UV-induced DNA damage in human keratinocytes: quantitation and correlation with long-term survival.
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/cm2 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/cm2), 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/cm2. Moreover, we demonstrated a correlation between the dose of UV and cell survival. The D37 (dose that reduced clonogenic survival to 37%) of UVB equaled 19 mJ/cm2, 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/cm2. 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.
Lisby
Steen
S
Department of Dermatology, University of Copenhagen, Bispebjerg Hospital, Denmark. sl05@bbh.hosp.dk
Gniadecki
Robert
R
Wulf
Hans Christian
HC
D002461:Cell Line, Transformed; D002470:Cell Survival / Q000528:radiation effects; D003584:Cytological Techniques; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D005914:Globins / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000502:physiology / Q000528:radiation effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014466:Ultraviolet Rays / Q000009:adverse effects*
D005914:Globins