CELL KINETIC CHARACTERIZATION OF GROWTH ARREST IN CULTURED HUMAN KERATINOCYTES

In this study we have performed a cell kinetic characterization of gro wth and growth arrest of keratinocytes derived from normal human skin. Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyurid ine (IdUrd) incorporation simultaneously. Normal human keratinocytes w ere grown in keratinocyte growth medium (KGM) and growth arrest was in duced by using either keratinocyte basal medium (KBM) or KGM supplemen ted with TGF-beta 1. It was found that human keratinocytes grown in KG M plus TGF-beta 1 were growth-arrested within 52 hours. The rate of Id Urd incorporation into DNA decreased by more than 95% after 52 hours a nd parallelled the decrease of cells in S-phase. Within 52 hours after addition of TGF-beta 1, 79% of the growth-arrested cells were in the G(0)/G(1)-phase of the cell cycle, a situation that approaches that of the normal epidermis. Growth arrest of human keratinocytes in KBM sho wed a similar decrease in the rate of IdUrd incorporation. However, th e decrease in IdUrd incorporation was not reflected in a decrease in c ells in S-phase, suggesting that the cells were blocked in G(0)/G(1), S or G(2)/M-phase rather than selectively in the physiological growth arrest state of G(0)/G(1). Secondly, we investigated the kinetics of t he cells when they were restimulated after growth arrest. We found tha t after termination of the growth arrest in KGM supplemented with TGF- beta I the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G(0)/G(1) population, suggesting that the cells are recruited as a cohort. After growth arrest induced by KBM, c ells also require 6 to 8 hours in KGM to initiate DNA synthesis, but t he cells are not recruited as a cohort. We conclude that growth arrest induced by TGF-beta 1 is the preferred system in which to study induc tion of keratinocyte proliferation, since it induces a state of quiesc ence that approaches that of normal human epidermis.