Abstract 5151: Protein kinase Cε, which is linked to development of squamous cell carcinomas, potentiates ultraviolet radiation-induced proliferation of hair follicle putative stem cells

Abstract

Abstract Protein kinase C epsilon (PKCε) is among the six PKC isoforms (α, δ, ε, η, μ, ζ) expressed in both mouse and human skin. 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) or initiation with 7,12-dimethylbenz[a]anthracene and tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Histologically, SCC in TG mice, like human SCC, is poorly differentiated and metastatic. To find clues about the mechanism by which PKCε may impart susceptibility to UVR-induced development of SCC, we compared the effects of UVR treatment of TG mice with their wildtype (WT) littermates on hair follicle putative stem cells (HSCs). HSCs in the mouse hair follicle are known to be the precursor cells for SCC in the mouse skin (Mol Carcinog. 46: 579-84, 2007). The cell surface markers CD34 and α6-integrin mark mouse hair follicle bulge cells, which have attributes of stem cells, including quiescence and multipotency. In this experiment, TG and WT mice were exposed to UVR (2kJ/m2, 3x weekly i.e, Monday, Wednesday, Friday) emitted by Kodacel-filtered FS-40 sun lamps. At 24 hr post last UVR (1, 2 or 4) exposures, mice were sacrificed and the dorsal skin removed for keratinocyte isolation. Keratinocytes were incubated for 30 minutes in the dark at 4oC with FITC-conjugated rat anti-human α6-integrin antibody at 10 μl per 106 cells and PE-conjugated rat anti-mouse CD34 antibody at 2 μg per 106 cells (FITC-α6-integrin and PE-CD34 antibodies; BD Biosciences). Flow cytometric analysis was done using a BD Biosciences FACS Calibur flow cytometer using a 488 nm laser as excitation. For both untreated and treated mice, the percent of double positive cells was higher in the TG than in WT mice. Both acute and chronic UVR treatment increased (2-fold) the number of double positive cell in both TG and WT mice. To examine the rate of proliferation of bulge region stem cells, a 5-bromo-2′-deoxyuridine labeling (BrdU) experiment was performed. Three-day old neonatal mice were injected twice daily for three days with 50 mg/kg of BrdU in PBS. At 8 weeks of age mice were sacrificed and the dorsal skin harvested for keratinocytes. The cells were stained for α6-integrin and CD34, fixed then stained for BrdU. In the WT mice, the percent of double positive cells maintaining BrdU label was 28.4 + 0.6% compared to 4.0 + 0.06% for the TG mice, an approximately 7-fold decrease in the TG mice. Similar results were obtained in a repeat experiment, indicating that the double positive cells in the TG mice cycle at a faster rate. This may contribute to the sensitivity of these transgenic animals to UVR-induced development of squamous cell carcinomas (Support: NIH grants CA102431 and CA35368 T32ES007015). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5151.