okadaic-acid and Carcinoma--Lewis-Lung

Cellular adherence and motility are processes that are controlled by focal adhesion assembly and disassembly. Consequently, the dynamics of focal adhesions regulate tumor cell metastasis and are influenced by the tyrosine phosphorylation state of paxillin. Metastatic LLC cells are more migratory and have reduced paxillin tyrosine phosphorylation as compared to nonmetastatic LLC cells. In nonmetastatic Lewis lung carcinoma (LLC) tumor cells, inhibition of the serine/threonine protein phosphatase-2A (PP-2A) activity results in increased motility that is associated with a reduction in the phosphotyrosine content of paxillin. Studies to determine if PP-2A can regulate protein tyrosine phosphatase activity showed that blocking PP-2A activity of nonmetastatic LLC-C8 tumor cells with okadaic acid reduces protein tyrosine phosphatase activity. Among the tyrosine phosphatases whose activity was inhibited upon PP-2A inhibition is Shp-2. In contrast, protein levels of Shp-2 are unaffected by PP-2A inhibition. While these results do not fully identify how inhibition of PP-2A results in tyrosine dephosphorylation of paxillin, they do demonstrate that PP-2A can link serine/threonine and tyrosine signaling pathways by regulating protein tyrosine phosphatases.

Topics: Animals; Blotting, Western; Carcinoma, Lewis Lung; Cytoskeletal Proteins; Down-Regulation; Enzyme Inhibitors; Intracellular Signaling Peptides and Proteins; Mice; Okadaic Acid; Paxillin; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotyrosine; Precipitin Tests; Protein Phosphatase 2; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatases; SH2 Domain-Containing Protein Tyrosine Phosphatases; src Homology Domains; Tumor Cells, Cultured

Cellular adhesion and motility, processes regulated by focal adhesion assembly and disassembly, can influence a tumor cell's ability to metastasize. Focal adhesion dynamics are, in turn, influenced by the serine and tyrosine phosphorylation state of paxillin. Using Lewis lung carcinoma (LLC) tumor variants, this study shows the importance of the serine/threonine protein phosphatase-2A (PP-2A) in maintaining adherence and restricting tumor cell motility, and modulating the serine and tyrosine phosphorylation of paxillin. Treating non-metastatic LLC-C8 tumor variants with okadaic acid to inhibit PP-2A activity resulted in cell rounding and increased motility. These effects on motility and adherence were accompanied by increased serine and decreased tyrosine phosphorylation of paxillin. These results suggest PP-2A regulation of paxillin phosphorylation may have a role in controlling tumor cell adherence and motility.

Topics: Animals; Carcinoma, Lewis Lung; Cell Movement; Cytoskeletal Proteins; Enzyme Inhibitors; Focal Adhesions; Mice; Neoplasm Proteins; Okadaic Acid; Paxillin; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphotyrosine; Protein Phosphatase 2; Protein Processing, Post-Translational

The role of protein kinase A (PKA) and protein phosphatases (PP) -1 and -2A in regulating the metastatic phenotype of Lewis lung carcinoma (LLC) cells was evaluated. The metastatic LLC-LN7 cells were more motile and invasive than were nonmetastatic LLC-C8 cells. Compared to the nonmetastatic cells, the LLC-LN7 cells had increased PKA activity and a deficiency in PP-2A. Nonmetastatic LLC-C8 cells became migratory and invasive when PP-1 and P-2A activities were inhibited with okadaic acid. This stimulation of LLC-C8 motility was tempered by PKA inhibition. Also examined was if the okadaic acid-stimulated LLC-C8 motility was associated with a change in the cytoskeletal organization to that typical of metastatic cells. Treatment of nonmetastatic LLC-C8 cells with okadaic acid caused a redistribution of F-actin toward the periphery of the cells, and eventually to a loss of the filamentous actin network. All of these effects were reversed upon removal of okadaic acid. Our results show that PP-1/2A maintain reduced motility and increased cytoskeletal organization within nonmetastatic LLC cells.

Topics: Actins; Animals; Carcinoma, Lewis Lung; Cell Movement; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Enzyme Inhibitors; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Tumor Cells, Cultured

Non-metastatic Lewis lung carcinoma cells (LLC-C8) become more motile when protein phosphatases (PP-1 and -2A) are inhibited by okadaic acid, attaining the same level of motility as metastatic LLC (LLC-LN7) variants. This stimulation of LLC-C8 motility was tempered when protein kinase A activity was inhibited. We examined whether the okadaic acid-stimulated LLC-C8 motility was associated with alterations in the cytoskeletal organization so that these non-metastatic cells acquire the rounded morphology and diffuse cytoskeletal organization previously described for metastatic LLC-LN7 cells. Non-metastatic LLC-C8 are typically adherent during culture, achieving a spread morphology. Treatment of non-metastatic LLC-C8 cells with okadaic acid resulted in a contraction of most of their extended processes, formation of spikes and membrane blebs within 10 min, and complete cell rounding within 20 min for most of the cells. While the overall level of F-actin was minimally affected by the okadaic acid, its uniform distribution shifted to localization toward the periphery of the rounded cells, often concentrating at a single focus. Immunofluorescent staining for vimentin showed a similar shift to the cell periphery and similar capping. After okadaic acid treatment, the filamentous network of microtubules in non-metastatic LLC-C8 cells disappeared and was replaced with a diffusely staining distribution of beta-tubulin. These results show that PP-1 and -2A maintain cytoskeletal organization and that inhibition of this control reduces cytoskeletal organization and increases tumor cell motility.

Topics: Actins; Animals; Carcinoma, Lewis Lung; Cell Movement; Cytoskeleton; Ethers, Cyclic; Isoenzymes; Okadaic Acid; Phosphoprotein Phosphatases; Staining and Labeling; Tubulin; Vimentin