Etoposides block cell division by interfering with the action of topoisomerase II, leaving enzyme-DNA double-strand breaks. We found that certain components of the trimeric DNA-dependent protein kinase influence cell survival following etoposide damage. Interestingly, either Ku70- or Ku80-deficient cell lines, but not mutant cell lines of the DNA-PK catalytic sub-unit (DNA-PKcs), were found to be hypersensitive to the effects of etoposide VP16. Ku70- and Ku80-deficient cells can be complemented to an etoposide resistant phenotype by introducing wildtype Ku70 or Ku80 cDNAs. Mutational analysis of introduced Ku70 cDNAs into murine embryonic stem cells deleted for Ku70 (-/-) showed that mutants where heterodimerization and DNA binding functions of Ku were disrupted, also blocked the restoration of etoposide resistance. In contrast with the differential etoposide sensitivity of DNA-PK mutants, both Ku- and DNA-PKcs-deficient cell lines showed G2 ionizing radiation-induced delays, a cell cycle phase where topoisomerase II function is critical. Thus, the topoisomerase II cleaved complexes may be an example of DNA lesions requiring the Ku heterodimer, but not DNA-PK for DNA repair.

Although post-translational modifications by the small ubiquitin-like modifiers (SUMO) are known to be important in DNA damage response, it is unclear whether they have a role in double-strand break (DSB) repair by non-homologous end joining (NHEJ). Here, we analyzed various DSB repair pathways upon inhibition of SUMO-mediated protein-protein interactions using peptides that contain the SUMO-interaction motif (SIM) and discriminate between mono- and SUMO-chain modifications. The SIM peptides specifically inhibit NHEJ as shown by in vivo repair assays and radio-sensitivity of cell lines deficient in different DSB repair pathways. Furthermore, mono-SUMO, instead of SUMO-chain, modifications appear to be involved in NHEJ. Immunoprecipitation experiments also showed that the SIM peptide interacted with SUMOylated Ku70 after radiation. This study is the first to show an important role for SUMO:SIM-mediated protein-protein interactions in NHEJ, and provides a mechanistic basis for the role of SIM peptide in sensitizing genotoxic stress of cancer cells.

Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans.

The purpose was to study the prognostic and predictive roles of DNA protein kinase catalytic subunit (DNA-PKcs), Ku70/80 and p53 for the effect of radiotherapy in breast cancer patients. Protein expressions of Ku70/80, DNA-PKcs and p53 were examined using immunohistochemistry in tumours from 224 breast cancer patients, who were randomised to receive post-operative radiotherapy or adjuvant chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil). One hundred and twenty-nine (60%) of the tumours had low expression of Ku70/80, 122 (57%) had low expression of DNA-PKcs and 65 (30%) had altered p53 expression. None of the proteins were indicative to the prognosis of local recurrence-free survival. Even though the expression of Ku70/80 and DNA-PKcs correlated well, they were not associated with treatment outcome in the same way. Low expression of Ku70/80 predicted good effect of radiotherapy (RR=0.31, 95% CI 0.13-0.76, p=0.01). In contrast, the greatest benefit of radiotherapy over chemotherapy was seen in patients with high DNA-PKcs expression (RR=0.25, 95% CI 0.07-0.84, p=0.02). Altered p53 expression predicted poor response to radiotherapy. We believe that the results reflect the different roles of DNA-PKcs and Ku70/80 in repair and cell death regulation after DNA damage. These differences could be of importance when developing drugs that target DNA repair.

It is well documented that unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of specific cellular target genes by acting in concert with a corepressor complex harboring histone deacetylase (HDAC) activity. To fully explore the cofactors that interact with the transcriptionally repressive form of TR, we biochemically isolated a multiprotein complex that assembles on a TR.retinoid X receptor (RXR) heterodimer in HeLa nuclear extracts and identified its polypeptide components by mass spectrometry. A subset of TR.RXR-associated polypeptides included NCoR, SMRT, TBL1, and HDAC3, which represent the core components of a previously described NCoR/SMRT corepressor complex. We also identified several polypeptides that constitute a DNA-dependent protein kinase (DNA-PK) enzyme complex, a regulator of DNA repair, recombination, and transcription. These polypeptides included the catalytic subunit DNA-PKcs, the regulatory subunits Ku70 and Ku86, and the poly(ADP-ribose) polymerase 1. Density gradient fractionation and immunoprecipitation analyses provided evidence for the existence of a high molecular weight TR.RXR.corepressor holocomplex containing both NCoR/SMRT and DNA-PK complexes. Chromatin immunoprecipitation studies confirmed that unliganded TR.RXR recruits both complexes to the triiodothyronine-responsive region of growth hormone gene in vivo. Interestingly, DNA-PKcs, a member of the phosphatidylinositol 3-kinase family, was found to phosphorylate HDAC3 when the purified TR.RXR.corepressor holocomplex was incubated with ATP. This phosphorylation was accompanied by a significant enhancement of the HDAC activity of this complex. Collectively, our results indicated that DNA-PK promotes the establishment of a repressive chromatin at a TR target promoter by enhancing the HDAC activity of the receptor-bound NCoR/SMRT corepressor complex.

Cervical carcinoma affects around 3400 women in the UK each year and advanced disease is routinely treated with radiation. As part of a programme to establish rapid and convenient methods of predicting tumour and patient responses to radiotherapy, we have examined the relationship between the pre-treatment expression of the Ku components of the DNA damage recognition complex DNA-PK and patient survival in cervical carcinoma. Using immunohistochemistry of formalin-fixed sections of tumour biopsies, antibodies to Ku70 and Ku80 stained identical regions of tumour and there was a high degree of correlation between the mean number of cells stained positive for the two components in 77 tumours (r = 0.82, P<0.001). In 53 tumours there was a borderline significant correlation between measurements of tumour radiosensitivity (surviving fraction at 2 gray: SF2) and Ku70 expression (r = 0.26, P = 0.057) and no correlation for Ku80 (r = 0.18, P = 0.19). However, all tumours with a low number of Ku70 or Ku80 positive cells were radiosensitive. Furthermore, using log-rank analysis there was significantly higher survival in the patients whose tumours had a low Ku70 expression (P = 0.046). This difference was also reflected with Ku80, but did not reach statistical significance (P = 0.087). The study suggests that lack of Ku protein leads to radiosensitivity in some tumours and that other factors are responsible for radiosensitive tumours with high Ku expression. It is likely that the most accurate prediction of treatment outcome will lie in assessing the expression of several proteins involved in the recognition and repair of DNA damage, one of which will be Ku.

The Ku antigen (70- and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PKcs) to DNA ends and to specific DNA sequences from which the kinase is activated. Ku and DNA-PKcs plays essential roles in double-stranded DNA break repair and V(D)J recombination and have been implicated in the regulation of specific gene transcription. In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a specific interaction between the 70-kDa subunit of Ku heterodimer and the homeodomain of HOXC4, a homeodomain protein expressed in the hematopoietic system. Unexpectedly, a similar interaction with Ku was observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins. Ku-homeodomain binding was mediated through the extreme C terminus of Ku70 and was abrogated by amino acid substitutions at Lys595/Lys596. Ku binding allowed the recruitment of the homeodomain to DNA ends and dramatically enhanced the phosphorylation of homeodomain-containing proteins by DNA-PK. These results suggest that Ku functions as a substrate docking protein for signaling by DNA-PK to homeodomain proteins from DNA ends.

Chemical castration improves responses to radiotherapy in prostate cancer, but the mechanism is unknown. We hypothesized that this radiosensitization is caused by castration-mediated down-regulation of nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). To test this, we enrolled 48 patients with localized prostate cancer in two arms of the study: either radiotherapy first or radiotherapy after neoadjuvant castration treatment. We biopsied patients at diagnosis and before and after castration and radiotherapy treatments to monitor androgen receptor, NHEJ, and DSB repair in verified cancer tissue. We show that patients receiving neoadjuvant castration treatment before radiotherapy had reduced amounts of the NHEJ protein Ku70, impaired radiotherapy-induced NHEJ activity, and higher amounts of unrepaired DSBs, measured by γ-H2AX foci in cancer tissues. This study demonstrates that chemical castration impairs NHEJ activity in prostate cancer tissue, explaining the improved response of patients with prostate cancer to radiotherapy after chemical castration.

The majority of antigen receptor diversity in mammals is generated by V(D)J recombination. During this process DNA double strand breaks are introduced at recombination signals by lymphoid specific RAG1/2 proteins generating blunt ended signal ends and hairpinned coding ends. Rejoining of all DNA ends requires ubiquitously expressed DNA repair proteins, such as Ku70/86 and DNA ligase IV/XRCC4. In addition, the formation of coding joints depends on the function of the scid gene encoding the catalytic subunit of DNA-dependent protein kinase, DNA-PK(CS), that is somehow required for processing of coding end hairpins. Recently, it was shown that purified RAG1/2 proteins can cleave DNA hairpins in vitro, but the same activity was also described for a protein complex of the DNA repair proteins Nbs1/Mre11/Rad50. This leaves the possibility that either protein complex might be involved in coding end processing in V(D)J recombination. We have therefore analyzed V(D)J recombination in cells from patients with Nijmegen breakage syndrome, carrying a mutation in the nbs1 gene. We find that V(D)J recombination frequencies and the quality of signal and coding joining are comparable to wild-type controls, as analyzed by a cellular V(D)J recombination assay. In addition, we did not detect significant differences in CDR3 sequences of endogenous Ig lambdaL and kappaL chain gene loci cloned from peripheral blood lymphocytes of an NBS patient and of healthy individuals. These findings suggest that the Nbs1/Mre11/Rad50 complex is not involved in coding end processing of V(D)J recombination.

Murine preB lymphocytes grow in tissue culture in the presence of stromal cells and interleukin 7 (IL-7), and can be induced to differentiate to surface-immunoglobulin-positive B cells in vitro by withdrawal of IL-7. Upon differentiation, proliferation ceases, and upregulation of Rag-1 and Rag-2 expression, and induction of V(D)J immunoglobulin-gene rearrangements occur. DNA-dependent protein kinase (DNA-PK) is required for effective V(D)J recombination and repair of DNA double-strand breaks. The holoenzyme comprises a catalytic subunit (DNA-PKcs) and the Ku heterodimer (Ku70/Ku80). We have analyzed expression of Ku70, Ku80 and DNA-PKcs upon induction of differentiation in preB cells derived from wild-type, severe combined immunodeficiency (SCID) and Rag-2-/- mice. Protein levels of Ku80 and Ku70 moderately decrease after induction in all three cell types. A distinct polypeptide that crossreacts with anti-Ku Ig appears in the cytoplasm of wild-type and Rag-2-/- cells, but not of SCID cells. In mouse preB cells, Ku70 and Ku80 are present in the nuclei and cytoplasm before and after onset of differentiation. In vivo, Ku70 is predominantly expressed in V(D)J-recombination-active, early-preB and CD4-/CD8- thymocyte cell populations. Upon differentiation, protein levels of DNA-PKcs are unaltered. DNA-PK activity, which is not detectable in SCID cells, increases in wild-type and Rag-2-/- cells more than twofold shortly after induction of differentiation, then falls back to about 50% of starting levels.

Reperfusion of ischemic tissue causes an immediate increase in DNA damage, including base lesions and strand breaks. Damage is reversible in surviving regions indicating that repair mechanisms are operable. DNA strand breaks are repaired by nonhomologous end joining in mammalian cells. This process requires DNA-dependent protein kinase (DNA-PK), composed of heterodimeric Ku antigen and a 460,000 Da catalytic subunit (DNA-PKcs). In this study, a rabbit spinal cord model of reversible ischemia was used to demonstrate the effect of acute CNS injury on the activity and expression of DNA-dependent protein kinase. The DNA-binding activity of Ku antigen, analyzed by an electrophoretic mobility shift assay, increased during reperfusion after a short ischemic insult (15 min of occlusion), from which the animals recover neurological function. After severe ischemic injury (60 min of occlusion) and reperfusion that results in permanent paraplegia, Ku DNA binding was reduced. Protein levels of the DNA-PK components-Ku70, Ku80, and DNA-PKcs-were monitored by immunoblotting. After 60 min of occlusion, the amount of DNA-PKcs and the enzyme poly(ADP-ribose) polymerase (PARP) decreased with the same time course during reperfusion. Concurrently 150 and 120 kDa fragments were immunostained by an anti-DNA-PKcs monoclonal antibody. This antibody was shown to cross-react with alpha-fodrin breakdown products. The 120 kDa fodrin peptide is associated with caspase-3 activation during apoptosis. Both DNA-PKcs and PARP are also substrates for caspase-3-like activities. The results are consistent with a model in which after a short ischemic insult, DNA repair proteins such as DNA-PK are activated. After severe ischemic injury, DNA damage overwhelms repair capabilities, and cell death programs are initiated.

All components of the double-stranded DNA break (DSB) repair complex DNA-dependent protein kinase (DNA-PK), including Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs), were found in the radiosensitive spermatogonia. Although p53 induction was unaffected, spermatogonial apoptosis occurred faster in the irradiated DNA-PKcs-deficient scid testis. This finding suggests that spermatogonial DNA-PK functions in DNA damage repair rather than p53 induction. Despite the fact that early spermatocytes lack the Ku proteins, spontaneous apoptosis of these cells occurred in the scid testis. The majority of these apoptotic spermatocytes were found at stage IV of the cycle of the seminiferous epithelium where a meiotic checkpoint has been suggested to exist. Meiotic synapsis and recombination during the early meiotic prophase induce DSBs, which are apparently less accurately repaired in scid spermatocytes that then fail to pass the meiotic checkpoint. The role for DNA-PKcs during the meiotic prophase differs from that in mitotic cells; it is not influenced by ionizing radiation and is independent of the Ku heterodimer.

Human ovarian carcinoma cells (C70 and C200) made resistant to cisplatin from A2780 cells demonstrated an approximately 20-fold resistance to the drug. These same cell lines showed no collateral resistance (as compared with the wild-type) to a novel glutathione S-transferase pi-activated prodrug [gamma-glutamyl-alpha-amino-beta[2-ethyl-N,N,N',N'-tetrakis (2-chloroethyl) phosphorodiamidate]-sulfonyl-propionyl-(R)-(-) phenylglycine; TLK286]. Previous results have shown a direct correlation between levels of GST pi expression and cytotoxicity for TLK286 (L. A. Rosario et al., Mol. Pharmacol., 58: 167-174, 2000.). However, protein levels of the isozyme were identical in wild-type C70 and C200 cell lines. In analyzing the DNA repair capacity of C70 and C200, an altered expression of the DNA-dependent protein kinase (DNA-PK) complex (catalytic subunit DNA-PKcs, and the heterodimers Ku70 and Ku80) was found. In C70 and C200 cells, DNA-PKcs was overexpressed at both the transcript and protein levels, whereas amounts of Ku70 and Ku80 were higher only at the level of protein expression. TLK286 in either its parent or activated form inhibited the catalytic kinase activity of purified DNA-PK with an IC50 value of approximately 1 microM. Coimmunoprecipitation of Ku70 after TLK286 treatment of purified DNA-PK and C70 cells showed a drug-induced destabilization of the protein-protein interaction between the catalytic subunit and the Ku heterodimer. Overall, these results implicate inhibition of DNA-PK as a component of TLK286 cytotoxicity and provide a rationale for its use in the clinical management of cisplatin-resistant ovarian cancer.

The average length of telomere repeats at the ends of chromosomes in most normal human somatic cells has been found to decrease by 50-200 base pairs with each cell division. The loss of telomere repeats has been causally linked to replicative senescence by the demonstration that overexpression of the enzyme telomerase can result in the elongation or maintenance of telomeres and immortalization of somatic cells with a diploid and apparently normal karyotype. Major questions that remain are related to the actual mechanism by which telomere shortening induces replicative senescence and the importance of telomere shortening and replicative senescence in the homeostasis of cells in renewal tissues and aging. This perspective is concerned with the consequences of telomere shortening at individual chromosomes in individual cells. Experimental evidence indicates that short telomeres accumulate prior to senescence and that replicative senescence is not triggered by the first telomere to reach a critical minimal threshold length. These observations are compatible with limited repair of short telomeres by telomerase-dependent or telomerase-independent DNA repair pathways. Deficiencies in telomere repair may result in accelerated senescence and aging as well as genetic instability that facilitates malignant transformation. Examples of molecules that may have a role in the repair of telomeric DNA prior to replicative senescence include ATM, p53, PARP, DNA-PK, Ku70/80, the human hRad50-hMre11-p95 complex, BRCA 1 and 2 and the helicases implicated in Bloom's and Werner's syndrome.

The Ku70-Ku80 ring complex encloses DNA ends to facilitate telomere maintenance and DNA break repair. Many studies focus on the ring-forming regions of subunits Ku70 and Ku80. Less is known about the Ku70 C-terminal tail, which lies outside the ring. Our results suggest that this region is responsible for dynamic sumoylation of Yku70 upon DNA association in budding yeast. Mutating a cluster of five lysines in this region largely eliminates Yku70 sumoylation. Chromatin immunoprecipitation analyses show that yku70 mutants with these lysines replaced by arginines exhibit reduced Ku-DNA association at both telomeres and internal DNA breaks. Consistent with this physical evidence, Yku70 sumoylation deficiency is associated with impaired ability to block DNA end resection and suppression of multiple defects caused by inefficient resection. Correlating with these, yku70 mutants with reduced sumoylation levels exhibit shorter telomeres, increased G overhang levels, and altered levels of non-homologous end joining. We also show that diminution of sumoylation does not affect Yku70 protein levels or its interactions with protein and RNA partners. These results suggest a model whereby Yku70 sumoylation upon DNA association strengthens Ku-DNA interaction to promote multiple functions of Ku.

Hepatocellular carcinoma (HCC) is among the most lethal and prevalent cancers in the human population. The initiation and progression of HCC is closely associated with chronic liver inflammation. Recent research indicates that nonhomologous end joining (NHEJ), one of the DNA repair mechanisms, autophagy and senescence are all involved in the pathogenesis of HCC induced by carcinogens or oxidative stress. DNA repair proteins including XRCC6/KU70 and XRCC5/KU80 are the critical NHEJ factors that play pivotal roles in genome-maintenance issues such as DNA replication and repair, telomere maintenance and chromosomal instability. Our studies indicate that a deficiency of toll-like receptor 4 (TLR4)-mediated immune activities results in a decreased expression of XRCC5 and XRCC6 in response to insult by the carcinogen diethylnitrosamine (DEN). This effect causes a failure in DNA repair, and promotes the transformation of precancerous hepatocytes and HCC development. Ectopic expression of XRCC6 protects against HCC initiation and progression by restoring the cellular senescent response and activation of immune networks, which induces an effective autophagic degradation, removes the accumulated reactive oxygen species (ROS), decreases DNA damage, attenuates proliferation, and promotes programmed cell death in TLR4-deficient livers. Our work indicates that repairing DNA damage by XRCC6 reverses TLR4-deficiency-worsened HCC development via restoring immunity to support senescence and autophagy in liver cells.

The Ku protein is essential for the repair of a majority of DNA double-strand breaks in mammalian cells. The purpose of this study was to investigate the relationship between the expression of Ku70/80 and sensitivity to radiation in cancer cell lines of the head and neck. The sensitivity to radiation in various head and neck cancer cell lines (AMC-HN-1 to -9) was analyzed by colony forming assay. Of the nine cell lines examined, the most radiosensitive cell line (AMC-HN-3) and the most radioresistant cell line (AMC-HN-9) were selected for this experiments. The expression of Ku70/80 was examined after irradiation using real time PCR, Western blotting and immunofluorescence in two different cell lines. Cell cycle distribution after irradiation were analysed. A differential radioresponse was demonstrated by expression of Ku70/80 in AMC-HN-3 and AMC-HN-9 cells. While the expression of Ku70 was slightly increased in the radioresistant AMC-HN-9 cell line, the expression of Ku80 was remarkably increased, suggesting a correlation between Ku80 expression and radiation resistance. Overexpression of Ku80 plays an important role in the repair of DNA damage induced by radiation. Ku80 expression may provide an effective predictive assay of radiosensitivity in head and neck cancers.

OBJECTIVE

Doxorubicin (DOX) is an effective anti-cancer therapeutic, but is associated with both acute and late-stage cardiotoxicity. Children are particularly sensitive to DOX-induced heart failure. Here, the impact of p53 inhibition on acute vs. late-stage DOX cardiotoxicity was examined in a juvenile model.

RESULTS

Two-week-old MHC-CB7 mice (which express dominant-interfering p53 in cardiomyocytes) and their non-transgenic (NON-TXG) littermates received weekly DOX injections for 5 weeks (25 mg/kg cumulative dose). One week after the last DOX treatment (acute stage), MHC-CB7 mice exhibited improved cardiac function and lower levels of cardiomyocyte apoptosis when compared with the NON-TXG mice. Surprisingly, by 13 weeks following the last DOX treatment (late stage), MHC-CB7 exhibited a progressive decrease in cardiac function and higher rates of cardiomyocyte apoptosis when compared with NON-TXG mice. p53 inhibition blocked transient DOX-induced STAT3 activation in MHC-CB7 mice, which was associated with enhanced induction of the DNA repair proteins Ku70 and Ku80. Mice with cardiomyocyte-restricted deletion of STAT3 exhibited worse cardiac function, higher levels of cardiomyocyte apoptosis, and a greater induction of Ku70 and Ku80 in response to DOX treatment during the acute stage when compared with control animals.

CONCLUSIONS

These data support a model wherein a p53-dependent cardioprotective pathway, mediated via STAT3 activation, mitigates DOX-induced myocardial stress during drug delivery. Furthermore, these data suggest an explanation as to how p53 inhibition can result in cardioprotection during drug treatment and, paradoxically, enhanced cardiotoxicity long after the cessation of drug treatment.

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks recognition and repair. However, Ku is expressed also on the surface of different types of cells along with its intracellular pool within the nucleus and the cytoplasm. Participation of membrane-associated Ku in cell-cell interaction has been reported recently. Here, we describe a novel function of cell-surface Ku as an adhesion receptor for fibronectin (Fn). The role of Ku in cell adhesion was investigated by comparing the Ku80 deficient Chinese hamster ovary (CHO) cell line, xrs-6, with clones transfected stably with either the hamster or human Ku80 cDNA. Ku expression in transfectant cells resulted in a significant increased adhesion on Fn and type IV collagen as compared to control cells. The observed increase in cell adhesion relied on Ku cell-surface expression, since antibodies directed against Ku70 or Ku80 subunit inhibited adhesion on Fn of Ku80, but not control vector, transfected xrs-6 cells. In addition, both Ku70 and Ku80 present a structural relationship with integrin I (or A) domains and the A1 and A3 domains of von Willebrand factor, domains known to be involved in Fn binding. Both Ku70 and Ku80 exhibit a complete set of residues compatible in their position and chemical nature with the formation of a metal ion-dependent adhesion (MIDAS) site implicated in ligand binding and integrin activation. Taken together, these functional and structural approaches support a new role for Ku as an adhesion receptor for Fn.

When explanted into culture, normal human cells exhibit a finite number of cell divisions before entering a proliferative arrest termed replicative senescence. To identify genes essential for entry into replicative senescence, we performed an RNA interference (RNAi)-based loss-of-function screen and found that suppression of the Never in Mitosis Gene A (NIMA)-related protein kinase gene NEK4 disrupted timely entry into senescence. NEK4 suppression extended the number of population doublings required to reach replicative senescence in several human fibroblast strains and resulted in decreased transcription of the cyclin-dependent kinase inhibitor p21. NEK4-suppressed cells displayed impaired cell cycle arrest in response to double-stranded DNA damage, and mass spectrometric analysis of Nek4 immune complexes identified a complex containing DNA-dependent protein kinase catalytic subunit [DNA-PK(cs)], Ku70, and Ku80. NEK4 suppression causes defects in the recruitment of DNA-PK(cs) to DNA upon induction of double-stranded DNA damage, resulting in reduced p53 activation and H2AX phosphorylation. Together, these observations implicate Nek4 as a novel regulator of replicative senescence and the response to double-stranded DNA damage.

BACKGROUND

Accelerated aging has been proposed as a pathologic mechanism of various chronic diseases, including COPD. This concept has almost exclusively been approached by analyses of individual markers. We investigated whether COPD is associated with accelerated aging using a panel of markers representing various interconnected aspects of the aging process.

METHODS

Lung function, leukocyte telomere length, lymphocyte gene expression of anti-aging (sirtuin 1, total klotho, and soluble klotho [Sklotho]), senescence (p16/21), and DNA repair (Ku70/80 and TERF2) proteins, and markers of systemic inflammation and oxidative stress were determined in 160 patients with COPD, 82 smoking subjects, and 38 never-smoking control subjects.

RESULTS

Median levels for telomere length, Sklotho, Ku70, and sirtuin 1 gene expression were lower (respectively, 4.4, 4.6, and 4.7 kbp for telomere length; 74%, 82%, and 100% for Sklotho; 88%, 92%, and 100% for Ku70 and 70%, 92%, and 100% for sirtuin 1, all P < .05) in patients compared with the smoking and never-smoking control groups. P21 gene expression was higher in patients compared with smoking control subjects. Telomere length correlated with Ku70 gene expression (r = 0.15, P = .02). After correction for age, smoking history, systemic inflammation, and oxidative stress, telomere length and p21 were the only markers that remained independently associated with lung function. In separate groups, only telomere length remained associated with lung function parameters.

CONCLUSIONS

Markers of the aging mechanism represent distinct molecular aspects of aging. Among them, different markers were altered in COPD, but only telomere length was consistently associated with lung function, and seems a useful marker for expressing accelerated aging in COPD.

Cyclin E protein levels and associated kinase activity rise in late G(1) phase, reach a peak at the G(1)/S transition, and quickly decline during S phase. The cyclin E/Cdk2 complex has a well-established function in regulating two fundamental biological processes: cell cycle progression and DNA replication. However, cyclin E expression is deregulated in a wide range of tumors. Our recent reports have uncovered a critical role for cyclin E, independent of Cdk2, in the cell death of hematopoietic tumor cells exposed to genotoxic stress. An 18-kD C-terminal fragment of cyclin E, p18-cyclin E, which is generated by caspase-mediated cleavage in hematopoietic cells during genotoxic stress-induced apoptosis has a critical role in the amplification of the intrinsic apoptotic pathway. By interacting with Ku70, p18-cyclin E liberates Bax, which participates in the amplification of apoptosis by sustaining a positive feedback loop targeting mitochondria. This process is independent of p53 function and new RNA or protein synthesis. Therefore, cyclin E emerges as an arbiter of the genotoxic stress response by regulating a finite physiological balance between cell proliferation and death in hematopoietic cells.

Cell death linked to DNA damage has been implicated in various diseases caused by environmental stress and infection. Severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. Accumulation of DNA damage has been proposed to be a principal mechanism of infection, inflammation, cancer, and aging. The most deleterious form of DNA damage is double-strand breaks (DSBs), where ataxia-telangiectasia-mutated (ATM) is the main transducer of the double-strand DNA break signal. Once the DNA is damaged, the DNA repair protein Ku70/80 translocates into the nucleus, a process which may be mediated by ataxia-telangiectasia-mutated, a member of the phosphoinositide-3-kinase-like family. The function and stability of Artemis may also be regulated by ataxia-telangiectasia-mutated through its phosphorylation upon the occurrence of DNA damage. Interestingly, both Artemis and Ku70/80 are substrates of DNA-dependent protein kinase (DNA-PK), another member of the phosphoinositide-3-kinase-like family. In this review, we show how Ku and Artemis function in the DNA damage response and the ataxia-telangiectasia-mutated signaling pathway and discuss potential applications of agents targeting these DNA damage response molecules in the treatment of inflammation and cancer.

Mammalian mitochondrial protein extracts possess DNA end-binding (DEB) activity. Protein binding to a 394 bp double-stranded DNA molecule was measured using an electrophoretic mobility shift assay. Mitochondrial DEB activity was highly specific for linear DNA. Inclusion of a vast excess of non-radioactive circular DNA did not disrupt binding to radioactive f394. In contrast, binding was abolished by the inclusion of linear competitor DNA. In mammals, nuclear DEB activity is due to Ku, a hetero-dimer composed of the Ku70 and Ku86 proteins. To determine whether mitochondrial DEB activity was also due to Ku, protein extracts were prepared from the Chinese hamster XR-V15B cell line, which lacks this protein. As anticipated, nuclear extracts prepared from these cells lacked DEB activity. In contrast, mitochondrial extracts prepared from these cells had wild-type levels of DEB activity, demonstrating that this latter activity is not a consequence of nuclear contamination. Although the nuclear and mitochondrial DEB activities are independent of each other, they are nevertheless closely related, since mitochondrial DEB activity was 'supershifted' by both anti-Ku70 and anti-Ku86 antisera. The nuclear DEB protein Ku plays an essential role in nuclear DNA double-strand break repair. The DEB activity described herein may therefore play a similar role in mitochondrial DNA repair.