DNA-dependent Protein Kinase Activity Research Articles

Androgen receptor in Sertoli cells regulates DNA double-strand break repair and chromosomal synapsis of spermatocytes partially through intercellular EGF-EGFR signaling.

Spermatogenesis does not progress beyond the pachytene stages of meiosis in Sertoli cell-specific AR knockout (SCARKO) mice. However, further evidence of meiotic arrest and underlying paracrine signals in SCARKO testes is still lacking. We utilized co-immunostaining of meiotic surface spreads to examine the key events during meiotic prophase I. SCARKO spermatocytes exhibited a failure in chromosomal synapsis observed by SCP1/SCP3 double-staining and CREST foci quantification. In addition, DNA double-strand breaks (DSBs) were formed but were not repaired in the mutant spermatocytes, as revealed by γ-H2AX staining and DNA-dependent protein kinase (DNA-PK) activity examination. The later stages of DSB repair, such as the accumulation of the RAD51 strand exchange protein and the localization of mismatch repair protein MLH1, were correspondingly altered in SCARKO spermatocytes. Notably, the expression of factors that guide RAD51 loading onto sites of DSBs, including TEX15, BRCA1/2 and PALB2, was severely impaired when either AR was down-regulated or EGF was up-regulated. We observed that some ligands in the epidermal growth factor (EGF) family were over-expressed in SCARKO Sertoli cells and that some receptors in the EGF receptor (EGFR) family were ectopically activated in the mutant spermatocytes. When EGF-EGFR signaling was repressed to approximately normal by the specific inhibitor AG1478 in the cultured SCARKO testis tissues, the arrested meiosis was partially rescued, and functional haploid cells were generated. Based on these data, we propose that AR in Sertoli cells regulates DSB repair and chromosomal synapsis of spermatocytes partially through proper intercellular EGF-EGFR signaling.

DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

Successes and Challenges of PARP Inhibitors in Cancer Therapy.

Successes and Challenges of PARP Inhibitors in Cancer Therapy.

Abstract 1859: Discerning the molecular basis of DNA-PK pro-tumorigenic functions and translational capacity as a therapeutic target in prostate cancer

Abstract Prostatic adenocarcinoma (PCa) is dependent on androgen receptor (AR) signaling at all stages of disease, as AR activation induces both cell proliferation and survival. Though organ-confined disease can be treated, the response is transient and reactivation of AR results in incurable state of disease (castration resistant prostate cancer, CRPC). Thus, there is a significant need to discern the mechanisms by which aberrant AR activity arises, and to develop new means to clinically target advanced disease. Recently, AR activation was found to promote tumor cell survival and proliferation through a feed-forward loop involving the repair factor DNA-dependent protein kinase (DNA-PK). Emerging new data correlates elevated DNA-PK expression with poor prognosis and identifies DNA-PK as a driver of metastatic signaling. Although the role of DNA-PK activity in DNA repair is well characterized, the underpinning mechanisms and consequences of DNA-PK in transcriptional regulation are understudied. Data to be discussed will build on our new preliminary DNA-PK interactome data to identify sequence-specific transcription factors found in complex with DNA-PK. In order to molecularly dissect DNA-PK transcriptional events, the DNA-PK cistrome and transcriptome will be defined using clinically relevant models, resulting in a detailed understanding of DNA-PK mediated transcriptional regulation. The correlation of high expression of DNA-PK with poor prognosis in advanced PCa, as well as the known roles of DNA-PK in cancer-relevant pathways make DNA-PK a viable therapeutic target for PCa. Despite the current clinical assessment of DNA-PK inhibitors in multiple tumor types, there are no existing trials targeting PCa. Additional studies that assess the efficacy of current DNA-PK inhibitors in PCa will be presented and used to determine the best clinical space for their use. Citation Format: Emanuela Dylgjeri, Jonathan Goodwin, Karen Knudsen. Discerning the molecular basis of DNA-PK pro-tumorigenic functions and translational capacity as a therapeutic target in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1859. doi:10.1158/1538-7445.AM2015-1859

Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt.

The inflammasome activates caspase-1 and the release of interleukin-1β (IL-1β) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2(-/-)/Apc(Min/+) than in APC(Min/+) mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1β and were primarily mediated by a non-bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK-mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2(-/-) mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers.

Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model.

In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.

Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit

DNA double strand breaks (DSBs) can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK). DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ). Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.

DNA-dependent protein kinase plays a role in inflammation independent of DNA-damage repair function and its inhibition blocks allergic inflammation in mice and modulates human CD4+ T cell function without causing SCID (HYP7P.264)

Abstract We reported that DNA- dependent protein kinase (DNA-PK) is critical for the expression of NF-κB-dependent genes in TNF-α -treated glioblastoma cells including VCAM-1. Here we show that DNA-PK protein level and function are critical for VCAM-1 expression in TNF-α-treated human endothelial and mouse lung smooth muscle cells and are required for monocytes adhesion. Interestingly, DNA-PK activation and subsequent VCAM-1 expression in presence of TNF occurred independently of DNA breaks/repair. Administration of the DNA-PK inhibitor, NU7441, and DNA-PK heterozygosity reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness (AHR), and OVA-specific IgE production in asthma model of mice. Such effects correlated with a marked reduction in lung VCAM-1 expression and production of several inflammatory cytokines. Remarkably, such protection occurred without causing SCID. These results were confirmed in a chronic model of asthma using house dust mite. DNA-PK inhibition reduced Th2 cytokines production without affecting Th1 cytokines production and cell proliferation in CD3/CD28-stimulated human CD4+T cells potentially by blocking gata-3 expression. In mouse CD4+T cells, DNA-PK inhibition, in vitro, severely blocked CD3/CD28-induced gata-3 and t-bet expression and prevented differentiation of Th1 and Th2 cells. Thus, our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.

Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase

DNA-dependent protein kinase (DNA-PK) mediates double stranded DNA break repair, V(D)J recombination, and immunoglobulin class switch recombination, as well as innate immune and pro-inflammatory responses. However, there is limited information regarding the role of DNA-PK in adaptive immunity mediated by dendritic cells (DCs), which are the primary antigen-presenting cells in allergic asthma. Here we show that house dust mite induces DNA-PK phosphorylation, which is a marker of DNA-PK activation, in DCs via the generation of intracellular reactive oxygen species. We also demonstrate that pharmacological inhibition of DNA-PK, as well as the specific deletion of DNA-PK in DCs, attenuates the induction of allergic sensitization and Th2 immunity via a mechanism that involves the impaired presentation of mite antigens. Furthermore, pharmacological inhibition of DNA-PK following antigen priming similarly reduces the manifestations of mite-induced airway disease. Collectively, these findings suggest that DNA-PK may be a potential target for treatment of allergic asthma.

Pioglitazone restores IGFBP-3 levels through DNA PK in retinal endothelial cells cultured in hyperglycemic conditions.

Previously, we reported that pioglitazone prevented insulin resistance and cell death in type 2 diabetic retina by reducing TNFα and suppressor of cytokine signaling 3 (SOCS3) levels. Numerous reports suggest prominent vasoprotective effects of insulin growth factor binding protein-3 (IGFBP-3) in diabetic retinopathy. We hypothesized that pioglitazone protects against retinal cell apoptosis by regulating IGFBP-3 levels, in addition to reducing TNFα. The current study explored potential IGFBP-3 regulatory pathways by pioglitazone in retinal endothelial cells cultured in high glucose. Primary human retinal endothelial cells (REC) were grown in normal (5 mM) and high glucose (25 mM) and treated with pioglitazone for 24 hours. Cell lysates were processed for Western blotting and ELISA analysis to evaluate IGFBP-3, TNFα, and cleaved caspase 3 protein levels. Our results show that treatment with pioglitazone restored the high glucose-induced decrease in IGFBP-3 levels. This regulation was independent of TNFα actions, as reducing TNFα levels with siRNA did not prevent pioglitazone from increasing IGFBP-3 levels. Pioglitazone required protein kinase A (PKA) and DNA-dependent protein kinase (DNA PK) activity to regulate IGFBP-3, as specific inhibitors for each protein prevented pioglitazone-mediated normalization of IGFBP-3 in high glucose. Insulin growth factor binding protein-3 activity was increased and apoptosis decreased by pioglitazone, which was eliminated when serine site 156 of IGFBP-3 was mutated suggesting a key role of this phosphorylation site in pioglitazone actions. Our findings suggest that pioglitazone mediates regulation of IGFBP-3 via activation of PKA/DNA PK pathway in hyperglycemic retinal endothelial cells.

The mechanisms responsible for the radiosensitizing effects of sorafenib on colon cancer cells.

Colorectal cancer is one of the most common malignancies in the world, and is generally treated more effectively by chemoradiotherapy rather than radiotherapy or chemotherapy alone. Targeted radiosensitizers are often used in order to enhance the radiosensitivity of tumor cells. The aim of the present study was to identify the mechanism of radiosensitization by sorafenib in colorectal cancer. Three human colorectal adenocarcinoma cell lines (HCT116, HT29 and SW480) were treated with sorafenib alone or radiation followed by sorafenib. In vitro tests were performed using colony forming assays, FACS analysis, immunohistochemistry, tumor cell motility assays, invasion assays and endothelial tube formation assays. Sorafenib enhanced the anti-proliferative effects of radiation, reducing colony formation, increasing G2/M arrest and enhancing radiation-induced apoptosis by reactive oxygen species. Sorafenib also inhibited the repair of radiation-induced DNA damage by blocking the activation of DNA-dependent protein kinase. Combination treatment significantly inhibited tumor cell migration, tumor cell invasion and vascular endothelial growth factor-mediated angiogenesis in vitro. Taken together, our results provide a scientific rationale for the use of sorafenib with radiotherapy in colon cancer and suggest a clinical utility for this approach.

Cellular deficiency of Werner syndrome protein or RECQ1 promotes genotoxic potential of hydroquinone and benzo[a]pyrene exposure.

The 5 known RecQ helicases in humans (RECQ1, BLM, WRN, RECQL4, and RECQ5) have demonstrated roles in diverse genome maintenance mechanisms but their functions in safeguarding the genome from environmental toxicants are poorly understood. Here, we have evaluated a potential role of WRN (mutated in Werner syndrome) and RECQ1 (the most abundant homolog of WRN) in hydroquinone (HQ)- and benzo[a]pyrene (BaP)-induced genotoxicity. Silencing of WRN or RECQ1 expression in HeLa cells increased their sensitivity to HQ and BaP but elicited distinct DNA damage response. The RECQ1-depleted cells exhibited increased replication protein A phosphorylation, Chk1 activation, and DNA double-strand breaks (DSBs) as compared to control or WRN-depleted cells following exposure to BaP treatment. The BaP-induced DSBs in RECQ1-depleted cells were dependent on DNA-dependent protein kinase activity. Notably, loss of WRN in RECQ1-depleted cells ameliorated BaP toxicity. Collectively, our results provide first indication of nonredundant participation of WRN and RECQ1 in protection from the potentially carcinogenic effects of BaP and HQ.

Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase.

Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK – an endogenous cytoprotective cascade.

Structure of the herpes simplex virus 1 genome: manipulation of nicks and gaps can abrogate infectivity and alter the cellular DNA damage response.

The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA-modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or adding random sequences to the 3' ends of DNA, producing 3' flaps, did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5' flaps in the DNA following treatment resulted in a dramatic reduction (95 to 100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5' flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5'-flapped DNA was restored in cells that do not express DNA-PKcs and in cells cotransfected with the immediate early protein ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-dependent protein kinase (DNA-PK) and the nonhomologous end joining (NHEJ) repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5' flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection. For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest, and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes adopt a configuration suitable for lytic DNA replication. This study demonstrates that virion DNA activates the cellular DDR kinase, DNA-PK, and that this response is inhibitory to viral infection. Furthermore, we show that HSV-1 ubiquitin ligase, ICP0, plays an important role in counteracting the negative effects of DNA-PK activation. These findings support the notion that DNA-PK is antiviral and suggest that the fate of incoming viral DNA has important consequences for the progression of lytic infection. This study underscores the complex evolutionary relationships between HSV and its host.

RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex.

RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.

Dual and/or selective DNA-PK, PI3K inhibition and isoform selectivity of some new and known 2-amino-substituted-1,3-benzoxazines and substituted-1,3-naphthoxazines

The 2-morpholino-substituted-benzoxazines 7a and 7b were used in the synthesis of 2-morpholino-di-O-benzyl, O-pyridin-2yl, 3-yl and 4yl-methoxy)-1,3-benzoxazines 8a–8d, and N-(2-morpholino-4-oxo-4H-benz[e][1,3]oxazin-7-yl)-N-(pyridin-2-and-3-ylmethyl)acetamides 8e and 8f. The DNA-dependent protein kinase (DNA-PK) and phosphatidylinositol 3-kinase (PI3K) α, β, γ, and δ isoforms were studied for the new compounds 8a–8f and PI3K for the 18 previously synthesized compounds 9–26. The most active DNA-PK inhibitors were the 2-morpholino-O-substituted linear or angular naphthoxazine compounds 18–20 and 21–22 which showed potent and selective DNA-PK activity (IC50 from 0.01 to 2.43 µM) over PI3K. 8-(2-(4-Methylpiperazin-1-yl)ethoxy)-2-morpholino compound 13, and 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy) compound 25 showed selective DNA-PK inhibition. 2-morpholino-8-substituted-benzoxazine 9 (8-ph) and 10–12 (8-(pyridine-2-, 3-, or 4-ylmethoxy) showed high-to-moderate inhibition of PI3K and DNA-PK. A similar pattern for DNA-PK nonselectivity over PI3K was observed for compounds with 7,8-O-bis-substituted 8a, 8c, and 8d. No DNA-PK selectivity over PI3K was observed regardless whether the substitution was phenyl, pyridin-2-ylmethoxy, pyridin-3-ylmethoxy, and pyridin-4-ylmethoxy.

Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair

Purpose: From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. Materials and methods: DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. Results: Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37 °C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. Conclusion: It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.

DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin

DNA-dependent protein kinase (DNA-PK) plays a key role in the repair of DNA double-strand breaks (DSBs) that are probably the most deleterious form of DNA damage. Inhibition of DNA-PK has been considered as an attractive approach to decrease resistance to therapeutically induced DNA DSBs. Ionizing radiation (IR) and doxorubicin, which induce DSBs, are used in the treatment of breast cancer. We determined the cellular concentration of DNA-PK and other DSB-activated kinases: ATM and ATR and the effect of DNA-PK inhibition by NU7441 on DNA repair, cell cycle, and survival after IR or doxorubicin treatment in three human breast cancer cell lines (MCF-7, MDA-MB-231, and T47D) representing different breast cancer subtypes. T47D cells had the highest expression of DNA-PKcs, ATM, and ATR and the most rapid rate of DNA DSB repair. IR caused a 10- to 16-fold increase in DNA-PK activity and two to threefold induction of ATM in all 3 cell lines. NU7441 inhibited IR-induced DNA-PK activity in all cell lines with IC50s in the range 0.17-0.25 μM. NU7441 retarded the repair of DSB and significantly increased the sensitivity of all cell lines to IR (4- to 12-fold) and doxorubicin (3- to 13-fold). The greatest sensitization by NU7441 was observed in MDA-MB-231 cells. NU7441 affected the cell cycle distribution in all studied cell lines; increasing accumulation of cells in G2/M phase after DNA damage. Our data indicate that DNA-PK might be an effective target for chemo- and radio-potentiation in breast cancer and suggest that further development of DNA-PK inhibitors for clinical use is warranted.

DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation

The DNA-dependent protein kinase (DNA-PK) may function as a key signaling kinase in various cellular pathways other than DNA repair. Using a two-dimensional gel electrophoresis approach and stable DNA double-strand break-mimicking molecules (Dbait32Hc) to activate DNA-PK in the nucleus and cytoplasm, we identified 26 proteins that were highly phosphorylated following DNA-PK activation. Most of these proteins are involved in protein stability and degradation, cell signaling and the cytoskeleton. We investigated the relationship between DNA-PK and the cytoskeleton and found that the intermediate filament (IF) vimentin was a target of DNA-PK in vitro and in cells. Vimentin was phosphorylated at Ser459, by DNA-PK, in cells transfected with Dbait32Hc. We produced specific antibodies and showed that Ser459-P-vimentin was mostly located at cell protrusions. In migratory cells, the vimentin phosphorylation induced by Dbait32Hc was associated with a lower cellular adhesion and migration capacity. Thus, this approach led to the identification of downstream cytoplasmic targets of DNA-PK and revealed a connection between DNA damage signaling and the cytoskeleton.

Werner Syndrome Helicase Has a Critical Role in DNA Damage Responses in the Absence of a Functional Fanconi Anemia Pathway

Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2(-/-) cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia-telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2(-/-) cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia-deficient tumors by their sensitization to DNA cross-linking agents is discussed.