Abstract
To find clues about the mechanism by which kinase C epsilon (PKCε) may impart susceptibility to ultraviolet radiation (UVR)-induced development of cutaneous squamous cell carcinomas (SCC), we compared PKCε transgenic (TG) mice and their wild-type (WT) littermates for (1) the effects of UVR exposures on percent of putative hair follicle stem cells (HSCs) and (2) HSCs proliferation. The percent of double HSCs (CD34+ and α6-integrin or CD34+/CD49f+) in the isolated keratinocytes were determined by flow cytometric analysis. Both single and chronic UVR treatments (1.8 kJ/m2) resulted in an increase in the frequency of double positive HSCs in PKCε TG mice as compared to their WT littermates. To determine the rate of proliferation of bulge region stem cells, a 5-bromo-2′-deoxyuridine labeling (BrdU) experiment was performed. In the WT mice, the percent of double positive HSCs retaining BrdU label was 28.4 ± 0.6% compared to 4.0 ± 0.06% for the TG mice, an approximately 7-fold decrease. A comparison of gene expression profiles of FACS sorted double positive HSCs showed increased expression of Pes1, Rad21, Tfdp1 and Cks1b genes in TG mice compared to WT mice. Also, PKCε over expression in mice increased the clonogenicity of isolated keratinocytes, a property commonly ascribed to stem cells.
Highlights
The multistage model of mouse skin carcinogenesis is a useful system in which biochemical events unique to initiation, promotion, or progression steps of carcinogenesis can be studied and related to cancer formation. 12-O-Tetradecanoylphorbol13-acetate (TPA), a component of croton oil, is a potent mouse skin tumor promoter [1, 2]
We have reported that epidermal PKCε levels dictate the susceptibility of PKCε transgenic (TG) mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposures to ultraviolet radiation (UVR) [8] or initiation with 7, 12-dimethylbenz[a]anthracene (DMBA) and tumor promotion with 12-O-tetradecanoylphorbol-13acetate (TPA) [9]
We present for the first time that (1) UVR exposures increased the number of double positive hair follicle stem cells (HSCs) in TG mice, (2) the percent of double positive HSCs retaining bromo-2-deoxyuridine labeling (BrdU) label in the WT mice was 7-fold more than the TG mice, indicating that the double positive cells in the TG mice cycle at a faster rate, (3) the keratinocytes from PKCε TG mice have higher proliferating potential compared to their WT littermates, and (4) a comparison of gene expression profile of Fluorescence assisted cell sorting G-CSF (FACS)-sorted HSCs showed an increase expression of Pes1, Rad21, Tfdp1, and Cks1b genes in TG mice compared to their WT littermates
Summary
The multistage model of mouse skin carcinogenesis is a useful system in which biochemical events unique to initiation, promotion, or progression steps of carcinogenesis can be studied and related to cancer formation. 12-O-Tetradecanoylphorbol13-acetate (TPA), a component of croton oil, is a potent mouse skin tumor promoter [1, 2]. 12-O-Tetradecanoylphorbol13-acetate (TPA), a component of croton oil, is a potent mouse skin tumor promoter [1, 2]. A major breakthrough in understanding the mechanism of TPA tumor promotion has been the identification of protein kinase C (PKC), as its major intracellular receptor [3]. PKCε is among the six PKC isoforms (α, δ, ε, η, μ, and ξ) expressed in both mouse and human skin [7]. We have reported that epidermal PKCε levels dictate the susceptibility of PKCε transgenic (TG) mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposures to ultraviolet radiation (UVR) [8] or initiation with 7, 12-dimethylbenz[a]anthracene (DMBA) and tumor promotion with 12-O-tetradecanoylphorbol-13acetate (TPA) [9]. SCC in TG mice, like human SCC, is poorly differentiated and metastatic [10]
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