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The capacity of cloned metastatic Lewis lung carcinoma cells (LLC-LN7) to invade through reconstituted basement membrane-coated filters was reduced after incubation with 1α,25-dihydroxyvitamin D [1,25(OH)D]. This was observed at doses as low as 10 m 1,25(OH)D. The 1,25(OH)D-treated cells also had reduced levels of protein kinase A (PKA) activity and an increase in the level of polymerized actin, properties that have previously been demonstrated for less metastatic LLC variants. In addition, levels of the intermediate filament protein vimentin increased in 1,25(OH)D-treated LLC-LN7 tumor cells. In contrast, the levels and distribution of tubulin were not affected by 1,25(OH)D. The possibility that the decline in PKA activity was involved in the 1,25(OH)D modulation of the cytoskeletal components was evaluated. To accomplish this, LLC-7 transfectants whose PKA levels were blocked due to expression of a mutated PKA R subunit (LN7-REV) were incubated with 1,25(OH)D and their levels of F-actin were measured. In the absence of 1,25(OH)D treatment, the PKA-defective LN7-REV cells had an increased level of polymerized actin as compared to the wild-type LLC-LN7 cells. This level of F-actin was minimally affected by 1,25(OH)D, suggesting that PKA activity is required for 1,25(OH)D modulation of actin polymerization. These studies show that 1,25(OH)D can reduce PKA activity in tumor cells, and that this reduction in PKA may be an intermediate signal through which 1,25(OH)D affects the cytoskeleton and diminishes tumor invasiveness.