ACTIVATION OF THE PROTEIN-KINASE-A SIGNAL-TRANSDUCTION PATHWAY BY GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR OR BY GENETIC MANIPULATION REDUCES CYTOSKELETAL ORGANIZATION IN LEWIS LUNG-CARCINOMA VARIANTS

Granulocyte-macrophage colony-stimulating factor (GM-CSF) that is prod uced by metastatic Lewis lung carcinoma (LLC-LN7) cells functions as a n autocrine stimulator of tumor-cell motility through protein kinase A (PKA) signal transduction. This GM-CSF-mediated enhancement of LLC-LN 7 cell motility coincides with a reduction in the level of polymerized F-actin. In contrast, non-metastatic LLC-C8 tumor cells, which have a diminished level of PKA signaling, do not produce GM-CSF and do not r espond to exogenous GM-CSF, since they remain non-motile and retain a high content of filamentous actin. The capacity of PKA to regulate the cytoskeletal organization of tumor cells was further studied with the use of LLC variants that had been stably transfected to over-express the C-alpha subunit of PKA (CEV cells) or to express a mutant cAMP-res istant PKA R(I alpha) subunit resulting in a defective PKA (REV cells) . When compared with wild-type metastatic LLC-LN7 cells, in which the F-actin staining was too diffuse to be clearly visualized microscopica lly, the PKA-defective REV-LN7 transfectants had an increased level of F-actin. In comparison with the wild-type non-metastatic LLC-C8 cells , which had a high content of F-actin, the CEV-C8 transfectants that o ver-expressed PKA activity had a reduced level of F-actin. The reduced polymerization of actin in these CEV-C8 transfectants was accompanied by reduced levels of the intermediate filament protein vimentin and a shift in the distribution both of F-actin and of vimentin to the peri phery of the cells. These results show reduced cytoskeletal organizati on in metastatic LLC-LN7 cells as compared with that of non-metastatic LLC-C8 cells, and indicate that elevation of PKA activity, either by autologous GM-CSF or by genetic manipulation, diminishes cytoskeletal organization. (C) 1994 Wiley-Liss, Inc.