Introduction • Skin’s exposure to Uv radiation mediates formation of pyrimidine dimers repaired by nucleotide excision repair (NER). NER deficiencies can lead to cancer . this is manifested by high incidence of melanoma and non-melanoma skin cancers in Xeroderma Pigmentosum C patients, harboring mutations in NER DNA damage recognition protein XPC . however, XPC was also linked to base excision repair (BER) . the phenotype of XPC cells encompasses photosensitivity and accumulation of DNA damage. therefore, we aim to develop screening method to identify RNAi and chemical products reversing the XPC phenotype and enabling repair of Uv-induced DNA damage. Methods • Screening procedure consists of transfecting or treating XPC fibroblasts or keratinocytes with either RNAi kinome or chemical library, Uv irradiation then assessment of reversal. Uv irradiation is subjected to platting format and disposition variables. therefore, evaluation of XPC cells survival following different irradiation formats by Mtt will allow the choice of the optimal one. As for readout for phenotype reversal the first is cell viability. hits from the screen should increase survival if the XPC cells are photosensitive to start with. the second readout is repair of accumulated pyrimidine dimers detected by immunocytochemistry necessitating pretreatment with denaturing agent to allow access to the damaged sites. Positive hits should allow NER restoration.
Results and Conclusion • Our results identified UvB lamp as optimal source for damage induction with no disposition issues in 96 well plates. however, XPC fibroblasts were not photosensitive compared to normal ruling out the use of viability as a readout for fibroblasts but not keratinocytes. For immunocytochemistry, the pretreatment of fixed cells with 3M hCL allowed the optimal assessment of DNA damage. In addition, damage accumulation was measured at the single cell level rather than whole well fluorescence by using Cellinsight technology. the next step will be the performance of the chemical and RNAi screens. Once hits that enhance NER are identified by phenotype reversal their effects on BER will be tested. Finally, treatments in 3D skin models to mimic physiologic conditions will be carried out. Products that reduce DNA damage can decrease cancer incidence in XPC patients and possibly delay its onset in normal individuals.
Keywords: skin cancer; XPC; screening; NER; UV radiation