Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells. (C) 2008 by Radiation Research Society.

Apoptosis is initiated in human keratinocytes exposed to signalling factors from microbeam irradiated cells.

PURPOSE: There is now no doubt that bystander signalling from irradiated cells occurs and causes a variety of responses in cells not targeted by the ionizing track. However, the mechanisms underlying these processes are unknown and the relevance to radiotherapy and risk assessment remains controversial. Previous research by our laboratory has shown bystander effects in a human keratinocyte cell line, HPV-G cells, exposed to medium from gamma irradiated HPV-G cells. The aim of this work was to investigate if similar mechanisms to those identified in medium transfer experiments occurred in these HPV-G cells when they are in the vicinity of microbeam irradiated cells. Demonstration of a commonality of mechanisms would support the idea that the process is not artifactual. MATERIALS AND METHODS: HPV-G cells were plated as two separate populations on mylar dishes. One population was directly irradiated using a charged particle microbeam (1 - 10 protons). The other population was not irradiated. Bystander factor-induced apoptosis was investigated in both populations following treatment by monitoring the levels of reactive oxygen species and mitochondrial membrane potential using fluorescent probes. Expression of the anti-apoptotic protein, bcl-2, and cytochrome c were determined, as well as apoptosis levels. RESULTS: Microbeam irradiation induced increases in reactive oxygen species and decreases in mitochondrial membrane potential at 6 h post-exposure, increased expression of bcl-2 and cytochrome c release at 6.5 h and increased apoptosis at 24 h. CONCLUSION: This study shows that similar bystander signalling pathways leading to apoptosis are induced following microbeam irradiation and following medium transfer. This demonstrates that the mechanisms involved are common across different radiation qualities and conditions and indicates that they may be relevant in vivo.

Neuropeptide YY1 receptor in human dental pulp cells of noncarious and carious teeth

Aim To determine the distribution of the NPY Y1 receptor in carious and noncarious human dental pulp tissue using immunohistochemistry. A subsidiary aim was to confirm the presence of the NPY Y1 protein product in membrane fractions of dental pulp tissue from carious and noncarious teeth using western blotting. Methodology Twenty two dental pulp samples were collected from carious and noncarious extracted teeth. Ten samples were processed for immunohistochemistry using a specific antibody to the NPY Y1 receptor. Twelve samples were used to obtain membrane extracts which were electrophoresed, blotted onto nitrocellulose and probed with NPY Y1 receptor antibody. Kruskal-Wallis one-way analysis of variance was employed to test for overall statistical differences between NPY Y1 levels in noncarious, moderately carious and grossly carious teeth. Results Neuropeptide Y Y1 receptor immunoreactivity was detected on the walls of blood vessels in pulp tissue from noncarious teeth. In carious teeth NPY Y1 immunoreactvity was observed on nerve fibres, blood vessels and inflammatory cells. Western blotting indicated the presence and confirmed the variability of NPY Y1 receptor protein expression in solubilised membrane preparations of human dental pulp tissue from carious and noncarious teeth. Conclusions Neuropeptide Y Y1 is expressed in human dental pulp tissue with evidence of increased expression in carious compared with noncarious teeth, suggesting a role for NPY Y1 in modulation of caries induced pulpal inflammation. © 2008 International Endodontic Journal.

A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6

Lung T lymphocytes are important in pulmonary immunity and inflammation. it has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5% +/- 1.9% (n = 11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8 +/- 2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells. (C) 2008 Elsevier B.V. All rights reserved.

Upregulation of Oxidative Stress Markers in Human Microvascular Endothelial Cells by Complexes of Serum Albumin and Digestion Products of Glycated Casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95 degrees C for 5 h to give AGE-casein (AGE-Cas). Simulated stomach and small intestine digestion of AGE-Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)-LMM-AGE-Cas complexes. Stimulation of human microvascular endothelial cells with BSA-LMM-AGE-Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin-3 (AGE-113), tumor necrosis factor alpha, and a marker of the mitogen-activated protein kinase pathway (MAPK-1), as well as p65NF-kappa B activation. Cells treated with LMM digestion products of AGE-Cas significantly increased AGE-R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA-LMM-AGE-Cas and LMM-AGE-Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and down-stream inflammatory pathways. AGE-R3 may protect against these effects.

Adenosine induces histamine release from human bronchoalveolar lavage mast cells

Previous studies have shown that in vitro adenosine enhances histamine release from activated human lung mast cells obtained by enzymic dispersion of lung parenchyma. However, adenosine alone has no effect on histamine release from these cells. Given the evidence for direct activation of mast cells after endobronchial challenge with adenosine and previous studies indicating that mast cells obtained at bronchoalveolar lavage are a better model for asthma studies than those obtained by enzymic dispersion of lung tissue, the histamine-releasing effect of adenosine was examined on lavage mast cells. Bronchoalveolar lavage fluid was obtained from patients attending hospital for routine bronchoscopy (n = 54). Lavage cells were challenged with adenosine or adenosine receptor agonists (20 min, 37 degrees C) and histamine release determined using an automated fluorometric assay. Endogenous adenosine levels were also measured in lavage fluid (n = 9) via an HPLC method. Adenosine alone caused histamine release from ravage mast cells in 37 of 54 patients with a maximal histamine release of 20.56 +/- 2.52% (range 5.2-61 %). The adenosine receptor agonists (R)-N-6-(2-phenylisopropyl)adenosine, 5'-N-ethylcarboxamido-adenosine and CGS21680 also induced histamine release from lavage mast cells. Preincubation of lavage mast cells with the adenosine receptor antagonist xanthine amine congener caused significant inhibition of the response to adenosine (P = 0.007). There was an inverse correlation between endogenous adenosine levels in the lavage fluid and the maximal response to in vitro adenosine challenge of the lavage cells. The findings of the present study indicate a means by which adenosine challenge of the airways can induce bronchoconstriction and support a role for adenosine in the pathophysiology of asthma. The results also suggest that cells obtained from bronchoalveolar ravage fluid may provide the ideal model for the testing of novel, adenosine receptor, targeted therapies for asthma.

Substance P induces histamine release from human pulmonary mast cells.

Substance P elicits histamine release from human skin and rodent mast cells. Since neuropeptide-mediated reflexes may be important in asthma, we examined the ability of substance P to stimulate human mast cells obtained at bronchoalveolar lavage (BAL). BAL samples were obtained at routine bronchoscopy from 35 non-preselected patients. Histamine release experiments were performed in a standard manner using substance P and the calcium ionophore A23187. Both substance P (50 μM) and A23187 caused histamine release (median 26.7%, range 6.2–62.8% and 32.1%, 7.7–56.8% respectively) which was significantly greater (P < 0.0001) than the spontaneous release (median 15.6%, range 4.1–33.4%), i.e. that in the absence of any stimulus. Substance P induced histamine release was via an energy dependent process and was blocked by preincubation with antimycin A. A significant correlation was observed between substance P induced release and spontaneous release but was not observed with A23187 induced release. Mast cell counts correlated significantly with substance P induced release but not with spontaneous or A23187 induced release. The substance P induced histamine secretion was elicited at similar concentrations to those used with rodent and human skin mast cells. Asthma is associated with increased numbers of mast cells which have both increased spontaneous and stimulated secretory responses. Thus, in vivo, the bronchoconstrictor action of substance P may in part result from activation of mast cells in the bronchial lumen.

Anti-apoptotic effects of Z alpha-1 antitrypsin in human bronchial epithelial cells.

α1-antitrypsin (α1-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of α1-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z α1-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved.

Control, M variant α1-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-κB activation and induced expression of a selection of pro- and anti-apoptotic genes.

Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-κB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-κB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies.

The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Persistent measles virus infection of mouse neural cells lacking known human entry receptors

Aims: Infection of the mouse central nervous system with wild type (WT) and vaccine strains of measles virus (MV) results in lack of clinical signs and limited antigen detection. It is considered that cell entry receptors for these viruses are not present on murine neural cells and infection is restricted at cell entry.

Methods: To examine this hypothesis, virus antigen and caspase 3 expression (for apoptosis) was compared in primary mixed, neural cell cultures infected in vitro or prepared from mice infected intracerebrally with WT, vaccine or rodent neuroadapted viruses. Viral RNA levels were examined in mouse brain by nested and real-time reverse transcriptase polymerase chain reaction.

Results: WT and vaccine strains were demonstrated for the first time to infect murine oligodendrocytes in addition to neurones despite a lack of the known MV cell receptors. Unexpectedly, the percentage of cells positive for viral antigen was higher for WT MV than neuroadapted virus in both in vitro and ex vivo cultures. In the latter the percentage of positive cells increased with time after mouse infection. Viral RNA (total and mRNA) was detected in brain for up to 20 days, while cultures were negative for caspase 3 in WT and vaccine virus infections.

Conclusions: WT and vaccine MV strains can use an endogenous cell entry receptor(s) or alternative virus uptake mechanism in murine neural cells. However, viral replication occurs at a low level and is associated with limited apoptosis. WT MV mouse infection may provide a model for the initial stages of persistent MV human central nervous system infections.