REGULATION OF CYTOSKELETAL ORGANIZATION IN TUMOR-CELLS BY PROTEIN PHOSPHATASE-1 AND PHOSPHATASE-2A

Non-metastatic Lewis lung carcinoma cells (LLC-C8) become more motile when protein phosphatases (PP-I and -2A) are inhibited by okadaic acid , attaining the same level of motility as metastatic LLC (LLC-LN7) var iants. This stimulation of LLC-C8 motility was tempered when protein k inase A activity was inhibited. We examined whether the okadaic acid-s timulated LLC-C8 motility was associated with alterations in the cytos keletal organization so that these non-metastatic cells acquire the ro unded morphology and diffuse cytoskeletal organization previously desc ribed for metastatic LLC-LN7 cells. Non-metastatic LLC-C8 ave typicall y adherent during culture, achieving a spread morphology. Treatment of non-metastatic LLC-C8 cells with okadaic acid resulted in a contracti on of most of their extended processes, formation of spikes and membra ne blebs within 10 min, and complete cell rounding within 20 min for m ost of the cells. While the overall level of F-actin was minimally aff ected by the okadaic acid, its uniform distribution shifted to localiz ation toward the periphery of the rounded cells, often concentrating a t a single focus. Immunofluorescent staining for vimentin showed a sim ilar shift to the cell periphery and similar capping. After okadaic ac id treatment, the filamentous network of microtubules in non-metastati c LLC-C8 cells disappeared and was replaced with a diffusely staining distribution of beta-tubulin. These results show that PP-I and -2A mai ntain cytoskeletal organization and that inhibition of this control re duces cytoskeletal organization and increases tumor cell motility. (C) 1995 Wiley-Liss, Inc.