ToxSci Advance Access published online on June 12, 2003
Toxicological Sciences, doi:10.1093/toxsci/kfg154
Toxicological Sciences © Society of Toxicology 2003; all rights reserved
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1 Institute of Environmental Health Sciences, Wayne State University, Detroit MI 48201, USA
* To whom correspondence should be addressed. E-mail: r.novak{at}wayne.edu.
Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging and cancer. Sensitivity to oxidative DNA damage and DNA repair efficiency were examined using a series of human breast epithelial cell lines, MCF-10A, MCF-10AT and MCF-10ATG3B with progressively elevated Ras protein. Breast epithelial cells were treated with H2O2, in the absence or presence of the DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C). DNA strand breaks were assessed by the mean olive tail moment (µm) using the alkaline single-cell gel electrophoresis (Comet) assay. In untreated cells, the mean olive tail moment values were 4.3±0.7, 8.3±1.1 and 7.1±0.6 µm in the MCF-10A, MCF-10AT and MCF-10ATG3B cells, respectively. Five min H2O2 treatment produced concentration-dependent DNA damage, with the MCF-10A cells most susceptible and the tumorigenic MCF-10ATG3B cells the least susceptible. Treatment with 100 µM H2O2 resulted in an
© 2003 Society of Toxicology
Genetic Toxicology
Oxidative DNA Damage and Repair in a Cell Lineage Model of Human Proliferative Breast Disease (PBD)
Abstract 
17-, 6- and 4.5-fold increase in mean olive tail moment values in the MCF-10A, MCF-10AT and MCF-10ATG3B cells, respectively, compared to untreated cells. The HCC1937 tumor cell line responded in a manner comparable to the MCF-10ATG3B cells treated with H2O2. HU/Ara-C pre-treatment resulted in an 1.5-fold increase in olive tail moment values in all three cell lines. Protein levels of antioxidant enzymes, including catalase, copper/zinc superoxide dismutase (Cu/Zn SOD) and manganese SOD (MnSOD) were determined in order to examine a potential mechanism for increased resistance to H2O2-mediated DNA damage. Levels of these enzymes increased progressively, with highest expression in MCF-10ATG3B cells. Increased cellular resistance also coincided with marked decreases in p53 protein levels. These results demonstrate, that in this cell lineage, sensitivity to oxidative DNA damage by H2O2 decreases with tumorigenicity (i.e MCF-10A vs MCF-10ATG3B), and show that DNA repair, altered Ras and p53 expression or compensatory mechanisms involving elevated antioxidant enzymes are involved in mediating these effects.
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