Cytoskeletal reorganization induced by retinoic acid treatment of human endometrial adenocarcinoma (RL95-2) cells is correlated with alterations in protein kinase C-alpha.

We have shown previously that treatment of human endometrial adenocarcinoma (RL95-2) cells with either 13-cis or all-trans retinoic acid results in reorganization of actin filaments, indicating reversion to a stationary phenotype. In the present study, we investigated the role of protein kinase C (PKC) in this process. Treatment of cells with PKC inhibitors (staurosporine, bisindolylmaleimide, or G¿6976) resulted in morphological alterations and reorganization of actin filaments similar to retinoic-acid-treated cells. For example, RL95-2 cells treated with staurosporine flattened, exhibited cell surface extensions and some actin filaments. Bisindolylmaleimide-treated cells flattened, and actin filaments reorganized similar to retinoic-acid-treated cells. RL95-2 cells treated with G¿6976, which inhibits only PKC, alpha, beta and gamma, exhibited many cell surface extensions and some actin filament reorganization. We then investigated whether retinoic acid affected the subcellular localization of PKC-alpha. In control cells, PKC-alpha was mainly evident as diffuse cytoplasmic immunostaining, with a small percentage of total PKC-alpha also evident in the plasma membrane. Retinoic acid treatment dramatically altered PKC-alpha localization, since a more distinct cytoplasmic and perinuclear staining pattern was apparent. Western blot analysis confirmed these results, since the amount of cytosolic PKC-alpha increased following retinoic acid treatment. Thus, retinoic-acid-induced endometrial differentiation may be associated with alterations in PKC-alpha localization and signaling.