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Kinetics of withdrawal from the cell cycle in cultured human epidermal keratinocytes.

In keratinizing epithelia one of the earliest changes in the process of terminal differentiation is cessation of replication or withdrawal from the cell cycle. In this report, we measured the loss of colony-forming ability, and confirmed that withdrawal from the cell cycle is a specific event that occurs during maturation of the keratinocyte in culture. In addition, the rate of withdrawal was assayed by labeling cultures for 24 h with [14C]dThd and then measuring the fraction of labeled cells that undergo repeated cycles of DNA synthesis. These additional cycles of replication were measured by feeding BrdUrd to the cultures and quantitating the distribution of 14C-labeled DNA in unsubstituted and BrdUrd-substituted DNA in CsCl density gradients. The results show that the fraction of 14C-labeled DNA undergoing replication decreases exponentially by 23% every 24 h. This cessation of replication could not be explained by a reduced level of replication in the entire culture since during each day of the experiment about 8% of the total DNA underwent replication. The exponential decrease in replication of 14C-labeled DNA represents withdrawal from the cell cycle. Since the doubling time for keratinocytes is approximately 24 h, these results suggest that following each cycle of replication, there is a probability of 0.23 that postreplicated cells will withdraw from the cell cycle.