Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells.
DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)-t-butyl-4-hydroxyanisole or N-acetyl-l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PK(pThr2609)) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine (SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PK(pThr2609) was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs.
Bacsi
Attila
A
Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA.
Kannan
Subbaraj
S
Lee
Myung-Soog
MS
Hazra
Tapas K
TK
Boldogh
Istvan
I
D000111:Acetylcysteine / Q000494:pharmacology; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D045744:Cell Line, Tumor; D002699:Chlorambucil / Q000494:pharmacology*; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000187:drug effects* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology*; D006801:Humans; D018384:Oxidative Stress / Q000502:physiology; D013997:Time Factors
D002699:Chlorambucil; D002083:Butylated Hydroxyanisole; D051747:DNA-Activated Protein Kinase; D000111:Acetylcysteine