phalloidine and lysophosphatidic-acid

Migration of cells involves a complex signaling network. The aim of the present study was to elucidate the impact of Rho-kinase (ROK) on G protein-coupled receptor-induced migration of human transitional cell carcinoma cells in an in vitro experimental setting.. Intracellular calcium concentration ([Ca(2+)](i)) was measured with the indicator dye Fura-2 in response to lysophosphatidic acid, thrombin and sphingosine-1-phosphate. Phospholipase C activity was determined in myo-[(3)H]inositol- (0.5 μCi/ml) labeled cells. Migration was performed using a Boyden chamber. Transient transfection of a dominant-negative mutant of ROK was done with calcium phosphate. For staining of actin filaments, tetramethylrhodamine isothiocyanate-conjugated phalloidin was used.. Lysophosphatidic acid, thrombin and sphingosine-1-phosphate cause increases in [Ca(2+)](i), cellular responses being accompanied by an enhancement of phospholipase C activity and sensitive to the G(i) inhibitor pertussis toxin. Agonists potently stimulated migration of T24 and J82 cells. Inhibition of Rho proteins by Clostridium difficile toxin B abrogated cell migration. Inhibition of ROK using HA1077 and Y-27632 mimicked the properties of toxin B. Expression of a ROK mutant drastically reduced migration.. G protein-coupled receptors potently stimulated cell migration in T24 and J82 cells. Rho proteins and ROK play a pivotal role in this signaling cascade. Rho and ROK may be putative targets for new therapy options in bladder cancer.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Amides; Bacterial Proteins; Bacterial Toxins; Calcium; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Movement; Fura-2; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Lysophospholipids; Mutation; Pertussis Toxin; Phalloidine; Protein Kinase Inhibitors; Pyridines; rho GTP-Binding Proteins; rho-Associated Kinases; Type C Phospholipases

To present an algorithm based on Hough transform for recognition and extraction of linear stress fibers formed on exposure to lysophosphatidic acid (LPA).. A ridge set of head points with lower shoulders is calculated, followed by a thinning process shrinking long, narrow regions to regions of single pixel thickness, then converted into a rectangular map whose value is the number of regional points in the path of a straight line at the angle and intercept determined by two coordinates. The location of the maximum in the map is sought, and the corresponding line with an unlimited length is constructed from the paired coordinates. We removed the line before repeating the process for the next longest straight line, continuing until all lines with reasonable lengths are extracted.. Application of the algorithm to the stress fiber images of DOV13 cells stained with Texas red-phalloidin on LPA and AG1478 demonstrates close matches between stress fibers in the original images and linear lines.. An algorithm for recognition of linear stress fibers formed on exposure to LPA is described and applications to stress fiber images using DOV13 cells with Texas red-phalloidin staining are demonstrated.

Topics: Algorithms; Cell Line, Tumor; Humans; Lysophospholipids; Phalloidine; Stress Fibers

Rho GTPases participate in a wide variety of signal transduction pathways regulating the actin cytoskeleton, gene expression, cellular migration and proliferation. The aim of this study was to evaluate the role of Rho GTPases in signal transduction pathways during acinus formation in a human salivary gland (HSG) cell line initiated by extracellular matrix (ECM; Matrigel) alone or in combination with epidermal growth factor, basic fibroblast growth factor and lysophosphatidic acid (LPA). Immunohistochemical and Western blotting analyses showed that HSG cells contained RhoA, RhoB, Rac1 and Cdc42 proteins. All growth factors enhanced the effects of ECM on acinus formation, in a pathway dependent on PI3-kinase and Rho GTPases. The role of ROCK, a major RhoA effector, seemed limited to cortical actin polymerization. LPA stimulated cell migration and acinus formation in a PI3-kinase-independent pathway. The results suggest that Rho proteins are important for epithelial-mesenchymal interactions during salivary gland development.

Topics: Actins; Cell Culture Techniques; Cell Line; Epidermal Growth Factor; Extracellular Matrix; Fibroblast Growth Factor 2; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Humans; Immunohistochemistry; Lysophospholipids; Microscopy, Confocal; Monomeric GTP-Binding Proteins; Phalloidine; Rhodamines; Salivary Glands; Signal Transduction