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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., (© 2017 Royal Australian and New Zealand College of Ophthalmologists.)

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-β 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-β pre-treatment. This ability of HCMV to overcome the potently anti-viral effects of IFN-β in ND10 expression deficient cells provides evidence that ND10 component upregulation is a key mediator of the anti-viral activity of IFN-β.

The cohesin complex is an evolutionarily conserved multi-subunit protein complex which regulates sister chromatid cohesion during mitosis and meiosis. Additionally, the cohesin complex regulates DNA replication, DNA repair, and transcription. The core of the complex consists of four subunits: SMC1A, SMC3, RAD21, and STAG1/2. Loss-of-function mutations in many of these proteins have been implicated in human developmental disorders collectively termed "cohesinopathies." Through clinical exome sequencing (CES) of an 8-year-old girl with a clinical history of global developmental delay, microcephaly, microtia with hearing loss, language delay, ADHD, and dysmorphic features, we describe a heterozygous de novo variant (c.205C>T; p.(Arg69*)) in the integral cohesin structural protein, STAG2. This variant is associated with decreased STAG2 protein expression. The analyses of metaphase spreads did not exhibit premature sister chromatid separation; however, delayed sister chromatid cohesion was observed. To further support the pathogenicity of STAG2 variants, we identified two additional female cases from the DECIPHER research database with mutations in STAG2 and phenotypes similar to our patient. Interestingly, the clinical features of these three cases are remarkably similar to those observed in other well-established cohesinopathies. Herein, we suggest that STAG2 is a dosage-sensitive gene and that heterozygous loss-of-function variants lead to a cohesinopathy., (© 2017 Wiley Periodicals, Inc.)

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).

The aims of the study are to detect whether there are any possible effects of chronic carbon monoxide (CO) exposure on the argyrophilic nucleolar-organizing region (AgNOR)-associated protein synthesis and evaluate any possible relationship between the amount of AgNOR protein and the level of myocardial injury also and between AgNOR and histopathological evaluation methods. Adult male albino Wistar rats (n = 18) were randomly divided into three groups (groups A, B, and C). Group A served as control, while groups B and C were rats exposed to CO gas chronically (1000 and 3000 ppm CO concentration with a flow rate of 4 L/min for 30 min/day for 7 days, respectively). Total AgNOR area/nuclear area (TAA/NA) and the mean AgNOR numbers for each myocyte nucleus were determined. There were significant differences among all groups for TAA/NA ratio. These differences were not significant for mean AgNOR numbers. According to the histopathological evaluation scores, there were significant differences between the groups. The differences were significant among the groups for loss of sarcomere pattern. A strong positive correlation between histopathological injury scores and TAA/NA ratio was found (Rsq = 0.48; p = 0.002), however, the correlation was not significant for mean AgNOR numbers (Rsq = 0.08; p = 0.25). In conclusion, TAA/NA ratio can be used as an indicator for obtaining information about the level of myocardial damage instead of histopathological evaluation scores., (© The Author(s) 2015.)

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.

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 βIII and MAP2 adding to the confusion about their origin. Concomitant with their appearance in reactive cells MAP2 and Tubulin βIII 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., (© 2015 International Society of Neuropathology.)

β1 Integrin-mediated cell-extracellular matrix interactions allow cancer cell survival and confer therapy resistance. It was shown that inhibition of β1 integrins sensitizes cells to radiotherapy. Here, we examined the impact of β1 integrin targeting on the repair of radiation-induced DNA double-strand breaks (DSBs). β1 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. β1 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 β1 integrin pathway. Intriguingly, combined inhibition of β1 integrin and PARP using Olaparib was significantly more effective than either treatment alone in non-irradiated and irradiated HNSCC cells. Here, we support β1 integrins as potential cancer targets and highlight a regulatory role for β1 integrins in the repair of radiogenic DNA damage via classical NHEJ. Further, the data suggest combined targeting of β1 integrin and PARP as promising approach for radiosensitization of HNSCC.

Human Gb3/CD77 synthase (α1,4-galactosyltransferase, P(k) synthase), encoded by A4GALT gene, is known for synthesis of Gal(α1-4)Gal moiety in globotriaosylceramide (Gb3Cer, CD77, P(k) blood group antigen), a glycosphingolipid of the globo series. Recently, it was shown that c.631C > G mutation in A4GALT, which causes p.Q211E substitution in the open reading frame of the enzyme, broadens the enzyme specificity, making it able also to synthesize Gal(α1-4)GalNAc moiety, which constitutes the defining terminal disaccharide of the NOR antigen (carried by two glycosphingolipids: NOR1 and NOR2). Terminal Gal(α1-4)Gal disaccharide is also present in another glycosphingolipid blood group antigen, called P1, which together with P(k) and NOR comprises the P1PK blood group system. Despite several attempts, it was never clearly shown that P1 antigen is synthesized by Gb3/CD77 synthase, leaving open an alternative hypothesis that there are two homologous α1,4-galactosyltransferases in humans. In this study, using recombinant Gb3/CD77 synthase produced in insect cells, we show that the consensus enzyme synthesizes both the P(k) and P1 antigens, while its p.Q211E variant additionally synthesizes the NOR antigen. This is the first direct biochemical evidence that Gb3/CD77 synthase is able to synthesize two different glycosphingolipid antigens: P(k) and P1, and when p.Q211E substitution is present, the NOR antigen is also synthesized., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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).

This work was performed on the offspring of 5 outbred female albino rats to give a comparative immunohistochemical evaluation of doublecortin (DCX) and NeuN expression in the neurons of the cerebellar cortex and nucleus interpositus in the early postnatal ontogenesis (postnatal days 2-15). DCX expression was detected in postmitotic neurons of the external granular layer and migrating neurons of the cerebellar cortex. At postnatal days 2 and 7 DCX expression in neocerebellum was higher than in paleocerebellum. NeuN expression was found to appear in migrating granule neurons, and reach the maximum in mature neurons of internal granular layer. DCX expression was not detected in Purkinje cells and in the nucleus interpositus of the cerebellum. In neurons of the nucleus interpositus the expression of NeuN progressively increased from postnatal days 2 to 15. Thus, a comparative immunohistochemical study of the dynamics of the expression of the pair of molecular markers studied proved to be an effective way of the assessment of the development of granular neurons of the cerebellum in early postnatal ontogenesis.

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.

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.

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.

Background: Nucleolus organizer regions (NORs) consist of the rRNA coding gene family (rDNA) in the cell nucleus. The argyrophilic proteins are selectively stained with silver nitrate and bind these regions. It was reported that NOR (rDNA) activity decreases in human lymphocytes, fibroblasts and bone marrow with age. However, to our knowledge there have not been any studies related to the NORs in oral epithelial cells of healthy individuals., Aim: Our aim is to detect any correlation between age and Total AgNOR area/Total nucleus area (TAA/TNA) values in buccal epithelial cells of healthy individuals., Methods: Oral epithelial cells from 50 healthy individuals (age range of 2-80 years old) were spread onto a clean glass slide, air dried and fixed. Then the AgNOR staining protocol was performed on these cells. TAA/TNA ratio and AgNOR dots were calculated using software. From each person 50 oral epithelial cells were evaluated., Results: Statistically significant correlations were found between mean TAA/TNA values and age (Rsq = 0.534, p < 0.001 for linear and Rsq = 0.728, p < 0.0001 for polynominal regression), and between AgNOR number and age (Rsq = 0.621, p < 0.001 for linear and Rsq = 0.693, p < 0.0001 for polynominal regression)., Conclusion: There is a significant correlation between age and AgNOR amount (ribosome biosynthesis rate) in buccal epithelial cells of healthy individuals. AgNORs in buccal epithelial cells may be used for detection of age.

Purpose: Hippocampal glucose hypometabolism has been implicated in the pathogenesis of temporal lobe epilepsy (TLE). However, the underlying pathophysiological basis for this hypometabolism remains elusive. The aim of this study was to investigate the relationship between hippocampal hypometabolism and the histological changes seen in rats after systemic pilocarpine treatment., Methods: (18)F-fluorodeoxyglucose (FDG) small-animal positron emission tomography (microPET) was performed on day zero (untreated), day seven (latent) and day sixty (chronic phase) after the initial status epilepticus. The microPET imaging data were correlated with the immunoreactivity of neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP) in the hippocampus at each time point., Results: (18)F-FDG-microPET images showed the hippocampus presented with persistent hypometabolism during epileptogenesis and partly recovered in the chronic phase. Hippocampal glucose uptake defects correlate with NeuN immunoreactivity in the latent phase and GFAP immunoreactivity in the chronic phase., Conclusions: Severe glucose hypometabolism in the hippocampus during the latent phase correlates with neuronal cell loss. The partial recovery of hippocampal glucose uptake in the chronic phase may be due to astrogliosis.

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.

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.

To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

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.

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., (© 2014 British Society for Neuroendocrinology.)

We have previously reported that in response to early life stress, such as maternal hyperthyroidism and maternal separation (MS), the rat hypothalamic vasopressinergic system becomes up-regulated, showing enlarged nuclear volume and cell number, with stress hyperresponsivity and high anxiety during adulthood. The detailed signaling pathways involving cell death/survival, modified by adverse experiences in this developmental window remains unknown. Here, we report the effects of MS on cellular density and time-dependent fluctuations of the expression of pro- and anti-apoptotic factors during the development of the hypothalamus. Neonatal male rats were exposed to 3 h-daily MS from postnatal days 2 to 15 (PND 2-15). Cellular density was assessed in the hypothalamus at PND 21 using methylene blue staining, and neuronal nuclear specific protein and glial fibrillary acidic protein immunostaining at PND 36. Expression of factors related to apoptosis and cell survival in the hypothalamus was examined at PND 1, 3, 6, 9, 12, 15, 20 and 43 by Western blot. Rats subjected to MS exhibited greater cell-density and increased neuronal density in all hypothalamic regions assessed. The time course of protein expression in the postnatal brain showed: (1) decreased expression of active caspase 3; (2) increased Bcl-2/Bax ratio; (3) increased activation of ERK1/2, Akt and inactivation of Bad; PND 15 and PND 20 were the most prominent time-points. These data indicate that MS can induce hypothalamic structural reorganization by promoting survival, suppressing cell death pathways, increasing cellular density which may alter the contribution of these modified regions to homeostasis.

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.

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.

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.

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.

Argyrophilic nucleolar organizing region associated proteins (AgNORs) play roles in cell proliferation and a variety of diseases. We attempted to determine whether decreased NOR protein synthesis causes human hair loss. We studied 21 healthy males who suffered hair loss on the frontal/vertex portion of the head. Hair root cells from normal and hair loss sites were stained for AgNOR. One hundred nuclei per site were evaluated and the AgNOR number and NORa/TNa proportions of individual cells were determined using a computer program. The cells from normal sites had significantly higher AgNOR counts than those from hair loss sites. Also, the cells from the normal sites had significantly higher NORa/TNa than cells from the hair loss sites. In the normal sites, the cells demonstrated more NOR protein synthesis than cells in hair loss sites. Therefore, decreased NOR protein synthesis appears to be related to hair loss in humans.

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.

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., (©2012 AACR.)

The aim of this study was to examine the preventive and therapeutic effects of thymoquinone (TQ) against cholestatic oxidative stress and liver damage in common bile duct ligated rats. A total of 24 male Sprague-Dawley rats were divided into three groups: control, bile duct ligation (BDL) and BDL + received TQ; each group contain 8 animals. The rats in TQ treated groups were given TQ (50 mg/kg body weight) once a day orally for 2 weeks starting 3 days prior to BDL operation. To date, no more biochemical and histopathological changes on common bile duct ligated rats by TQ treatment have been reported. The application of BDL clearly increased the tissue hydroxyproline (HP) content, malondialdehyde (MDA) levels and decreased the antioxidant enzyme [superoxide dismutase (SOD), glutathione peroxidase (GPx)] activities. TQ treatment significantly decreased the elevated tissue HP content, and MDA levels and raised the reduced of SOD, and GPx enzymes in the tissues. The changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in BDL group. Treatment of BDL with TQ attenuated alterations in liver histology. The immunopositivity of alpha smooth muscle actin and proliferating cell nuclear antigen in BDL were observed to be reduced with the TQ treatment. The present study demonstrates that oral administration of TQ in bile duct ligated rats maintained antioxidant defenses and reduces liver oxidative damage and ductular proliferation. This effect of TQ may be useful in the preservation of liver function in cholestasis.

Background: Human SA/STAG proteins, homologues of the yeast Irr1/Scc3 cohesin, are the least studied constituents of the sister chromatid cohesion complex crucial for proper chromosome segregation. The two SA paralogues, SA1 and SA2, show some specificity towards the chromosome region they stabilize, and SA2, but not SA1, has been shown to participate in transcriptional regulation as well. The molecular basis of this functional divergence is unknown., Methodology/principal Findings: In silico analysis indicates numerous putative nuclear localization (NLS) and export (NES) signals in the SA proteins, suggesting the possibility of their nucleocytoplasmic shuttling. We studied the functionality of those putative signals by expressing fluorescently tagged SA1 and SA2 in the yeast Saccharomyces cerevisiae. Only the N-terminal NLS turned out to be functional in SA1. In contrast, the SA2 protein has at least two functional NLS and also two functional NES. Depending on the balance between these opposing signals, SA2 resides in the nucleus or is distributed throughout the cell. Validation of the above conclusions in HeLa cells confirmed that the same N-terminal NLS of SA1 is functional in those cells. In contrast, in SA2 the principal NLS functioning in HeLa cells is different from that identified in yeast and is localized to the C-terminus., Conclusions/significance: This is the first demonstration of the possibility of non-nuclear localization of an SA protein. The reported difference in the organization between the two SA homologues may also be relevant to their partially divergent functions. The mechanisms determining subcellular localization of cohesins are only partially conserved between yeast and human cells.

Based on the role of Ku80 in mediating radiation-induced DNA repair, we investigated Ku80 expression in human lung cancers of different pathological types and evaluated the effect of radiotherapy on Ku80 expression levels in a mouse model. We used immunohistochemistry and real-time PCR to determine Ku80 protein and mRNA levels, respectively. We inoculated nude mice with A549 cells and subjected the tumor-bearing mice to varying doses of irradiation. Lung carcinoma tissue exhibited higher Ku80 mRNA and protein levels when compared with normal tissue. Among the tumor subtypes, lung adenocarcinoma and lung squamous carcinoma showed higher levels of Ku80 protein and mRNA, compared with small-cell lung carcinoma. There was a dose-dependent and time-dependent increase in Ku80 mRNA levels in nude mice that were inoculated with A549 cells and exposed to varying doses of irradiation. Ku80 may play an important role in the DNA damage response pathway. Higher Ku80 levels in lung squamous carcinoma and adenocarcinoma may explain their lower radiosensitivity when compared with small-cell lung carcinoma. Ku80 expression levels could be useful in predicting radiosensitivity of lung tumors and inhibition of Ku80 may be an interesting target to improve radiosensitivity in lung cancer patients.

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.

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action of the multimeric DNA-PK repair complex. The components of this complex are the Ku autoantigens, XRCC5 and XRCC6 (Ku80 and Ku70, respectively). How this repair complex responds to perturbed Cu homeostasis and Cu-mediated oxidative stress has not been investigated. We previously reported that XRCC5 expression is altered in response to cellular Cu levels, with low Cu inhibiting XRCC5 expression and high Cu levels enhancing expression. In this study we further investigated the interaction between XRCC5 and Cu. We report that cytosolic XRCC5 is increased in response to Cu, but not zinc, iron, or nickel, and the level of cytosolic XRCC5 correlates with protection against oxidative damage to DNA. These observations were made in both HeLa cells and fibroblasts. Cytosolic XRCC5 interacted with the Cu chaperone and detoxification protein human Atox1 homologue (HAH), and down regulation of XRCC5 expression using siRNA led to enhanced HAH expression when cells were exposed to Cu. XRCC5 could also be purified from cytosolic extracts using a Cu-loaded column. These findings provide further evidence that cytosolic XRCC5 has a key role in protection against DNA oxidation from Cu, through either direct sequestration or signaling through other Cu-detoxification molecules. Our findings have important implications for the development of therapeutic treatments targeting Cu in neurodegeneration and/or cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Longevity genes attenuate the aging process, but their expression in the brain during aging remains unknown. Loss of the majority of heteromeric brain nicotinic acetylcholine receptors (nAChRs) results in premature brain aging, and altered regulation of longevity genes could be involved. Using in situ hybridization, the expression of SIRT1, Ku70, Nampt, p53, forkhead Box O3 (FoxO3), and mitochondria uncoupling protein 5 (UCP5) was determined in neocortex and hippocampus of young adult 3-month and middle-aged 18-month-old wild-type (WT), and age-matched mice lacking β2* heteromeric nAChRs (β2-/-). Age-related structural changes were detected in WT mice. In particular, cortical thickness was decreased but neuronal density increased, and hippocampal volume increased with age. In contrast, young β2-/- mice exhibited increased cortical neuronal density, and with age, cortical thickness decreased more dramatically, and hippocampal volume did not increase. Thus, young β2-/- mice exhibited cortical signs of aging, and aging was accelerated at 18 months. The longevity genes probed exhibited similar expression patterns in frontal brain structures, with strong expression in hippocampus, medial habenula (MHb), and cortex. In WT mice, age significantly decreased expression of all genes except SIRT1 in cortical structures, and a similar pattern was detected in the MHb. Genotype had no effect on expression in young adults in either cortex or MHb, but increased mRNA expression of SIRT1, Nampt, and Ku70 was detected in cortex, hippocampus, and MHb of aged β2-/- mice compared with WT mice. This is the first study to determine age-related expression of survival genes in forebrain areas. Although, structural changes indicative of accelerated aging are evident in young β2-/- mice, the data suggest that nAChRs do not directly regulate expression of survival genes. However, loss of β2* nAChRs could result in augmented cellular stress, which indirectly increases expression of SIRT1, Nampt, and Ku70 as an adaptive response to provide protection against neurodegeneration., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

In patients with HIV/AIDS, neuropathic pain is a common neurological complication. Infection with the HIV itself may lead to neuropathic pain, and painful symptoms are enhanced when patients are treated with nucleoside reverse transcriptase inhibitors (NRTIs). The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current studies, we tested the role of TNFα in antiretroviral drug-induced neuropathic pain. We administered 2',3'-dideoxycytidine (ddC, one of the NRTIs) systemically to induce mechanical allodynia. We found that ddC induced overexpression of both mRNA and proteins of GFAP and TNFα in the spinal dorsal horn. TNFα was colocalized with GFAP in the spinal dorsal horn and with NeuN in the DRG. Knockdown of TNFα with siRNA blocked the mechanical allodynia induced by ddC. Intrathecal administration of glial inhibitor or recombinant TNF soluble receptor, reversed mechanical allodynia induced by ddC. These results suggest that TNFα is involved in NRTI-induced neuropathic pain., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Boron neutron capture reaction (BNCR) is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He) particle and a recoiled lithium nucleus (7Li). These particles have the characteristics of high linear energy transfer (LET) radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells., Methods: Chinese hamster ovary (CHO-K1) cells and a DNA double-strand break (DSB) repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells), were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX) and 53BP1 foci using immunofluorescence intensity., Results: Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness) estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity., Conclusion: Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have an advantage over gamma-ray sensitive cells in BNCR.

The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c-Cbl--known for its roles in regulating lymphocyte signalling--as a modulator of dendritic cell activation. In c-Cbl-deficient dendritic cells, Toll-like receptor-induced expression of proinflammatory factors, such as interleukin-12, is increased, correlating with a greater potency of dendritic-cell-based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)-κB activity. In addition, c-Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF-κB. Our data indicate that c-Cbl has a crucial, RING-domain-dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.

Objective: To evaluate the impact on the AgNORs expression and apoptosis in the prostate of the hamster-Mesocricetus auratus (HMA) submitted to the application of finasteride., Methods: Twenty male rodents of the species HMA (n = 20) were randomly assigned to groups of ten animals: Finasteride group (n = 10) and the Control group (n = 10). In the finasteride group 7.14 ng / mL finasteride was subcutaneously (SC) administered on the back of the animals three times a week for 90 days. AgNOR expression was evaluated as a marker of cell proliferation and apoptosis as a marker of cell death., Results: The expression of AgNORs was lower in the finasteride group, 2.846 ± 0.877 vs. 3.68 ± 1.07 argyrophilic regions per square micrometer (µm2) in the control group, p = <0.0001. Apoptosis was more frequent in the finasteride group, 53.62 ± 1.389 versus 14.76 ± 2.13 per µm2 in the control group, p = 0.0408., Conclusion: We observed decreased expression of AgNORs and promotion of apoptosis in the prostate of rodents treated with finasteride.

Objectives: To investigate whether BCL-2 expression would improve MVP/IGF-1R prediction of clinical outcome in cervix carcinoma patients treated by radiochemotherapy, and suggest possible mechanisms behind this effect., Methods: Fifty consecutive patients, who achieved complete response to treatment, from a whole series of 60 cases suffering from non-metastatic localized cervical carcinoma, were prospectively included in this study from July 1999 to December 2003. Follow-up was closed in January 2011. All patients received pelvic radiation (45-64.80 Gy in 1.8-2 Gy fractions) with concomitant cisplatin at 40 mg/m2/week doses followed by brachytherapy. Oncoprotein expression was studied by immunohistochemistry in paraffin-embedded tumour tissue., Results: No relation was found between BCL-2 and clinicopathological variables. High MVP/IGF-1R/BCL-2 tumour expression was strongly related to poor local and regional disease-free survival (P<0.0001), distant disease-free survival (P=0.010), disease-free survival (P<0.0001), and cause-specific survival (P<0.0001). NHEJ repair protein Ku70/80 expression was significantly repressed in tumours overexpressing all three oncoproteins (P=0.047). No differences were observed in proliferation (Ki67 expression) or P53 alteration., Conclusions: BCL-2, MVP, and IGF-1R overexpression were related to poorer clinical outcome in cervical cancer patients who achieved clinical complete response to radiochemotherapy. The NHEJ repair protein Ku70/80 expression could be involved in the regulation of these oncoproteins., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: hMLH1 is one of the major proteins of the mammalian mismatch repair system. It has been suggested that the mismatch repair machinery could be linked to p53, a tumor suppressor protein. The AgNOR technique is used to assess cell proliferation. The aim was to compare the immunoexpression of hMLH1 and p53, and AgNOR number in oral leukoplakias with different degrees of dysplasia., Methods: Sixty-two samples were evaluated by immunohistochemistry for hMLH1 and p53, and AgNOR technique, being 17 without dysplasia, 19 with mild dysplasia, 16 with moderate dysplasia, and 10 with severe dysplasia., Results: hMLH1 immunoexpression showed decreasing indexes, while p53 and AgNOR showed increasing indexes from lesions with lower degrees of dysplasia to lesions with more severe dysplasia. An inverse correlation between hMLH1 and both p53 and AgNOR, and a direct correlation between p53 and AgNOR were observed., Conclusions: Alterations in the immunoexpression pattern of hMLH1 and p53 seemed to be early events in oral carcinogenesis. During acquisition of a more dysplastic phenotype, keratinocytes may show diminished capacity of DNA repair and tumor suppression, as well as higher cellular proliferation, and these pathways can be somehow interconnected., (© 2011 John Wiley & Sons A/S.)

The nuclear autoantigen SP100 (speckled protein 100) is reported to control cellular gene expression, cell growth and differentiation. To investigate its relevance in brain tumors, we investigated SP100 expression and function in human glioblastomas and meningiomas. SP100 was expressed in both tumors at the mRNA and protein levels in situ and in vitro, however, expression in meningioma samples and meningioma cells exceeded that in glioblastoma samples and cultivated cells significantly. Moreover, whereas nearly all meningioma cells were SP100-immunopositive, only part of the glioblastoma cells were SP100 stainable. In vitro, SP100 was upregulated by interferon-α and -γ in both malignant cell types. To study its functional role, SP100 was overexpressed in glioblastoma cells. This SP100 overexpression reduced considerably the glioblastoma cell proliferation and migration to fetal calf serum. We conclude that SP100 expression reduces malignancy of brain tumors. Since meningiomas show a generally higher SP100 expression, this may be one of the factors explaining their lower malignancy compared to glioblastomas.

Background: DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive., Methodology/principal Findings: In the present study, we characterized the roles of DNA-PK on recombinant adeno-associated virus DNA replication. Inhibition of DNA-PK by a DNA-PK inhibitor or siRNA targeting DNA-PKcs significantly decreased replication of AAV in MO59K and 293 cells. Southern blot analysis showed that replicated rAAV DNA formed head-to-head or tail-to-tail junctions. The head-to-tail junction was low or undetectable suggesting AAV-ITR self-priming is the major mechanism for rAAV DNA replication. In an in vitro replication assay, anti-Ku80 antibody strongly inhibited rAAV replication, while anti-Ku70 antibody moderately decreased rAAV replication. Similarly, when Ku heterodimer (Ku70/80) was depleted, less replicated rAAV DNA were detected. Finally, we showed that AAV-ITRs directly interacted with Ku proteins., Conclusion/significance: Collectively, our results showed that that DNA-PK enhances rAAV replication through the interaction of Ku proteins and AAV-ITRs.

We studied the distribution of glial fibrillary acidic protein (GFAP) and neuron-specific nuclear histone protein NeuN in sulci of the brain cortex during the pre- and postnatal ontogeny. The expression of GFAP during morphogenetic development of the sulci and gyri is cyclic. After functional reorientation of GFAP in human fetuses at weeks 28-30 it ceases to be a marker of morphogenetically active glia and becomes a marker of glial cells exclusively. Redistribution of NeuN expression in different layers of sulci during their formation was found: enhanced expression of NeuN in the cortical layer 6 of sulci and its reduced expression in the upper layers were noted, whereas outside the cortical sulci NeuN expression was similar in all layers. At weeks 24-25 of gestation, NeuN serves as a marker of ingrowth of secondary visual fibers from the dorsal thalamus.

Purpose: Telomere dysfunction is believed to be a significant factor in carcinogenesis. To elucidate the carcinogenesis mechanism in gastric cancer, the expression of telomeric proteins and changes in telomere length were investigated during multistage carcinogenesis of gastric cancer., Methods: Tissue samples were obtained during surgical operations from the normal gastric mucosa of 10 patients, the precancerous lesions of 15 patients, the gastric cancer tissues (GC) of 20 patients, and of tumors due to gastric cancer with lymph node metastasis (GCLM) from 5 patients. The expression of TRF1, TRF2, and TIN2 proteins was measured by Western blotting, while the expression of TERT, KU70, and BRCA1 proteins was detected using the immunohistochemical method. The mean telomere length was determined by Southern blotting., Results: Compared with normal gastric mucosa tissues, the expression of TRF1, TRF2, and TIN2 proteins was significantly higher in precancerous lesions, GC, and GCLM (P < 0.01). The expression of TRF1, TRF2, and TIN2 proteins was significantly higher in GC and GCLM than in precancerous lesions (P < 0.01). The expression of TERT and Ku70 proteins in precancerous lesions and GC tissues was significantly higher than that in normal gastric mucosa tissues (P < 0.01). The expression of TERT and Ku70 proteins in GC tissues was significantly higher than in precancerous lesions (P < 0.01). In normal gastric mucosa, the BRCA1 protein was primarily located in the cell nucleus. In precancerous lesions and GC, the expression of the BRCA1 protein was apparent in the cell cytoplasm. The mean telomere length in precancerous lesions, GC, and GCLM was significantly shorter than that in normal gastric mucosa tissues (P < 0.05). The mean telomere length in GC and GCLM was significantly shorter than that in precancerous lesions (P < 0.05). The mean telomere length in all tissue samples was inversely correlated with the level of TRF1, TRF2, TIN2, TERT, and Ku70 proteins., Conclusions: Our results suggest that the over-expression of telomeric proteins, TRF1, TRF2, TIN2, TERT, and Ku70, and the transposition of the BRCA1 protein may work together to reduce the telomere length in precancerous lesions and gastric cancer, and could contribute to the multistage carcinogenesis of gastric cancer. These findings offer new insight into the mechanism of carcinogenesis in gastric cancer.

Purpose: We have recently shown that breast tumors express high levels of TrkA compared with normal breast tissues, with TrkA overexpression enhancing breast cancer cell invasion in vitro and metastasis in animal models. In this study, we tried to identify molecules involved in TrkA overexpression-mediated biological effects in breast cancer cells., Experimental Design: We used a proteomic-based approach to identify proteins involved in TrkA overexpression-stimulated invasion of MDA-MB-231 breast cancer cells. Proteins from control and TrkA overexpressing cells were separated using a cup-loading two-dimensional electrophoresis system before MALDI and LC-MS/MS mass spectrometry analysis., Results: Among several putative regulated proteins, Ku86 was found increased in TrkA overexpressing cells. Moreover, Ku86 was co-immunoprecipitated with TrkA, suggesting the interaction of these two proteins in TrkA overexpressing cells. Interestingly, inhibition with small-interfering RNA and neutralizing antibodies showed that Ku86 was required for TrkA-stimulated cell invasion., Conclusions and Clinical Relevance: These data allowed the identification of Ku86 as a new player involved in metastasis in breast cancer cells. Our findings suggest that TrkA and its down stream signaling pathways should be regarded as potential new targets for the development of future breast cancer therapy., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The prognosis in papillary thyroid cancer (PTC) is usually good. Ten-year survival can be seen in 90-98% of patients. Immunohistochemical study (antigen K-67) ascertained that a female patient with PTC had a low number of proliferating cells, which is usually seen in the favorable course of the disease. However, in the presented case, PTC was highly aggressive and showed a significant invasive growth, provided regional and distant metastases, rapidly progressed and, despite the performed surgical treatment, the patient died due to disease progression 3 months after surgery. This discrepancy between the number of proliferating cells and the aggressive course of PTC should be explained by the high expression of argyrophilic nucleolar organizer region associated proteins nucleofozmin and nucleolin, detected by immunohistochemical study, which is known to cause an increase in the rate of a mitotic cycle rate and to promote intercellular adhesion and enhancement of invasive growth and metastatic spread. Various factors involved in the regulation of proliferation of cells and their capacity for invasion and metastasis should be studied to make the most objective estimation of the degree of malignancy of a tumor and its prognosis.

Background: Sp100 is a permanent ProMyelocytic Leukaemian nuclear bodies (PML NB)-associated protein, and has been reported to participate in the regulation of transcriptional activity, apoptosis and other cellular biological processes. The aim of the present study was to explore the expression of Sp100 and its potential clinical implications in laryngeal cancer., Material/methods: The mRNA and protein levels of Sp100 in 96 laryngeal cancer samples and paired normal epithelium were examined by RT-PCR, Western blot and immuno-histochemical staining. The correlation of Sp100 expression with clinicopathological features of these patients was assessed by Chi-squared test., Results: The coherent low Sp100 expression of both transcriptional and translational levels were confirmed in the malignant tissues compared to the normal mucosa, and the expression was down-regulated among the well-, moderately- and poorly-differentiated cancer cells accordingly. Moreover, immuno-histochemical staining demonstrated that Sp100 showed a predominantly nuclear pattern in well-differentiated cancer cells, and diffuse cellular distribution in cytoplasm in poorly-differentiated cancer cells. Besides histological type of cancer cells, other clinicopathological characteristics, including age, sex, T classification, lymph node metastasis, distant metastasis and clinical stage, showed no significant correlation with Sp100 low expression., Conclusions: Our finding provides an important clue to further understanding the role of Sp100 in the initiation and progression of tumorgenesis.

Repair of DNA strand breaks induced during lymphoid antigen receptor rearrangement involves non-homologous end-joining (NHEJ). We investigated NHEJ in the aetiology of lymphoproliferative disorders (LPDs) and the disease subtypes therein through real-time quantitative RT-PCR gene expression analysis. Lower expression of XRCC6 and MRE11A was observed in all tumours, with higher expression of both XRCC4 and RAD50 observed only in multiple myeloma (MM). Hierarchical clustering enabled tumours to be clearly distinguished from controls, and by morphological sub-type. We postulate this identifies targets worthy of investigation in the genetic predisposition, pathogenesis and prognosis of lymphoid malignancies.

Multiple molecular resistance mechanisms reduce the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small cell lung cancer (NSCLC). We previously demonstrated that amphiregulin (Areg) inhibits gefitinib-induced apoptosis in NSCLC cells by inactivating the proapoptotic protein BAX. In this part of the investigation, we studied the molecular mechanisms leading to BAX inactivation. We show that Areg prevents gefitinib-mediated acetylation of Ku70. This augments the BAX-Ku70 interaction and therefore prevents BAX-mediated apoptosis. Accordingly, Areg or Ku70 knock down restore BAX activation and apoptosis in gefitinib-treated H358 cells in vitro. In addition, overexpression of the histone acetyltransferase (HAT) CREB-binding protein (CBP) or treatments with histone deacetylase (HDAC) inhibitors sensitize H358 cells to gefitinib. Moreover, a treatment with vorinostat, a HDAC inhibitor strongly sensitized tumors to gefitinib in vivo. These findings suggest new prospects in combining both HDAC and epidermal growth factor receptor inhibitors for the treatment of NSCLC.