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PubMed1991

Expression of the Ku protein during cell proliferation.

The Ku protein is a DNA-binding nuclear protein complex composed of 86 kDa and 70 kDa subunits. Recently, in vitro studies suggested a role of the Ku protein in the activation of gene transcription. We studied the expression of these proteins during cell proliferation by Northern blot hybridizations using specific cDNA probes and by immunofluorescence and immunoblot analysis using specific monoclonal antibodies. The genes coding for both subunits were activated during late G1-phase in the transition of human PHA-stimulated lymphocytes from quiescent (G0 phase) to proliferative (S phase) state. These genes were inactivated when human leukemia cells HL60 were differentiated into monocytes upon treatment with the phorbol ester TPA. Changes at the protein level were significantly smaller than changes at the mRNA levels in both cell systems, suggesting a high stability of the Ku protein. Immunofluorescence studies demonstrated nucleolar as well as nuclear localization of the Ku protein in quiescent lymphocytes and during the early G1-phase; during the late G1, S and G2 phases, the Ku protein was only localized in discrete structures in the nucleoplasm. These results demonstrate that the gene expression for the Ku protein is associated with the proliferative state of the cells and that the nucleolar localization of the Ku protein is cell-cycle-dependent.

Antigens, Nuclear↗
PubMed1992

The Ku complex is modulated in response to viral infection and other cellular changes.

The complex of Ku with DNA is demonstrated to have multiple forms as assayed by gel retardation analysis. In CV1 cells this variation of complex can be modulated in response to viral infection with SV40. By Western blot analysis, a correlation can be made between modification of the complex formed on DNA in response to viral infection with variation of the 85 kDa subunit of Ku. Modification of the 85 kDa subunit can also be seen when cells are exposed to various extracellular stimuli including variation in serum levels, PMA and CaPO4.

Amino Acid Sequence↗
PubMed1993

Characterization of the 130-bp repeat enhancer element of the rat ribosomal gene: functional interaction with transcription factor E1BF.

The 130-bp repetitive element (RE) of the rat rDNA (ribosomal RNA-encoding gene) intergenic spacer stimulated the synthesis of rRNA four- to sixfold, in comparison with that of the promoter alone, both in vivo and in vitro, when ligated to the rat rDNA promoter. The addition of increasing amounts of highly purified E1BF (enhancer-1 binding factor), which binds to the rat rDNA promoter and an upstream nonrepetitive enhancer element [Zhang and Jacob, Mol. Cell. Biol. 10 (1990) 5177-5186], to an in vitro transcription system resulted in enhancement of rDNA transcription from the recombinant plasmids containing the promoter or promoter-RE. However, E1BF-mediated stimulation of transcription under the influence of the RE continued at higher concentrations of E1BF than did the control transcription from the promoter alone. The binding affinity of E1BF for the RE was comparable to its affinity for the nonrepetitive far upstream enhancer element previously characterized in our laboratory. The sequences protected by E1BF in the RE differed from those protected by UBF (upstream control element-binding factor), a well characterized pol I transcription factor. These data suggest that E1BF belongs to a class of transcription factors which interact with the promoter and spacer cis-acting RE to modulate rDNA transcription.

Animals↗
PubMed1996

Restoration of X-ray and etoposide resistance, Ku-end binding activity and V(D) J recombination to the Chinese hamster sxi-3 mutant by a hamster Ku86 cDNA.

Ku is a heterodimeric protein composed of 86 and 70 kDa subunits that binds preferentially to the double-stranded ends of DNA. Recent molecular characterization of ionizing-radiation sensitive (IRs) mutants belonging to the XRCC5 complementation group demonstrated the involvement of Ku in DNA double-strand break (DSB) repair and lymphoid V(D)J recombination. Here, we describe the isolation of a full-length hamster cDNA encoding the large subunit of the Ku heterodimer and demonstrate that the stable expression of this cDNA can functionally restore IR, Ku DNA end-binding activity and V(D)J recombination proficiency in the Chinese hamster IRs sxi-3 mutant. Moreover, we also demonstrate that sxi-3 cells are hypersensitive to etoposide, a DNA topoisomerase II inhibitor, and that resistance to this drug was restored by the Ku86 cDNA. These experiments suggest that a defect in the large subunit of the heterodimeric Ku protein is the sole factor responsible for the known defects of sxi-3 cells and our data of further support the role of Ku in DNA DSB repair and V(D)J recombination.

Amino Acid Sequence↗
PubMed2004

Bax-inhibiting peptide derived from mouse and rat Ku70.

Bax is a proapoptotic protein that plays a key role in the induction of apoptosis. Ku70 has activities to repair DNA damage in the nucleus and to suppress apoptosis by inhibiting Bax in the cytosol. We previously designed peptides based on the amino acid sequence of Bax-binding domain of human Ku70, and showed that these peptides bind Bax and inhibit cell death in human cell lines. In the present report, we examined the biological activities of other pentapeptides, VPTLK and VPALR, derived from mouse and rat Ku70. Cells in culture accumulated FITC-labeled VPTLK and VPALR, indicating that these peptides are cell permeable (human, mouse, rat, and porcine cells were examined). These peptides bound to Bax and suppressed cell death in various cell types including primary cultured cells. These data suggest that such Bax inhibiting peptides from three mammalian species may be used to protect healthy cells from apoptotic injury under pathological conditions.

Amino Acid Sequence↗
PubMed2004

Coexposure to benzo[a]pyrene plus UVA induced DNA double strand breaks: visualization of Ku assembly in the nucleus having DNA lesions.

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant with potential carcinogenicity. It has been shown that BaP, upon UVA irradiation, synergistically induced oxidative DNA damage, but other DNA damage was not confirmed. In this study, we examined whether coexposure to BaP plus UVA induces double strand breaks (DSBs) using xrs-5 cells, deficient in the repair of DSBs (Ku80 mutant), and whether Ku translocates involving the formation of DSBs. BaP plus UVA had a significant cytotoxic effect on CHO-K1 cells and an even more drastic effect on Ku80-deficient, xrs-5 cells, suggesting that the DSBs were generated by coexposure to BaP plus UVA. The DSBs were repaired in CHO-K1 cells within 30 min, but not in xrs-5 cells, indicating the involvement of a non-homologous end joining, which needs Ku proteins. Furthermore, we succeeded in visualizing that Ku80 rapidly assembled to the exposed region, in which DSBs might be generated, and clarified that the presence of both Ku70 and Ku80 was important for their accumulation.

Animals↗
PubMed2004

Ku80 is required but not sufficient for Galpha13-mediated endodermal differentiation in P19 embryonic carcinoma cells.

We have shown that a constitutively active Galpha13 (Galpha13Q226L) induces differentiation in P19 embryonic carcinoma cells to an endodermal phenotype. In this report, we demonstrate that Ku, a heterodimer of p80 (Ku80) and p70 (Ku70), is upregulated in P19 cells overexpressing Galpha13Q226L. Ku is the regulatory subunit of the DNA-dependent protein kinase and is primarily involved in DNA repair and recombination. Ku80 also is a somatostatin receptor. We show that while overexpression of Ku80 drastically reduced P19 cell proliferation, it was not sufficient to induce endodermal differentiation. However, coexpression of Galpha13Q226L and an antisense Ku80 abrogated the retarded growth rate and endodermal differentiation observed in cells expressing only Galpha13Q226L. Overexpression of Galpha13Q226L or Ku80 downregulated RNA polymerase I-mediated transcriptional activity and overexpression of antisense Ku80 restored the activity to control level. These results suggest that Ku80 is required for Galpha13-mediated endodermal differentiation in P19 cells.

Animals↗
PubMed2005

Enhancement of radiosensitivity in H1299 cancer cells by actin-associated protein cofilin.

Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity.

Actin Cytoskeleton↗
PubMed2006

The use of telomere biology to identify and develop superior nitrone based anti-oxidants.

We have employed a biological chemistry approach to dissect the mechanisms underpinning cellular responses to oxidant stress and to develop biologically relevant anti-oxidants. We have used telomere biology to define cellular stress responses and have observed telomere independent, p21- and p16-dependent stasis following oxidative insult in human fibroblasts. This was accompanied by a [corrected] reduction in XRCC5 expression and a reduction in [corrected] SIRT 1 expression. Using these markers in conjunction with senescence-associated beta-galactosidase expression, we have developed and screened novel nitrone based anti-oxidant compounds. We have identified functional compounds that are unsuitable for use in primary human cells. This has allowed subsequent identification of suitably structured compounds that act as superior biological anti-oxidants, which have potential for use in clinical interventions.

Antioxidants↗
PubMed2006

Regulation of the alpha-fetoprotein promoter: Ku binding and DNA spatial conformation.

This work shows that the proximal promoter of the mouse Afp gene contains a Ku binding site and that Ku binding is associated with down-regulation of the transcriptional activity of the Afp promoter. The Ku binding site is located in a segment able to adopt a peculiar structured form, probably a hairpin structure. Interestingly, the structured form eliminates the binding sites of the positive transcription factor HNF1. Furthermore, a DNAse hypersensitive site is detected in footprinting experiments done with extracts of AFP non-expressing hepatoma cells. These observations suggest that the structured form is stabilised by Ku and is associated with extinction of the gene in AFP non-expressing hepatic cells.

Animals↗
PubMed2004

Examination of surface-bound Ku-DNA complexes in an aqueous environment using MAC mode atomic force microscopy.

In the development of biosensors, it is essential to understand how the signal-transducing element may perturb surface-bound proteins and nucleic acids. The tip of the atomic force microscope is such an element in atomic force microscopy. In this paper, we describe the influence of tip-sample interactions on the measured height of the DNA repair protein, Ku, that has been adsorbed onto a mica surface which was submerged in aqueous solution. We find that the measured height of the Ku molecule depends critically on whether or not it is associated with DNA. Additionally, we observed that the conditions (time and concentration) under which Ku is incubated with DNA, affect the appearance (number and type) of the DNA-Ku complexes observed.

Biosensing Techniques↗
PubMed2004

The truncation of Ku86 in human lymphocytes.

The Ku heterodimer, which consists of Ku70 and Ku86 subunits, is a major sensor of DNA breaks. A truncated form of Ku86 lacking its C-terminus, termed Ku86 variant, has been detected in extracts from different human cells. Here we report that in human lymphocytes the Ku86 variant is not present in vivo but is generated in vitro upon cell lysis by a trypsin-like protease. The resulting Ku86 variant exists exclusively in complexes with Ku70, which possess strong affinity to DNA double strand termini. In different blood donors the levels of Ku86 variant correlated with the magnitude of radiation induced DNA breaks.

Adult↗
PubMed2007

Camptothecin acts synergistically with imatinib and overcomes imatinib resistance through Bcr-Abl independence in human K562 cells.

In this study, we have tried to find new targets and effective drugs for imatinib-resistant chronic myelogenous leukemia (CML) cells displaying loss of Bcr-Abl kinase target dependence. The imatinib-resistant K562/R1, -R2 and -R3 cells showed profound declines of Bcr-Abl level and concurrently exhibited up-regulation of Bcl-2 and Ku70/80, and down-regulation of Bax, DNA-PKcs and BRCA1, suggesting that loss of Bcr-Abl after exposure to imatinib might be accompanied by other cell survival mechanism. K562/R3 cells were more sensitive to camptothecin (CPT)- and radiation-induced apoptosis than K562 cells, indicating hypersensitivity of imatinib-resistant cells to DNA damaging agents. Moreover, when K562 cells were treated with the combination of imatinib with CPT, the level of Bax and the cleavage of PARP-1 and DNA-PK were significantly increased in comparison with the effects of each drug. Therefore, our study suggests that CPT can be used to treat CML with loss of Bcr-Abl expression.

Antigens, Nuclear↗
PubMed2004

Sir-mediated repression can occur independently of chromosomal and subnuclear contexts.

Epigenetic mechanisms silence the HM mating-type loci in budding yeast. These loci are tightly linked to telomeres, which are also repressed and held together in clusters at the nuclear periphery, much like mammalian heterochromatin. Yeast telomere anchoring can occur in the absence of silent chromatin through the DNA end binding factor Ku. Here we examine whether silent chromatin binds the nuclear periphery independently of telomeres and whether silencing persists in the absence of anchorage. HMR was excised from the chromosome by inducible site-specific recombination and tracked by real-time fluorescence microscopy. Silent rings associate with the nuclear envelope, while nonsilent rings move freely throughout the nucleus. Silent chromatin anchorage requires the action of either Ku or Esc1. In the absence of both proteins, rings move throughout the nucleoplasm yet remain silent. Thus, transcriptional repression can be sustained without perinuclear anchoring.

Antigens, Nuclear↗
PubMed2005

DNA repair, genome stability, and aging.

Aging can be defined as progressive functional decline and increasing mortality over time. Here, we review evidence linking aging to nuclear DNA lesions: DNA damage accumulates with age, and DNA repair defects can cause phenotypes resembling premature aging. We discuss how cellular DNA damage responses may contribute to manifestations of aging. We review Sir2, a factor linking genomic stability, metabolism, and aging. We conclude with a general discussion of the role of mutant mice in aging research and avenues for future investigation.

Animals↗
PubMed2005

Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii.

Rickettsia conorii, a strictly intracellular and category C priority bacterial pathogen (NIAID), invades different mammalian cells. Although some signaling events involved in bacterial entry have been documented, the bacterial and host proteins mediating entry were not known. We report the identification of the Ku70 subunit of DNA-dependent protein kinase (DNA-PK) as a receptor involved in R. conorii internalization. Ku70 is recruited to R. conorii entry sites, and inhibition of Ku70 expression impairs R. conorii internalization. Bacterial invasion is dependent on the presence of cholesterol-enriched microdomains containing Ku70. R. conorii infection stimulates the ubiquitination of Ku70. In addition, the ubiquitin ligase c-Cbl is recruited to R. conorii entry foci, and downregulation of endogenous c-Cbl blocks bacterial invasion and Ku70 ubiquitination. An affinity chromatography approach identified the rickettsial protein rOmpB as a ligand for Ku70. This is the first report of a receptor-ligand interaction involved in the internalization of any rickettsial species.

Actins↗
PubMed2006

Down-regulation of PARP-1, but not of Ku80 or DNA-PKcs', results in higher gene targeting efficiency.

The viability of non-homologous end-joining (NHEJ)-defective mice suggests that homologous recombination (HR) might take over its role in DNA repair. To test this hypothesis, we examined gene targeting frequencies (TF) in DNA-PK(cs), Ku80 and poly(ADP-ribose) polymerase (PARP-1) nullizygous cells. We observed a 3-fold TF increase in PARP-1 knockout embryonic stem (ES) cells, which is consistent with the predicted role of PARP-1 as a switch between HR and NHEJ. To a lesser extent, such effect could be reproduced upon chemical inhibition of PARP-1. However, TF was not enhanced in Ku80- or DNA-PK(cs)-defective cells. Our study also suggests an unexpected involvement of DNA-PK(cs) in HR.

Animals↗