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PubMed2006

The nuclear DNA repair protein Ku70/80 is a tumor-associated antigen displaying rapid receptor mediated endocytosis.

To be of therapeutic relevance, a tumor-associated antigen should be expressed on the surface of neoplastic cells but not, or to a significantly lower extent, on cells of non-transformed nature. The Ku heterodimer (Ku70/80) is involved in DNA double strand break recognition and repair and is ubiquitously expressed in the nucleus of all cells. However, its exclusive nuclear localization has been reassessed by studies that demonstrate Ku to be expressed on the surface of tumor cell lines, displaying functions in cell adhesion, migration and invasion. In this study, we add another feature to the pluripotent role of Ku70/80 by showing that, upon binding the novel human recombinant antibody INCA-X, the Ku70/80 heterodimer is internalized into pancreatic carcinoma cells. The receptor-mediated endocytosis of Ku70/80 is rapid (t(1/) (2) 12 min) and extensive (90% of the receptor pool inside the cell after 100 min) as measured by rotating radioimmunoassay. Ku70/80 was also successfully used as a port of entry for cytotoxic payloads to tumor cells of various origin, as determined by indirect immunotoxin administration of a saporin-conjugated, secondary anti-human antibody. Thus, the internalization properties of the Ku70/80 suggest a potential role of this tumor associated antigen in selective drug-delivery in several human malignancies.

Antigens, Neoplasm↗
PubMed2003

Role of Pin2/TRF1 in telomere maintenance and cell cycle control.

Telomeres are specialized structures found at the extreme ends of chromosomes, which have many functions, including preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Deregulation of telomere length has been implicated in cancer and ageing. Telomere maintenance is tightly regulated by telomerase and many other telomere-associated proteins and is also closely linked to cell cycle control, especially mitotic regulation. However, little is known about the identity and function of the signaling molecules connecting telomere maintenance and cell cycle control. Pin2/TRF1 was originally identified as a protein bound to telomeric DNA (TRF1) and as a protein involved in mitotic regulation (Pin2). Pin2/TRF1 negatively regulates telomere length and importantly, its function is tightly regulated during the cell cycle, acting as an important regulator of mitosis. Recent identification of many Pin2/TRF1 upstream regulators and downstream targets has provided important clues to understanding the dual roles of Pin2/TRF1 in telomere maintenance and cell cycle control. These results have led us to propose that Pin2/TRF1 functions as a key molecule in connecting telomere maintenance and cell cycle control.

Amino Acid Sequence↗
PubMed2006

Molecular dynamics simulation and automated docking of the pro-apoptotic Bax protein and its complex with a peptide designed from the Bax-binding domain of anti-apoptotic Ku70.

Bax, a multi-domain protein belonging to the large family of Bcl-2 proteins, has a pivotal role for the initiation of the cytochrome c-mediated apoptosis, a vital physiologic process to eliminate damaged or unwanted cells. In response to specific stimuli Bax translocates from cytosol to mitochondria outer membrane where a process of oligomerization occurs with pore formation through which cytochrome c and other death molecules escape. The pro-death action of Bax is regulated by the interaction with other pro-survival proteins. However, the conformational changes and the structural details necessary for homo and hetero interaction with other regulating proteins are largely unknown. This article reports a combined investigation of molecular dynamics (MD) simulation and automated docking that evidence the molecular regions of Bax involved in the binding with anti-apoptotic exapeptide (Bip) designed from Ku70, a subunit of the protein complex essential for non-homologous DNA repair but that inhibits also the Bax translocation to mitochondria. Since Bip suppresses apoptosis induced by several anti-cancer drugs, it appears relevant to achieve a better understanding of the structural and dynamical aspects that characterize the Bip-Bax complex in view of potential therapeutic implications. The present results show that the Bax region with the highest affinity for Bip is located in proximity of BH3 homology domain of Bax and also involves the alpha-helices 1 and 8. Moreover, the comparison of essential motions of Bax at 300 and 400 K before and after the formation of the complex with Bip evidences how the binding with the exa-peptide affects the collective motions of specific molecular districts of Bax considered to have functional relevance.

Amino Acid Sequence↗
PubMed2007

PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

Amino Acid Sequence↗
PubMed2003

The non-homologous end-joining pathway is not involved in the radiosensitization of mammalian cells by heat shock.

A synergistic increase in cell killing is observed when a heat-shock is administered prior to, during, or immediately after exposure to ionizing radiation (IR). This phenomenon, known as heat-radiosensitization, is believed to be mediated by inhibition of repair of radiation-induced double strand breaks (DSB) when cells are exposed to temperatures above 42 degrees C. However, the mechanism by which heat inhibits DSB repair is unclear. The bulk of radiation-induced DSBs are repaired via the non-homologous end-joining pathway (NHEJ). Several reports indicate that the Ku70 and Ku80 subunits of the mammalian DNA-dependent protein kinase (DNA-PK), a complex involved in NHEJ, appear to be susceptible to a heat-induced loss of DNA-binding activity, with Ku80 representing the heat-sensitive component. Since the heat-induced loss and subsequent recovery of Ku-DNA binding activity correlates well with heat-radiosensitization, a role for Ku80 and NHEJ in heat-radiosensitization has been proposed. However, direct evidence implicating Ku80 (and NHEJ) in heat-radiosensitization has been indeterminate. In this study, we demonstrate that equitoxic heat treatments at 42.5-45.5 degrees C induce a similar amount of aggregation of Ku80 in human U-1 melanoma cells. These data suggest that the time-temperature-dependent relationship between heat lethality and Ku80 aggregation are similar. However, the aggregation/disaggregation of Ku80 and its transient or permanent inactivation is unrelated to heat-radiosensitization. When survival curves were obtained for irradiated or irradiated and heated Ku80(-/-) mouse embryo fibroblasts (MEFs) and compared with survival curves obtained for wild-type (WT) cells, we found that heat-radiosensitization was not reduced in the Ku80(-/-) cells, but actually increased. Thus, our findings indicate that Ku80 is not essential for heat-radiosensitization. Non-involvement of Ku-dependent or Ku-independent NHEJ pathways in heat-radiosensitization was confirmed by comparing clonogenic survival between DNA ligase IV-defective and WT human cells. Our data therefore implicate homologous recombination in inhibition of repair of radiation-induced DSBs and as a target for heat-radiosensitization.

Animals↗
PubMed2006

Assembly of nuclear matrix-bound protein complexes involved in non-homologous end joining is induced by inhibition of DNA topoisomerase II.

Topoisomerases maintain the DNA structure by relieving the torsional stress and alleviating other topological problems occurring in DNA during transcription and replication. Topoisomerase II appears to have a close association with the family of proteins involved in the organization of chromatin in a series of loops on the proteinaceous chromosomal matrix. Beyond its physiological functions, topoisomerase II is the target for some of the most active anticancer drugs. Inhibition of the topoisomerase II function can result in DNA double-strand breaks (DSBs) and, thus, lead to chromosomal translocations. The earliest event during DSB repair is phosphorylation of histone H2AX at S139 (so-called gammaH2AX) which is believed to serve as a focal point for the assembly of repair proteins at the DSB. In this work, we have demonstrated the formation of gammaH2AX foci in two human cell lines--K562 and HeLa--after suppression of topoisomerase II activity with etoposide. Furthermore, these foci remained visible at nuclear matrices and colocalized with the major components of non-homologous end joining (NHEJ) system of DSBs repair. Thus, inhibition of topoisomerase II activity triggers assembly of NHEJ complexes at the nuclear matrix.

Antigens, Nuclear↗
PubMed2007

Epigenetic reprogramming of liver cells in tamoxifen-induced rat hepatocarcinogenesis.

Tamoxifen, a nonsteroidal anti-estrogen, is a potent genotoxic hepatocarcinogen in rats, with both tumor initiating and promoting properties. Recently it has been demonstrated that genotoxic carcinogens, in addition to exerting genotoxic effects, often cause epigenetic alterations and these induced epigenetic changes may play important mechanistic role in carcinogenesis. In the present study, we investigated the role of tamoxifen-induced epigenetic changes in hepatocarcinogenic process. The results of the study showed that exposure of female F344 rats to tamoxifen resulted in progressive loss of CpG methylation in regulatory sequences of long interspersed nucleotide elements (LINE-1) and prominent increase in expression of LINE-1 elements and c-myc proto-oncogene. The accumulation of tamoxifen-induced DNA lesions was accompanied by the decreased level of Rad51, Ku70, and DNA polymerase beta (Polbeta) proteins that play a crucial role in maintenance of genomic stability. Furthermore, feeding rats with tamoxifen-containing diet led to increased regenerative cell proliferation, as indicated by the increased level of Ki-67 and proliferating cell nuclear antigen (PCNA) proteins. These data indicate that exposure of animals to genotoxic hepatocarcinogen tamoxifen led to early phenotypical alterations in livers characterized by emergence of epigenetically reprogrammed cells with a specific cancer-related epigenetic phenotype prior to tumor formation.

Animals↗
PubMed2002

Heterogeneous expression of Ku70 in human tissues is associated with morphological and functional alterations of the nucleus.

Ku70 is a subunit of DNA-protein kinase complex and involved in diverse intranuclear events including the repair of double-stranded DNA breaks. Ku70 is rich in the interphase nucleus of cultured cells. In human tissues, however, the distribution of Ku70 has not yet been systematically examined. To characterize the difference of Ku70 distribution between cells of human tissues and cultured cells, the expression of Ku70 was examined in various normal and neoplastic human tissues by immunohistochemistry and immunoblot. In addition, the role of Ku70 in the cellular response against ionizing radiation (IR) was analysed in fibroblasts after exposure to 5 Gy IR and apoptotic indices were examined in Ku70-overexpressed fibroblasts from an ataxia telangiectasia patient and in normal fibroblasts, before and after irradiation. In contrast to cultured cells, Ku70 was not detected in some interphase cells of human tissues and was distributed heterogeneously, even in the same nucleus. Ku70 expression was strikingly low in terminally differentiated cells such as neutrophils, eosinophils, glomerular capillary endothelial cells and fibroblasts, and was absent in spermatids. In spermatocytes, Ku70 was tightly integrated with chromosome filaments, unlike other somatic cells under mitosis. After exposure to IR, Ku70 expression was not increased in ataxia telangiectasia fibroblasts, but was significantly increased in normal fibroblasts. Most of the increased Ku70 was of soluble nuclear protein fraction. Furthermore, overexpression of Ku70 increased radiation resistance both in ataxia telangiectasia fibroblasts and normal fibroblasts. The presented data indicate that the distribution of Ku70 in cells of human tissues is closely associated with the cell cycle, cellular differentiation, nuclear shape and the process of repair of DNA damage caused by IR.

Adult↗
PubMed1993

Site-specific proteolytic cleavage of Ku protein bound to DNA.

Ku protein, a relatively abundant nuclear protein associated with DNA of mammalian cells, is known to be a heterodimer with subunits of 85 and 72 kDa which binds in vitro to DNA ends and subsequently translocates along the molecule. The functional role played by this protein in the cell, however, remains to be elucidated. We have observed here that Ku protein, purified from cultured monkey cells, is the target of specific endoproteolysis in vitro, by which the 85 kDa subunit is cleaved at a precise site while the 72 kDa subunit remains intact. This cleavage releases an 18 kDa polypeptide and converts Ku protein into a heterodimer composed of the 72 kDa subunit associated with a 69 kDa fragment from the 85 kDa subunit. The proteolyzed form of Ku protein, denoted Ku', has DNA binding properties similar to those of Ku protein. The proteolytic mechanism, which is inhibited by leupeptin and chymostatin, is extremely sensitive to ionic conditions, in particular to pH, being very active at pH 7.0 and completely inhibited at pH 8.0. In addition, cleavage occurs only when Ku protein is bound to DNA, not free in solution. We suggest that in vivo, such proteolysis might be necessary for Ku protein function at some stage of the cell cycle.

Animals↗
PubMed2004

Efficient gene targeting in Kluyveromyces lactis.

Integration of a DNA fragment in a host genome requires the action of a double-strand break (DSB) repair mechanism. Homologous recombination (HR) is initiated by binding of Rad52p to DNA ends and results in targeted integration. Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non-homologous end joining (NHEJ). In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces lactis shows variable, but in general low, gene targeting efficiency. To study and to improve gene targeting efficiency, K. lactis has been used as a model. The KlRAD51, KlRAD52 and KlKU80 genes have been isolated and deletion mutants for these genes have been constructed. Efficiency of gene targeting was determined at the KlADE2 locus using targeting constructs with different lengths of homologous flanking sequences. In wild-type K. lactis, the gene targeting efficiency ranged from 0% with 50 to 88% with 600 bp flanks. The Klku80 mutant, however, showed >97% gene targeting efficiency independently of the size of the homologous flanks. These results demonstrate that deletion of the NHEJ mechanism results in a higher gene targeting efficiency. Furthermore, increased gene targeting efficiency was achieved by the transformation of wild-type K. lactis with the KlADE2 deletion construct in the presence of excess small DNA fragments. Using this method, PCR-generated deletion constructs containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene replacement.

Antigens, Nuclear↗
PubMed1995

The possible involvement of DNA-dependent protein kinase in the phosphorylation of P1 protein in cell-free DNA replication of Xenopus eggs.

We prepared two types of antibodies: one directed against an oligopeptide corresponding to the C-terminal portion of P1 protein and the other against an oligopeptide corresponding to the C-terminal portion of the 80-kDa subunit of the Ku antigen (p80 Ku) essential for DNA-dependent protein kinase (DNA-PK) activity. Immunoprecipitation and immunoblot of sperm nuclei preincubated in Xenopus egg extracts by anti-P1 antibody showed that Xenopus P1 protein is a phosphoprotein with two phosphorylated forms: a hyperphosphorylated form extractable with Triton X-100 and a hypophosphorylated form resistant to Triton X-100. The immunodepletion of extracts with anti-p80 Ku IgG-bound beads caused the hyperphosphorylated form to disappear but hardly affected the hypophosphorylated form of P1 protein. DNA replication was stimulated by immunodepletion of the extract with anti-p80 Ku IgG-bound beads. These findings suggest that DNA-PK down-regulates DNA replication through inhibition of hyperphosphorylation of P1 protein during S phase in this cell-free system.

Amino Acid Sequence↗
PubMed1998

Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks.

DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

Animals↗
PubMed1999

Defining functional domains of Ku80: DNA end binding and survival after radiation.

The Ku heterodimeric protein (Ku80/Ku70) is an essential component of the double-strand break DNA repair pathway in mammalian cells. We have recently defined a central region within Ku80 that is required for heterodimerization with Ku70. We now identified a core region within Ku80 (amino acids 210 to 531) that is necessary for binding of Ku to DNA ends. Interaction with Ku70 and DNA end binding are important for Ku80 function in vivo, since Ku80 mutants lacking DNA end binding activity were unable to restore radiation resistance in Ku80 deficient fibroblast cell lines. However, Ku80 mutants were identified that retained DNA end binding activity but were unable to restore radiation survival, thus pointing to additional functional properties of Ku80. An N-terminal deletional mutant of Ku80 was able to suppress wild type Ku80 function for radiation survival in several cell lines, thus demonstrating dominant negative function.

Animals↗
PubMed1999

The E-box of the human glycophorin B promoter is involved in the erythroid-specific expression of the GPB gene.

Previous studies performed on the glycophorin B (GPB) expression demonstrated that this gene is expressed in erythroid cells only and that the ubiquitous factor Ku70 is involved in the process. Here, we investigated the contribution of the -70 E-box sequence toward the GPB promoter expression. We found that the E-box bound two factors, the USF1/USF2 protein and an unidentified ubiquitous protein which was named factor U. Site-directed mutagenesis performed on the -70 E-box showed that the USF factor had an activating effect in CAT assays. Conversely, mutation of the -70 E-box that impaired the binding of factor U led to a positive CAT activity in nonerythroid cells and thus to the loss of the erythroid-specific expression of the GPB gene. This indicates that, in addition to the Ku70 factor, the extinction of the GPB promoter expression in nonerythroid cells depends also on the repressing effect of the factor U.

Antigens, Nuclear↗
PubMed2000

Interferon-alpha signaling promotes nucleus-to-cytoplasmic redistribution of p95Vav, and formation of a multisubunit complex involving Vav, Ku80, and Tyk2.

Interferons (IFNs) are a family of hormone-like secretory proteins with multiple phenotypical changes, including gene expression and morphological alterations. Earlier studies have shown that IFN-activated Tyk2 kinase physical associates with p95Vav (Vav), a proto-oncogene gene product expressed in hematopoietic cells. Since Tyk2 is a cytoplasmic kinase and Vav is believed to be localized in the nuclear compartment, here we explored the possibility of Vav redistribution in IFN-alpha-activated cells, using the U266 human myeloma cell line as a model system. Using biochemical assays and in situ confocal microscopy, we demonstrate that IFN-alpha treatment triggers a rapid (10 min) translocation of Vav from the nuclear compartment to the cytoplasm. In addition, we also show the existence of IFN-alpha-induced physical interaction between Vav and Ku80, Ku80, and Tyk2, and among Vav, Ku80, and Tyk2 in the cytoplasmic compartment of IFN-stimulated cells. The observed IFN-alpha-induced association among Vav, Ku80, and Tyk2 was dependent on cellular tyrosine kinase activity. Since recently Vav has been shown to promote the GDP/GTP exchange activity of the cytoskeleton signaling molecule small GTPase Rac1 and activates its downstream signaling, our present findings raise the possibility of involvement of the small GTPase in IFN signaling leading to its biological effects, including cytoskeleton reorganization.

Antigens, Nuclear↗
PubMed2000

Ku70 can translocate to the nucleus independent of Ku80 translocation and DNA-PK autophosphorylation.

Ku plays an important role in multiple nuclear processes, e.g., DNA repair, chromosome maintenance, and transcriptional regulation. Although some evidence suggests that the nuclear translocation of Ku plays a key role in regulating the function of Ku, the mechanism is poorly understood. Using the site-directed mutagenesis technique, we demonstrate here that Ku70 can translocate to the nucleus without heterodimerization with Ku80. The nuclear accumulation of Ku70 mutants of the nuclear localization signal, which retained their binding ability with Ku80, was diminished. On the other hand, Ku70 mutants which lacked the ability to bind with Ku80 could translocate to the nuclei. Human Ku70, when transfected, accumulated within the nuclei of hamster xrs-6 cells which had undetectable DNA-PK activity and Ku80. Ku70 and Ku80 mutants of DNA-PK phosphorylation sites showed normal heterodimerization and nuclear translocation. These findings also support the idea that Ku70 can translocate to the nucleus independent of DNA-PK autophosphorylation.

Active Transport, Cell Nucleus↗
PubMed2001

Repression of the HSP70B promoter by NFIL6, Ku70, and MAPK involves three complementary mechanisms.

We have studied mechanisms of HSP70 gene regulation at 37 degrees C by the cellular factors NF-IL6 and Ku70. As both factors repress HSF1, we first examined whether phosphorylation on serine 303 and 307 of HSF1 by MAPK and GSK3, which has known to inhibit HSF1, was involved in the repression. However, repression by NF-IL6 was found using HSF1 mutants S303G and S307G refractory to the effects of MAPK and GSK3. We then examined whether NF-IL6 repressed HSP70B by a mechanism resembling Ku proteins. However, in Ku-deficient cells, NF-IL6 was still able to displace HSF1 from heat shock element (HSE) and repressed HSF1 activation. In addition, activation of the HSP 70B promoter by wild type, S303G, or S307G HSF1 was observed to be much more pronounced in Ku-deficient cells. In vitro translated Ku70 interacted with HSF1 by binding to and displacing it from HSE. These data indicate that the repression of the HSP70B promoter by NF-IL6, Ku70, and MAPK occurs independently of each other and involves three complementary mechanisms.

Amino Acid Substitution↗
PubMed2002

Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells.

In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields. Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser(15)), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays. It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G(1) cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression. A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields. Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7%. However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression.

Animals↗