CELL KINETIC CHARACTERIZATION OF THE EPIDERMAL GROWTH-FACTOR DEPENDENT BALB MK LINE USING FLOW CYTOMETRIC ANALYSIS OF DNA CONTENT AND IODODEOXYURIDINE INCORPORATION/

Epidermal hyperproliferation (psoriasis, wound repair) is the result o f quiescent (G(0)) keratinocytes being recruited into the cell cycle. A detailed characterization of the cell cycle kinetic parameters of th e mouse keratinocyte line (Balb/MK) has been carried out with the aid of bivariate iododeoxyuridine (IdUrd) and DNA analysis using flow cyto metry, in order to establish whether it might provide a useful model f or the study of the biochemical events controlling recruitment into th e cell cycle. Balb/MK keratinocytes were cultured using low Ca2+ Dulbe cco's modified Eagle's medium/F12 in the presence of 10% dialysed feta l bovine serum and 4 ng/ml epidermal growth factor (EGF). IdUrd labell ing followed by flow cytometric analysis of trypsinized cells showed t hat about 95% of the population were actively cycling, with a cell cyc le time of around 14 h and no significant contact inhibition. Omission of serum and EGF followed by IdUrd pulse-labelling indicated that the cells progressively withdrew from the cycle and, after 16 h, less tha n 10% incorporated IdUrd. Subsequent restimulation with serum resulted in a synchronized cohort of cells being recruited. Entry into the S p hase of the cell cycle (IdUrd incorporation) started at 8 h and was ma ximal between 12 h and 16 h after stimulation. Restimulation with EGF alone reached a growth fraction of 87% after 24 h continuous labelling compared with 97% using serum together with EGF. Epidermal growth fac tor already showed a significant stimulation at 10 pg/ml compared with the controls. There is a broad plateau centred on 5 ng/ml, followed b y a slight decline above this level. We conclude that the use of a cel l line with defined cell cycle kinetic parameters which can be switche d between the quiescent and cycling states in a fully defined medium w ill provide an ideal model for biochemical studies of the relevant sig nal transduction pathways in keratinocytes.