Human chronic lymphocytic leukemia B cells can escape DNA damage-induced apoptosis through the nonhomologous end-joining DNA repair pathway

Abstract

Nonhomologous end-joining (NHEJ) DNA factors maintain genomic stability through their DNA double-strand break (DSB) repair and telomere-associated activities. Unrepaired or misrepaired DSBs can lead to apoptotic death or chromosomal damage. The B cells of some B-chronic lymphocytic leukemia (B-CLL) patients are resistant to radiation-induced apoptosis in vitro. We show here that the novel DNA-dependent protein kinase (DNA-PK) inhibitor, NU7026 (2-(morpholin-4-yl)-benzo[h]chomen-4-one), and the phosphatidylinositol 3 (PI-3) kinase inhibitor, wortmannin, restored sensitivity to DNA damage-induced apoptosis of otherwise resistant cells. These resistant malignant B cells also escaped DSB-induced apoptosis following exposure to etoposide or neocarzinostatin. We found that at 15 minutes after irradiation, the levels of NHEJ (as measured by an in vitro DSB end-ligation assay) and DNA-PK catalytic subunit (DNA-PKcs) activity were, respectively, 2-fold and 4-fold higher in radioresistant than in radio- IntroductionMammalian cells respond to a variety of genotoxic stresses, including exposure to ionizing radiation and topoisomerase inhibitors, and endogenous reactive oxygen species, via 2 mechanisms for maintaining genome integrity: DNA repair and apoptosis. Inaccurate repair or a lack of repair of double-strand breaks (DSBs) can lead to apoptosis 1 or to mutations or large-scale genomic instability through the generation of nonlethal chromosomal aberrations. 2 DSBs are repaired by homologous recombination (HR) and by nonhomologous end-joining (NHEJ). NHEJ is the predominant mechanism in higher eukaryotes, whereas single-celled organisms (such as yeast) rely more heavily on HR. DNA-dependent protein kinase (DNA-PK) is a key component of the NHEJ pathway. 3 DNA-PK is a nuclear serine/threonine protein kinase, comprising a 460-kDa catalytic subunit, DNA-PKcs, and a DNA-binding subunit, the Ku autoantigen (a Ku70 and Ku80 protein dimer). The Ku70/Ku80 heterodimer binds the free DNA ends generated by DSBs and recruits the catalytic subunit of the complex. [4][5][6] In vitro, this active DNA-PK complex can phosphorylate many DNA-bound proteins including protein 53 (p53), Ku, X-ray cross-complementing group 4 (XRCC4), and DNA-PKcs 3,7,8 and can bind to XRCC4 and ligase IV, which join together the ends of the broken DNA strands. [8][9][10] Little is known about the role of the NHEJ repair pathway in apoptosis. Studies suggesting that DNA-PK plays a role in apoptosis remain controversial and principally relate to the interaction of this protein with p53. 11 The results from some studies indicate that DNA-PK activates and phosphorylates p53 12,13 and murine double mutant 2 (MDM2) 14 after DNA damage. However, other groups reported that DNA-PKcs-defective mice and DNAPKcs-defective cell lines display normal p53-mediated apoptosis responses. 15,16 DNA-PK activity has been implicated in resistance to nitrogen mustard therapy in leukemia cells, 17,18 but further studies are necessary to understand how these cells escape apoptosis aft...

DNA Damage Response/Signaling and Genome (In)Stability as the New Reliable Biological Parameters Defining Clinical Feature of CLL

“…Thus, we evaluated whether altered telomeres in resistant cells could be revealed by assaying classical DNA damage double-strand break (DSB) signaling and testing for the induction of telomere dysfunction-induced foci (TIF). This hypothesis has been supported by our previous results showing that resistant cells were able to upregulate non-homologous end-joining and in particular, by evidencing an upregulation of the activity of Ku heterodimer DNA end-binding (Deriano et al, 2005). Both, Ku80 and Ku70 have been identified in telomeric complexes, thus emphasizing the deregulation of these factors also at the telomeres in resistant cells.…”

“…We addressed an in vitro assay enabling us to measure the overall activity and fidelity of non-homologous endjoining (NHEJ) DNA repair and the activities of two essential components of NHEJ heterodimer Ku70/Ku80 and DNA-PKcs. Accelerated DNA repair, an increased activity of Ku DNA end-binding as well as an increased kinase activity of DNA-PKcs were observed in resistant cells (Deriano et al, 2005). Moreover, this upregulation of NHEJ was found to be error-prone and thus potentially mutagenic since large DNA deletions occurred at sites of repair (Deriano et al, 2006).…”

“…In this way, we tested whether cell resistance to -rays-induced apoptosis would be validated by the same resistance induced by these drugs. Effectively, we reported (Deriano et al, 2005), that these cells were resistant to all tested DNA damaging agents concluding that the resistance to apoptosis should underscore a defect in DNA damage repair/signaling. DNA repair has already been postulated as the mechanism causing drug resistance in CLL (Panasci et al, 2001;rev.…”

NK314 potentiates antitumor activity with adult T-cell leukemia-lymphoma cells by inhibition of dual targets on topoisomerase IIα and DNA-dependent protein kinase

“…[27][28][29] Various agents for inhibiting DNA-PK have been identified and clinical trials have been conducted using both subclinical and clinical approaches. [42][43][44][45][46][47] As for ATL cells, it was reported that transcription factor Tax binds to DNA-PKcs and activates DNA-PK. However, the Tax-DNA-PK complex was incapable of repairing new DNA DSBs induced by genotoxic stimuli in ATL cells.…”

DNA repair of myeloma plasma cells correlates with clinical outcome: the effect of the nonhomologous end-joining inhibitor SCR7

“…Many inhibitors targeting DNA repair pathways (PARP1, DNA-PK, ATM, ATR, MGMT, APE) or cell-cycle checkpoints (CHK1, CHK2) have now been developed and might be useful to induce tumor cell apoptosis in combination with DNA damage-inducing drugs. [25][26][27][28][29][30] Herein, to further investigate the mechanistic basis for the link between DNA repair efficiency and response to antimyeloma therapy, we studied major DNA repair mechanisms in MM cell lines and malignant bone marrow plasma cells (BMPCs) from patients with MM before antimyeloma therapy. We found that BMPCs from responders to melphalan therapy are characterized by slower rates of NER and DSBs repair (DSB/R) compared with nonresponders.…”