Poly(ADP-ribose) polymerase 3 (PARP-3) is a newly characterized PARP. In contrast to the two best-studied nuclear PARPs, PARP-1 and PARP-2, PARP-3 activity is apparently not stimulated by DNA damage. However, our previous work has demonstrated that PARP-3 interacts with several DNA damage response proteins, including Ku70/Ku80, DNA-PK, and PARP-1, suggesting that it contributes to the DNA damage response. Furthermore, a possible function for PARP-3 in the regulation of gene expression has been inferred from our observations that it associates with polycomb group proteins, which are responsible for epigenetic modifications leading to gene silencing. In this report, we extend our characterization of PARP-3 by revealing its distribution in the tissues and cell types of adult cynomolgous monkeys using a well-characterized PARP-3 polyclonal antibody. This study is the first to demonstrate that PARP-3 is genuinely expressed in most of the examined tissues. However, its expression is highly restricted to specific cell types of each tissue, indicating that PARP-3 expression is tightly regulated. One of the key findings of this study is that PARP-3 is highly expressed in the nuclei of epithelial cells forming the ducts of prostate, salivary glands, liver, and pancreas and in the neurons of terminal ganglia.

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.

We have previously shown that local infection of tobacco plants with tobacco mosaic virus (TMV) or oilseed rape mosaic virus (ORMV) results in a systemic increase in the homologous recombination frequency (HRF). Here, we analyzed what other changes in the genome are triggered by pathogen infection. For the analysis of HRF, mutation frequency (MF) and microsatellite instability (MI), we used three different transgenic Arabidopsis lines carrying β-glucuronidase (GUS)-based substrates in their genome. We found that local infection of Arabidopsis with ORMV resulted in an increase of all three frequencies, albeit to differing degrees. The most prominent increase was observed in microsatellite instability. The increase in HRF was the lowest, although still statistically significant. The analysis of methylation of the 35S promoter and transgene expression showed that the greater instability of the transgene was not attributed to these changes. Strand breaks brought about a significant increase in non-treated tissues of infected plants. The expression of genes associated with various repair processes, such as KU70, RAD51, MSH2, DNA POL α and DNA POL δ, was also increased. To summarize, our data demonstrate that local ORMV infection destabilizes the genome in systemic tissues of Arabidopsis plants in various ways resulting in large rearrangements, point mutations and microsatellite instability.

S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks.

Mesenchymal stem cells (MSC) have been identified as having great potential as autologous cell therapeutics to treat traumatic brain injury and spinal injury as well as neuronal and cardiac ischemic events. All future clinical applications of MSC cell therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to systematically alter the protein expression of therapeutically beneficial biomolecules in harvested MSC cells with high fidelity in a single transfection event. We have developed a delivery platform on the basis of the use of a solid gold nanoparticle that has been surface modified to produce a fusion containing a zwitterionic, pentapeptide designed from Bax inhibiting peptide (Ku70) to enhance cellular uptake and a linearized expression vector to induce enhanced expression of brain-derived neurotrophic factor (BDNF) in rat-derived MSCs. Ku70 is observed to effect >80% transfection following a single treatment of femur bone marrow isolated rat MSCs with efficiencies for the delivery of a 6.6 kbp gene on either a Au nanoparticle (NP) or CdSe/ZnS quantum dot (QD). Gene expression is observed within 4 d by optical measurements, and secretion is observed within 10 d by Western Blot analysis. The combination of being able to selectively engineer the NP, to colocalize biological agents, and to enhance the stability of those agents has provided the strong impetus to utilize this novel class of materials to engineer primary MSCs.

Acetylation status of DNA end joining protein Ku70 dictates its function in DNA repair and Bax-mediated apoptosis. Despite the knowledge of HDACs and HATs that are reported to modulate the acetylation dynamics of Ku70, very little is known about proteins that critically coordinate these key modifications. Here, we demonstrate that nuclear matrix-associated protein scaffold/matrix-associated region-binding protein 1 (SMAR1) is a novel interacting partner of Ku70 and coordinates with HDAC6 to maintain Ku70 in a deacetylated state. Our studies revealed that knockdown of SMAR1 results in enhanced acetylation of Ku70, which leads to impaired recruitment of Ku70 in the chromatin fractions. Interestingly, ionizing radiation (IR) induces the expression of SMAR1 and its redistribution as distinct nuclear foci upon ATM-mediated phosphorylation at serine 370. Furthermore, SMAR1 regulates IR-induced G2/M cell cycle arrest by facilitating Chk2 phosphorylation. Alternatively, SMAR1 provides radioresistance by modulating the association of deacetylated Ku70 with Bax, abrogating the mitochondrial translocation of Bax. Thus, we provide mechanistic insights of SMAR1-mediated regulation of repair and apoptosis via a complex crosstalk involving Ku70, HDAC6 and Bax.

BACKGROUND

The thermophilic filamentous fungus Myceliophthora thermophila has many suitable characteristics for industrial biotechnology and could be a promising new chassis system for synthetic biology, particularly the ATCC 42464 strain, whose genome was sequenced in 2011. However, metabolic engineering of this strain using genetic approaches has not been reported owing to a lack of genetic tools for this organism.

RESULTS

In the present study, we developed a high efficiency Agrobacterium tumefaciens mediated transformation system for M. thermophila, including an approach for targeted gene deletion using green fluorescence protein (GFP) as a marker for selection. Up to 145 transformants per 10(5) conidia were obtained in one transformation plate. Moreover, a ku70 deletion mutant was constructed in the ATCC 42464 background using the tools developed in present study and subsequently characterized. The ku70 deletion construct was designed using resistance to phosphinothricin as the selection marker. Additionally, a GFP-encoding cassette was incorporated that allowed for the selection of site-specific (no fluorescence) or ectopic (fluorescence) integration of the ku70 construct. Transformants with ectopically integrated ku70 deletion constructs were therefore identified using the fluorescent signal of GFP. PCR and Southern blotting analyses of non-fluorescent putative ku70 deletion transformants revealed all 11 tested transformants to be correct deletions. The deletion frequency in a pool of 116 transformants analyzed was 58 %. Moreover, the homologous rate improved about 3 folds under ku70 mutant using the pyrG as a test gene to disrupt in M. thermophila.

CONCLUSIONS

We successfully developed an efficient transformation and target gene disruption approach for M. thermophila ATCC 42464 mediated by A. tumefaciens. The tools and the ku70 deletion strain developed here should advance the development of M. thermophila as an industrial host through metabolic engineering and accelerate the elucidation of the mechanism of rapid cellulose degradation in this thermophilic fungus.

The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

The sequencing of the human genome has led to the identification of many genes whose functions remain to be determined. Because of conservation of genetic function, microbial systems have often been used for identification and characterization of human genes. We have investigated the use of the Escherichia coli SOS induction assay as a screen for yeast and human genes that might play a role in DNA metabolism and/or in genome stability. The SOS system has previously been used to analyze bacterial and viral genes that directly modify DNA. An initial screen of meiotically expressed yeast genes revealed several genes associated with chromosome metabolism (e.g., RAD51 and HHT1 as well as others). The SOS induction assay was then extended to the isolation of human genes. Several known human genes involved in DNA metabolism, such as the Ku70 end-binding protein and DNA ligase IV, were identified, as well as a large number of previously unknown genes. Thus, the SOS assay can be used to identify and characterize human genes, many of which may participate in chromosome metabolism.

Heat shock prior, during, or immediately after ionizing radiation synergistically increases cell killing, a phenomenon termed hyperthermic radiosensitization. Recently, we have shown a constitutive DNA-binding factor in rodent cells that is inactivated by heat shock to be identical to Ku autoantigen. Ku, consisting of an Mr 70,000 (Ku70) and an Mr 86,000 (Ku80) subunit, is a heterodimeric nuclear protein and is the DNA-binding regulatory component of the mammalian DNA-dependent protein kinase DNA-PK. Recent genetic and biochemical studies indicate the involvement of Ku and DNA-PK in DNA double-strand break repair and V(D)J recombination. On the basis of these findings, we propose that heat-induced loss of the DNA-binding activity of Ku may lead to hyperthermic radiosensitization. To test this hypothesis, we examined and compared the DNA-binding activity of Ku, the DNA-PK kinase activity, and hyperthermic radiosensitization in rodent cells immediately after heat shock and during post-heat shock recovery at 37 degrees C. Our results show that the heat-induced loss of Ku-DNA binding activity correlates well with an increased radiosensitivity of the heat-shocked cells, and furthermore, the loss of synergistic interaction between heat and radiation parallels the recovery of the DNA-binding activity of Ku. On the other hand, the heat-induced decrease of DNA-PK activity did not correlate with hyperthermic radiosensitization. Our data, for the first time, provide evidence for a role of Ku protein in modulating the cellular response to combined treatments of heat shock and ionizing radiation.

By studying the role of mismatch repair in hypermutation at the immunoglobulin loci, the field of antibody hypermutation has been integrated into the larger area of DNA repair. Trans-acting factors - Ku70, Ku80 and possibly SWAP-70 - have been identified for the temporally related but not mechanistically related immunoglobulin heavy-chain class-switch.

Ku, the heterodimer of Ku70 and Ku86, plays crucial roles in non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. It has recently been reported that heterozygous disruption of the human KU86 locus results in haploinsufficient phenotypes, including retarded growth, increased radiosensitivity, elevated p53 levels and shortened telomeres. In this paper, however, we show that heterozygous inactivation of either the KU70 or KU86 gene does not cause any defects in cell proliferation or DSB repair in human somatic cells. Moreover, although these heterozygous cell lines express reduced levels of both Ku70 and Ku86, they appear to maintain overall genome integrity with no elevated p53 levels or telomere shortening. These results clearly indicate that Ku haploinsufficiency is not a commonly observed phenomenon in human cells. Our data also suggest that the impact of KU70/KU86 mutations on telomere metabolism varies between cell types in humans.

Despite the known longevity of human hematopoietic stem and progenitor cells (HSC), numerous functional impairments of these cells can be observed in an age-dependent manner. However, the molecular alterations associated with aging of HSC are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from newborn, young and old healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and evaluated by FACS analysis. We performed cDNA macroarray analyses on a first set of CD34+ samples (n=13). We found the genes encoding KU-antigen 70 kD (KU70), microsomal glutathione S-transferase 1 (MGST1) and BCL2-interacting killer (BIK) to possess age-related mRNA expression levels. KU70 is a DNA repair gene and part of the DNA-dependent protein kinase (DNA-PK) complex. Its expression was negatively correlated with donor age showing highest expression levels in newborn, 2.6-fold lower levels in young and 6.3-fold lower levels in old donors. The transcription levels of MGST1, a gene protecting against oxidative stress, progressively increased with age. Expression was lowest in newborn, 2.6-fold higher in young and 4.3-fold higher in old donors. BIK is a proapoptotic gene and its expression was positively correlated with donor age: lowest in newborn, 1.8-fold higher in young and 4.1-fold higher in old donors. These findings were confirmed with an independent, second set of CD34+ samples (n=16) by means of quantitative real-time RT-PCR. Elucidation of age-dependent molecular alterations in healthy HSC facilitate a better understanding of functional impairments in hematopoiesis and may become valuable for anti-aging drug development and the emerging field of regenerative medicine.

The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of gamma-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM target in the DSB response. These data show that the same genetic pathways control how cells respond to single DSBs and to multiple lesions induced by whole-cell DNA damage.

The genetic factors that determine immune responsiveness and DNA repair capacity are reviewed as major elements influencing the life span. Within this framework two sets of new data obtained in mice and humans are reported and discussed. As to the first set, the role of immune response genes was investigated in Biozzi mice genetically selected for high (H) or low (L) antibody response. After 15-20 generations of assortative mating, H and L mice exhibited almost complete intraline homozygosity and interline polymorphism including distinct H-2 haplotypes, such as q in H and s in L mice. In order to analyze the impact of quantitative trait loci (QTL) on the antibody response as well as on the DNA repair capacity in cells of the immune system independently of the selected H-2 haplotype, congenic Hq and Hs as well as Lq and Ls mice were produced and analysed. Both the antibody response and DNA repair capacity were found to be independent of the H-2 haplotype and determined by QTL. As to the second set of data, DNA repair was also studied in irradiated peripheral blood mononuclear cells (PBMC) from ageing humans. The levels of ku 70, ku 80, DNA-PKcs, phosphorylated ku 80 as well as the DNA-binding activity of the ku70/ku 80 heterodimer were determined in the cytoplasmic and nuclear extracts obtained, before and after irradiation, from young and elderly subjects. The results of this study suggest that the decreased DNA repair capacity in PBMC from elderly subjects may be related to impaired migration of the phosphorylated ku 80 from the cytoplasm to the nucleus. This finding helps to elucidate questions related to the impairment of DNA repair during ageing.

DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)-t-butyl-4-hydroxyanisole or N-acetyl-l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PK(pThr2609)) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine (SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PK(pThr2609) was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs.

We have investigated the effects of 2 weeks of insulin-like growth factor-1 (IGF-1) supplementation (5 μg/kg per day) and 6 weeks of exercise training (60% of the maximal oxygen consumption [VO₂ max]) on neurogenesis, DNA damage/repair, and sirtuin content in the hippocampus of young (3 months old) and old (26 months old) rats. Exercise improved the spatial memory of the old group, but IGF-1 supplementation eliminated this effect. An age-associated decrease in neurogenesis was attenuated by exercise and IGF-1 treatment. Aging increased the levels of 8-oxo-7,8-dihydroguanine (8-oxoG) and the protein Ku70, indicating the role of DNA damage in age-related neuropathology. Acetylation of 8-oxoguanine DNA glycosylase (OGG1) was detected in vivo, and this decreased with aging. However, in young animals, exercise and IGF-1 treatment increased acetylated (ac) OGG1 levels. Sirtuin 1 (SIRT1) and SIRT3, as DNA damage-associated lysine deacetylases, were measured, and SIRT1 decreased with aging, resulting in a large increase in acetylated lysine residues in the hippocampus. On the other hand, SIRT3 increased with aging. Exercise-induced neurogenesis might not be a causative factor of increased spatial memory, because IGF-1 plus exercise can induce neurogenesis in the hippocampus of older rats. Data revealed that the age-associated increase in 8-oxoG levels is due to decreased acetylation of OGG1. Age-associated decreases in SIRT1 and the associated increase in lysine acetylation, in the hippocampus, could have significant impact on function and thus, could suggest a therapeutic target.

Roscovitine has been reported to have anti-proliferative properties and is in process of undergoing clinical trials. In addition to its intrinsic anticancer properties, it has recently been suggested that roscovitine may also enhance the activity of traditional chemo- and radio- therapies in certain cancer cell lines. The purpose of this study was to define the activity of roscovitine in increasing radiosensitivity of human non-small cell lung cancer (NSCLC) cell line A549 cells in vitro. A549 cells were exposed to ionizing radiation (IR) of gamma-ray with or without roscovitine pretreatment. Clonogenic assay was performed and cell cycle and apoptosis were analyzed by flow cytometry. Expression of PARP, Ku70 and Ku80 proteins was detected by Western blot. The active form of caspase-3 positive cells were measured by flow cytometry. Our results showed that roscovitine caused dose-dependent apoptosis in A549 cells. Pretreatment with minimally toxic concentration of roscovitine significantly radiosensitized A549 cells by inhibiting colony formation. We then examined potential mechanisms that may contribute to the enhanced radiation response induced by roscovitine. Our results showed that the combination treatment significantly induced apoptosis in A549 cells compared to roscovitine or IR treatment alone. Meanwhile, in the co-treatment group, the percentage of cells with the active form of caspase-3 was markedly increased, while roscovitine or IR alone had little effect. Roscovitine decreased S phase cells when used alone or in sequential combination with IR. Furthermore, this combination treatment blocked DNA repair process after IR, indicated by down regulation of Ku70 and Ku80 proteins, while the singly used treatment did not. Taken together, these results suggest that roscovitine has the potential to act as a radio-sensitizer in A549 cells by promoting caspase-3 activity and increasing apoptosis, affecting cell cycle distribution and impairing DNA repair process.

The DNA-dependent protein kinase plays a critical role in mammalian DNA double strand break (DSB) repair and in specialized recombination, such as lymphoid V(D)J recombination. Its regulatory subunit Ku (dimer of the Ku70 and Ku80 protein) binds to DNA and recruits the kinase catalytic sub-unit, DNA-PKcs. We show here that three different strains deficient in either the Ku80 (xrs-6) or DNA-PKcs (V-3, scid) component of DNA-PK are markedly sensitive (3.5- to 5-fold) to a group of DNA cross-linking agents, the nitrogen mustards (NMs) (melphalan and mechlorethamine) as compared to their parental cell line. Importantly, the level of hypersensitivity to these drugs was close to the level of hypersensitivity observed for radiomimetic agents that create DSBs in DNA (bleomycin and neocarzinostatin). In addition, sensitivity to NMs was restored to the parental level in the xrs-6 cell line stably transfected with the human Ku80 gene (xrs-6/Ku80), showing unequivocally that DNA-PK is involved in this phenotype. These results indicate that a function of the whole DNA-PK protein complex is involved in the cellular response to NMs and suggest that the repair of DNA interstrand cross-links induced in DNA by NMs involved a DNA-PK dependent pathway that shares common features with DNA DSBs repair.

Mantle cell lymphomas (MCL) are characterized by their aggressive behavior and poor response to chemotherapy regimens. We report here evidence of increased in vitro radiation sensitivity in two cell lines that we have generated from two MCL patients (UPN1 and UPN2). However, despite their increased radiation sensitivity, UPN2 cells were totally resistant to apoptotic cell death, whereas UPN1 cells underwent massive apoptosis 6 h after irradiation. The frequency of induced chromosomal abnormalities was higher in UPN1 as compared to UPN2. Distinct mechanisms have been found to contribute to this phenotype: a major telomere shortening (UPN1 and UPN2), deletion of one ATM allele and a point mutation in the remaining allele in UPN2, mutation of p53 gene (UPN1 and UPN2) with absence of functional p53 as revealed by functional yeast assays. After irradiation, Ku70 levels in UPN1 increased and decreased in UPN2, whereas in the same conditions, DNA-PKcs protein levels decreased in UPN1 and remained unchanged in UPN2. Thus, irradiation-induced apoptotic cell death can occur despite the nonfunctional status of p53 (UPN1), suggesting activation of a unique pathway in MCL cells for the induction of this event. Overall, our study demonstrates that MCL cells show increased radiation sensitivity, which can be the result of distinct molecular events. These findings could clinically be exploited to increase the dismal response rates of MCL patients to the current chemotherapy regimens.

Functional inactivation of the p53 gene and robust DNA repair capacity may be among the salient causes of radioresistance in tumor cells. We expressed the wild-type (wt) p53 gene in a p53-mutant human epidermoid carcinoma cell line, A431, using an adenoviral vector [adenovirus-p53 (Ad-p53), INGN 201], examined its radiosensitivity, and correlated p53 status and radiosensitivity with cellular repair functions. Using clonogenic survival assays and the terminal deoxynucleotidyl transferase-mediated nick end labeling assay for apoptosis, we demonstrated that preirradiation treatment with Ad-p53 significantly increased the radiosensitivity of A431 cells over controls. Induction of p53 expression using a construct where p53 expression was under the control of an inducible promoter also significantly increased radiosensitivity of H1299 lung tumor cells, which are otherwise null for p53. These results did not correlate with radiation-induced apoptosis but did correlate with functional impairment of DNA repair and suppressed expression of several repair-related genes, such as Ku70, DNA-dependent protein kinase, ataxia telangiectasia mutated, and X-ray-sensitive complementation group 4. Normal human fibroblast MRC-9 cells showed no impairment in the repair capability due to Ad-p53 despite the suppression of some repair genes. Expression of Ku70, which is known to mediate diverse cellular functions, correlated with the differential effects of p53 on radiosensitivity in the normal and tumor cells.

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.

DNA-dependent protein kinase (DNA-PK) consists of a 460 kDa subunit that contains the catalytic domain (DNA-PKcs) complexed with two polypeptides of 70 kDa and 80 kDa (Ku70 and Ku80) which comprise the Ku autoantigen. DNA-PKcs requires association with DNA via Ku for catalytic activation and is implicated in double strand break repair, V(D)J recombination and transcription. We have utilised a cell-free system of concentrated Xenopus laevis egg extracts to investigate the regulation and possible functions of DNA-PK. Recently, we have shown that this system can reproduce events of apoptosis, including activation of an apoptotic protease that cleaves poly(ADP-ribose) polymerase. Here, we report that DNA-PK is rapidly inactivated with the onset of apoptosis in this system. Loss of activity is concomitant with cleavage of the catalytic subunit, whereas the Ku subunits are stable. Cleavage and inactivation of DNA-PKcs is prevented by prior addition of the anti-apoptotic protein Bcl-2 or inhibition of an apoptotic protease that has characteristics of the CPP-32/Ced-3 family of cysteine proteases that cleave poly(ADP-ribose) polymerase. These results suggest that cleavage and inactivation of DNA-PKcs prevents this factor from functioning in DNA repair, recombination or transcriptional regulation during apoptosis.

Oxidative stress caused by reactive oxygen species (ROS) induces DNA base modifications and DNA strand breaks. In this study, the protective effect of baicalein against H(2)O(2)-induced DNA damage was investigated in V79-4 Chinese hamster fibroblast cells. H(2)O(2) treatment increased the levels of intracellular ROS and DNA double-strand breaks (DSBs) and decreased the level of Ku70 protein and the phosphorylation (activation) of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which are involved in the repair of DSBs by nonhomologous end joining. Baicalein effectively scavenged intracellular ROS induced by H(2)O(2), reduced DSBs, and rescued Ku70 protein level and phosphorylation of DNA-PKcs. In cellular response to DNA base damage, 8-oxoguanine DNA glycosylase 1 (OGG1) plays a vital role in the removal of 8-oxoguanine (8-OxoG), which is formed mainly by oxidative stress. Baicalein significantly decreased the levels of 8-OxoG induced by H(2)O(2), and this correlated with increases in OGG1 promoter activity and OGG1 mRNA and protein expression. The phosphorylated form of Akt kinase, which is a regulator of OGG1, was sharply decreased by H(2)O(2), but was prevented by baicalein. A specific Akt inhibitor abolished the cytoprotective effects of baicalein, suggesting that OGG1 induction by baicalein involves the Akt pathway. In conclusion, baicalein exerted protective effects against DNA damage induced by oxidative stress by activating DNA repair systems and scavenging ROS.