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

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 2 h 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 3–4 h, 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.

Introduction

The nitrogen mustard (NM)-type of alkylating agents, such as mechlorethamine, melphalan, and cyclophosphamides are widely used in treating a variety of human malignancies. Although these agents have been used for chemotherapy more then 30 years, the factors responsible for acquired resistance of tumor cells to these agents are still poorly understood. Cytotoxicity of NMs has commonly been ascribed to various intracellular effects, including damage to proteins and lipids, DNA alkylation, inter-strand cross-links and DNA dsbs are the major source of cytotoxicity. Resistance to Cbl has not been associated with increased expression of P-glycoprotein and/or MDR-related protein (of which both reduce intracellular drug levels), or topoisomerase II, but is partly due to anti-apoptotic proteins (Roy et al., 2000), increased expression/activity of glutathione-S-transferases (GST) (Horton et al., 1999), and enhanced DNA repair (Muller et al., 1999, Panasci et al., 2001, Panasci et al., 2002).

To examine the role of DNA repair in acquired drug resistance, we have generated chlorambucil (Cbl)-resistant variants (A2780/100) from A2780 cells by selection with escalating doses of Cbl (Horton et al., 1999). A2780/100 cells are ∼10-fold more resistant to the cytotoxic effect of Cbl than the parent cells, based on LD₅₀ values (Horton et al., 1999, Roy et al., 2000), and show cross-resistance to other NMs (e.g., melphalan), nitrosoureas (e.g., BCNU) and also to cisplatin, and ionizing radiation (IR) (Horton et al., 1999). We expected that this effect was due to increased expression of anti-apoptotic proteins, Bcl-x_L, Mcl-1 (Roy et al., 2000), and/or GSTμ, which could detoxify the drug (Horton et al., 1999). Surprisingly, modulation of Bcl-x_L, Bax and GSTμ levels by ectopic expression did not significantly alter Cbl cytotoxicity either in A2780 or A2780/100 cells (Horton et al., 1999, Roy et al., 2000), suggesting that the primary mechanism of drug resistance in A2780/100 cells is not due to enhanced drug detoxification or overexpression of anti-apoptotic proteins.

Cbl induces DNA monoalkyl adducts, which are then converted into inter-strand cross-links (Lawley and Phillips, 1996). Distinct DNA repair pathways, including base excision (BER) and recombinational repair could be involved in the repair of these DNA lesions (Mitra and Kaina, 1993, Panasci et al., 2002). Our data show that resistance to NMs in A2780/100 cells was not associated with increased expression of BER proteins such as MPG, APE1, DNA β-polymerase and MGMT or ERCC-4, which is consistent with the scenario that the monoalkyl adducts subject to repair via BER pathway are readily converted into DNA cross-links (Mitra et al., 2002, Roy et al., 2000).

Because DNA cross-links are lethal, inhibition of their repair represents one strategy to overcome tumor cell resistance to DNA cross-linking alkylating agents. Hypersensitivity of cells to NMs and/or mitomycin C is found in several cell lines deficient in XRCC-4, Ku70, Ku86, DNA-PKcs and Artemis (Panasci et al., 2001, Panasci et al., 2002). For example, Ku86-deficient cells showed significant sensitivity to NMs (Caldecott and Jeggo, 1991).

Repair of DNA cross-links may involve dsb intermediates, which are repaired in mammals primarily via nonhomologous end-joining (NHEJ) process. NHEJ requires DNA-PK complex, consisting of the catalytic subunit DNA-PKcs, Ku70 and Ku86 heterodimer (Gottlieb and Jackson, 1993, Jeggo, 1997). DNA-PKcs is a member of the large phosphatidylinositol 3-kinase-related kinase (PI3K) family, along with the ataxia telangiectasia gene product (ATM) and RAD-3-related kinase. Ku70/86 is essential for DNA-PKcs function in vivo, and likely acts by promoting the recruitment of DNA-PKcs to DNA ends, leading to its activation. Ku70/86 also has limited DNA helicase activity and can mediate structural transitions in flanking DNA sequences for binding of DNA-PK. In addition, a recently identified protein, Artemis, mutated in human severe combined immunodeficiency disease, interacts with DNA-PKcs to induce an exonuclease activity, necessary for processing the DNA ends (Ma et al., 2002, Moshous et al., 2001). In addition to its important role in NHEJ, DNA-PKcs is implicated in protein modification (e.g., phosphorylation of c-Fos, c-Jun, c-Myc, p53, heat shock protein) and control of cell cycle (Durocher and Jackson, 2001, Hartley et al., 1995, Lees-Miller et al., 1990, Lees-Miller et al., 1992, Nilsson et al., 1999).

No studies published to date have examined whether oxidative stress affects the activity of DNA-PKcs. We show here that expression of DNA-PKcs, Ku70 and Ku86 are similar in both drug-sensitive and -resistant cells. In drug-sensitive cells, a low kinase activity of DNA-PKcs due to sustained ROS production causes enhanced sensitivity to Cbl, and other agents. We show here for the first time that ROS may inhibit repair of DNA dsbs by altering kinase activity of DNA-PKcs, and that the induction of mild chronic oxidative stress in tumor cells could enhance their drug sensitivity during chemotherapy.