Reduced DNA double strand breaks in chlorambucil resistant cells are related to high DNA-PKcs activity and low oxidative stress.
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.
Department of Microbiology and Immunology, Sealy Center for Molecular Sciences, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA. sboldogh@utmb.edu
Istvan
I
Boldogh
Gargi
G
Roy
Myung Soog
MS
Lee
Attila
A
Bacsi
Tapas K
TK
Hazra
Kishor K
KK
Bhakat
Gokul C
GC
Das
Sankar
S
Mitra
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D045744:Cell Line, Tumor; D002699:Chlorambucil / Q000494:pharmacology*; D003114:Colony-Forming Units Assay; D020552:Comet Assay; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage / Q000235:genetics*; D004273:DNA, Neoplasm / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008970:Molecular Weight; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects*; D011494:Protein Kinases / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D004273:DNA, Neoplasm; D017382:Reactive Oxygen Species; D002699:Chlorambucil; D004247:DNA; D011494:Protein Kinases; D020169:Caspases