Concurrence of replicative senescence and elevated expression of p16(INK4A) with subculture-induced but not calcium-induced differentiation in normalhuman oral keratinocytes

Primary normal human oral keratinocytes (NHOKs) undergo differentiation in the presence of calcium concentrations higher than 0.15 mM in vitro, which is useful in investigating the mechanisms involved in the differentiation o f epithelial cells. Serial subculture of NHOKs to the postmitotic stage als o induces terminal differentiation. However, the detailed mechanisms of bot h differentiation processes remain substantially unknown. To investigate th e molecular differences in these processes, NHOKs were induced to different iate by exposure to 1.2 mM of calcium and by serial subculture to the postm itotic stage. To study whether the cells were induced to differentiate and to undergo replicative senescence, the amount of cellular involucrin and th e expression of senescence-associated beta-galactosidase (SA-beta-gal) were measured respectively. The expression of replicative senescence-associated genes and the activity of telomerase from the differentiated cells were al so determined. Both calcium treatment and serial subculture to the postmito tic stage notably elevated the cellular involucrin. The percentage of SA-be ta-gal-positive cells was significantly elevated by the continued subcultur e, but such changes were not observed in keratinocytes exposed to calcium. The concentration of cellular p16(INK4A) protein was progressively increase d by the continued subculture but was not changed by calcium treatment. On the other hand, the concentrations of cellular p53 were similar in both dif ferentiation processes. However, telomerase activity was lost in NHOKs that had undergone differentiation by both calcium treatment and serial subcult ure. The results indicate that calcium-induced differentiation of NHOKs has similar characteristics to their serial subculture-induced differentiation , but that the differentiation processes are not identical, because calcium -induced differentiation does not concur with either replicative senescence or the gradually increased concentration of p16(INK4A). (C) 2000 Published by Elsevier Science Ltd.