Flow cytometric analysis of DNA damage in nucleoids from cultured human breast epithelial cells treated with hydrogen peroxide.
Flow cytometric analysis of nucleoids is useful to investigate nuclear matrix-DNA associations in cells. We have now applied this technique to examine whether differences in nucleoid structure can be detected between tumor and normal-like human breast epithelial cells. We have previously shown that MCF-7 tumor and MCF-10A normal-like human breast cells exhibit different levels of endogenous oxidative DNA damage as well as differences in response to hydrogen peroxide. We therefore examined whether flow cytometric analysis of nucleoids can be used to detect both endogenous DNA damage and DNA damage induced by hydrogen peroxide in these cells. Nucleoids were prepared by lysis of MCF-7 and MCF-10A cells with a high-salt buffer. The size of the DNA loops around the nuclear matrix core was detected by measuring the forward light-scatter signal in the presence of ethidium bromide. The relaxation and supercoiling of DNA in the presence of low and high concentrations of ethidium bromide, respectively, was similar in MCF-7 tumor and MCF-10A normal-like cells. After treatment of cells with 100 microM and higher of hydrogen peroxide, there was a statistically significant increase in the forward light scatter from nucleoids prepared in the presence of high concentrations of ethidium bromide, and this increase was similar in both cell lines. The changes in the forward light scatter signal with increasing hydrogen peroxide concentration did not mimic the patterns of increases in single strand breaks or formation of 5-hydroxymethyl-2 '-deoxyuridine that we reported previously. This indicates that the flow cytometric technique probably detects other types of changes induced by hydrogen peroxide.
Djuric
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Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA.
KuKuruga
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Everett-Bauer
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CK
Nakeff
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AN
D001940:Breast / Q000648:ultrastructure*; D002467:Cell Nucleus / Q000737:chemistry* / Q000187:drug effects; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D004249:DNA Damage; D004278:DNA, Superhelical / Q000737:chemistry / Q000187:drug effects; D004847:Epithelial Cells / Q000648:ultrastructure*; D004996:Ethidium / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004278:DNA, Superhelical; D004247:DNA; D006861:Hydrogen Peroxide; D004996:Ethidium