TY - JOUR
T1 - Reduced DNA double strand breaks in chlorambucil resistant cells are related to high DNA-PKcs activity and low oxidative stress
AU - Boldogh, Istvan
AU - Roy, Gargi
AU - Lee, Myung Soog
AU - Bacsi, Attila
AU - Hazra, Tapas K.
AU - Bhakat, Kishor K.
AU - Das, Gokul C.
AU - Mitra, Sankar
N1 - Funding Information:
We thank Dr. David Konkel and Mardelle Susman for helpful scientific discussions and editorial help in assembling the manuscript. This research is supported by National Institute of Health (CA84461, I.B.) and National Institute of Environmental Health and Sciences Center (ES06676, I.B.), UTMB, Galveston, Texas.
PY - 2003/11/15
Y1 - 2003/11/15
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.
AB - 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.
KW - DNA double strand breaks
KW - DNA-PK
KW - Oxidative stress
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U2 - 10.1016/j.tox.2003.08.013
DO - 10.1016/j.tox.2003.08.013
M3 - Article
C2 - 14599773
AN - SCOPUS:0242299705
SN - 0300-483X
VL - 193
SP - 137
EP - 152
JO - Toxicology
JF - Toxicology
IS - 1-2
ER -