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Assessing DNA Cross‐Linking and Repair in Human Leukemia Cells
Author(s) -
Le Phuong M.,
Millard Julie T.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.754.1
Subject(s) - comet assay , dna damage , dna , chemistry , cytotoxicity , dna repair , microbiology and biotechnology , cancer cell , biochemistry , in vitro , biology , genetics , cancer
The anti‐tumor effects of bifunctional alkylating agents have been recognized for more than 50 years, notably in the frontline chemotherapeutic drug, nitrogen mustard and its derivatives. These compounds are double‐edged swords: killing both cancerous and non‐cancerous cells by forming cytotoxic DNA adducts via interstrand and intrastrand cross‐links. Intrastrand crosslinks can be readily removed by nucleotide excision repair mechanisms while interstrand crosslinks (ICLs) pose a challenging block to DNA replication, transcription, and repair and are hence a potentially lethal event. Using human leukemia (HL‐60) cells as an in vitro model, we are investigating the cytotoxicity and DNA repair of ICLs induced by nitrogen mustard (HN2), epichlorohydrin (ECH), and 1,2,3,4‐diepoxybutane (DEB)‐the active form of the anti‐ovarian cancer drug Ovastat. The median lethal dose (LD 50 ) of each compound was determined via the AlamarBlue assay over a wide a range of drug concentrations. DNA cross‐linking activity and repair were quantified via single cell gel electrophoresis (the comet assay). Drug‐induced ICL formation retarded the mobility of hydrogen peroxide‐treated DNA in an electric field, creating a comet‐like migration pattern at the individual cell level. The shorter and brighter the comet tail was, the more crosslinks were present. Preliminary data showed a consistent cytotoxicity trend after a 12‐hour or 24‐hour drug treatment with HN2 > ECH ≥ DEB. Comet assay analysis from treating cells with equitoxic doses revealed that the percentage decrease in tail moment, a combined parameter that includes the comet tail length and fluorescence intensity, varied between different drugs, corresponding to their different cross‐linking potentials. Interestingly, cross‐linking activity was maintained in some, but not all, compounds at 24 hours compared to 12 hours, suggesting differential rates of DNA repair. With these quantitative investigations, we hope to correlate the toxicity of these bifunctional alkylating agents with their cross‐linking specificity. Our ultimate goal is to elucidate the underlying mechanism by which ICLs induced by one drug are more susceptible to removal and repair than another. These findings can improve our understanding of dose‐dependent drug cocktail administration and the long‐term effects of these chemotherapeutic drugs. Support or Funding Information This research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.