Reduced DNA double strand breaks in chlorambucil resistant cells are related to high DNA-PKcs activity and low oxidative stress

Istvan Boldogh, Gargi Roy, Myung Soog Lee, Attila Bacsi, Tapas Hazra, Kishor K. Bhakat, Gokul C. Das, Sankar Mitra

Research output: Contribution to journalArticle

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Abstract

Modulation of DNA repair represents a strategy to overcome acquired drug resistance of cells to genotoxic chemotherapeutic agents, including nitrogen mustards (NM). These agents induce DNA inter-strand cross-links, which in turn produce double strand breaks (dsbs). These breaks are primarily repaired via the nonhomologous end-joining (NHEJ) pathway. A DNA-dependent protein kinase (DNA-PK) complex plays an important role in NHEJ, and its increased level/activity is associated with acquired drug resistance of human tumors. We show in this report that the DNA-PK complex has comparable levels and kinase activity of DNA-PK catalytic subunit (DNA-PKcs) in a nearly isogenic pair of drug-sensitive (A2780) and resistant (A2780/100) cells; however, treatment with chlorambucil (Cbl), a NM-type of drug, induced differential effects in these cells. The kinase activity of DNA-PKcs was increased up to 2h after Cbl treatment in both cell types; however, it subsequently decreased only in sensitive cells, which is consistent with increased levels of DNA dsbs. The decreased kinase activity of DNA-PKcs was not due to a change in its amount or the levels of Ku70 and Ku86, their subcellular distribution, cell cycle progression or caspase-mediated degradation of DNA-PK. In addition to DNA cross-links, Cbl treatment of cells causes a 2.2-fold increase in the level of reactive oxygen species (ROS) in both cell types. However, the ROS in A2780/100 cells were reduced to the basal level after 3-4h, while sensitive cells continued to produce ROS and undergo apoptosis. Pre-treatment of A2780 cells with the glutathione (GSH) precursor, N-acetyl-L-cysteine prevented Cbl-induced increase in ROS, augmented the kinase activity of DNA-PKcs, decreased the levels of DNA dsbs and increased cell survival. Depletion in GSH from A2780/100 cells by L-buthionine sulfoximine (BSO) resulted in sustained production of ROS, lowered DNA-PKcs kinase activity, enhanced levels of DNA dsbs, and increased cell killing by Cbl. We propose that oxidative stress decreases repair of DNA dsbs via lowering kinase activity of DNA-PKcs and that induction of ROS could be the basis for adjuvant therapies for sensitizing tumor cells to nitrogen mustards and other DNA cross-linking drugs.

Original languageEnglish (US)
Pages (from-to)137-152
Number of pages16
JournalToxicology
Volume193
Issue number1-2
DOIs
StatePublished - Nov 15 2003

Fingerprint

Chlorambucil
Oxidative stress
Double-Stranded DNA Breaks
Catalytic Domain
Oxidative Stress
DNA-Activated Protein Kinase
Reactive Oxygen Species
Phosphotransferases
DNA
Mechlorethamine
Cells
Polynucleotide 5'-Hydroxyl-Kinase
Pharmaceutical Preparations
Joining
Tumors
Repair
Buthionine Sulfoximine
Drug Resistance
Catalytic DNA
Acetylcysteine

Keywords

  • DNA double strand breaks
  • DNA-PK
  • Oxidative stress

ASJC Scopus subject areas

  • Toxicology

Cite this

Reduced DNA double strand breaks in chlorambucil resistant cells are related to high DNA-PKcs activity and low oxidative stress. / Boldogh, Istvan; Roy, Gargi; Lee, Myung Soog; Bacsi, Attila; Hazra, Tapas; Bhakat, Kishor K.; Das, Gokul C.; Mitra, Sankar.

In: Toxicology, Vol. 193, No. 1-2, 15.11.2003, p. 137-152.

Research output: Contribution to journalArticle

Boldogh, Istvan ; Roy, Gargi ; Lee, Myung Soog ; Bacsi, Attila ; Hazra, Tapas ; Bhakat, Kishor K. ; Das, Gokul C. ; Mitra, Sankar. / Reduced DNA double strand breaks in chlorambucil resistant cells are related to high DNA-PKcs activity and low oxidative stress. In: Toxicology. 2003 ; Vol. 193, No. 1-2. pp. 137-152.
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N2 - Modulation of DNA repair represents a strategy to overcome acquired drug resistance of cells to genotoxic chemotherapeutic agents, including nitrogen mustards (NM). These agents induce DNA inter-strand cross-links, which in turn produce double strand breaks (dsbs). These breaks are primarily repaired via the nonhomologous end-joining (NHEJ) pathway. A DNA-dependent protein kinase (DNA-PK) complex plays an important role in NHEJ, and its increased level/activity is associated with acquired drug resistance of human tumors. We show in this report that the DNA-PK complex has comparable levels and kinase activity of DNA-PK catalytic subunit (DNA-PKcs) in a nearly isogenic pair of drug-sensitive (A2780) and resistant (A2780/100) cells; however, treatment with chlorambucil (Cbl), a NM-type of drug, induced differential effects in these cells. The kinase activity of DNA-PKcs was increased up to 2h after Cbl treatment in both cell types; however, it subsequently decreased only in sensitive cells, which is consistent with increased levels of DNA dsbs. The decreased kinase activity of DNA-PKcs was not due to a change in its amount or the levels of Ku70 and Ku86, their subcellular distribution, cell cycle progression or caspase-mediated degradation of DNA-PK. In addition to DNA cross-links, Cbl treatment of cells causes a 2.2-fold increase in the level of reactive oxygen species (ROS) in both cell types. However, the ROS in A2780/100 cells were reduced to the basal level after 3-4h, while sensitive cells continued to produce ROS and undergo apoptosis. Pre-treatment of A2780 cells with the glutathione (GSH) precursor, N-acetyl-L-cysteine prevented Cbl-induced increase in ROS, augmented the kinase activity of DNA-PKcs, decreased the levels of DNA dsbs and increased cell survival. Depletion in GSH from A2780/100 cells by L-buthionine sulfoximine (BSO) resulted in sustained production of ROS, lowered DNA-PKcs kinase activity, enhanced levels of DNA dsbs, and increased cell killing by Cbl. We propose that oxidative stress decreases repair of DNA dsbs via lowering kinase activity of DNA-PKcs and that induction of ROS could be the basis for adjuvant therapies for sensitizing tumor cells to nitrogen mustards and other DNA cross-linking drugs.

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