phalloidine and Cell-Transformation--Neoplastic

SSeCKS (pronounced essex) encodes a major protein kinase C substrate, the expression of which is down-regulated in src- and ras-transformed rodent fibroblasts but not in raf-transformed rodent fibroblasts (X. Lin et al., Mol. Cell. Biol., 15: 2754-2762, 1995). Using a panel of ras-transformed or revertant Rat-6 cells that exhibit selective parameters of transformation, we show that down-regulation of SSeCKS correlates with anchorage-independent growth. Cotransfection of NIH3T3 fibroblasts with an SSeCKS expression plasmid decreased 6-30-fold the ability of a v-src expressor plasmid to induce colonies in soft agar. To differentiate between possible tumor suppressive or growth-inhibitory effects of SSeCKS, we developed conditionally transformed cell lines (expressing ts72v-src) with tetracycline-regulated SSeCKS expression. SSeCKS suppressed the ability of v-src to induce increased cellular refractility, focus formation, soft agar colony formation, in vitro invasiveness in Matrigel, and growth in low serum (0.5%) but did not inhibit cell proliferation in high serum (10%) at the permissive (35 degrees C) temperature for src kinase activity. However, at the nonpermissive (39.5 degrees C) temperature, SSeCKS induced growth arrest. SSeCKS expression did not affect: (a) the protein level, in vivo or in vitro kinase activity of ts72src; (b) the activity of jun NH2-terminal kinase; and (c) the level of mitogen-activated protein kinase (extracellular signal-regulated kinase 2) protein. However, extracellular signal-regulated kinase 2 activity was induced 5-10-fold by SSeCKS in the presence of active src. SSeCKS reversed the ability of v-src to decrease the formation of vinculin-associated adhesion plaques, actin-based stress fibers, and filopodia structures. These data suggest a tumor suppressive role for SSeCKS via the control of cytoskeletal architecture and cell signaling.

Topics: 3T3 Cells; A Kinase Anchor Proteins; Animals; Apoptosis; Blotting, Western; Cell Cycle Proteins; Cell Division; Cell Transformation, Neoplastic; Down-Regulation; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Neoplastic; Mice; Mitogens; Neoplastic Stem Cells; Oncogene Protein pp60(v-src); Phalloidine; Protein Kinase C; Transcription, Genetic; Tumor Cells, Cultured; Vinculin

The effect of transforming growth factor beta (TGF-beta) on morphology, actin cytoskeleton organization, anchorage-dependent proliferation, anchorage-independent proliferation, and alpha-fetoprotein secretion in a series of related hepatocyte-derived cell lines was measured. We reported previously that TGF-beta 1 partially suppressed the transformed phenotype of a ras- transformed SV40-immortalized hepatocyte cell line designated NR4. In the present study, the CWSV14 cell line, which was derived by transfecting rat hepatocytes with SV40 DNA, was analyzed at low (14LP), mid (14MP), and high (14HP) passage. CWSV14 cells are weakly tumorigenic at low passage and spontaneously transform with in vitro passaging. Tumor cell lines (14T1 and 14T2) derived from hepatocellular carcinomas produced by CWSV14 cells were also analyzed. TGF-beta 1 partially suppressed the transformed phenotype of 14HP, 14T1 and 14T2 cells but had no effect on cell proliferation in these cells. In contrast, TGF-beta 1 induced apoptosis in 14LP and 14MP cells. Studies using both the NR4 and 14T1 cells showed that suppression of the transformed phenotype also could be induced by TGF-beta 2 or TGF-beta 3. We conclude that TGF-beta-induced suppression of the transformed phenotype can be observed in ras-and spontaneously transformed hepatocyte cell lines. It is also apparent that the effect of TGF-beta 1 on hepatocyte cell lines differs depending upon the state of progression toward malignancy; that is, while TGF-beta 1 suppressed the transformed phenotype in highly transformed and tumor cell lines representing late stages in progression, it induced apoptosis in weakly or moderately transformed cell lines representing early stages in progression.

Topics: Actins; alpha-Fetoproteins; Animals; Apoptosis; Cell Division; Cell Transformation, Neoplastic; DNA; Fluorescence; Liver; Phalloidine; Phenotype; Rats; Time Factors; Transforming Growth Factor beta

The cytoskeletons of two established chick embryo cell (CEC) lines were examined by fluorescence and electron microscopy and compared with those of control cells and cells transformed by Rous sarcoma virus (RSV). In normal CEC, many stress fibers were observed. On the other hand, stress fibers were disorganized in nontransformed spontaneously established CEC, non-tumorigenic CEC partially transformed with a chemical carcinogen, and tumorigenic RSV-transformed CEC. In the normal CEC, actin filaments formed several bundles along the processes of the cell. Stereo-images of the peripheral region revealed bundles of filaments which were located along the attached side to the substrate. A fine well preserved network of filaments was also observed. On the other hand, in spontaneously established, partially transformed and RSV-transformed CEC, a fine network of filaments, but no actin cables, was found. These results support previous evidence that the cytoskeletal changes themselves are not directly related to the transformation or tumorigenicity of cells.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Cytoskeleton; Detergents; Microscopy, Electron; Microscopy, Fluorescence; Phalloidine

Topics: Animals; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Liver Neoplasms; Neoplasm Transplantation; Oligopeptides; Phalloidine; Rats