Multivariate flow cytometry of epidermal regeneration provoked by a skin irritant and a tumor promoter.
The DNA content and the changes in cellular and nuclear size of isolated regenerating mouse epidermal basal cells were studied after topical application of the skin irritant cantharidin and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to the back skin of hairless mice. The DNA and protein contents of isolated basal cells were stained with propidium iodide and fluorescein isothiocyanate, respectively, and analysed by flow cytometry using the total protein fluorescence as an estimate of cell size and the DNA fluorescence pulse width as an estimate of nuclear size. Transmission electron microscopy was used to identify cells sorted from regions in the bivariate DNA/protein distributions. The results showed that both chemicals induced an increase in cellular as well as nuclear size of the basal cells. The increase in size was higher in TPA treated than in cantharidin treated animals, and the bivariate DNA/protein distributions of TPA treated cells differed from those of cantharidin treated cells in that two subpopulations of cycling keratinocytes could be identified. These deviations are probably related to the higher proliferative response observed after TPA treatment and the possibility that proliferative subpopulations in epidermis respond differently to TPA. It may reflect mechanisms providing for a growth advantage of initiated cells, important in tumor promotion. About 8% of the cells in the suspensions from treated animals were non-cycling non-keratinocytes, probably infiltrating leukocytes. The results indicate a strong correlation between rapid regenerative cell cycle progression, i.e., reduced G1 transit time and increased cellular and nuclear size.(ABSTRACT TRUNCATED AT 250 WORDS)
Kirkhus
B
B
Institute of Pathology, National Hospital, Oslo, Norway.
Glasø
M
M
Clausen
O P
OP
D000818:Animals; D001973:Bromodeoxyuridine; D002193:Cantharidin / Q000494:pharmacology; D002469:Cell Separation; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D051379:Mice; D008812:Mice, Hairless; D008854:Microscopy, Electron; D010703:Phorbol Esters / Q000494:pharmacology; D011419:Propidium; D011506:Proteins / Q000032:analysis; D012038:Regeneration; D012867:Skin / Q000166:cytology* / Q000187:drug effects; D012879:Skin Physiological Phenomena
D010703:Phorbol Esters; D011506:Proteins; D011419:Propidium; D004247:DNA; D001973:Bromodeoxyuridine; D016650:Fluorescein-5-isothiocyanate; D002193:Cantharidin