cis-Dichlorodiammineplatinum(II) (CDDP; cisplatin) is commonly used in combination with ionizing radiation (IR) in the treatment of various malignancies. In vitro, many observations suggest that acquisition of CDDP resistance in cell lines confers cross-resistance to IR, but the molecular mechanisms involved have not been well documented yet. We report here the selection and characterization of a murine CDDP-resistant L1210 cell line (L1210/3R) that exhibits cross-resistance to IR because of an increased capacity to repair double-strand breaks compared with parental cells (L1210/P). In resistant cells, electrophoretic mobility shift assays revealed an increased DNA-end binding activity that could be ascribed, by supershifting the retardation complexes with antibodies, to the autoantigen Ku. The heterodimeric Ku protein, composed of 86-kDa (Ku80) and 70-kDa (Ku70) subunits, is the DNA-targeting component of DNA-dependent protein kinase (DNA-PK), which plays a critical role in mammalian DNA double-strand breaks repair. The increased Ku-binding activity in resistant cells was associated with an overexpression affecting specifically the Ku80 subunit. These data strongly suggest that the increase in Ku activity is responsible for the phenotype of cross-resistance to IR. In addition, these observations, along with previous results from DNA-PK- mutant cells, provide evidence in favor of a role of Ku/DNA-PK in resistance to CDDP. These results suggest that Ku activity may be an important molecular target in cancer therapy at the crossroad between cellular responses to CDDP and IR.

Radiation-induced DNA double-stand breaks (DSBs) lead to numerous biological effects. To elucidate the molecular mechanisms involved in cellular responses to low dose and low dose-rate radiation, it is informative to clarify the roles of DSB repair related genes. In higher vertebrate cells, there are at least two major DSB repair pathways, namely non-homologous end-joining (NHEJ) and homologous recombination (HR). Here, it is shown that in chicken DT40 cells irradiated with gamma-rays at a low dose-rate (2.4 cGy/day), the growth delay in NHEJ-related KU70- and PRKDC (encoding DNA-PKcs)-defective cells were remarkably higher than in cells defective for the HR-related RAD51B and RAD54 genes. DNA-PKcs- defective human M059J cells also showed an obvious growth delay when compared to control M059K cells. RAD54(-/-)KU70(-/-) cells demonstrated their highest degree of growth delay after an X-irradiation with a high dose-rate of 0.9 Gy/min. However they showed a lower degree of growth delay than that seen in KU70(-/-) and PRKDC(-/-/-) cells exposed to low dose-rate irradiation. These findings indicate that cellular responses to low dose-rate radiation are remarkably different from those to high dose-rate radiation. The fact that both DT40 and mammalian NHEJ-defective cells were highly sensitive to low dose-rate radiation, provide a foundation for the concept that NHEJ-related factors may be useful as molecular markers to predict the sensitivity of humans to low dose-rate radiation.

DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70-Ku80 that binds to double-stranded DNA ends. Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency. Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect. Expression of Ku(449-732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival. The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.

Hyperthermia has a radiosensitizing effect, which is one of the most important biological bases for its use in cancer therapy with radiation. Although the mechanism of this effect has not been clarified in molecular terms, possible involvement of either one or both of two major DNA double-strand break (DSB) repair pathways, i.e. nonhomologous end joining (NHEJ) and homologous recombination (HR), has been speculated. To test this possibility, we examined cells of the chicken B-lymphocyte cell line DT40 and its derivatives lacking NHEJ and/or HR: KU70(-/-), DNA-PKcs(-/-/-), RAD54(-/-) and KU70(-/-)/RAD54(-/-). Radiosensitization by hyperthermia could be seen in all of the mutants, including KU70(-/-)/RAD54(-/-), which lacked both NHEJ and HR. Therefore, radiosensitization by hyperthermia cannot be explained simply by its inhibitory effects, if any, on NHEJ and/or HR alone. However, in NHEJ-defective KU70(-/-) and DNA-PKcs(-/-/-), consisting of two subpopulations with distinct radiosensitivity, the radiosensitive subpopulation, which is considered to be cells in G(1) and early S, was not sensitized. Substantial sensitization was seen only in the radioresistant subpopulation, which is considered to be cells in late S and G(2), capable of repairing DSBs through HR. This observation did not exclude possible involvement of NHEJ in G(1) and early S phase and also suggested inhibitory effects of hyperthermia on HR. Thus partial contribution of NHEJ and HR in radiosensitization by hyperthermia, especially that depending on the cell cycle stage, remains to be considered.

Although exposure to asbestos fibers is associated with the development of lung cancer, the underlying mechanism(s) remains unclear. Using human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells, we previously showed that UICC chrysotiles can malignantly transform these cells in a stepwise fashion before they become tumorigenic in nude mice. In the present study we used cDNA expression arrays to screen differentially expressed genes among the tumorigenic cells. A total of 15 genes were identified, 11 of which were further confirmed by northern blot. Expression levels of these genes were then determined among transformed BEP2D cells at different stages of the neoplastic process, including non-tumorigenic cells that were resistant to serum-induced terminal differentiation, early and late passage transformed BEP2D cells, five representative tumor cell lines and fused tumorigenic-control cell lines which were no longer tumorigenic. A consistent 2- to 3-fold down-regulation of the DCC (deleted in colon cancer), Ku70 and heat shock protein 27 genes were detected in all the independently generated tumor cell lines while expression levels in early transformants as well as in the fusion cell lines remained normal. In contrast, all the tumor cell lines examined demonstrated 2- to 4-fold overexpression of the insulin receptor and its signal transduction genes. Differential expression of these genes was completely restored in the fusion cell lines examined. No alteration in c-jun or EGF receptor expression was found in any of the cell lines. Our data suggest that activation of the insulin receptor pathway and inactivation of DCC and Ku70 may cooperate in malignant transformation of BEP2D cells induced by asbestos.

OBJECTIVE

Breast cancer is one of the major killers worldwide. Aberrant double-stranded break (DSB) repair leads to genomic instability, which is a hallmark of malignant cells. Double-stranded breaks are repaired in two pathways: homologous recombination (HR) and non-homologous DNA end joining (NHEJ). It is not known whether these repair pathways are affected in sporadic breast tumours.

METHODS

In the present work the distribution of genotypes and frequency of alleles of the Ku70, A46922G (rs132793) polymorphism and Ligase IV, A6008G (Ile591Val) (rs2232641) polymorphism in breast cancer women were investigated. The genetic polymorphism analysis was performed using a DNA ABI PRISM 377 sequence detection system (Applied Biosystems) in 135 sporadic breast cancer cases.

RESULTS

The distribution of the genotypes of the A46922G polymorphism of Ku70 in patients differed significantly (p < 0.05) from those predicted by the Hardy-Weinberg equilibrium. There were significant differences in the frequencies of alleles between the breast cancer subjects and controls (p < 0.05). However, the distribution of the genotypes of the A6008G polymorphism of Ligase IV in both controls and patients did not differ significantly (p>> 0.05) from that predicted by the Hardy-Weinberg distribution.

CONCLUSIONS

The results support the hypothesis that the A46922G polymorphism of the Ku70 gene may be associated with the incidence of breast cancer in women from the Lodz region of Poland.

The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5' deoxyribosephosphate (5'dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5'dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role.

Radiation therapy is one of the most promising therapeutic strategies in unresectable esophageal squamous cell carcinoma (ESCC). The histone deacetylase (HDAC) inhibitor has been shown to enhance radiosensitivity. Valproic acid (VPA) is a well-known drug used to treat seizure disorders and epilepsy, and has been shown to inhibit HDACs. We recently reported that a clinically safe dose of VPA enhances radiation‑induced cytotoxicity in human ESCC cells. However, the mechanism of radiosensitizing effect of VPA has not yet been confirmed. The present study examined the effect of VPA on DNA double-strand break (DSB) repair after radiation in the human ESCC cell lines KES, TE9 and TE11 by examining H2AX phosphorylation (γH2AX) levels as a marker of radiation‑induced DSBs. The present study also examined whether VPA inhibited radiation-induced DNA DSB repair by suppressing non-homologous end joining (NHEJ), focusing particularly on the acetylation of Ku70. VPA was shown to prolong γH2AX levels after irradiation in all three ESCC cell lines. Moreover, prolonged γH2AX foci formation after irradiation was also observed by immunocytochemistry following VPA pretreatment in KES and TE9 cells. VPA was shown to induce Ku70 acetylation after irradiation in all three ESCC cell lines. Our results suggest that VPA prolonged radiation‑induced DSBs by inhibiting NHEJ in DSB repair pathways in ESCC. VPA could therefore be used as an effective radiosensitizer in ESCC radiotherapy.

The promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic 'RVxF' motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC)-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway.

Plant Ku genes were identified very recently in Arabidopsis thaliana, and their roles in repair of double-stranded break DNA and maintenance of telomere integrity were scrutinized. In this study, the cDNAs encoding Ku70 (VrKu70) and Ku80 (VrKu80) were isolated from mung bean (Vigna radiata L.) hypocotyls. Both genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid implicating that exogenous auxins triggered the effects following their uptake by the cells. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca(2+) chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and alpha-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways.

Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell.

Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1-418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1-418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1-418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1-418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411-418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is important for the physiological function of Rad52 not only at a late stage following irradiation, but also at an early stage.

This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5°C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.

The promoter of the KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) forming G-quadruplex structures that are recognized by nuclear proteins: PARP-1, Ku70 and hnRNPA1. Here we have studied the interaction between hnRNPA1 (and its derivative UP1) and the G-quadruplexes of KRAS by EMSA, FRET and CD experiments. FRET and CD showed that hnRNPA1/UP1 is able to unfold the G-quadruplexes of KRAS and facilitate the quadruplex to duplex transformation. This finding strengthens our previous hypothesis that the transcription regulation of KRAS is mediated by G-quadruplex structures. Against this background we designed G4-decoy oligonucleotides specific for KRAS that exhibit a strong antiproliferative effect in pancreatic cancer cells.

Indefinite proliferation of eukaryotic cells depends on telomerase that counteracts the depletion of DNA from chromosome termini due to the end replication problem. The requirement for telomerase can, under certain conditions, be circumvented by employing homologous recombination-based mechanisms for telomere maintenance. Whereas yeast and mammalian cells lacking telomerase appear to readily adopt alternative telomere lengthening (ALT), in Arabidopsis ALT is inhibited by the Ku heterodimer. Although we failed to establish ALT cell lines from Ku-proficient lines, ALT can be efficiently induced in cells derived from mutants deficient for telomerase reverse transcriptase (TERT) and Ku70. In this study, we describe the growth performance, genome stability and long-term maintenance of telomeric DNA in Arabidopsis ku70 tert ALTcultures. ALT activation increases karyotype stability in the majority of tert ku70 cell lines, which contrasts with ongoing chromosomal rearrangements detected in survival tert cultures that lack any detectable telomeric sequences. Curiously, ku70 tert ALT lines and tert survivor cultures proliferate at a similar rate, although the latter display remarkable chromosomal abnormalities, including giant circular and linear megachromosomes that seem to arise by fusions of multiple chromosomes. This observation underlies the extraordinary tolerance of plant cells to telomere dysfunction.

We showed that the drug sensitivity of multidrug-resistant (MDR) cells could be enhanced by fractionated irradiation. The molecular changes associated with fractionated radiation-induced chemosensitization were characterized. Irradiated cells of the multidrug-resistant CEM/MDR sublines (CEM/MDR/IR1, 2 and 3) showed a loss of P-glycoprotein (P-gp) and concurrent reduction of Ku DNA binding and DNA-PK activities with decreased level of Ku70/80 and increased level of DNA-PKcs, and these changes were followed by an increased susceptibility to anticancer drugs. These irradiated MDR cells also exhibited the reduction of other chemoresistance-related proteins, including BCL2, NF-kappaB, EGFR, MDM2 and Ku70/80, and the suppression of HIF-1alpha expression induced by hypoxia. In contrast, fractionated irradiation increased the levels of these proteins and induced drug resistance in the parental drug-sensitive CEM cells. These results suggest that the chemoresistance-related proteins are differentially modulated in drug-sensitive and MDR cells by fractionated irradiation, and the optimized treatment with fractionated radiation could lead to new chemoradiotherapeutic strategies to treat multidrug-resistant tumors.

The human immunodeficiency virus type 1 (HIV-1) viral protein R (vpr) gene is an evolutionarily conserved gene among the primate lentiviruses. Several functions are attributed to Vpr including the ability to cause cell death, cell cycle arrest, apoptosis and DNA damage. The Vpr domain responsible for DNA damage as well as the mechanism(s) through which Vpr induces this damage is unknown. Using site-directed mutagenesis, we identified the helical domain II within Vpr (aa 37-50) as the region responsible for causing DNA damage. Interestingly, Vpr Delta(37-50) failed to cause cell cycle arrest or apoptosis, to induce Ku70 or Ku80 and to suppress tumor growth, but maintained its capability to activate the HIV-1 LTR, to localize to the nucleus and to promote nonhomologous end-joining. In addition, our cytogenetic data indicated that helical domain II induced chromosomal aberrations, which mimicked those induced by cisplatin, an anticancer agent. This novel molecular mimicry function of Vpr might lead to its potential therapeutic use as a tumor suppressor.

Deformed Epidermal Autoregulatory Factor 1 (DEAF1) is a transcription factor linked to suicide, cancer, autoimmune disorders and neural tube defects. To better understand the role of DEAF1 in protein interaction networks, a GST-DEAF1 fusion protein was used to isolate interacting proteins in mammalian cell lysates, and the XRCC6 (Ku70) and the XRCC5 (Ku80) subunits of DNA dependent protein kinase (DNA-PK) complex were identified by mass spectrometry, and the DNA-PK catalytic subunit was identified by immunoblotting. Interaction of DEAF1 with Ku70 and Ku80 was confirmed to occur within cells by co-immunoprecipitation of epitope-tagged proteins, and was mediated through interaction with the Ku70 subunit. Using in vitro GST-pulldowns, interaction between DEAF1 and the Ku70 subunit was mapped to the DEAF1 DNA binding domain and the C-terminal Bax-binding region of Ku70. In transfected cells, DEAF1 and Ku70 colocalized to the nucleus, but Ku70 could not relocalize a mutant cytoplasmic form of DEAF1 to the nucleus. Using an in vitro kinase assay, DEAF1 was phosphorylated by DNA-PK in a DNA-independent manner. Electrophoretic mobility shift assays showed that DEAF1 or Ku70/Ku80 did not interfere with the DNA binding of each other, but DNA containing DEAF1 binding sites inhibited the DEAF1-Ku70 interaction. The data demonstrates that DEAF1 can interact with the DNA-PK complex through interactions of its DNA binding domain with the carboxy-terminal region of Ku70 that contains the Bax binding domain, and that DEAF1 is a potential substrate for DNA-PK.

Previous research showed that the consumption of heat-killed E. coli O157:H7 bacteria resulted in an increase in the level of DNA damage in intestine, liver and spleen cells. We hypothesized that certain bacterial components released from heat-killed bacteria trigger this response. We analysed the possibility that bacterial components [such as lipopolysaccharides (LPS)] could induce changes in the level of proteins involved in cell proliferation, DNA repair and DNA methylation in distal spleen tissues of mice. Four-week-old male mice were provided water supplemented with whole heat-killed E. coli O157:H7 bacteria or components of bacteria (DNA, RNA, proteins and LPS). Spleen cells responded to exposure to whole heat-killed bacteria and LPS with an alteration in the level of PCNA proteins, DNA methylation proteins (DNMT1, DNMT3A, DNMT3B, and MeCP2) and DNA repair proteins (APE1 and KU70). Other bacterial components analysed in this study mostly did not alter protein expression. The data suggest that LPS is a bacterial component capable of inducing molecular changes in naïve spleen cells of hosts exposed to it.

Ornithine decarboxylase (ODC) antizyme targets ODC for ubiquitin-independent proteosome degradation, thereby inhibiting polyamine synthesis. It has been shown to regulate DNA methylation and has tumor suppressor activity. Increasing evidence suggested that antizyme may also have ODC-independent functions. Here, we report that antizyme plays a role in DNA double-strand break repairs. A zinc-inducible human antizyme gene expression vector was transfected into UM1 human oral squamous cancer cells that do not express endogenous antizyme. The resultant upregulated genes were screened by cDNA arrays and confirmed by quantitative real-time polymerase chain reaction. DNA-dependent protein kinase including its catalytic subunit DNA-PKcs and regulatory subunit Ku70, two key proteins of the DNA damage repair machinery, was significantly upregulated after ectopic expression of antizyme. Consistently, we found that UM1 cells are sensitive to gamma irradiation and deficient in DNA damage repairs, as shown by radio-sensitivity and Comet assays. Ectopic expression of antizyme increased radio-resistance of UM1 cells and restored their capacity of DNA damage repairs to the level of UM2 cells that have an identical genetic background but express endogenous antizyme. Plasmid end-joining assays confirmed that antizyme enhances the ability of UM1 cells to repair DNA double-strand breaks by the nonhomologous end-joining pathway.

Prenatal exposure to low-dose radiation increases the risk of microcephaly and/or mental retardation. Microcephaly is also associated with genetic mutations that affect the non-homologous end-joining pathway of DNA double-strand break repair. To examine the link between these two causal factors, we characterized the neural developmental effects of acute radiation exposure in mouse littermate embryos harboring mutations in the Ku70 and p53 genes. Both low-dose radiation exposure and Ku70 deficiency induced morphologically indistinguishable cortical neuronal apoptosis. Irradiated Ku70-deficient embryos displayed anatomical damage indicative of increased radiosensitivity in the developing cerebral cortex. Deleting the p53 gene not only rescued cortical neuronal apoptosis at all levels but also restored the in vitro growth of Ku70-deficient embryonic fibroblasts despite the presence of unrepaired DNA/chromosomal breaks. The results confirm the role of DNA double-strand breaks as a common causative agent of apoptosis in the developing cerebral cortex. Furthermore, the findings suggest a disease mechanism by which the presence of endogenous DNA double-strand breaks in the newly generated cortical neurons becomes radiomimetic when DNA end joining is defective. This in turn activates p53-dependent neuronal apoptosis and leads to microcephaly and mental retardation.

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.

The channel structure of the Ku protein elegantly reveals the mechanistic basis of sequence-independent DNA-end binding, which is essential to genome integrity after exposure to ionizing radiation or in V(D)J recombination. However, contradicting evidence indicates that this protein is also involved in the regulation of gene expression and in other regulatory processes with intact chromosomes. This computational study predicts that a putative DNA binding domain of this protein, the SAP domain, can form DNA-bound complexes with relatively high affinities (ΔG ≈ -20 kcal mol(-1)). The binding modes are searched by low frequency vibration modes driven by the fully flexible docking method while binding affinities are calculated by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. We find this well defined 5 kDa domain with a helix-extended loop-helix structure is suitable to form favorable electrostatic and hydrophobic interactions with either the major groove or the minor groove of DNA. The calculation also reveals the sequence specified binding preference which may relate to the observed pause sites when Ku translocates along DNA and the perplex binding of Ku with circular DNA.

Loss of cardiomyocytes occurs with aging and contributes to cardiovascular complications. In the present study, we highlighted the role of clusterin, a protein that has recently been associated with the protection of cardiomyocytes from apoptosis. Clusterin protects cardiac cells against damage from myocardial infarction, transplant, or myocarditis. Clusterin can act directly or indirectly on apoptosis by regulating several intracellular pathways. These pathways include (1) the oxidant and inflammatory program, (2) insulin growth factor 1 (IGF-1) pathway, (3) KU70 / BCL-2-associated X protein (BAX) pathway, (4) tumor necrosis factor alpha (TNF-α) pathway, (5) BCL-2 antagonist of cell death (BAD) pathway, and (6) mitogen-activated protein kinase (MAPK) pathway. Given the key role of clusterin in preventing loss of cardiac tissue, modulating the expression and function of this protein carries the potential of improving cardiovascular care in the future.