Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology.
The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.
Kobayashi
N
N
Department of Dermatology, Nara Medical University, Kashihara, Japan. nobuk@naramed-u.ac.jp
Katsumi
S
S
Imoto
K
K
Nakagawa
A
A
Miyagawa
S
S
Furumura
M
M
Mori
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002478:Cells, Cultured; D004249:DNA Damage / Q000528:radiation effects*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D010860:Pigments, Biological / Q000502:physiology*; D011740:Pyrimidine Dimers / Q000032:analysis / Q000276:immunology; D012867:Skin / Q000166:cytology / Q000528:radiation effects; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D010860:Pigments, Biological; D011740:Pyrimidine Dimers