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Carcinogenesis, Vol 18, 1401-1405, Copyright © 1997 by Oxford University Press

ARTICLES

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

Q Yang, M Hergenhahn and H Bartsch
Deutsches Krebsforschungszentrum (DKFZ), Division Toxicology and Cancer Risk Factors, Heidelberg, Germany.

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.
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Q. Yang, M. Hergenhahn, A. Weninger, and H. Bartsch
Cigarette smoke induces direct DNA damage in the human B-lymphoid cell line Raji
Carcinogenesis, September 1, 1999; 20(9): 1769 - 1775.
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