Sensitive 32P-HPLC technique shows base sequence dependent differences in photolesion repair in human keratinocytes

Understanding the basis for individual susceptibility to skin cancer requires an understanding of the factors contributing to tumorigenesis. One such factor is the ability of the cell to repair DNA lesions induced following insult to the genome. Currently, research in this field is hampered by the lack of a suitably sensitive and specific method for the detection of DNA lesions. Developed previously 32P-HPLC in vitro analysis is applied in this study to measure UVB-induced dipyrimidine photolesions in human keratinocyte cultures. The high sensitivity of this method permitted the detection of individual cyclobutane pyrimidine dimers and 6-4 photoproducts in cells irradiated with UVB at doses below one minimal erythema dose. Using this technique one could detect approximately a 2-fold difference in a base sequence repair of photolesions. The rates of repair in the chromosomally unstable HaCaT keratinocyte cell line and in cultured primary human keratinocytes were compared. The presented data indicate the potential of the 32P-HPLC method for the study of DNA repair in cultured cells as well as for biomonitoring studies in humans.