Nuclear domain 10 components upregulated via interferon during human cytomegalovirus infection potently regulate viral infection.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that causes life-threatening disease in immunocompromised and immunonaïve individuals. Type I interferons (IFNs) are crucial molecules in the innate immune response to HCMV and are also known to upregulate several components of the interchromosomal multiprotein aggregates collectively referred to as nuclear domain 10 (ND10). In the context of herpesvirus infection, ND10 components are known to restrict gene expression. This raises the question as to whether key ND10 components (PML, Sp100 and hDaxx) act as anti-viral IFN-stimulated genes (ISGs) during HCMV infection. In this study, analysis of ND10 component transcription during HCMV infection demonstrated that PML and Sp100 were significantly upregulated whilst hDaxx expression remained unchanged. In cells engineered to block the production of, or response to, type I IFNs, upregulation of PML and Sp100 was not detected during HCMV infection. Furthermore, pre-treatment with an IFN-ß neutralizing antibody inhibited upregulation of PML and Sp100 during both infection and treatment with HCMV-infected cell supernatant. The significance of ND10 components functioning as anti-viral ISGs during HCMV infection was determined through knockdown of PML, Sp100 and hDaxx. ND10 knockdown cells were significantly more permissive to HCMV infection, as previously described but, in contrast to control cells, could support HCMV plaque formation following IFN-ß pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-ß in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-ß.

Human limbal neurospheres prevent photoreceptor cell death in a rat model of retinal degeneration.

BACKGROUND: The culture of retinal progenitors from an accessible adult stem cell source such as the limbus could provide a useful autologous source of retinal cell therapies. The human corneoscleral limbus contains multipotent stem cells that can be cultured as floating neurospheres. Previous work in rodents has demonstrated neuronal and photoreceptor differentiation from limbal neurosphere cultures. Here, this study has examined undifferentiated cultured adult human limbal neurospheres as donor cells for retinal cell therapies by transplantation into a rat model of retinal degeneration. METHODS: Gene expression in limbal neurospheres was examined by immunostaining and western blot. Human limbal neurospheres were transplanted into the subretinal space of Royal College of Surgeon's rats. Rats were monitored by optical coherence tomography for 6 weeks then processed for retinal histology. RESULTS: Human limbal neurospheres expressed the neural lineage markers, Nestin, sex determining region box-2 and N-cadherin, and the retinal transcription factors microphthalmia-associated transcription factor, sex determining region box-2 and orthodentical homeobox-2. Human limbal neurospheres could be cultured to express NeuN, neurofilament and rhodopsin. Rats receiving saline or no injection underwent complete degeneration of the retinal outer nuclear layer after 3 weeks. In contrast, rats injected with human limbal neurospheres or retinal pigment epithelial cells maintained the outer nuclear layer for up to 6 weeks. Gene expression in transplanted limbal neurospheres was inconsistent with the production of mature retinal pigment epithelial or photoreceptor cells. CONCLUSIONS: Human limbal neurospheres represent an accessible source of autologous donor cells for the treatment of retinal diseases.

The detection of curcumins' antitumoral effects via argyrophilic nucleolar organizing region-associated protein synthesis in mice with ehrlich's ascitic carcinoma.

BACKGROUND: Curcumin is a polyphenol compound that has antioxidant, anticancer, anti-inflammatory, anti-hyperlipidemic and antimicrobial effects. Nucleolar-organizing regions are the sites of the gene on chromosomes. The present study was aimed to show the antitumoral effect of curcumin via AgNOR protein synthesis in Ehrlich's ascitic carcinoma (EAC) bearing mice. METHODS: Twenty three mice with EAC were randomly divided into 3 groups as positive control (n = 7), group 2 (n = 8) and 3 (n = 8) treated intraperitoneally with curcumin (25 mg/kg) and (50 mg/kg), respectively. The animals were sacrificed on Day 16, the solid tumors were removed out. Then, total AgNOR area/nuclear area (TAA/NA) and the mean AgNOR number were estimated for each mice. RESULT: Statistically significant differences were determined among the whole groups for TAA/NA ratio (p = 0.000), conversely mean AgNOR number (p = 0.361). When comparingthe two groups; while no difference was determined between the control and curcumin (25 mg/kg) groups (p = 0.061), the significant differences were detected between the control and curcumin (50 mg/kg) groups (p = 0.000) and between curcumin (25 mg/kg) and curcumin (50 mg/kg) groups (p = 0.000) for TAA/NA ratio. However, there was no significant difference for the mean AgNOR number in double comparison of the groups. CONCLUSIONS: The current study showed that curcumin had a crucial function against cancer development. Also, both AgNOR values might be used as biomarkers for detection of the most reliable therapeutic dose selection of cancer treatment (Tab. 3, Fig. 2, Ref. 27).

Complete loss of STAG2 expression is an indicator of good prognosis in patients with bladder cancer.

Stromal antigen 2 (STAG2) is an important member of cohesin, a conserved complex holding the sister chromatid together. Recent whole-genome sequencing studies have identified that genetic alterations of stag2 are common in bladder cancer (BC). The prognostic implications of STAG2 expression in BC remain unclear; we therefore analyzed its associations with the histopathological characteristics and clinical outcome in a Chinese population. We used immunohistochemistry assay to determine STAG2 protein expression in tumor tissues from 125 BC patients. STAG2 expression was analyzed according to clinicopathological features and patients' survival. Univariable and multivariable analyses were performed to identify predictors for recurrence-free survival (RFS) and cancer-specific survival (CSS). STAG2 expression was detected in 79.2 % of BC tissues, and 20.8 % of the tumor tissues had a complete loss of STAG2 protein expression. This STAG2-negative result was associated with a lower tumor histological grade with P = 0.009. The log-rank analysis revealed that the complete loss of STAG2 expression was associated with a lower risk of recurrence (P = 0.023) and a diminished risk of death (P = 0.034), especially in the subgroup of muscle-invasive BC (P = 0.043 for RFS and P = 0.087 for CSS). In multivariable Cox regression models, the loss of STAG2 expression remained a beneficial factor for RFS and CSS of BC patients. Univariate and multivariate analyses' results indicated that the complete loss of STAG2 expression was predictive for better RFS and CSS, suggesting its potential value as a prognostic biomarker.

Altered Loyalties of Neuronal Markers in Cultured Slices of Resected Human Brain Tissue.

Organotypic cultures from normal neocortical tissue obtained at epilepsy surgery show a severe injury response. This response involves both neuronal degeneration and the proliferation of reactive cells. A salient feature of the reactive cells is the co-expression of microglial and astrocytic markers. Surprisingly, the reactive cells also began to express neuronal markers Tubulin ßIII and MAP2 adding to the confusion about their origin. Concomitant with their appearance in reactive cells MAP2 and Tubulin ßIII expression disappeared from neurons. While NeuN expression decreased significantly, it did not entirely disappear from many neurons. Moreover, it was not observed in reactive cells, showing that NeuN is a reliable marker of neurons.

Targeting of ß1 integrins impairs DNA repair for radiosensitization of head and neck cancer cells.

ß1 Integrin-mediated cell-extracellular matrix interactions allow cancer cell survival and confer therapy resistance. It was shown that inhibition of ß1 integrins sensitizes cells to radiotherapy. Here, we examined the impact of ß1 integrin targeting on the repair of radiation-induced DNA double-strand breaks (DSBs). ß1 Integrin inhibition was accomplished using the monoclonal antibody AIIB2 and experiments were performed in three-dimensional cell cultures and tumor xenografts of human head and neck squamous cell carcinoma (HNSCC) cell lines. AIIB2, X-ray irradiation, small interfering RNA-mediated knockdown and Olaparib treatment were performed and residual DSB number, protein and gene expression, non-homologous end joining (NHEJ) activity as well as clonogenic survival were determined. ß1 Integrin targeting impaired repair of radiogenic DSB (γH2AX/53BP1, pDNA-PKcs T2609 foci) in vitro and in vivo and reduced the protein expression of Ku70, Rad50 and Nbs1. Further, we identified Ku70, Ku80 and DNA-PKcs but not poly(ADP-ribose) polymerase (PARP)-1 to reside in the ß1 integrin pathway. Intriguingly, combined inhibition of ß1 integrin and PARP using Olaparib was significantly more effective than either treatment alone in non-irradiated and irradiated HNSCC cells. Here, we support ß1 integrins as potential cancer targets and highlight a regulatory role for ß1 integrins in the repair of radiogenic DNA damage via classical NHEJ. Further, the data suggest combined targeting of ß1 integrin and PARP as promising approach for radiosensitization of HNSCC.

May argyrophilic nucleolar organizing region-associated protein synthesis be used for selecting the most reliable dose of drugs such as rhamnetin in cancer treatments?

BACKGROUND: Rhamnetin is a flavonoid that has antioxidant, anti-inflammatory and anti-cancer effects. Nucleolar-organizing regions are the ribosomal genes region. We aimed to identify whether rhamnetin has an effect on cell proliferation and whether AgNOR proteins may be used for the detection of therapeutic benefits of the drugs and new metabolites, which have the potential of being used for cancer treatments. METHODS: Twenty-four mice with Ehrlich's ascites carcinoma (EAC) were randomly assigned to three main groups as positive control, and groups 2 and 3 treated intraperitoneally with rhamnetin (100 µg/kg and 200 µg/kg, respectively). All the animals were sacrificed on day16, 24 h after the last dose; the tumors, which developed at the site of injection were removed. Then, mean AgNOR number and total AgNOR area/nuclear area (TAA/NA) were detected for each mouse. RESULTS: Significant differences were detected among all groups for mean AgNOR number (p = 0.000) and TAA/NA ratio (p = 0.000). While the difference between positive control and Rhamnetin (100 µg/kg) group was not significant (p = 0.387), there are significant differences between positive control and Rhamnetin (200 µg/kg) group (p = 0.000) and between Rhamnetin (100 µg/kg) and Rhamnetin (200 µg/kg) groups (p = 0.000) for TAA/NA ratio. CONCLUSION: Rhamnetin has an important role in preventing cancer formation. Our study showed that mean AgNOR numbers and TAA/NA values may be used also as biomarkers for evaluating the success rate of the performed therapeutic strategy and accurate dose selection for the management of the disease (Tab. 3, Fig. 3, Ref. 45).

Ku80 Counters Oxidative Stress-Induced DNA Damage and Cataract Formation in the Human Lens.

PURPOSE: Oxidative stress in the human lens leads to a wide range of damage including DNA strand breaks, which are likely to contribute to cataract formation. The protein Ku80 is a fundamental component of the nonhomologous end-joining pathway that repairs DNA double strand breaks. This study investigates the putative impact of Ku80 in cataract prevention in the human lens. METHODS: The present study used the human lens epithelial cell line FHL124 and whole human lens organ culture. Targeted siRNA was used to deplete Ku80, with Western blot and immunocytochemistry employed to assess Ku80 expression levels. Oxidative stress was induced with hydrogen peroxide and DNA strand breaks measured by alkaline comet assay and γH2AX foci counts. Visual quality of whole human lenses was measured with image analysis software. RESULTS: Expression of Ku80 was predominately found in the cell nucleus of both FHL124 cells and native human lens epithelium. Treatment of FHL124 cells and whole lens cultures with siRNA targeted against Ku80 resulted in a significant knockdown at the protein level. Application of oxidative stress (30 µM H2O2) created more DNA strand breaks when added to Ku80 knockdown cells than in scrambled siRNA control cells as determined by the alkaline comet assay and the number of γH2AX foci. In whole lens cultures, exposure to 1 mM H2O2 resulted in more lens opacity in Ku80 knockdown lenses than match-paired controls. CONCLUSIONS: Depletion of Ku80 in the lens through acute change or a consequence of aging is likely to increase levels of DNA strand breaks, which could negatively influence physiological function and promote lens opacity. It is therefore feasible that Ku80 plays a role in retarding cataract formation.

Alpha-Linolenic Acid-Induced Increase in Neurogenesis is a Key Factor in the Improvement in the Passive Avoidance Task After Soman Exposure.

Exposure to organophosphorous (OP) nerve agents such as soman inhibits the critical enzyme acetylcholinesterase (AChE) leading to excessive acetylcholine accumulation in synapses, resulting in cholinergic crisis, status epilepticus and brain damage in survivors. The hippocampus is profoundly damaged after soman exposure leading to long-term memory deficits. We have previously shown that treatment with three sequential doses of alpha-linolenic acid, an essential omega-3 polyunsaturated fatty acid, increases brain plasticity in naïve animals. However, the effects of this dosing schedule administered after a brain insult and the underlying molecular mechanisms in the hippocampus are unknown. We now show that injection of three sequential doses of alpha-linolenic acid after soman exposure increases the endogenous expression of mature BDNF, activates Akt and the mammalian target of rapamycin complex 1 (mTORC1), increases neurogenesis in the subgranular zone of the dentate gyrus, increases retention latency in the passive avoidance task and increases animal survival. In sharp contrast, while soman exposure also increases mature BDNF, this increase did not activate downstream signaling pathways or neurogenesis. Administration of the inhibitor of mTORC1, rapamycin, blocked the alpha-linolenic acid-induced neurogenesis and the enhanced retention latency but did not affect animal survival. Our results suggest that alpha-linolenic acid induces a long-lasting neurorestorative effect that involves activation of mTORC1 possibly via a BDNF-TrkB-mediated mechanism.

Prognostic significance of Ku80 in pT2N0M0 esophageal squamous cell carcinoma after Ivor-Lewis esophagectomy.

Recent studies have shown that Ku80, a DNA repair protein, was involved in progression of malignant tumors. This study aimed to clarify the clinicopathological significance and prognostic value of Ku80 in pT2N0M0 esophageal squamous cell carcinoma (ESCC). We enrolled 217 patients with pT2N0M0 midthoracic ESCC who had undergone Ivor-Lewis esophagectomy. The expression profile of Ku80 was examined by immunohistochemistry. The results were correlated with the clinicopathological variables, overall survival (OS) and disease-free survival (DFS), in pT2N0M0 ESCC patients. The expression of Ku80 were higher in ESCC tissues than the corresponding health esophageal mucosa (P < 0.001). Clinically, the Ku80 expression levels were significantly related to tumor size (P = 0.018), differentiation degree (P = 0.010), and tumor-node-metastasis (TNM) stage (P = 0.001). Subsequent multivariate analysis demonstrated that tumor size, differentiation degree, TNM stage, and Ku80 expression were independent prognostic factors for the OS and the DFS of pT2N0M0 ESCC patients. Our data indicated that Ku80 expression level associates with key clinicopathological features and is an independent predictor of the OS and the DFS in pT2N0M0 ESCC patients.

By downregulating Ku80, hsa-miR-526b suppresses non-small cell lung cancer.

Ku80 is involved in DNA double-strand breaks (DSBs) repair. Ku80 is overexpressed in lung cancer tissues, yet, molecular mechanisms have not been examined. We identified that miRNA, hsa-miR-526b, is bound to the 3'-UTR of Ku80 mRNA, thus decreasing Ku80 expression in NSCLC cells. Hsa-miR-526b was downregulated in NSCLC tissues compared with corresponding non-tumorous tissues, and its expression was inversely correlated with Ku80 upregulation. Overexpression of Ku80 and downregulation of hsa-miR-526b were associated with poor clinical outcomes of NSCLC patients. Hsa-miR-526b suppressed NSCLC cell proliferation, clonogenicity, and induced cell cycle arrest and apoptosis. Hsa-miR-526b inhibited xenografts and orthotopic lung tumor growth. Further, Ku80 knockdown in NSCLC cells suppressed tumor properties in vitro and in vivo similar to hsa-miR-526b overexpression. In agreement, Ku80 restoration partially reversed cell cycle arrest and apoptosis induced by hsa-miR-526b in NSCLC cells in vitro and in vivo. In addition, hsa-miR-526b overexpression or Ku80 knockdown increased p53 and p21CIP1/WAF1 expression. These findings reveal that hsa-miR-526b is a potential target in cancer therapy.

Efficacy of argyrophilic nucleolar organizing region analysis using computer-assisted and manual in oral leukoplakia: A comparative study.

OBJECTIVE: Nucleolar organizer regions are loops of DNA containing ribosomal RNA genes and presumably are associated with ribosomal RNA activity, protein synthesis, and cell proliferation. Argyrophilic nucleolar organizer region (AgNOR) count has been suggested as an objective method in differentiating dysplastic lesions from non-dysplastic lesions. MATERIALS AND METHODS: This descriptive study was done on archival paraffin blocks (n = 60), consisting of 10 normal human oral epithelium, 22 cases of non-dysplastic leukoplakia (NDLK), and 28 cases of dysplastic leukoplakia (DLK). The AgNORs were counted with the aid of a manual using conventional light microscopy and photographs of the same were taken and analyzed using Image Pro Express 6.0 (Media Cybernetic Inc., USA) for windows. RESULTS: The mean AgNOR count per nucleus was found to be higher in patients with DLK as compared to NDLK and controls using both manual counting and image analysis method and on comparing both the techniques, image analysis provide a more accurate reflection of AgNOR counts than manual counting. CONCLUSION: To conclude, reliability of computerized image technique of AgNOR count is the most appropriate marker to differentiate between dysplastic and NDLK. Computer-assisted image analysis system was found to be an effective tool in achieving high reproducibility as compare to manual.

Chemotherapy enhances cross-presentation of nuclear tumor antigens.

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8⁺ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens.

Effects of chronic oestradiol, progesterone and medroxyprogesterone acetate on hippocampal neurogenesis and adrenal mass in adult female rats.

Both natural oestrogens and progesterone influence synaptic plasticity and neurogenesis within the female hippocampus. However, less is known of the impact of synthetic hormones on hippocampal structure and function. There is some evidence that the administration of the synthetic progestin, medroxyprogesterone acetate (MPA) is not as beneficial as natural progesterone and can attenuate oestrogen-induced neuroprotection. Although the effects of oestradiol have been well studied, little is known about the effects of natural and synthetic progestins alone and in combination with oestradiol on adult neurogenesis in females. In the present study, we investigated the effects of chronic oestradiol, progesterone, MPA and the co-administration of each progestin with oestradiol on neurogenesis within the dentate gyrus of adult ovariectomised female rats. Twenty-four hours after a bromodeoxyuridine (BrdU; 200 mg/kg) injection, female rats were repeatedly administered either progesterone (1 or 4 mg), MPA (1 or 4 mg), oestradiol benzoate (EB), progesterone or MPA in combination with EB (10 µg), or vehicle for 21 days. Rats were perfused on day 22 and brain tissue was analysed for the number of BrdU-labelled and Ki67 (an endogenous marker of cell proliferation)-expressing cells. EB alone and MPA + EB significantly decreased neurogenesis and the number of surviving BrdU-labelled cells in the dorsal region of the dentate gyrus, independent of any effects on cell proliferation. Furthermore, MPA (1 and 4 mg) and MPA + EB treated animals had significantly lower adrenal/body mass ratios and reduced serum corticosterone (CORT) levels. By contrast, progesterone + EB treated animals had significantly higher adrenal/body mass ratios and 1 mg of progesterone, progesterone + EB, and EB significantly increased CORT levels. The results of the present study demonstrate that different progestins alone and in combination with oestradiol can differentially affect neurogenesis (via cell survival) and regulation of the hypothalamic-pituitary-adrenal axis. These findings have implications for women using hormone replacement therapies with MPA for both neuroprotection and stress-related disorders.

An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle.

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8(+) T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

Suppression of Ku80 correlates with radiosensitivity and telomere shortening in the U2OS telomerase-negative osteosarcoma cell line.

Ku70/80 heterodimer is a central element in the nonhomologous end joining (NHEJ) DNA repair pathway, Ku80 playing a key role in regulating the multiple functions of Ku proteins. It has been found that the Ku80 protein located at telomeres is a major contributor to radiosensitivity in some telomerase positive human cancer cells. However, in ALT human osteosarcoma cells, the precise function in radiosensitivity and telomere maintenance is still unknown. The aim of this study was to investigate the effects of Ku80 depletion in the U2OS ALT cell line cell line. Suppression of Ku80 expression was performed using a vector-based shRNA and stable Ku80 knockdown in cells was verified by Western blotting. U2OS cells treated with shRNA-Ku80 showed lower radiobiological parameters (D0, Dq and SF2) in clonogenic assays. Furthermore, shRNA-Ku80 vector transfected cells displayed shortening of the telomere length and showed less expression of TRF2 protein. These results demonstrated that down-regulation of Ku80 can sensitize ALT cells U2OS to radiation, and this radiosensitization is related to telomere length shortening.

Loss of expression of the double strand break repair protein ATM is associated with worse prognosis in colorectal cancer and loss of Ku70 expression is associated with CIN.

Repair of double strand DNA breaks (DSBs) is pivotal in maintaining normal cell division and disruption of this system has been shown to be a key factor in carcinogenesis. Loss of expression of the DSB repair proteins have previously been shown to be associated with poorer survival in colorectal cancer. We wished to ascertain the relationship of altered expression of the DSB repair proteins γ-H2AX (gamma-H2AX), ATM and Ku70 with biological and clinico-pathological features of colorectal cancer. 908 tumours from the VICTOR clinical trial of stage II/III colorectal cancer were analysed for expression of γ-H2AX, ATM and Ku70 using immunohistochemistry. Expression levels were correlated with CIN and with disease-free survival, correcting for microsatellite instability, BRAF/KRAS mutation status, Dukes stage, chemo/radiotherapy, age, gender and tumour location. Down-regulated Ku70 expression was associated with chromosomal instability (p=0.029) in colorectal cancer. Reduced ATM expression was an independent marker of poor disease-free survival (HR=1.67, 95% CI 1.11-2.50, p=0.015). For Ku70, further studies are required to investigate the potential relationship of non-homologous end joining with chromosomal instability. Loss of ATM expression might serve as a biomarker of poor prognosis in colorectal cancer.

Baicalein (5,6,7-trihydroxyflavone) reduces oxidative stress-induced DNA damage by upregulating the DNA repair system.

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.

The role of XRCC6 T-991C functional polymorphism in renal cell carcinoma.

BACKGROUND: The DNA non-homologous end-joining repair gene XRCC6 (Ku70) plays a key role in both the DNA double-strand break (DSB) repair and cell cycle arrest. Defects in DSB repair capacity can lead to genomic instability. We hypothesized that a variant in the XRCC6 gene was associated with susceptibility to renal cell carcinoma (RCC). MATERIALS AND METHODS: In a hospital-based case-control study of 92 patients with RCC and 580 cancer-free controls, the frequency matched by age and sex, the associations of XRCC6 promoter T-991C (rs5751129), promoter G-57C (rs2267437), promoter A-31G (rs132770), and intron 3 (rs132774) polymorphisms with RCC risk were investigated in a Taiwanese population. At the same time, 30 adjacent renal tissue samples were tested to estimate the XRCC6 mRNA expression by real-time quantitative reverse transcription. RESULTS: Compared with the TT genotype, the TC genotype had a significantly increased risk of RCC [adjusted odds ratio=2.24, 95% confidence interval=1.25-4.08, p=0.0175]. The in vivo mRNA expression in renal tissues revealed a statistically significant lower XRCC6 mRNA expression in samples with TC/CC genotypes compared to those with the TT genotype (p=0.0039). CONCLUSION: These evidence suggests that the XRCC6 T-991C genotype together with its mRNA expression are involved in the etiology of RCC and may be a marker for susceptibility to RCC in the population of Taiwan.

Aurora A inhibitor (MLN8237) plus vincristine plus rituximab is synthetic lethal and a potential curative therapy in aggressive B-cell non-Hodgkin lymphoma.

PURPOSE: Aurora A and B are oncogenic serine/threonine kinases that regulate mitosis. Overexpression of Auroras promotes resistance to microtubule-targeted agents. We investigated mechanistic synergy by inhibiting the mitotic spindle apparatus in the presence of MLN8237 [M], an Aurora A inhibitor with either vincristine [MV] or docetaxel [MD] in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The addition of rituximab [R] to MV or MD was evaluated for synthetic lethality. EXPERIMENTAL DESIGN: Aggressive B-NHL cell subtypes were evaluated in vitro and in vivo for target modulation and anti-NHL activity with single agents, doublets, and triplets by analyzing cell proliferation, apoptosis, tumor growth, survival, and mechanisms of response/relapse by gene expression profiling with protein validation. RESULTS: MV is synergistic whereas MD is additive for cell proliferation inhibition in B-NHL cell culture models. Addition of rituximab to MV is superior to MD, but both significantly induce apoptosis compared with doublet therapy. Mouse xenograft models of mantle cell lymphoma showed modest single-agent activity for MLN8237, rituximab, docetaxel, and vincristine with tumor growth inhibition (TGI) of approximately 10% to 15%. Of the doublets, MV caused tumor regression, whereas TGI was observed with MD (approximately 55%-60%) and MR (approximately 25%-50%), respectively. Although MV caused tumor regression, mice relapsed 20 days after stopping therapy. In contrast, MVR was curative, whereas MDR led to TGI of approximately 85%. Proliferation cell nuclear antigen, Aurora B, cyclin B1, cyclin D1, and Bcl-2 proteins of harvested tumors confirmed response and resistance to therapy. CONCLUSIONS: Addition of rituximab to MV is a novel therapeutic strategy for aggressive B-NHL and warrants clinical trial evaluation.