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 isolat ed regenerating mouse epidermal basal cells were studied after topical application of the skin irritant cantharidin and the tumor promoter 1 2-O-tetradecanoylphorbol-13-acetate (TPA) to the back skin of hairless mice. The DNA and protein contents of isolated basal cells were stain ed with propidium iodide and fluorescein isothiocyanate, respectively, and analysed by flow cytometry using the total protein fluorescence a s 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 distr ibutions. The results showed that both chemicals induced an increase i n cellular as well as nuclear size of the basal cells. The increase in size was higher in TPA treated than in cantharidin treated animals, a nd the bivariate DNA/protein distributions of TPA treated cells differ ed from those of cantharidin treated cells in that two subpopulations of cycling keratinocytes could be identified. These deviations are pro bably related to the higher proliferative response observed after TPA treatment and the possibility that proliferative subpopulations in epi dermis respond differently to TPA. It may reflect mechanisms providing for a growth advantage of initiated cells, important in tumor promoti on. 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 cellula r and nuclear size. The increase in cellular size was observed simulta neously in all cell cycle phases, well before the wave of cells enteri ng S phase 16 h after application. This suggests that an increase in s ize may be essential for the subsequent initiation of DNA synthesis an d the reduction in cell cycle time. The results support the hypothesis that the increase in cell size may be caused by gene activation or po st-transcriptional processes rather than an increase due to an initial block in cell cycle progression.