DNA-dependent Protein Kinase Activity Research Articles

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

Inhibition of DNA damage repair by artificial activation of PARP with siDNA

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.

HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration

Human immunodeficiency virus-1 (HIV-1) has infected more than 60 million people and caused nearly 30 million deaths worldwide, ultimately the consequence of cytolytic infection of CD4(+) T cells. In humans and in macaque models, most of these cells contain viral DNA and are rapidly eliminated at the peak of viraemia, yet the mechanism by which HIV-1 induces helper T-cell death has not been defined. Here we show that virus-induced cell killing is triggered by viral integration. Infection by wild-type HIV-1, but not an integrase-deficient mutant, induced the death of activated primary CD4 lymphocytes. Similarly, raltegravir, a pharmacologic integrase inhibitor, abolished HIV-1-induced cell killing both in cell culture and in CD4(+) T cells from acutely infected subjects. The mechanism of killing during viral integration involved the activation of DNA-dependent protein kinase (DNA-PK), a central integrator of the DNA damage response, which caused phosphorylation of p53 and histone H2AX. Pharmacological inhibition of DNA-PK abolished cell death during HIV-1 infection in vitro, suggesting that processes which reduce DNA-PK activation in CD4 cells could facilitate the formation of latently infected cells that give rise to reservoirs in vivo. We propose that activation of DNA-PK during viral integration has a central role in CD4(+) T-cell depletion, raising the possibility that integrase inhibitors and interventions directed towards DNA-PK may improve T-cell survival and immune function in infected individuals.

Integration triggers death

That HIV cripples the immune system by killing CD4+ T cells has long been known. It now emerges that the protein DNA-PK, activated by viral integration into the host-cell genome, is the agent of this death response. See Letter p.376 It is known that CD4+ helper T cells, key immune regulators, are eliminated by HIV-1 infection, but how the virus causes cell death has been unclear. Here Gary Nabel and colleagues show that HIV-1 integration is both necessary and sufficient to trigger CD4+ T-cell death, and that integration triggers cell death through activation of DNA-dependent protein kinase (DNA-PK), part of the DNA repair mechanism, and phosphorylation of p53. This work may help to explain how reservoirs of virus are established in HIV-infected subjects, and suggests that treatment with integrase or DNA-PK inhibitors might prolong CD4 cell survival and delay the development of AIDS.

DNA Ligases I and III Cooperate in Alternative Non-Homologous End-Joining in Vertebrates

Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated. Here, we take advantage of the powerful genetics of the DT40 chicken B-cell system to delineate the roles of LIG1 and LIG3 in alternative NHEJ. Our results expand the functions of LIG1 to alternative NHEJ and demonstrate a remarkable ability for LIG3 to backup DSB repair by NHEJ in addition to its essential function in the mitochondria. Together with results on DNA replication, these observations uncover a remarkable and previously unappreciated functional flexibility and interchangeability between LIG1 and LIG3.

Inactivation of DNA–Dependent Protein Kinase Promotes Heat–Induced Apoptosis Independently of Heat–Shock Protein Induction in Human Cancer Cell Lines

The inhibition of DNA damage response pathway seems to be an attractive strategy for cancer therapy. It was previously reported that in rodent cells exposed to heat stress, cell growth was promoted by the activity of DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair. The absence of a functioning DNA-PK was associated with down regulation of heat shock protein 70 (HSP70). The objective of this study is thus to investigate the role of DNA-PK inhibition in heat-induced apoptosis in human cell lines. The inhibitors of phosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser2056, such as NU7026 and NU7441, were utilized. Furthermore, knock down of DNA-PKcs was carried out using small interfering RNA (siDNA-PKcs). For heat exposure, cells were placed in water bath at 44°C for 60 min. Apoptosis was evaluated after 24 h incubation flow cytometrically. Proteins were extracted after 24 h and analyzed for HSP70 and HSP40 expression by Western blotting. Total RNA was extracted 6 h after treatment and analyzed using a GeneChip® microarray system to identify and select the up-regulated genes (≥1.5 fold). The results showed an enhancement in heat-induced apoptosis in absence of functioning DNA-PKcs. Interestingly, the expression levels of HSP70 and HSP40 were elevated in the absence of DNA-PKcs under heat stress. The results of genetic network analysis showed that HSPs and JUN genes were up-regulated independently of DNA-PKcs in exposed parent and knock out cells. In the presence of functioning DNA-PKcs, there was an observed up-regulation of anti-apoptotic genes, such as NR1D1, whereas in the absence of DNA-PKcs the pro-apoptotic genes, such as EGR2, were preferentially up-regulated. From these findings, we concluded that in human cells, the inactivation of DNA-PKcs can promote heat-induced apoptosis independently of heat-shock proteins.

Insulin signalling mechanisms for triacylglycerol storage

Insulin signalling is uniquely required for storing energy as fat in humans. While de novo synthesis of fatty acids and triacylglycerol occurs mostly in liver, adipose tissue is the primary site for triacylglycerol storage. Insulin signalling mechanisms in adipose tissue that stimulate hydrolysis of circulating triacylglycerol, uptake of the released fatty acids and their conversion to triacylglycerol are poorly understood. New findings include (1) activation of DNA-dependent protein kinase to stimulate upstream stimulatory factor (USF)1/USF2 heterodimers, enhancing the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c); (2) stimulation of fatty acid synthase through AMP kinase modulation; (3) mobilisation of lipid droplet proteins to promote retention of triacylglycerol; and (4) upregulation of a novel carbohydrate response element binding protein β isoform that potently stimulates transcription of lipogenic enzymes. Additionally, insulin signalling through mammalian target of rapamycin to activate transcription and processing of SREBP1c described in liver may apply to adipose tissue. Paradoxically, insulin resistance in obesity and type 2 diabetes is associated with increased triacylglycerol synthesis in liver, while it is decreased in adipose tissue. This and other mysteries about insulin signalling and insulin resistance in adipose tissue make this topic especially fertile for future research.

Steroidogenic Factor 1 (NR5A1) Maintains Centrosome Homeostasis in Steroidogenic Cells by Restricting Centrosomal DNA-Dependent Protein Kinase Activation

Steroidogenic factor 1 (SF-1 or NR5A1) is a nuclear receptor that controls adrenogenital cell growth and differentiation. Adrenogenital primordial cells from SF-1 knockout mice die of apoptosis, but the mechanism by which SF-1 regulates cell survival is not entirely clear. Besides functioning in the nucleus, SF-1 also resides in the centrosome and controls centrosome homeostasis. Here, we show that SF-1 restricts centrosome overduplication by inhibiting aberrant activation of DNA-dependent protein kinase (DNA-PK) in the centrosome. SF-1 was found to be associated with Ku70/Ku80 only in the centrosome, sequestering them from the catalytic subunit of DNA-PK (DNA-PKcs). In the absence of SF-1, DNA-PKcs was recruited to the centrosome and activated, causing aberrant activation of centrosomal Akt and cyclin-dependent kinase 2 (CDK2)/cyclin A and leading to centrosome overduplication. Centrosome overduplication caused by SF-1 depletion was averted by the elimination of DNA-PKcs, Ku70/80, or cyclin A or by the inhibition of CDK2 or Akt. In the nucleus, SF-1 did not interact with Ku70/80, and SF-1 depletion did not activate a nuclear DNA damage response. Centriole biogenesis was also unaffected. Thus, centrosomal DNA-PK signaling triggers centrosome overduplication, and this centrosomal event, but not the nuclear DNA damage response, is controlled by SF-1.

An autonomous chromatin/DNA-PK mechanism for localized DNA damage signaling in mammalian cells

Rapid phosphorylation of histone variant H2AX proximal to DNA breaks is an initiating event and a hallmark of eukaryotic DNA damage responses. Three mammalian kinases are known to phosphorylate H2AX in response to DNA damage. However, the mechanism(s) for damage-localized phosphorylation remains incompletely understood. The DNA-dependent protein kinase (DNA-PK) is the most abundant H2AX-modifying kinases and uniquely activated by binding DNA termini. Here, we have developed a novel approach to examine enzyme activity and substrate properties by executing biochemical assays on intact cellular structures. We apply this approach to examine the mechanisms of localized protein modification in chromatin within fixed cells. DNA-PK retains substrate specificity and independently generates break-localized γH2AX foci in chromatin. In situ DNA-PK activity recapitulates localization and intensity of in vivo H2AX phosphorylation and requires no active cellular processes. Nuclease treatments or addition of exogenous DNA resulted in genome-wide H2AX phosphorylation, showing that DNA termini dictated the locality of H2AX phosphorylation in situ. DNA-PK also reconstituted focal phosphorylation of structural maintenance of chromatin protein 1, but not activating transcription factor 2. Allosteric regulation of DNA-PK by DNA termini protruding from chromatin constitutes an autonomous mechanism for break-localized protein phosphorylation that generates sub-nuclear foci. We discuss generalized implications of this mechanism in localizing mammalian DNA damage responses.

Dual Functions of the Homeoprotein DLX4 in Modulating Responsiveness of Tumor Cells to Topoisomerase II-Targeting Drugs

Topoisomerase II (TOP2)-targeting poisons such as anthracyclines and etoposide are commonly used for cancer chemotherapy and kill tumor cells by causing accumulation of DNA double-strand breaks (DSB). Several lines of evidence indicate that overexpression of TOP2A, the gene encoding topoisomerase IIα, increases sensitivity of tumor cells to TOP2 poisons, but it is not clear why some TOP2A-overexpressing (TOP2A-High) tumors respond poorly to these drugs. In this study, we identified that TOP2A expression is induced by DLX4, a homeoprotein that is overexpressed in breast and ovarian cancers. Analysis of breast cancer datasets revealed that TOP2A-high cases that also highly expressed DLX4 responded more poorly to anthracycline-based chemotherapy than TOP2A-high cases that expressed DLX4 at low levels. Overexpression of TOP2A alone in tumor cells increased the level of DSBs induced by TOP2 poisons. In contrast, DLX4 reduced the level of TOP2 poison-induced DSBs irrespective of its induction of TOP2A. DLX4 did not stimulate homologous recombination-mediated repair of DSBs. However, DLX4 interacted with Ku proteins, stimulated DNA-dependent protein kinase activity, and increased erroneous end-joining repair of DSBs. Whereas DLX4 did not reduce levels of TOP2 poison-induced DSBs in Ku-deficient cells, DLX4 stimulated DSB repair and reduced the level of TOP2 poison-induced DSBs when Ku was reconstituted in these cells. Our findings indicate that DLX4 induces TOP2A expression but reduces sensitivity of tumor cells to TOP2 poisons by stimulating Ku-dependent repair of DSBs. These opposing activities of DLX4 could explain why some TOP2A-overexpressing tumors are not highly sensitive to TOP2 poisons.

DNA-dependent protein kinase and DNA repair: relevance to Alzheimer's disease

The pathological hallmark of Alzheimer's disease (AD), the leading cause of senile dementia, involves region-specific neuronal death and an accumulation of neuronal and extracellular lesions termed neurofibrillary tangles and senile plaques, respectively. One of the biochemical abnormalities observed in AD is reduced DNA end-joining activity. The reduced capacity of post-mitotic neurons for some types of DNA repair is further compromised by aging. The predominant mechanism to repair double-strand DNA (dsDNA) breaks (DSB) is non-homologous end joining (NHEJ), which requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK is a holoenzyme comprising the p460 kDa DNA-PK catalytic subunit (DNA-PKcs) and the Ku heterodimer consisting of p86 (Ku 80) and p70 (Ku 70) subunits. Ku binds to DNA ends first and then recruits DNA-PKcs during NHEJ. However, in AD brains, reduced NHEJ activity has been reported along with reduced levels of DNA-PKcs and the Ku proteins, indicating a potential link between AD and dsDNA damage. Since age-matched control brains also show a reduction in these protein levels, whether there is a direct link between NHEJ ability and AD remains unknown. Possible mechanisms involving the role of DNA-PK in neurodegeneration, a benchmark of AD, are the focus of this review.

An Inhibitor of Nonhomologous End-Joining Abrogates Double-Strand Break Repair and Impedes Cancer Progression

DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end-joining (NHEJ). Inhibiting Ligase IV could result in amassing of DSBs, thereby serving as a strategy toward treatment of cancer. Here, we identify a molecule, SCR7 that inhibits joining of DSBs in cell-free repair system. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding but not that of T4 DNA Ligase or Ligase I. SCR7 inhibits NHEJ in a Ligase IV-dependent manner within cells,and activates the intrinsic apoptotic pathway. More importantly, SCR7 impedes tumor progression in mouse models and when coadministered with DSB-inducing therapeutic modalities enhances their sensitivity significantly. This inhibitor to target NHEJ offers a strategy toward the treatment of cancer and improvement of existing regimens.

Inhibition of DNA-dependent protein kinase reduced palmitate and oleate-induced lipid accumulation in HepG2 cells

The aim of this study is to investigate the involvement of DNA-dependent protein kinase (DNA-PK) in palmitate and oleate-induced lipid accumulation in hepatocytes. We treated HepG2 with free fatty acids (FFA) (0.33 mM palmitate and 0.66 mM oleate) mixture to induce lipid accumulation. Cellular lipid was determined by Nile Red staining followed by flow cytometry detection as well as phase contrast and fluorescence microscope examination. Cell viability was detected by MTT assay. Apoptosis was detected by DAPI staining. Lipogenic gene expression was examined by real-time PCR at mRNA level and Western blotting at protein level. Promoter transcriptional activity was measured by dual luciferase assay. FFA treatment neither affected HepG2 cells viability nor induced DNA fragmentation, while induced cellular lipid accumulation was associated by the upregulation of sterol regulatory element-binding protein-1 (SREBP1) and fatty acid synthase (FAS) at both mRNA and protein levels. Interestingly, we also found that both the protein phosphatase 2A (PP2A) protein expression and DNA-PK activity were increased in these cells. Inhibition of PP2A by okadaic acid, knockdown of DNA-PK by siRNA or inhibition of DNA-PK by specific DNA-PK inhibitors curtailed the FFA-induced upregulations of the SREBP1 mRNA expression and the nuclear active SREBP1 protein expression, and reduced FFA-induced upregulation of FAS promoter transcriptional activity and lipid accumulation. This is the first time suggesting that inhibition of DNA-PK reduced FFA-induced lipid accumulation in hepatocytes. This finding might help us better understand non-alcoholic steatohepatitis pathogenesis.

Chemoproteomic Profiling Identifies Changes in DNA-PK as Markers of Early Dengue Virus Infection

Many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used RNA- and protein-based profiling methods that monitor steady-state gene expression. Mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. Taking dengue virus 2 (DV2) infection as a model system, we utilized reactive ATP- and ADP-acyl phosphates as chemical proteomic probes to detect changes in host kinase function that occur within the first hour of infection. The DNA-dependent protein kinase (DNA-PK) was discovered as a host enzyme with significantly elevated probe labeling within 60 min of DV2 infection. Increased probe labeling was associated with increased DNA-PK activity in nuclear lysates and localization of DNA-PK in nucleoli. These effects on DNA-PK were found to require a postfusion step of DV2 entry and were recapitulated by transfection of cells with RNA corresponding to stem loop B of the DV2 5' untranslated region. Upon investigation of the potential downstream consequences of these phenomena, we detected a modest but significant reduction in the interferon response induced by DV2 in cells partially depleted of the Ku80 subunit of DNA-PK. These findings identify changes in DNA-PK localization and activity as very early markers of DV2 infection. More broadly, these results highlight the utility of chemoproteomic profiling as a tool to detect changes in protein function associated with different cell states and that may occur on very short time scales.

Chemosensitization of Cancer Cells by KU-0060648, a Dual Inhibitor of DNA-PK and PI-3K

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 μmol/L (MCF7 cells) and 0.17 μmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 μmol/L (MCF7 cells) and more than 10 μmol/L (SW620 cells). Five-day exposure to 1 μmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.

Heat shock protein 90α (HSP90α), a substrate and chaperone of DNA-PK necessary for the apoptotic response

The "apoptotic ring" is characterized by the phosphorylation of histone H2AX at serine 139 (γ-H2AX) by DNA-dependent protein kinase (DNA-PK). The γ-H2AX apoptotic ring differs from the nuclear foci patterns observed in response to DNA-damaging agents. It contains phosphorylated DNA damage response proteins including activated Chk2, activated ATM, and activated DNA-PK itself but lacks MDC1 and 53BP1, which are required to initiate DNA repair. Because DNA-PK can phosphorylate heat shock protein 90α (HSP90α) in biochemical assays, we investigated whether HSP90α is involved in the apoptotic ring. Here we show that HSP90α is phosphorylated by DNA-PK on threonines 5 and 7 early during apoptosis and that both phosphorylated HSP90α and DNA-PK colocalize in the apoptotic ring. We also show that DNA-PK is a client of HSP90α and that HSP90α is required for full DNA-PK activation, γ-H2AX formation, DNA fragmentation, and apoptotic body formation. In contrast, HSP90 inhibition by geldanamycin markedly enhances TRAIL-induced DNA-PK and H2AX activation. Together, our results reveal that HSP90α is a substrate and chaperone of DNA-PK in the apoptotic response. The response of phosphorylated HSP90α to TRAIL and its localization to the γ-H2AX ring represent epigenetic features of apoptosis that offer insights for studying and monitoring nuclear apoptosis.

Functional Interplay between Caspase Cleavage and Phosphorylation Sculpts the Apoptotic Proteome

Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation.

Novel Survivin Inhibitor YM155 elicits Cytotoxicity in Glioblastoma Cell Lines with Normal or Deficiency DNA-Dependent Protein Kinase Activity

Pediatric glioblastoma is a malignant disease with an extremely poor clinical outcome. Patients usually suffer from resistance to radiation therapy, so targeted drug treatment may be a new possibility for glioblastoma therapy. Survivin is also overexpressed in glioblastoma. YM155, a novel small-molecule survivin inhibitor, has not been examined for its use in glioblastoma therapy. The human glioblastoma cell line M059K, which expresses normal DNA-dependent protein kinase (DNA-PK) activity and is radiation-resistant, and M059J, which is deficient in DNA-PK activity and radiation-sensitive, were used in the study. Cell viability, DNA fragmentation, and the expression of survivin and securin following YM155 treatment were examined using MTT (methylthiazolyldiphenyl-tetrazolium) assay, ELISA assay, and Western blot analysis, respectively. YM155 caused a concentration-dependent cytotoxic effect, inhibiting the cell viability of both M059K and M059J cells by 70% after 48 hours of treatment with 50 nM YM155. The half-maximal inhibitory concentration (IC50) was around 30-35 nM for both cell lines. Apoptosis was determined to have occurred in both cell lines because immunoreactive signals from the DNA fragments in the cytoplasm were increased 24 hours after treatment with 30 nM YM155. The expression of survivin and securin in the M059K cells was greater than that measured in the M059J cells. Treatment with 30 nM YM155, for both 24 and 48 hours, significantly suppressed the expression of survivin and securin in both cell lines. The novel survivin inhibitor YM155 elicits potent cytotoxicity in glioblastoma cells in vitro via DNA-PK-independent mechanisms. YM155 could be used as a new therapeutic agent for the treatment of human glioblastomas.

Abstract 3122: The DNA-PK inhibitor NU7441 inhibits non-homologous end joining, enhances radio- and chemosensitivity and increases dependence on homologous recombination in hepatocellular carcinoma cell lines

Abstract Background DNA double-strand breaks (DSBs), the most cytotoxic lesions induced by ionizing radiation (IR) and anticancer drugs such as topoisomerase II poisons (e.g., doxorubicin), are repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). DNA-dependent protein kinase (DNA-PK), which initiates NHEJ, is up-regulated in hepatocellular carcinoma (HCC) (GEO profiles), possibly contributing to anticancer therapy resistance. To assess DNA-PK as a potential therapeutic target for chemo- and radio-sensitisation in HCC we determined the effect of the DNA-PK inhibitor, NU7441, on DSB repair and cytotoxicity in HCC cells. Methods DNA-PK protein levels and activation by IR (Western blot), DSB levels (y-H2AX foci), HR (RAD51 foci), cell growth (DAPI fluorescence) and cytotoxicity (colony formation) following exposure to IR or doxorubicin was determined in a panel of 6 hepatoma cell lines (HepG2, Hep3B, Huh7, SNU-182, SNU475 and PLC/PRF/5). Results DNA-PK protein concentration and activity did not vary significantly across the panel (±24% and ±37%, respectively) but there were cell-specific sensitivities to IR and doxorubicin (e.g. HepG2 2-fold more resistant than Hep3B). NU7441 significantly sensitised all cells to both doxorubicin (average PF50 4.3±3.0) and IR (average PF50 3.9±1.1) in growth inhibition assays and significantly reduced survival (4.8 to 3.3-fold) in colony forming assays. Following exposure to IR, NU7441 significantly delayed yH2AX focus clearance in all cell lines (e.g. only 13% cleared at 4 hr compared to 50% in control), but increased RAD51 focus formation (3-fold). Conclusion While chemo- and radio-sensitivity in HCC cells was not dependent on DNA-PK expression or activity, NU7441 causes greater than 2-fold chemo- and radiosensitisation in all cells. This was accompanied by a substantial reduction in the rapid phase of DNA repair (NHEJ-dependent) and a shift to a greater reliance on the slower HR repair. DNA-PK inhibitors may have potential as chemo- and radio-sensitisors in hepatoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3122. doi:1538-7445.AM2012-3122

DNA‐DEPENDENT PROTEIN KINASE IS CRITICAL FOR VCAM‐1 EXPRESSION UPON TNF TREATMENT THROUGH PHOSPHORYLATION OF p50 NF‐kappa B: A CRITICAL INVOLVEMENT IN VASCULAR AND LUNG INFLAMMATION

NF‐κB is critical for vascular cell adhesion molecule 1 (VCAM‐1) expression during vascular inflammation. We show that DNA‐dependent protein kinase (DNA‐PK) is required for VCAM‐1 expression in response to TNF‐α in an established cell line and in primary endothelial cells. DNA‐PK deficiency, achieved by DNA‐PKcs heterozygosity, reduced VCAM‐1 expression in the aorta in an animal model of septic shock and blocked airway neutrophilia in an animal model of acute lung injury. Interestingly, such effect unravels a novel function for DNA‐PK independent of its role in immune competence. Phosphorylation and nuclear translocation of p65 NF‐κB (p65) was insufficient for VCAM‐1 expression in response to TNF‐α. The requirement for p50 NF‐κB (p50) was associated with its interaction with and phosphorylation by DNA‐PK, which appears to be dominant over the requirement for p65 activation. Additionally, DNA‐PK activity appeared to increase the κB‐site binding ability of p50/p50 and p50/p65 dimers to VCAM‐1 promoter. Serine 20 in p50 was found to be phosphorylated by DNA‐PK. Re‐establishing wild‐type p50, but not its S20/A mutant, in p50‐knockout cells reversed VCAM‐1 expression. Together, these results elucidate a novel mechanism for the regulation of VCAM‐1 expression by DNA‐PK through phosphorylation of p50 and its critical involvement in vascular and lung inflammation.