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 of quiescent (G0) keratinocytes being recruited into the cell cycle. A detailed characterization of the cell cycle kinetic parameters of the mouse keratinocyte line (Balb/MK) has been carried out with the aid of bivariate iododeoxyuridine (IdUrd) and DNA analysis using flow cytometry, in order to establish whether it might provide a useful model for the study of the biochemical events controlling recruitment into the cell cycle. Balb/MK keratinocytes were cultured using low Ca2+ Dulbecco's modified Eagle's medium/F 12 in the presence of 10% dialysed fetal bovine serum and 4 ng/ml epidermal growth factor (EGF). IdUrd labelling followed by flow cytometric analysis of trypsinized cells showed that about 95% of the population were actively cycling, with a cell cycle time of around 14h 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 16h, less than 10% incorporated IdUrd. Subsequent restimulation with serum resulted in a synchronized cohort of cells being recruited. Entry into the S phase of the cell cycle (IdUrd incorporation) started at 8 h and was maximal between 12 h and 16h 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 factor already showed a significant stimulation at 10 pg/ml compared with the controls. There is a broad plateau centred on 5 ng/ml, followed by a slight decline above this level. We conclude that the use of a cell line with defined cell cycle kinetic parameters which can be switched between the quiescent and cycling states in a fully defined medium will provide an ideal model for biochemical studies of the relevant signal transduction pathways in keratinocytes.
van Hooijdonk
C A
CA
Department of Dermatology, University Hospital Nijmegen, the Netherlands.
van Erp
P E
PE
Freund
R L
RL
de Jongh
G J
GJ
Schalkwijk
J
J
van de Kerkhof
P C
PC
Mier
P D
PD
D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D004247:DNA / Q000032:analysis; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D007065:Idoxuridine / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects; D051379:Mice
D004815:Epidermal Growth Factor; D004247:DNA; D007065:Idoxuridine