Discrimination between DNA-protein and DNA-DNA crosslinks using proteinase K in the alkaline single cell gel (SCG) assay

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  • Hayashi Hiroyuki
    Toxicology Laboratory, Pharmaceutical Development Dept., Meiji Seika Kaisha, Ltd.
  • Imai Masako
    Toxicology Laboratory, Pharmaceutical Development Dept., Meiji Seika Kaisha, Ltd.
  • Shindo Yasuhiro
    Toxicology Laboratory, Pharmaceutical Development Dept., Meiji Seika Kaisha, Ltd.

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The alkaline (pH>13) SCG assay, with post-lysis treatment of DNA with proteinase K (PK), to discriminate between DNA-protein and DNA-DNA crosslinks was evaluated using mouse lymphoma L5178Y tk+/- cells exposed in vitro to formaldehyde or cisplatinum. Formaldehyde specifically induces DNA-protein crosslinks, whereas cisplatinum induces inter- and intra-strand DNA crosslinks. In the absence of treatment with PK after lysis, formaldehyde induced a dose-dependent significant decrease in DNA migration. The use of PK after lysis to remove residual protein prevented the reduction in DNA migration. In contrast, cisplatinum induced a decrease in tail moment, with and without PK treatment after lysis. These results indicate that post-lysis treatment with PK can be expected as a supplementary method in the SCG assay to discriminate between DNA-protein and DNA-DNA crosslinks.

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