Differential expression of the cyclin-dependent kinase inhibitor P27 in primary hepatocytes in early-mid G1 and G1/S transitions

P27, an inhibitor of cyclin-dependent kinases, plays an important role in t he control of cell adhesion and contact inhibition-dependent cell cycle reg ulation, Hepatocytes, maintained in primary culture, offer a model of synch ronized primary epithelial cells which retain a differentiated profile whil e stimulated to proliferate, We therefore investigated the pattern of endog enous p27 expression in cyclin rat hepatocytes isolated by collagenase perf usion followed by mitogenic stimulation, P27 mas expressed in whole normal liver and freshly isolated hepatocytes. We then observed a sharp decrease i n p27 levels, concomitant with the progression in early-mid G1, followed by reaccumulation in late G1 and the G1/S transition, Immunochemistry and Brd U labelling demonstrated nuclear localization of p27 and its expression in cells engaged in both G1 and S phase, P27 was detected in late G1 in comple xes containing cyclins D1, E and A, Cyclin E- and A-associated kinase activ ities, however, mere detected at the G1/S transition and depletion experime nts confirmed that most active complexes were free of p27, Phosphorylated f orms of p27 mere detected in unstimulated and stimulated hepatocytes in bot h early-mid G1 and G1/S, Finally, two-dimensional gel electrophoresis showe d evidence for several forms of p27 with a distinct profile of distribution in quiescent and stimulated hepatocytes, Collectively, our data offer a mo del in which p27 shows a biphasic profile of accumulation, with the early d ecrease possibly involved in the progression through early and mid G1, In c ontrast with most cell types tested so far, the late G1 accumulation did no t impair formation of active cyclin E- and a associated kinases, and thus G 1/S transition.