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Heat-mediated reduction of apoptosis in UVB-damaged keratinocytes in vitro and in human skin ex vivo - PubMed

  • ️Fri Jan 01 2016

Heat-mediated reduction of apoptosis in UVB-damaged keratinocytes in vitro and in human skin ex vivo

Leslie Calapre et al. BMC Dermatol. 2016.

Abstract

Background: UV radiation induces significant DNA damage in keratinocytes and is a known risk factor for skin carcinogenesis. However, it has been reported previously that repeated and simultaneous exposure to UV and heat stress increases the rate of cutaneous tumour formation in mice. Since constant exposure to high temperatures and UV are often experienced in the environment, the effects of exposure to UV and heat needs to be clearly addressed in human epidermal cells.

Methods: In this study, we determined the effects of repeated UVB exposure 1 kJ/m(2) followed by heat (39 °C) to human keratinocytes. Normal human ex vivo skin models and primary keratinocytes (NHEK) were exposed once a day to UVB and/or heat stress for four consecutive days. Cells were then assessed for changes in proliferation, apoptosis and gene expression at 2 days post-exposure, to determine the cumulative and persistent effects of UV and/or heat in skin keratinocytes.

Results: Using ex vivo skin models and primary keratinocytes in vitro, we showed that UVB plus heat treated keratinocytes exhibit persistent DNA damage, as observed with UVB alone. However, we found that apoptosis was significantly reduced in UVB plus heat treated samples. Immunohistochemical and whole genome transcription analysis showed that multiple UVB plus heat exposures induced inactivation of the p53-mediated stress response. Furthermore, we demonstrated that repeated exposure to UV plus heat induced SIRT1 expression and a decrease in acetylated p53 in keratinocytes, which is consistent with the significant downregulation of p53-regulated pro-apoptotic and DNA damage repair genes in these cells.

Conclusion: Our results suggest that UVB-induced p53-mediated cell cycle arrest and apoptosis are reduced in the presence of heat stress, leading to increased survival of DNA damaged cells. Thus, exposure to UVB and heat stress may act synergistically to allow survival of damaged cells, which could have implications for initiation skin carcinogenesis.

Keywords: Apoptosis; DNA damage; Heat stress; Keratinocytes; UVB; p53.

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Figures

Fig. 1
Fig. 1

Effects of UVB and/or heat stress on DNA damage and apoptosis in keratinocytes in vitro and in an ex vivo skin model. Bar graphs of mean ± SD percent keratinocytes that (a and d) harboured DNA damage (CPD), (b and e) were apoptotic, or (c and f) harboured DNA damage and expressed p53 in (a–c) NHEK or (d–f) skin per field of view. Statistically significant differences are indicated with *, ** or *** for p-values <0.05, <0.01 or ≤0.001 respectively

Fig. 2
Fig. 2

Exposure to UVB radiation and heat stress induced expression of genes with roles in cell survival. Hierarchal clustering analysis of 28 differentially expressed genes associated with biological changes observed in UVB and/or heat exposed ex vivo keratinocytes. Upregulation of cell-survival associated genes (red) and downregulation of apoptosis genes (green) are prominently clustered in UVB plus heat treated samples. Genes annotated by a pink box are known downstream targets of the p53 protein

Fig. 3
Fig. 3

Exposure to UVB plus heat induced a significant decrease in acetylated p53 and an increase in SIRT1 protein levels in NHEK and in skin models. a Immunohistochemical staining of CPD (red), p53-a382 or SIRT1-p (green) and DAPI (blue) in untreated or UVB and/or heat treated ex vivo skin keratinocytes. Inset images are an enlarged view of SIRT1/DNA damaged (CPD positive) keratinocytes, which are also indicated by red arrows. Broken lines denote the epidermal/dermal border. Scale bar (white line) =100 μm. b–e Bar graphs of mean ± SD percent keratinocytes with CPD and positive for (b and c) p53-a382 or (d and e) SIRT1-p per field of view in (b and d) primary NHEK or (c and e) in skin models. Statistically significant differences are indicated with * for p-values <0.05

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