Oral Presentation 2015 Annual Meeting of the Australasian Society for Dermatology Research

UVR and ROS damage protection mediated by DCT expression in human melanocytic cells (#6)

Stephen A Ainger 1 , Xuan L Yong 1 , Dubravka Skalamera 2 , Brian Gabrielli 2 , J. Helen Leonard 3 , Richard A Sturm 1
  1. Dermatology Research Centre, University of Queensland, School of Medicine, Translational Research Institute, Brisbane, Queensland, Australia
  2. University of Queensland, Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia
  3. QIMR, Berghofer Medical Research Institute, Brisbane, Queensland, Australia

The melanogenic enzyme Dopachrome Tautomerase (DCT) is involved in the formation of the photoprotective skin pigment eumelanin, and has also been shown to have a role in response to apoptotic stimuli and oxidative stress. To examine the effect of DCT on UVB DNA damage responses and survival pathways in melanocytic cells, knockdown experiments using melanoma cells, MC1R locus genotyped primary melanoblasts (MB) in monoculture and in co-culture with primary keratinocytes were carried out. Transduction with lentiviral vectors was used for DCT ablation and overexpression, and included MC1R WT and RHC homozygous strains known to be deficient in DCT. We found survival was reduced by DCT ablation and by UVB over time, whereas increased DCT protein levels enhanced cellular survival. DCT ablation reduced p53/pp53 proteins in most cases, while RHC MB cells displayed unchanged or decreased pp53. Overexpression of DCT in MB cells resulted in increased or unchanged p53/pp53 levels. Knockdown of DCT in melanoma cell lines and WT MC1R primary MB cells reduces the cell’s ability to survive after UVB exposure, and alters DNA damage response protein expression, which reduces the cell’s ability to repair UV-induced DNA damage. When compared to WT MC1R MB cells, RHC MC1R variant cells display vastly reduced expression of melanogenic proteins such as DCT, affecting sensitivity to UVB radiation and DNA repair pathways. When comparing co-cultures of WT MC1R-expressing MB cells with monocultures of MB cells only, a protective effect seems to be conferred by the keratinocytes to the MB, shown by increased cell survival when exposed to UVB.