Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells

Attila Bacsi, Subbaraj Kannan, Myung Soog Lee, Tapas Hazra, Istvan Boldogh

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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-PKpThr2609) 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-PKpThr2609 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.

Original languageEnglish
Pages (from-to)1650-1659
Number of pages10
JournalFree Radical Biology and Medicine
Volume39
Issue number12
DOIs
StatePublished - Dec 15 2005

Fingerprint

DNA-Activated Protein Kinase
Chlorambucil
Double-Stranded DNA Breaks
Cells
Modulation
DNA
Oxidative Stress
Oxidative stress
Threonine
DNA Damage
Buthionine Sulfoximine
Glucose Oxidase
Post Translational Protein Processing
Ionizing Radiation
Repair
Cysteine
Acetylcysteine
Catalytic Domain
Phosphotransferases
Antioxidants

Keywords

  • DNA dsbs repair
  • DNA-PK
  • Free radicals
  • Oxidative stress

ASJC Scopus subject areas

  • Medicine(all)
  • Toxicology
  • Clinical Biochemistry

Cite this

Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells. / Bacsi, Attila; Kannan, Subbaraj; Lee, Myung Soog; Hazra, Tapas; Boldogh, Istvan.

In: Free Radical Biology and Medicine, Vol. 39, No. 12, 15.12.2005, p. 1650-1659.

Research output: Contribution to journalArticle

Bacsi, Attila ; Kannan, Subbaraj ; Lee, Myung Soog ; Hazra, Tapas ; Boldogh, Istvan. / Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells. In: Free Radical Biology and Medicine. 2005 ; Vol. 39, No. 12. pp. 1650-1659.
@article{ca239c8396ec48ef8bcfe1ef7ba59a38,
title = "Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells",
abstract = "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-PKpThr2609) 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-PKpThr2609 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.",
keywords = "DNA dsbs repair, DNA-PK, Free radicals, Oxidative stress",
author = "Attila Bacsi and Subbaraj Kannan and Lee, {Myung Soog} and Tapas Hazra and Istvan Boldogh",
year = "2005",
month = "12",
day = "15",
doi = "10.1016/j.freeradbiomed.2005.08.013",
language = "English",
volume = "39",
pages = "1650--1659",
journal = "Free Radical Biology and Medicine",
issn = "0891-5849",
publisher = "Elsevier Inc.",
number = "12",

}

TY - JOUR

T1 - Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells

AU - Bacsi, Attila

AU - Kannan, Subbaraj

AU - Lee, Myung Soog

AU - Hazra, Tapas

AU - Boldogh, Istvan

PY - 2005/12/15

Y1 - 2005/12/15

N2 - 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-PKpThr2609) 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-PKpThr2609 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.

AB - 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-PKpThr2609) 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-PKpThr2609 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.

KW - DNA dsbs repair

KW - DNA-PK

KW - Free radicals

KW - Oxidative stress

UR - http://www.scopus.com/inward/record.url?scp=27844459434&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=27844459434&partnerID=8YFLogxK

U2 - 10.1016/j.freeradbiomed.2005.08.013

DO - 10.1016/j.freeradbiomed.2005.08.013

M3 - Article

C2 - 16298690

AN - SCOPUS:27844459434

VL - 39

SP - 1650

EP - 1659

JO - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

IS - 12

ER -