Epstein-Barr virus episomes as targets for cigarette smoke- and gamma-irradiation-induced DNA damage: studies on the EBNA-1 region by a new gene-specific technique.
Following our demonstration of cytochrome P450-independent DNA damage induced by aqueous solutions of cigarette smoke in human mucosal cells in vivo, and in a lymphoblastoid cell line, we have developed a new technique to demonstrate gene-region specific DNA damage, with the EBNA-1 gene present in multiple nuclear matrix-attached episomes in Raji cells serving as an amplified target. DNA was extracted from Raji cells treated by gamma-irradiation or aqueous solutions of cigarette smoke; adducted bases or other damage were removed chemically by depurination/alkali treatment. Single-strand breaks induced directly by cigarette smoke as well as DNA cleaved at the site of former adducts were end-labelled either with alpha-[32P]dCTP or with biotin-16-dUTP. With 32P-labelling, a dose-dependent increase in DNA labelling was seen for different concentrations of cigarette smoke; undiluted smoke produced a similar amount of damage as 22.4 Gy of gamma-irradiation. For isolation of DNA regions that contained biotin label at the sites of former damage, DNA was cut by restriction endonucleases and 3-kb-fragments including the target gene, EBV-EBNA-1, were isolated by agarose-gel electrophoresis. Those containing biotin were selected on streptavidin-coated magnetic beads. PCR amplification of the bound DNA revealed EBNA-1 DNA only when cells were pretreated with either cigarette smoke or gamma-irradiation. The presented method thus provides a new approach for detecting gene-specific damage in a readily accessible target, EBV episomes. The method is also potentially applicable for studying single-copy genes such as p53, the types of adducts involved, and quantitative aspects of DNA damage and its repair.
Yang
Q
Q
Deutsches Krebsforschungszentrum (DKFZ), Division Toxicology and Cancer Risk Factors, Heidelberg, Germany.
Hergenhahn
M
M
Bartsch
H
H
D004249:DNA Damage; D019309:Epstein-Barr Virus Nuclear Antigens; D005720:Gamma Rays; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D012906:Smoke / Q000009:adverse effects*; D014026:Nicotiana
D019309:Epstein-Barr Virus Nuclear Antigens; D012906:Smoke