UV‐induced DNA damage in human keratinocytes: Quantitation and correlation with long‐term survival

Abstract: Ultraviolet (UV) radiation has a major role in the pathogenesis of skin cancer due to its capacity to induce immunosuppression and DNA damage in cells. In this study, we describe the use of a novel extra‐long polymerase chain reaction (XL‐PCR) assay for detection of UV‐inducible DNA lesions in a human keratinocyte line (HaCaT cells). Ultraviolet B (UVB), in doses from 4 to 50 mJ/cm 2 resulted in a linear increase in the number of DNA lesions in the genome [range 0.3 ± 0.2 lesions−3.6 ± 0.7 lesions (mean ± SD)/10 kb]. At lower doses of UVB (<10 mJ/cm 2 ), 89 ± 13% lesions were repaired within 24 h of culture. At higher doses, more lesions remained unrepaired, but the repair efficacy expressed as a proportion of repaired lesions to the total amount of DNA lesions remained constant in the range 0–50 mJ/cm 2 . Moreover, we demonstrated a correlation between the dose of UV and cell survival. The D 37 (dose that reduced clonogenic survival to 37%) of UVB equaled 19 mJ/cm 2 , corresponding to the introduction of 1.4 lesions/10 kb. In contrast to UVB, UVA1 irradiation neither induced measurable DNA damage nor induced cell death in the doses up to 15 J/cm 2 . In conclusion, the non‐radioactive extra‐long (XL)‐based real‐time (RT)‐PCR assay system can be used to quantify the UV‐induced DNA damage in intact cells. The DNA lesions detected by this assay are mainly induced by short‐waved radiation in the UVB range, and unrepaired DNA lesions cause keratinocyte death or permanent cell‐cycle block.