B. Kirkhus et al., MULTIVARIATE FLOW-CYTOMETRY OF EPIDERMAL REGENERATION PROVOKED BY A SKIN IRRITANT AND A TUMOR PROMOTER, Cytometry, 13(3), 1992, pp. 267-274
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.