ELEVATED CAMP LEVELS INDUCE MULTILAYERING OF MDCK CELLS WITHOUT DISRUPTING CELL-SURFACE POLARITY

The effect of hormones on the morphology and cell surface polarity of the epithelial cell line MDCK was examined. When MDCK cells were seede d in high densities in media containing FCS a regular monolayer was fo rmed. However, in serum-free medium supplemented with insulin, transfe rrin, prostaglandin E1, hydrocortisone and triiodothyronine, the devel opment of a multilayer with intercellular lumina was observed. In horm one-depletion studies we identified PGE1 as the inducer of these multi layers. Since dibutyryl cyclic AMP and the phosphodiesterase inhibitor isobutyl methylxanthine could substitute for PGE1, we conclude that a n elevated intracellular cAMP level resulted in formation of the multi layer. Further analysis, by electron microscopy and immunocytochemistr y revealed a polarized organization of the multilayered cells. Junctio nal complexes, enclosing microvilli-rich membrane domains, were found at the apices of adjacent cells facing the medium and those surroundin g the intercellular lumina. Surprisingly, cells participitating in the formation of both the free surface and the surface of the intercellul ar lumen, exhibited two distinct membranes with microvilli, each separ ated by junctional complexes. Immunolocalization of membrane marker pr oteins demonstrated that an apical 114 kDa membrane protein was locali zed to the free cell surfaces, the same membrane domains where extensi ve microvilli were also observed. The distribution of a basolateral 58 kDa membrane protein was restricted to sites of cell contact. These r esults provided evidence that nontransformed epithelial MDCK cells for m multilayers in response to elevated cAMP levels; however, they retai n the potential of developing cell surface polarity.