Multivariate flow cytometry of epidermal regeneration provoked by a skin irritant and a tumor promoter

Abstract

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 12-0-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 DNNprotein 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 DNNprotein 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. The increase in cellular size was observed simultaneously in all cell cycle phases, well before the wave of cells entering S phase 16 h after application. This suggests that an increase in size may be essential for the subsequent initiation of DNA synthesis and the reduction in cell cycle time. The results support the hypothesis that the increase in cell size may be caused by gene activation or posttranscriptional processes rather than an increase due to an initial block in cell cycle progression.

Flow cytometric fluorescence pulse width analysis of etoposide-induced nuclear enlargement in HCT116 cells

“…Recently, researchers have examined the calf thymocyte nuclear size using fluorescence pulse width analysis (Hoffman 2009). However, the flow cytometric analysis of fluorescence pulse width for biological particle sizing remains unpopular, with limited published data (Hoffman 2009; Kirkhus et al 1992; Leary et al 1979).

Fig.

…”

The tumor promoting activity of the EP4 receptor for prostaglandin E2 in murine skin

“…IL-20 has been shown to play a role in skin inflammation, along with several related cytokines (Uto-Konomi et al, 2012). The regenerative hyperplasia seen in psoriasis also has been linked to IL-20 Wegenka, 2010); this may be relevant to the robust regenerative response of BK5.EP4 mice to DMBA-induced cell death, which in turn resembles the regenerative cell cycle progression in keratinocytes following TPA treatment (Kirkhus et al, 1992). Some of the other upregulated genes, including Kcnk2 (TREK-1), have been linked to ovarian and prostate cancer (Innamaa et al, 2013;Voloshyna et al, 2008).…”