinocytosis, with phosphoinositide 3-kinase inhibitor activation stimulating BCG uptake independently of Akt. Similarly, activated Ras strongly activated Pak1-dependent uptake of BCG. These results reveal that oncogenic activation of macropinocytosis determines BCG uptake by bladder cancer cells, implying that tumor responsiveness to BCG may be governed by the specific mutations present in the treated cancer cell.

Topics: Amiloride; BCG Vaccine; cdc42 GTP-Binding Protein; Cell Line, Tumor; Clathrin; Cytochalasin D; Dynamins; Humans; p21-Activated Kinases; Phosphatidylinositol 3-Kinases; Pinocytosis; PTEN Phosphohydrolase; rac1 GTP-Binding Protein; ras Proteins; Staurosporine; Urinary Bladder Neoplasms

Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of beta4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and beta4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion.

Topics: Actins; Animals; Carcinoma; Cell Line, Transformed; Cell Line, Tumor; Cells, Cultured; Clone Cells; Cytochalasin D; Epithelial Cells; Growth Substances; Hemidesmosomes; Humans; Integrin alpha3beta1; Keratinocytes; Kinetics; Nocodazole; Rats; Urinary Bladder Neoplasms

Changes of cell morphology and the state of differentiation are known to play important roles in embryogenesis as well as in carcinogenesis. Examples of particularly profound changes are the conversions of epithelial to mesenchymal cells; i.e., the dissociation of some or all polygonal, polar epithelial cells and their transformation into elongate, fibroblastoid cells of high motility. As an in vitro model system for such changes in cell morphology, we have used cell cultures of the rat bladder carcinoma-derived cell line NBT-II which, on exposure to inducing medium containing a commercial serum substitute (Ultroser G), show an extensive change in their organization (epithelial-mesenchymal transition): the junctions between the epithelial cells are split, the epithelial cell organization is lost, and the resulting individual cells become motile and assume a spindle-like fibroblastoid appearance. Using immunofluorescence microscopy and biochemical protein characterization techniques, we show that this change is accompanied by a redistribution of desmosomal plaque proteins (desmoplakins, desmoglein, plakoglobin) and by a reorganization of the cytokeratin and the actin-fodrin filament systems. Moreover, intermediate-sized filaments of the vimentin type are formed in the fibroblastoid cells. We demonstrate that the modulation of desmosomal proteins, specifically an increase in soluble desmoplakins, is a relatively early event in cell dissociation and in epithelial-mesenchymal transition. In this process, a latent period of 5 h upon addition of inducing medium precedes the removal of these desmosomal components from the plasma membrane. The transition, which is reversible, is dependent on continued protein synthesis and phosphorylation but not on the presence of the inducing medium beyond the initial 2-h period. We discuss the value of this experimental system as a physiologically relevant approach for studying the regulation of the assembly and disassembly of desmosomes and other intercellular adhesion structures, and as a model of the conversion of cells from one state of differentiation into another.

Topics: Animals; Cell Line; Cell Movement; Cytochalasin D; Cytoskeletal Proteins; Desmogleins; Desmoplakins; Desmosomes; Epithelial Cells; Fibroblasts; Fluorescent Antibody Technique; gamma Catenin; Immunoblotting; Kinetics; Phosphorylation; Rats; Tumor Cells, Cultured; Urinary Bladder Neoplasms