DNA Repair Protein Ku70 Research Articles

Conjugation of sulpiride with a cell penetrating peptide to augment the antidepressant efficacy and reduce serum prolactin levels

Depression ranks as the fourth most prevalent global disease, with suicide incidents occurring at a younger age. Sulpiride (SUL), an atypical antidepressant drug acting as a dopamine D2 receptor antagonist and possessing anti-inflammatory properties, exhibits limited ability to penetrate the blood brain barrier (BBB). This weak penetration hampers its inhibitory effect on prolactin release in the pituitary gland, consequently leading to hyperprolactinemia. In order to enhance the central nervous system efficacy of sulpiride and reduce serum prolactin levels, we covalently linked sulpiride to VPALR derived from the nuclear DNA repair protein ku70. In vivo study on depressive mice using intraperitoneal injection of VPALR-SUL demonstrated a significant increase in struggle time and total distance compared to those treated with only sulpiride while also reducing serum prolactin concentration. The pharmacokinetic study results showed that VPALR-SUL prolonged half-life and increased bioavailability. In conclusion, VPALR-SUL exhibited potential for enhancing sulpiride transport across the BBB, augmenting its antidepressant effects, and reducing serum prolactin levels. This study laid a foundation for improving sulpiride delivery and developing novel antidepressants.

Ku70 senses cytosolic DNA and assembles a tumor-suppressive signalosome.

The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5+Rab7+ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.

MTOR Inhibitor Rapalink-1 Prevents Ethanol-Induced Senescence in Endothelial Cells.

The cardiovascular risk factors, including smoking, ethanol, and oxidative stress, can induce cellular senescence. The senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase (MMPs). These pro-inflammatory molecules and MMPs promote the infiltration and accumulation of inflammatory cells in the vascular tissue, exacerbating vascular tissue inflammation. MMPs damage vascular tissue by degenerating the extracellular matrix. Consequently, these cellular and molecular events promote the initiation and progression of cardiovascular diseases. We used Rapalink-1, an mTOR inhibitor, to block ethanol-induced senescence. Rapalink-1 inhibited oxidative-stress-induced DNA damage and senescence in endothelial cells exposed to ethanol. It attenuated the relative protein expression of senescence marker P21 and improved the relative protein expression of DNA repair protein KU70 and aging marker Lamin B1. It inhibited the activation of NF-κB, MAPKs (P38 and ERK), and mTOR pathway proteins (mTOR, 4EBP-1, and S6). Moreover, Rapalink-1 suppressed ethanol-induced mRNA expression of ICAM-1, E-selectin, MCP-1, IL-8, MMP-2, and TIMP-2. Rapalink-1 also reduced the relative protein expression of MMP-2. In summary, Rapalink-1 prevented senescence, inhibited pro-inflammatory pathway activation, and ameliorated pro-inflammatory molecule expression and MMP-2.

The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

The cellular response to environmental stresses, such as heat and oxidative stress, is dependent on extensive trafficking of stress-signalling molecules between the cytoplasm and nucleus, which potentiates stress-activated signalling pathways, eventually resulting in cell repair or death. Although Ran-dependent nucleocytoplasmic transport mediated by members of the importin (IPO) super family of nuclear transporters is believed to be responsible for nearly all macromolecular transit between nucleus and cytoplasm, it is paradoxically known to be significantly impaired under conditions of stress. Importin 13 (IPO13) is a unique bidirectional transporter that binds to and releases cargo in a Ran-dependent manner, but in some cases, cargo release from IPO13 is affected by loading of another cargo. To investigate IPO13's role in stress-activated pathways, we performed cell-based screens to identify a multitude of binding partners of IPO13 from human brain, lung, and testes. Analysis of the IPO13 interactome intriguingly indicated more than half of the candidate binding partners to be annotated for roles in stress responses; these included the pro-apoptotic protein nuclear clusterin (nCLU), as well as the nCLU-interacting DNA repair protein KU70. Here, we show, for the first time, that unlike other IPOs which are mislocalised and non-functional, IPO13 continues to translocate between the nucleus and cytoplasm under stress, retaining the capacity to import certain cargoes, such as nCLU, but not export others, such as KU70, as shown by analysis using fluorescence recovery after photobleaching. Importantly, depletion of IPO13 reduces stress-induced import of nCLU and protects against stress-induced cell death, with concomitant protection from DNA damage during stress. Overexpression/FACS experiments demonstrate that nCLU is dependent on IPO13 to trigger stress-induced cell death via apoptosis. Taken together, these results implicate IPO13 as a novel functional nuclear transporter in cellular stress, with a key role thereby in cell fate decision.

Sensitization of cervical cancer cells to radiation by the cyclin-dependent kinase inhibitor dinaciclib.

Dinaciclib is a selective cyclin-dependent kinase inhibitor, but its radiosensitizing effect remains unclear. The aim of this study is to investigate the radiosensitizing effect of Dinaciclib on cervical cancer cells. Two cervical cancer cell lines, Hela and Siha, were selected, and the IC50 was determined by CCK8. The radiosensitizing effect of Dinaciclib was verified by plate cloning assay, and the G2/M phase arrest and apoptosis of IR cells were verified by flow cytometry. Immunofluorescence assay was used to verify the formation of γH2AX foci following DNA damage. Western blot was performed to detect cell cycle, apoptosis, autophagy, and DNA damage-related pathways. Dinaciclib increased the cell sensitivity to IR. IR induced G2/M phase arrest and apoptosis, and Dinaciclib enhanced this effect. Further, Dinaciclib delayed DNA repair, including non-homologous end joining repair and homologous recombination repair, and reduced the expression of DNA repair proteins Ku80 (SiHa cells), Ku70, and RAD51, as well as the expression of apoptotic marker Bcl-2. The expression of autophagy marker Beclin1 induced tumor cell death and increased the formation of DNA damage marker γH2AX foci. Dinaciclib improves the sensitivity of cervical cancer cells to IR by inducing cell cycle arrest, delaying DNA repair, and increasing apoptosis. However, further research is needed to unravel the complexity of DNA repair pathways.

Doxycycline Attenuated Ethanol-Induced Inflammaging in Endothelial Cells: Implications in Alcohol-Mediated Vascular Diseases.

Excess alcohol consumption is a potential risk factor for cardiovascular diseases and is linked to accelerated aging. Drug discovery to reduce toxic cellular events of alcohol is required. Here, we investigated the effects of ethanol on human umbilical vein endothelial cells (HUVECs) and explored if doxycycline attenuates ethanol-mediated molecular events in endothelial cells. Initially, a drug screening using a panel of 170 drugs was performed, and doxycycline was selected for further experiments. HUVECs were treated with different concentrations (300 mM and 400 mM) of ethanol with or without doxycycline (10 µg/mL). Telomere length was quantified as telomere to single-copy gene (T/S) ratio. Telomere length and the mRNA expression were quantified by qRT-PCR, and protein level was analyzed by Western blot (WB). Ethanol treatment accelerated cellular aging, and doxycycline treatment recovered telomere length. Pathway analysis showed that doxycycline inhibited mTOR and NFκ-B activation. Doxycycline restored the expression of aging-associated proteins, including lamin b1 and DNA repair proteins KU70 and KU80. Doxycycline reduced senescence and senescence-associated secretory phenotype (SASP) in ethanol-treated HUVECs. In conclusion, we report that ethanol-induced inflammation and aging in HUVECs were ameliorated by doxycycline.

Platinum complexes as inhibitors of DNA repair protein Ku70 and topoisomerase IIα in cancer cells.

Ku70 protein and topoisomerase IIα (Topo IIα) are promising targets of anticancer drugs, which play critical roles in DNA repair and replication processes. Three platinum(II) complexes, [PtCl(NH3)2(9-(pyridin-2-ylmethyl)-9H-carbazole)]NO3 (OPPC), [PtCl(NH3)2(9-(pyridin-3-ylmethyl)-9H-carbazole)]NO3 (MPPC), and [PtCl(NH3)2(9-(pyridin-4-ylmethyl)-9H-carbazole)]NO3 (PPPC), were designed as inhibitors of Ku70 and Topo IIα. Their antitumor activity and inhibitory efficacy on Ku70 and Topo IIα were investigated on cellular and molecular levels. OPPC exhibited high antiproliferative activity against various cancer cell lines, with acute toxicity to mice being lower than that of cisplatin. Moreover, OPPC could enter cancer cells effectively and cause DNA damage, which was evidenced by the enhanced expression of γ-H2AX, Chk1/2 phosphorylation, p53 and cell cycle arrest. OPPC also downregulated the DNA damage repair protein Ku70 and inhibited the formation of Ku70 foci-the central points or loci of Ku70, which would suppress DNA repair and induce a nonhomologous end joining response in cancer cells. More importantly, these complexes showed inhibition towards Topo IIα; in particular, OPPC was more effective than MPPC and PPPC. In the Topo IIα knockdown cells, Ku70 and Topo IIα were directly associated with the DNA damage and apoptotic response. The molecular docking provided detailed structural insights into the interactions of the complexes with Topo IIα. This study demonstrates that the cytotoxicity of these complexes is associated with the DNA damage and repair pathways mediated by Ku70 and Topo IIα; OPPC is an effective inhibitor of Ku70 and Topo IIα and restrains cancer cells via a mechanism utterly distinct from that of cisplatin.

Inhibition of DNA Repair Protein Ku70 in High-Glucose Environment Aggravates the Neurotoxicity Induced by Bupivacaine in SH-SY5Y Cells.

Bupivacaine, a common local anesthetic, causes serious nerve injury, especially in diabetic patients, as high glucose has been reported to enhance bupivacaine-induced neurotoxicity. However, the key regulator for synergism remains unknown. To our surprise, the expression of repair protein Ku70 is suppressed, while the high-glucose environment induces DNA oxidative damage in neurons. Here, we aim to investigate whether the inhibition of Ku70 by high-glucose conditions aggrandized bupivacaine-induced DNA damage. Consistent with previous results, bupivacaine induced reactive oxygen species production and upregulated Ku70 and cleaved caspase-3 expressions at both transcript and protein levels and ultimately caused nucleic acid damage and apoptosis in human neuroblastoma (SH-SY5Y) cells. High-glucose treatment inhibited the expression of Ku70 and enhanced bupivacaine-induced neurotoxicity. In contrast, the overexpression of Ku70 mitigated DNA damage and apoptosis triggered by bupivacaine and high glucose. In conclusion, our data indicated that local anesthetics may aggravate nerve toxicity in a high-glucose environment.

Inhibition of Ku70 in a high-glucose environment aggravates bupivacaine-induced dorsal root ganglion neurotoxicity

BackgroundBupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression. MethodsWe examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50 mM glucose, and the cells were then treated with BP for 3 h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting. ResultsCompared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions. ConclusionThese data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70.

Abstract 1567: Ku70, Ku80 and Rab5C as biomarkers for radiotherapy resistance in rectal tumors during neoadjuvant therapy

Abstract Rectal cancer is the second most common cancer in the large intestine, corresponding to one-third of the total cases of colorectal cancer. The neoadjuvant chemoradiation in rectal cancer treatment reduces the risk of local recurrence, but pathologic complete response is achieved only in 10-40% of patients. The response to neoadjuvant treatment is poorly understated in rectal tumors as well as the mechanisms associated with chemoradiation resistance. In order to identify the players involved with the therapy resistance, we performed proteomic analysis of biopsies from stage II and III rectal adenocarcinoma patients before neoadjuvant treatment and compared with residual tumor tissue removed by surgery after neoadjuvant therapy. Ku70, Ku80 and Rab5C showed a significant increased expression after the chemoradiation treatment. To better understand the mechanism associated with therapy resistance, we generated an ionizing radiation-resistant rectal adenocarcinoma cell line. These cells and also their secreted exosomes exhibit overexpression of the same three proteins identified in tissue samples from patients submitted to chemoradiation. Using this in vitro model, we identified that Rab5C modulates EGFR internalization promoted by irradiation, which was also essential to regulate the expression of the DNA repair proteins Ku70 and Ku80, and cell resistance to ionizing radiation. Taken together, these findings indicate that EGFR and Rab5C are potential targets to sensitize rectal cancer cells to neoadjuvant treatment. In addition, the presence of Ku70, Ku80 and Rab5C in circulating exosomes can be explored as biomarkers to follow resistance to radiotherapy in patients under neoadjunat therapy for rectal tumors. Citation Format: Vilma Regina Martins, Antuani Rafael Baptistella, Petrus Paulo Silva, Edson Kuatelela Cassinela, Fabio Marchi, Adriana Fp Leme, Maria Dirlei Begnami, Samuel Aguiar. Ku70, Ku80 and Rab5C as biomarkers for radiotherapy resistance in rectal tumors during neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1567.

Systems analysis of the prostate tumor suppressor NKX3.1 supports roles in DNA repair and luminal cell differentiation.

NKX3.1 is a homeobox transcription factor whose function as a prostate tumor suppressor remains insufficiently understood because neither the transcriptional program governed by NKX3.1, nor its interacting proteins have been fully revealed. Using affinity purification and mass spectrometry, we have established an extensive NKX3.1 interactome which contains the DNA repair proteins Ku70, Ku80, and PARP, thus providing a molecular underpinning to previous reports implicating NKX3.1 in DNA repair. Transcriptomic profiling of NKX3.1-negative prostate epithelial cells acutely expressing NKX3.1 revealed a rapid and complex response that is a near mirror image of the gene expression signature of human prostatic intraepithelial neoplasia (PIN). Pathway and network analyses suggested that NKX3.1 actuates a cellular reprogramming toward luminal cell differentiation characterized by suppression of pro-oncogenic c-MYC and interferon-STAT signaling and activation of tumor suppressor pathways. Consistently, ectopic expression of NKX3.1 conferred a growth arrest depending on TNFα and JNK signaling. We propose that the tumor suppressor function of NKX3.1 entails a transcriptional program that maintains the differentiation state of secretory luminal cells and that disruption of NKX3.1 contributes to prostate tumorigenesis by permitting luminal cell de-differentiation potentially augmented by defects in DNA repair.

Systems analysis of the prostate tumor suppressor NKX3.1 supports roles in DNA repair and luminal cell differentiation

NKX3.1 is a homeobox transcription factor whose function as a prostate tumor suppressor remains insufficiently understood because neither the transcriptional program governed by NKX3.1, nor its interacting proteins have been fully revealed. Using affinity purification and mass spectrometry, we have established an extensive NKX3.1 interactome which contains the DNA repair proteins Ku70, Ku80, and PARP, thus providing a molecular underpinning to previous reports implicating NKX3.1 in DNA repair. Transcriptomic profiling of NKX3.1-negative prostate epithelial cells acutely expressing NKX3.1 revealed a rapid and complex response that is a near mirror image of the gene expression signature of human prostatic intraepithelial neoplasia (PIN). Pathway and network analyses suggested that NKX3.1 actuates a cellular reprogramming toward luminal cell differentiation characterized by suppression of pro-oncogenic c-MYC and interferon-STAT signaling and activation of tumor suppressor pathways. Consistently, ectopic expression of NKX3.1 conferred a growth arrest depending on TNFα and JNK signaling. We propose that the tumor suppressor function of NKX3.1 entails a transcriptional program that maintains the differentiation state of secretory luminal cells and that disruption of NKX3.1 contributes to prostate tumorigenesis by permitting luminal cell de-differentiation potentially augmented by defects in DNA repair.

Systems analysis of the prostate tumor suppressor NKX3.1 supports roles in DNA repair and luminal cell differentiation.

NKX3.1 is a homeobox transcription factor whose function as a prostate tumor suppressor remains insufficiently understood because neither the transcriptional program governed by NKX3.1, nor its interacting proteins have been fully revealed. Using affinity purification and mass spectrometry, we have established an extensive NKX3.1 interactome which contains the DNA repair proteins Ku70, Ku80, and PARP, thus providing a molecular underpinning to previous reports implicating NKX3.1 in DNA repair. Transcriptomic profiling of NKX3.1-negative prostate epithelial cells acutely expressing NKX3.1 revealed a rapid and complex response that is a near mirror image of the gene expression signature of human prostatic intraepithelial neoplasia (PIN). Pathway and network analyses suggested that NKX3.1 actuates a cellular reprogramming toward luminal cell differentiation characterized by suppression of pro-oncogenic c-MYC and interferon-STAT signaling and activation of tumor suppressor pathways. Consistently, ectopic expression of NKX3.1 conferred a growth arrest depending on TNFα and JNK signaling. We propose that the tumor suppressor function of NKX3.1 entails a transcriptional program that maintains the differentiation state of secretory luminal cells and that disruption of NKX3.1 contributes to prostate tumorigenesis by permitting luminal cell de-differentiation potentially augmented by defects in DNA repair.

P53 inhibition exacerbates late-stage anthracycline cardiotoxicity.

Doxorubicin (DOX) is an effective anti-cancer therapeutic, but is associated with both acute and late-stage cardiotoxicity. Children are particularly sensitive to DOX-induced heart failure. Here, the impact of p53 inhibition on acute vs. late-stage DOX cardiotoxicity was examined in a juvenile model. Two-week-old MHC-CB7 mice (which express dominant-interfering p53 in cardiomyocytes) and their non-transgenic (NON-TXG) littermates received weekly DOX injections for 5 weeks (25 mg/kg cumulative dose). One week after the last DOX treatment (acute stage), MHC-CB7 mice exhibited improved cardiac function and lower levels of cardiomyocyte apoptosis when compared with the NON-TXG mice. Surprisingly, by 13 weeks following the last DOX treatment (late stage), MHC-CB7 exhibited a progressive decrease in cardiac function and higher rates of cardiomyocyte apoptosis when compared with NON-TXG mice. p53 inhibition blocked transient DOX-induced STAT3 activation in MHC-CB7 mice, which was associated with enhanced induction of the DNA repair proteins Ku70 and Ku80. Mice with cardiomyocyte-restricted deletion of STAT3 exhibited worse cardiac function, higher levels of cardiomyocyte apoptosis, and a greater induction of Ku70 and Ku80 in response to DOX treatment during the acute stage when compared with control animals. These data support a model wherein a p53-dependent cardioprotective pathway, mediated via STAT3 activation, mitigates DOX-induced myocardial stress during drug delivery. Furthermore, these data suggest an explanation as to how p53 inhibition can result in cardioprotection during drug treatment and, paradoxically, enhanced cardiotoxicity long after the cessation of drug treatment.

MicroRNA-124 (miR-124) Regulates Ku70 Expression and is Correlated with Neuronal Death Induced by Ischemia/Reperfusion

MicroRNAs are small, non-coding RNA molecules that regulate gene expression, and miR-124 is the most abundant miRNA in the brain. Studies have shown that miR-124 is clearly reduced in the ischemic brain after stroke; however, the role of miR-124 after stroke is less well studied. Using TargetScan, MicroCosm Targets version 5, and microRNA.org databases, we identified miR-124 as a possible regulator of the DNA repair protein Ku70. We validated that Ku70 is a target for miR-124 with a luciferase reporter activity assay. Moreover, adult rats subjected to focal cerebral ischemia exhibited a substantial reduction of miR-124 expression, which was inversely upregulated by Ku70 expression. In vivo treatment with miR-124 antagomir effectively enhanced Ku70 mRNA and protein levels in the ischemic region. Furthermore, knockdown of cerebral miR-124 reduced cell death and infarct size and improved neurological outcomes. Our data demonstrate that miR-124 is an endogenous regulator of Ku70 that improves ischemia/reperfusion (I/R)-induced brain injury and dysfunction.

Inhibiting UHRF1 expression enhances radiosensitivity in human esophageal squamous cell carcinoma

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) is a novel nuclear protein which is overexpressed in various cancers but not yet examined in esophageal squamous cell carcinoma (ESCC). The correlation between UHRF1 and the radioresistance in ESCC is still unclear. In the present study, the expression of UHRF1 was examined by immunohistochemistry in specimens of ESCC patients treated with radiotherapy. The results showed that UHRF1 was significantly overexpressed in ESCC specimens. Overexpression of UHRF1 correlated significantly with advanced T-stage, positive lymph node metastasis and poor differentiation. In addition, UHRF1 was associated with radiotherapy response, in which overexpression of UHRF1 was observed more frequently in the radioresistant group than in the effective group. At the molecular level, inhibition of UHRF1 by lentivirus-mediated shRNA targeting UHRF1 increased the radiosensitivity and apoptosis, while decreased radiation-induced G2/M phase arrest in TE-1 cells. Moreover, inhibition of UHRF1 resulted in higher residual γH2AX expression after irradiation, but not initial γH2AX. Further study showed that inhibition of UHRF1 down-regulated the endogenous expressions of DNA repair protein Ku70 and Ku80 in TE-1 cells, and significantly inhibited the increase of these proteins after irradiation. Above all, our data suggested that UHRF1 might play an important role in radioresistance of ESCC, and inhibition of UHRF1 can increase the radiosensitivity of TE-1 cells by altering cell cycle progression, enhancing apoptosis, and decreasing DNA damage repair capacity.

ABH2 Couples Regulation of Ribosomal DNA Transcription with DNA Alkylation Repair

Transcription has been linked to DNA damage. How the most highly transcribed mammalian ribosomal (rDNA) genes maintain genome integrity in the absence of transcription-coupled DNA damage repair is poorly understood. Here, we report that ABH2/ALKBH2, a DNA alkylation repair enzyme, is highly enriched in the nucleolus. ABH2 interacts with DNA repair proteins Ku70 and Ku80 as well as nucleolar proteins nucleolin, nucleophosmin 1, and upstream binding factor (UBF). ABH2 associates with and promotes rDNA transcription through its DNA repair activity. ABH2 knockdown impairs rDNA transcription and leads to increased single-stranded and double-stranded DNA breaks that are more pronounced in the rDNA genes, whereas ABH2 overexpression protects cells from methyl-methanesulfonate-induced DNA damage and inhibition of rDNA transcription. In response to massive alkylation damage, ABH2 rapidly redistributes from the nucleolus to nucleoplasm. Our study thus reveals a critical role of ABH2 in maintaining rDNA gene integrity and transcription and provides insight into the ABH2 DNA repair function.

Toll-like receptor 4 activity protects against hepatocellular tumorigenesis and progression by regulating expression of DNA repair protein Ku70 in mice

Hepatocellular carcinoma (HCC) is a devastating consequence of chronic inflammatory liver diseases. The goal of this study was to investigate whether Toll-like receptor 4 (TLR4) activity contributes to HCC initiation and progression in mice. A mouse model of diethylnitrosamine (DEN)-induced HCC was generated with wild-type and TLR4 mutant mice, and the development and progression of HCC and senescent responses were assessed using morphologic, immunological, and biochemical criteria. We found that genetic or pharmacologic blocking of TLR4 increased susceptibility to DEN-induced HCC carcinogenesis and progression, which was indicated by increases in number of tumor nodules, tumor volume, and animal death. The enhanced HCC was associated with a broad-spectrum reduction of immune response to DEN liver injury, as indicated by decreases in the liver-infiltrating F4/80+ macrophages, the apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase/NF-κB and IRF3 signaling activities, and the expression of inflammatory cytokines. Suppressed immune networks resulted in a halt of cellular senescence induction in TLR4 mutant liver tissue, which promoted proliferation and suppressed programmed cell death. Moreover, TLR4 mutation resulted in a suppressed capacity of DNA repair due to a decrease in TLR4-medicated expression of DNA repair proteins Ku70/80 in liver tissue and cells. Isotopic expression of Ku70 in TLR4 mutant mice restored senescence and interrupted the positive feedback loop of DNA damage and oxidative stress, which reversed TLR4 mutation-deteriorated HCC carcinogenesis and progression. TLR4 plays an integrated defense role against HCC carcinogenesis by enhancing the expression and function of DNA repair protein Ku70. Our studies provide novel insight into TLR4 activity in the regulation of HCC tumorigenesis, which may be useful for the prevention of HCC development.

Molecular characterization of DNA repair protein Ku70 from Vitis vinifera and its purification from transgenic tobacco

The DNA double strand break repair in plants is preferentially by non homologous end joining (NHEJ) pathway. A key protein of NHEJ pathway is Ku70. We have identified Ku70 homolog (VvKu70) from grapevine genome database. In this report we characterize a Ku70 homologue from Vitis vinifera cv. Mango. The VvKu70 expression was found to increase strongly in response to gamma radiation. The transcript level of VvKu70 was found to increase up to 36h in gamma irradiated shoots of grapevine. The expression of VvKu70 was found in many organs like stem, leaves and roots. A GFP fused VvKu70 protein was found to be nuclear localized which indicates that the VvKu70 is a nuclear localized protein. The VvKu70 identified by in silico approaches is present as a single copy number in V. vinifera cv. Mango genome. The VvKu70-GFP fused protein possesses ATPase activity and fails to bind dsDNA but binds ssDNA.

The Defect of Ku70 Affects Sensitivity to X-Ray and Radiation-Induced Caspase-Dependent Apoptosis in Lung Cells

The DNA repair protein Ku70 is a key player in chemoresistance to anticancer agents (e.g., etoposide) or radioresistance. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. Previously, we established and characterized Ku70-deficient murine lung epithelial (Ku70 -/- MLE) cells and found that these cells are more sensitive than Ku70 +/- MLE cells (control cells) to X-irradiation, as determined by clonogenic survival assay; however, the mechanism underlying this sensitivity remains unclear. In this study, we examined the mechanism by which X-irradiation triggers the death of Ku70 -/- MLE cells. Our results showed that Ku70 -/- MLE cells were more sensitive to radiation-induced apoptosis than control cells, although X-irradiation activated caspase-3 and caspase-7, and cleaved PARP in both cell lines. We also examined the expression level of phosphorylated H2AX (γH2AX), which is a marker of DSB, and observed the phosphorylation of H2AX and the elimination of γH2AX in both cell lines after X-irradiation. The elimination in Ku70 -/- MLE cells was slower than that in control cells, suggesting that DSB repair activity in the Ku70 -/- MLE cells is lower than that in control cells. These findings suggest that Ku70 might play a key role in the inhibition of apoptosis through the DSB repair pathway in lung epithelial cells. Our findings also suggest that these cell lines might be useful for the study of Ku70 functions and the Ku70-dependent DSB repair pathway in lung epithelial cells.