During lymphocyte Ag receptor gene assembly, DNA cleavage by the Rag proteins generates pairs of coding and signal ends that are normally joined into coding joints and signal joints, respectively, by the classical nonhomologous end-joining (NHEJ) pathway of DNA double strand break repair. Coding and signal ends can also be aberrantly joined to each other, generating hybrid joints, through NHEJ or through NHEJ-independent pathways, such as Rag-mediated transposition. Hybrid joints do not participate in the formation of functional Ag receptor genes and can alter the configuration of Ag receptor loci in ways that limit subsequent productive rearrangements. The formation of these nonfunctional hybrid joints occurs rarely in wild type lymphocytes, demonstrating that mechanisms exist to limit both the NHEJ-dependent and the NHEJ-independent joining of a signal end to a coding end. In contrast to wild-type cells, hybrid joint formation occurs at high levels in ataxia telangiectasia mutated (Atm)-deficient lymphocytes, suggesting that Atm functions to limit the formation of these aberrant joints. In this study, we show that hybrid joint formation in Atm-deficient cells requires the NHEJ proteins Artemis, DNA-PKcs, and Ku70, demonstrating that Atm functions primarily by modulating the NHEJ-dependent, and not the NHEJ-independent, joining of coding ends to signal ends.

OBJECTIVE

Increased expression of survivin has been identified as a negative prognostic marker in a variety of human cancers. We have previously shown that survivin is a radiation-resistance factor and that the therapeutic effect of survivin knock-down might result from an impaired DNA repair capacity. In this study, we aimed to elucidate an interrelationship between survivin's cellular localization and DNA double-strand break repair.

METHODS

Survivin's cellular distribution and nuclear complex formation were assayed by Western blotting of subcellular fractions, by immunofluorescence staining, and co-immunoprecipitation in SW480 colorectal cancer cells. DNA repair capacity was analyzed by kinetics of gamma-H2AX foci formation, and by DNA-dependent protein kinase (DNA-PKcs) assays in the presence of survivin-specific or nonspecific control siRNA.

RESULTS

Following irradiation, we observed a rapid nuclear accumulation of survivin and subsequent phosphorylation of the protein in the nucleus. Co-immunoprecipitation analyses from nuclear extracts revealed an interaction among survivin, Ku70, gamma-H2AX, MDC1, and DNA-PKcs that was confirmed by immunofluorescence co-localization in nuclear foci. Survivin knock down by siRNA resulted in an impaired DNA double strand break repair, as demonstrated by an increased detection of gamma-H2AX foci/nucleus at 60 min and a higher amount of residual gamma-H2AX foci at 24 hr postirradiation. Furthermore, we detected in survivin-depleted cells a hampered S2056 autophosphorylation of DNA-PKcs and a significantly decreased DNA-PKcs kinase activity.

CONCLUSIONS

These data indicate that nuclear survivin is linked to DNA double-strand break repair by interaction with members of the DNA double-strand breaks repair machinery, thus regulating DNA-PKcs activity.

Nuclear respiratory factor 1 (NRF-1) is one of the key transcriptional activators for nuclear-coded genes involved in mitochondrial biogenesis and function as well as for many housekeeping genes. A transcriptional co-activator PGC-1 and its related family member PRC have previously been shown to interact with NRF-1 and co-activate NRF-1. We show here that NRF-1 can also directly interact with poly(ADP-ribose) polymerase 1 (PARP-1) and co-purify the PARP-1.DNA-PK.Ku80.Ku70.topoisomerase IIbeta-containing protein complex. Our in vitro binding experiments show that DNA-binding/dimerization domain of NRF-1 and the N-terminal half of PARP-1, which contains two Zinc fingers and the auto-modification domain, are responsible for the interaction, and that this interaction occurs with or without PARP-1 poly(ADP-ribosyl)ation (PARylation). DNA-bound NRF-1 can form a complex with PARP-1, suggesting that NRF-1 can recruit the PARP-1.DNA-PK.Ku80.Ku70.topoisomerase IIbeta-containing protein complex to the promoter. PARP-1 can also PARylate the DNA-binding domain of NRF-1 and negatively regulate NRF-1.PARP-1 interaction. Transient transfection and chromatin immunoprecipitation experiments suggest that PARP-1 plays a role during transcriptional activation by NRF-1. Our finding identifies a new aspect of transcriptional regulation used by NRF-1.

During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G(1) phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-alpha, IKK-beta and IkappaB-alpha, increased expression of IkappaB-alpha, and suppressed nuclear translocation of NF-kappaB and its DNA binding activity. Dephosphorylation of NF-kappaB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-kappaB.

The ergot fungus Claviceps purpurea uses mainly the nonhomologous-end-joining (NHEJ) system for integration of exogenous DNA, leading to a low frequency of homologous integration (1-2%). To improve gene targeting efficiency we deleted the C. purpurea ku70 gene in two different strains: the pathogenic strain 20.1 and the apathogenic, ergot alkaloid producing strain P1. The mutants were not impaired in vegetative and pathogenic development nor alkaloid production. Gene targeting efficiency was significantly increased (50-60%) in the Deltaku70 mutants. The P1 Deltaku70 strain (producing ergotamine and ergocryptine) was used for targeted deletion of lpsA1, one of the two trimodular NRPS genes present in the alkaloid gene cluster, encoding D-lysergyl peptide synthetases involved in formation of the tripeptide moiety of ergopeptines. Mutants lacking the lpsA1 gene were shown to be incapable of producing ergotamine but were still able to produce ergocryptine, proving that LpsA1 is involved in ergotamine biosynthesis.

Mcl-1 is a unique antiapoptotic Bcl2 family member with a short half-life due to its rapid turnover through ubiquitination. We discovered that Ku70, a DNA double-strand break repair protein, functions as a deubiquitinase to stabilize Mcl-1. Ku70 knockout in mouse embryonic fibroblast (MEF) cells or depletion from human lung cancer H1299 cells leads to the accumulation of polyubiquitinated Mcl-1 and a reduction in its half-life and protein expression. Conversely, expression of exogenous Ku70 in Ku70(-/-) MEF cells restores Mcl-1 expression. Subcellular fractionation indicates that Ku70 extensively colocalizes with Mcl-1 in mitochondria, endoplasmic reticulum and nucleus in H1299 cells. Ku70 directly interacts with Mcl-1 via its C terminus (that is, aa 536-609), which is required and sufficient for deubiquitination and stabilization of Mcl-1, leading to suppression of apoptosis. Purified Ku70 protein directly deubiquitinates Mcl-1 by removing K48-linked polyubiquitin chains. Ku70 knockdown not only promotes Mcl-1 turnover but also enhances antitumor efficacy of the BH3-mimetic ABT-737 in human lung cancer xenografts. These findings identify Ku70 as a novel Mcl-1 deubiquitinase that could be a potential target for cancer therapy by manipulating Mcl-1 deubiquitination.

During cancer development, coordinated changes in cell motility and cell cycle progression are required for the gradual transformation of normal cells into cancer cells. Previous studies have shown that ARF6 is a critical regulator of epithelial cell integrity and motility via its role in membrane movement and actin-based cytoskeletal remodeling. Recently, we have found that ARF6 also plays a role during cell division. It localizes to the cleavage furrow and midbody of cells during mitosis, and its activity is regulated during cytokinesis. Here, we investigate the requirement for ARF6 during mitosis and find that depletion of ARF6 using RNA interference disrupts the completion of cytokinesis. This finding demonstrates that ARF6 is essential during the final stages of cytokinesis. In addition, we have identified Ku70, a DNA-binding protein that is required for DNA damage repair, as a new ARF6-interacting protein and found that it is part of a complex with ARF6, especially during mitosis. These results clarify the importance of ARF6 activity during cytokinesis and begin to reveal other molecules that may contribute to the function of ARF6.

Coilin is a nuclear protein that plays a role in Cajal body formation. The function of nucleoplasmic coilin is unknown. Here we report that coilin interacts with Ku70 and Ku80, which are major players in the DNA repair process. Ku proteins compete with SMN and SmB' proteins for coilin interaction sites. The binding domain on coilin for Ku proteins cannot be localized to one discrete region, and only full-length coilin is capable of inhibiting in vitro non-homologous DNA end joining (NHEJ). Since Ku proteins do not accumulate in CBs, these findings suggest that nucleoplasmic coilin participates in the regulation of DNA repair.

The present study is the first to show in pancreatic cancer (PC) the growth inhibition and apoptosis by novel MDM2 inhibitors (MI-319 & 219) through reactivation of p53 pathway. Our results highlight two new secondary targets of MDM2 inhibitor 'SIRT1' and Ku70. SIRT1 which has a role in ageing and cancer and is known to regulate p53 signaling through acetylation. Ku70 is a key component of non-homologous end joining machinery in the DNA damage pathway and is known to regulate apoptosis by blocking Bax entry into mitochondria. Growth inhibition and apoptosis by MI-219, MI-319 was accompanied by increase in levels of p53 along with p21(WAF1) and the proapoptotic Puma. SiRNA against p21(WAF1) abrogated the growth inhibition of PC cells confirming p21(WAF1) as a key player downstream of activated p53. Immunoprecipitation-western blot analysis revealed reduced association of MDM2-p53 interaction in drug exposed PC cells. In combination studies, the inhibitors synergistically augmented anti-tumor effects of therapeutic drug gemcitabine both in terms of cell growth inhibition as well as apoptosis. Surface plasmon resonance studies confirmed strong binding between MI-319 and Ku70 (K(D) 170 nM). Western blot revealed suppression of SIRT1 and Ku70 with simultaneous upregulation of acetyl-p53 (Lys379) and Bax. Co-Immunoprecipitation studies confirmed that MI-319 could disrupt Ku70-Bax and SIRT1-Bax interaction. Further, using wt-p53 xenograft of Capan-2, we found that oral administration of MI-319 at 300 mg/kg for 14 days resulted in significant tumor growth inhibition without any observed toxicity to the animals. No tumor inhibition was found in mut-p53 BxPC-3 xenografts. In light of our results, the inhibitors of MDM2 warrant clinical investigation as new agents for PC treatment.

Programmed cell death and DNA repair are two fundamental biological processes that play essential roles in cell fate and genetic transmission. The canonical role of Bcl-2 family members is the regulation of programmed cell death. Strikingly, numerous studies from different laboratories have shown that although Bcl-2 increases cell survival, it also inhibits all DNA repair systems, resulting in genome instability/variability. Bcl-2 affects the mechanistically distinct DNA repair systems via different mechanisms. These effects are generally independent of the regulation of apoptosis, revealing additional roles for Bcl-2. The targets of Bcl-2 include APE1, MSH2, PARP1, Ku70 and the oncosuppressor BRCA1. Targetting BRCA1 should be of particular importance because this might impact many essential cellular processes in which BRCA1 is involved, including homologous recombination (HR), non-homologous end joining (NHEJ), base excision repair, cell-cycle regulation, cell death, ubiquitination, inactivation of the X-chromosome, transcription, and protein translation. Beside the pathological consequences, inhibition of DNA repair by Bcl-2 can be, in contrast, advantageously used in some physiological situations: (1) repression of excessive unschedule HR, thus protecting against the accumulation of toxic HR intermediates and HR-dependent genome rearrangements; (2) inhibition of NHEJ might protect against retrovirus integration; (3) it has been proposed that inhibition of mismatch repair might also favors hypermutation at immunoglobulin genes. Finally, because Bcl-2 affects the maintenance of genome stability, one can suggest Bcl-2 might play a role in molecular evolution. Bcl-2 family members control cell death through complex stochiometric equilibriums. Incorporating DNA repair proteins to such an elaborate network should allow for a fine tuning of the coordinated control of cell viability and genetic stability/instability. Relationships between DNA repair and regulation of cell death represent exciting challenges for future prospects and are essential for the development of promising new strategies against cancer.

Ku70 protein, cooperating with Ku80 and DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs), is involved in DNA double-strand break (DNA DSB) repair and V(D)J recombination. Recent studies have revealed increased ionizing radiosensitivity in Ku70-deficient cells. The presented study, using a human squamous cell lung carcinoma cell line, demonstrated that introduction of an antisense Ku70 nucleic acid made the cells more radio- and chemosensitive than the parental cells. Ku70 protein expression was suppressed in the cells with antisense Ku70 construct when compared to the wild-type cells. A relatively small but statistically significant increase in radiosensitivity of the cells was achieved by the introduction of the antisense Ku70. The increased radiosensitivity in vitro was accompanied by an approximately two-fold increase in alpha and alpha/beta values in a linear-quadratic model. The antisense Ku70 increased the chemosensitivity of the cells to some DNA-damaging agents such as bleomycin and methyl methanesulfonate, but not to cisplatin, mitomycin C, and paclitaxel. This system provides us with partial suppression of Ku70, and will be a useful experimental model for investigating the physiological roles of the DNA DSB repair gene.

In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence. In contrast, cancer cells counteract the loss of telomeric DNA either by inducing the expression of telomerase or by activating the alternative lengthening of telomeres (ALT) pathway. ALT cells are characterized by heterogeneous telomeres and the presence of extrachromosomal circular double-stranded DNA molecules containing telomeric repeat sequences. These telomeric circles (t-circles) are though to be generated through a recombination process and utilized as templates for telomere elongation by rolling-circle-replication, although their precise mechanism of formation and role in telomere maintenance and cell proliferation is largely unknown. Here we show that shRNA-mediated knockdown of the Ku70/80 heterodimer, a factor with functions at both pathological and natural DNA ends, inhibits ALT cell growth and results in a significant decrease in the levels of t-circles without affecting overall telomere length. These findings demonstrate that non homology-based processes contribute to the maintenance of t-circles and proliferation of ALT cells.

Immunoglobulin (Ig) class switching is central to the maturation of the antibody response as IgG, IgA, and IgE are endowed with more diverse biological effector functions than IgM. It is induced upon engagement of CD40 on B lymphocytes by CD40L expressed by activated CD4+ T cells and exposure of B cells to T cell-secreted cytokines including interleukin-4 and transforming growth factor-beta. It begins with germ line IH-CH transcription and unfolds through class switch DNA recombination (CSR). We show here that the HoxC4 and Oct-1 homeodomain proteins together with the Ku70/Ku86 heterodimer bind as a complex to newly identified switch (S) regulatory ATTT elements (SREs) in the Igamma and Iepsilon promoters and downstream regions to dampen basal germ line Igamma-Cgamma and Iepsilon-Cepsilon transcriptions and repress CSR to Cgamma and Cepsilon. This mechanism is inactive in the Calpha1/Calpha2 loci because of the lack of SREs in the Ialpha1/Ialpha2 promoters. Accordingly, in resting human IgM+IgD+ B cells, HoxC4, Oct-1, and Ku70/Ku86 can be readily identified as bound to the Igamma and Iepsilon promoters but not the Ialpha1/Ialpha2 promoters. CD40 signaling dissociates the HoxC4.Oct-1. Ku complex from the Igamma and Iepsilon promoter SREs, thereby relieving the IH-CH transcriptional repression and allowing CSR to unfold. Dissociation of HoxC4.Oct-1. Ku from DNA is hampered by CD153 engagement, a CD40-signaling inhibitor. Thus, these findings outline a HoxC4.Oct-1. Ku-dependent mechanism of selective regulation of class switching to IgG and IgE and further suggest distinct co-evolution and shared CSR activation pathways in the Cgamma and Cepsilon as opposed to the Calpha1/Calpha2 loci.

BACKGROUND

The Tousled-like kinases are involved in chromatin assembly, DNA repair, transcription, and chromosome segregation. Previous evidence indicated that TLK1B can promote repair of plasmids with cohesive ends in vitro, but it was inferred that the mechanism was indirect and via chromatin assembly, mediated by its interaction with the chromatin assembly factor Asf1. We recently identified Rad9 as a substrate of TLK1B, and we presented evidence that the TLK1B-Rad9 interaction plays some role in DSB repair. Hence the relative contribution of Asf1 and Rad9 to the protective effect of TLK1B in DSBs repair is not known. Using an adeno-HO-mediated cleavage system in MM3MG cells, we previously showed that overexpression of either TLK1B or a kinase-dead protein (KD) promoted repair and the assembly of Rad9 in proximity of the DSB at early time points post-infection. This established that it is a chaperone activity of TLK1B and not directly the kinase activity that promotes recruitment of 9-1-1 to the DSB. However, the phosphorylation of Rad9(S328) by TLK1B appeared important for mediating a cell cycle checkpoint, and thus, this phosphorylation of Rad9 may have other effects on 9-1-1 functionality.

RESULTS

Here we present direct evidence that TLK1B can promote repair of linearized plasmids with incompatible ends that require processing prior to ligation. Immunodepletion of Rad9 indicated that Rad9 was important for processing the ends preceding ligation, suggesting that the interaction of TLK1B with Rad9 is a key mediator for this type of repair. Ligation of incompatible ends also required DNA-PK, as addition of wortmannin or immunodepletion of Ku70 abrogated ligation. Depletion of Ku70 prevented the ligation of the plasmid but did not affect stimulation of the fill-in of the ends by added TLK1B, which was attributed to Rad9. From experiments with the HO-cleavage system, we now show that Rad17, a subunit of the "clamp loader", associates normally with the DSB in KD-overexpressing cells. However, the subsequent release of Rad17 and Rad9 upon repair of the DSB was significantly slower in these cells compared to controls or cells expressing wt-TLK1B.

CONCLUSIONS

TLKs play important roles in DNA repair, not only by modulation of chromatin assembly via Asf1, but also by a more direct function in processing the ends of a DSB via interaction with Rad9. Inhibition of Rad9 phosphorylation in KD-overexpressing cells may have consequences in signaling completion of the repair and cell cycle re-entry, and could explain a loss of viability from DSBs in these cells.

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.

Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation. Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations. Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70. Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle. Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku. The nuclear translocation of Ku80 started during the late telophase/early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70. Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70. To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and defined to a region spanning nine amino acid residues (positions 561 - 569). The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97. All these findings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70.

To select the appropriate patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), it is important to gain a better understanding of the intracellular pathways leading to EGFR-TKI resistance, which is a common problem in patients with lung cancer. We recently reported that mutant KRAS adenocarcinoma is resistant to gefitinib as a result of amphiregulin and insulin-like growth factor-1 receptor overexpression. This resistance leads to inhibition of Ku70 acetylation, thus enhancing the BAX/Ku70 interaction and preventing apoptosis. Here, we determined the intracellular pathways involved in gefitinib resistance in lung cancers and explored the impact of their inhibition. We analyzed the activation of the phosphatidyl inositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) pathway in lung tumors. The activation of AKT was associated with disease progression in tumors with wild-type EGFR from patients treated with gefitinib (phase II clinical trial IFCT0401). The administration of IGF1R-TKI or amphiregulin-directed shRNA decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. The combination of PI3K/AKT inhibition with gefitinib restored apoptosis via Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which decreased the BAX/Ku70 interaction, inhibited AKT signaling and induced gefitinib-dependent apoptosis. The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in lung tumors with KRAS mutation, through the regulation of the BAX/Ku70 interaction. This finding suggests that combined treatments could improve the outcomes for this subset of lung cancer patients, who have a poor prognosis.

The filamentous ascomycete Botrytis cinerea is one of the most studied models for understanding the necrotrophic behaviour of phytopathogenic fungi. The genomes of two strains of B. cinerea have been sequenced (B05.10 and T4), which may contribute to elucidating the virulence polymorphism in this fungus. In this study, both strains were genetically modified in order to construct recipient strains designed to target genes that are hard to knock out. Deletions of BcKu70 gene in B05.10 strain and BcKu80 gene in T4 strain both affected the nonhomologous end-joining (NHEJ) DNA repair mechanism. NHEJ is responsible for the ectopic integration of gene replacement cassettes during fungal transformation and leads to a lower frequency of homologous recombination (HR). Ku deficiencies in B. cinerea did not disturb in vitro or in planta growth, but clearly improved HR efficiency for the putative sesquiterpene cyclase-encoding gene Cnd15, which was hard to knock out in a wild-type strain.

A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (γH2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of γH2AX foci after irradiating synchronized cell populations with (60)Co γ-rays. Our results show statistically significant differences in the number of γH2AX foci between the repair-deficient and -proficient cell line, with a higher amount of γH2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.

Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects-facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein contributing to genome stability via repair of DNA lesions via the base excision repair pathway. It also plays a role in gene expression regulation and RNA metabolism. Another, poorly characterized function is its ability to bind to negative calcium responsive elements (nCaRE) of some gene promoters. The presence of many functional nCaRE sequences regulating gene transcription can be envisioned, given their conservation within ALU repeats. To look for functional nCaRE sequences within the human genome, we performed bioinformatic analyses and identified 57 genes potentially regulated by APE1. We focused on sirtuin-1 (SIRT1) deacetylase due to its involvement in cell stress, including senescence, apoptosis, and tumorigenesis, and its role in the deacetylation of APE1 after genotoxic stress. The human SIRT1 promoter presents two nCaRE elements stably bound by APE1 through its N-terminus. We demonstrate that APE1 is part of a multiprotein complex including hOGG1, Ku70, and RNA Pol II, which is recruited on SIRT1 promoter to regulate SIRT1 gene functions during early response to oxidative stress. These findings provide new insights into the role of nCaRE sequences in the transcriptional regulation of mammalian genes.

OBJECTIVE

The objective of this study was to determine whether the expressions of the two components of DNA-dependent protein kinase, Ku70 and DNA-protein kinase catalytic subunit (DNA-PKcs), influence the response to radiotherapy (RT) and outcome of treatment of nondisseminated nasopharyngeal carcinoma (NPC) in patients who received definitive RT.

METHODS

Sixty-six patients with NPC who were treated with radiotherapy alone or with concurrent chemotherapy between June 1995 and December 2001 were divided into groups based on the levels of immunoreactivity for Ku70 and DNA-PKcs in pretreatment biopsy specimens. The overexpression of Ku70 or DNA-PKcs groups included patients whose biopsy specimens showed at least 50% immunopositive tumor cells; patients in which less than 50% of the tumor cells in the biopsy tissues were immunopositive were placed in the low Ku70 and DNA-PKcs groups. The immunoreactivities for Ku70 and DNA-PKcs were retrospectively compared with the sensitivity of the tumor to radiation and the patterns of therapy failure. Univariate analyses were performed to determine the prognostic factors that influenced locoregional control of NPC.

RESULTS

The 5-year locoregional control rate was significantly higher in the low Ku70 group (Ku-) (85%) than in the high Ku70 group (Ku+) (42%) (p = 0.0042). However, there were no differences in the metastases-free survival rates between the 2 groups (Ku70+, 82%; Ku70- 78%; p = 0.8672). Univariate analysis indicated that the overexpression of Ku70 surpassed other well-known predictive clinicopathologic parameters as an independent prognostic factor for locoregional control. Eighteen of 22 patients who had locoregional recurrences of the tumor displayed an overexpression of Ku70. No significant association was found between the level of DNA-PKcs expression and the clinical outcome.

CONCLUSIONS

Our data suggest that the level of Ku70 expression can be used as a molecular marker to predict the response to RT and the locoregional control after RT and concurrent chemotherapy in patients with nondisseminated NPC.