UVB light activates an S phase-dependent DNA damage response in human keratinocytes independent of oxidative stress.

Ultraviolet B (UVB; 280 to 320 nm) radiation damages DNA in epidermal cells and contributes to the development of skin cancer. In response, keratinocytes activate a DNA damage response (DDR) that regulates DNA repair through signaling pathways involving the protein kinases ATR (ataxia telangiectasia and Rad3-related), ATM (ataxia telangiectasia mutated), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Activation of these kinases promotes recruitment of DNA repair proteins, including phosphorylated H2AX and p53, to sites of DNA damage. Treatment of primary human neonatal foreskin epidermal keratinocytes and HaCaT keratinocytes with UVB (2.5 to 50 mJ/cm2) resulted in dose- and time-dependent activation of DDR proteins. In HaCaT cells, DDR signaling was most pronounced in S phase compared with cells in G0/G1 or G2/M, as determined by flow cytometry and cell cycle synchronization. Confocal microscopy revealed the accumulation of phosphorylated DNA-PKcs and phosphorylated H2AX in discrete subnuclear foci in S-phase cells, consistent with recruitment to sites of DNA double-strand breaks. UVB also induced oxidative stress in HaCaT cells, as reflected by increased intracellular reactive oxygen species (ROS). Depletion of intracellular glutathione using buthionine sulfoximine significantly enhanced UVB-induced ROS production but had little or no effect on activation of DDR signaling. These findings demonstrate that UVB induces cell cycle-dependent DNA damage in human keratinocytes that is independent of oxidative stress. Understanding mechanisms of UVB activation of the DDR in keratinocytes will be important for elucidating molecular pathways contributing to skin carcinogenesis.