Comparative Removal of Pyrimidine Dimers from Human Epidermal Keratinocytes In Vivo and In Vitro

Abstract

We have compared the kinetics for repair of UV-induced cyclobutane pyrimidine dimers in the DNA of keratinocytes in human skin and in cell culture. A small area of the buttocks of volunteers was exposed to UVB-irradiation and biopsies were taken at various time intervals. Epidermal keratinocytes in culture from the same subjects were exposed to UVC with doses chosen to elicit comparable yields of dimers in cellular DNA. The initial density of pyrimidine dimers and the kinetics of their removal were assessed utilizing the dimer-specific T4 endonuclease V and sedimentation of the unlabeled DNA through alkaline sucrose gradients. The position of DNA in the gradients was determined using a monoclonal antibody against random sequences of single-stranded DNA in a sensitive immunoassay. The initial dimer frequency was 3.9-6.7 per 10(8) daltons DNA. About 40% of the dimers were removed within 1 h, 70% in 6 h, and 90% in 24 h for both in vivo and in vitro samples. The early rapid removal phase may represent preferential repair of actively transcribed genes. Our findings reaffirm the usefulness and applicability of cell culture systems to model in vivo repair phenomena. The use of monoclonal antibodies to detect single-stranded DNA in alkaline sucrose gradients may be of value in a variety of studies involving human tissues in which it is not possible to use radioactive labeling of the DNA for the analysis.