The PARP-1 inhibitor Olaparib causes retention of γ-H2AX foci in BRCA1 heterozygote cells following exposure to gamma radiation

This article is made available through the Brunel Open Access Publishing Fund. Copyright © 2013 Emma C. Bourton et al. This is an open access article distributed under the Creative Commons Attribution Li-cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.A novel treatment for cancer patients with homozygous deletions of BRCA1 and BRCA2 is to use drugs that inhibit the enzyme poly(ADP-ribose) polymerase (PARP). Specific inhibition of PARP-1 can induce synthetic lethality in irradi- ated cancer cells while theoretically leaving normal tissue unaffected. We recently demonstrated in a cell survival assay that lymphoblastoid cells with mono-allelic mutations of BRCA1 were hypersensitive to gamma radiation in the pres- ence of the PARP-1 inhibitor Olaparib compared to normal cells and mono-allelic BRCA2 cells. To determine if the enhanced radiation sensitivity was due to a persistence of DNA strand breaks, we performed γ-H2AX foci analysis in cells derived from two normal individuals, three heterozygous BRCA1 and three heterozygous BRCA2 cell lines. Cells were exposed to 2 Gy gamma radiation in the presence or absence of 5 μM Olaparib. Using immunofluorescence and imaging flow cytometry, foci were measured in untreated cells and at 0.5, 3, 5 and 24 hours post-irradiation. In all lymphoblastoid cells treated with 2 Gy gamma radiation, there was a predictable induction of DNA strand breaks, with a modest but significant retention of foci over 24 hours in irradiated cells treated with Olaparib (ANOVA P < 0.05). However, in mono-allelic BRCA1 cells, there was a failure to fully repair DNA double-strand breaks (DSB) in the pres- ence of Olaparib, evidenced by a significant retention of foci at 24 hours’ post irradiation (t-Test P < 0.05). These data show that the cellular hypersensitivity of mono-allelic BRCA1 lymphoblastoid cells to gamma radiation in the presence of the Olaparib is due to the retention of DNA DSB. These data may indicate that patients with inherited mutations in the BRCA1 gene treated with radiotherapy and PARP-1 inhibitors may experience elevated radiation-associated normal tissue toxicity.Vidal Sassoon Foundation of America

Neisseria gonorrhoeae Infection Induces Altered Amphiregulin Processing and Release

Adhesion of the human pathogen Neisseria gonorrhoeae has established effects on the host cell and evokes a variety of cellular events including growth factor activation. In the present study we report that infection with N. gonorrhoeae causes altered amphiregulin processing and release in human epithelial cells. Amphiregulin is a well-studied growth factor with functions in various cell processes and is upregulated in different forms cancer and proliferative diseases. The protein is prototypically cleaved on the cell surface in response to external stimuli. We demonstrate that upon infection, a massive upregulation of amphiregulin mRNA is seen. The protein changes its subcellular distribution and is also alternatively cleaved at the plasma membrane, which results in augmented release of an infection-specific 36 kDa amphiregulin product from the surface of human cervical epithelial cells. Further, using antibodies directed against different domains of the protein we could determine the impact of infection on pro-peptide processing. In summary, we present data showing that the infection of N. gonorrhoeae causes an alternative amphiregulin processing, subcellular distribution and release in human epithelial cervical cells that likely contribute to the predisposition cellular abnormalities and anti-apoptotic features of N. gonorrhoeae infections

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

LightForm-group/formable: v0.1.21

&lt;h2&gt;[0.1.21] - 2023.11.09&lt;/h2&gt; &lt;h3&gt;Fixed&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Resolve numpy deprecation.&lt;/li&gt; &lt;/ul&gt

LightForm-group/matflow-damask: v0.1.22

[0.1.22] - 2021.11.09 Added Add saving of VE phases to a VTK file

LightForm-group/matflow-damask: v0.1.29

[0.1.29] - 2022.10.11 Fixed Fix bug in generate_volume_element_random_voronoi function where supplied orientations are not validated

LightForm-group/matflow-damask: v0.1.18

[0.1.18] - 2021.08.06 Added Add implementation of Taylor simulation task. Add implementation of task sample_texture method from_random. Changed Use new output map options (volume_data, phase_data, field_data and grain_data) for reading results from a simulation. Remove methods that made use of the DAMASK command line scripts. Add orientations_use_max_precision parameter to task/methods: generate_volume_element/random_voronoi, generate_volume_element/random_voronoi_from_orientations, sample_texture/from_random, generate_microstructure_seeds/random and simulate_volume_element_loading/CP_FFT

Enhanced γ-H2AX DNA damage foci detection using multimagnification and extended depth of field in imaging flow cytometry

Accurate and rapid methods for the detection of DNA damage foci in eukaryotic cells are central to DNA repair studies, which identify differences in DNA repair capacity in cell lines. Such assays have been important in delineating mechanisms of DNA repair in human cells. Previously we were the first to demonstrate the use of imaging flow cytometry for the detection of γ-H2AX foci in cells exposed to ionizing radiation causing the induction of DNA strand breaks. In this report we extend these studies and show an enhancement of foci quantitation and image resolution using next generation imaging flow cytometry with the Amnis ImagestreamX Mark II. We demonstrate using cell lines derived from normal individuals, and DNA double strand break repair defective cells that the number of foci observed is significantly increased when using 60× as compared to 40× magnification. Also, foci numbers and resolution is further increased with the application of the focus stacking (Extended Depth of Field-EDF) capacity activated. This report represents the first such demonstration of multimagnification and EDF for the enhanced quantitation of DNA damage in cells and provides a level of resolution, which near matches in situ microscopy methods for the detection of γ-H2AX foci.Bart's Charity, Cock Lane, London, UK . Grant Number: EC1A 9BU, 419/2071, and 419/218

Increased γ-H2AX and Rad51 DNA repair biomarker expression in human cell lines resistant to the chemotherapeutic agents nitrogen mustard and cisplatin.

Chemotherapeutic anticancer drugs mediate cytotoxicity by a number of mechanisms. However, alkylating agents which induce DNA interstrand cross links (ICL) are amongst the most effective anticancer agents and often form the mainstay of many anticancer therapies. The effectiveness of these drugs can be limited by the development of drug resistance in cancer cells and many studies have demonstrated that alterations in DNA repair kinetics are responsible for drug resistance. In this study we developed two cell lines resistant to the alkylating agents nitrogen mustard (HN2) and cisplatin (Pt). To determine if drug resistance was associated with enhanced ICL DNA repair we used immunocytochemistry and imaging flow cytometry to quantitate the number of gamma-H2AX and Rad51 foci in the nuclei of cells post drug exposure. Gamma-H2AX was used to evaluate DNA strand breaks caused by repair incision nucleases and Rad51 was used to measure the activity of homologous recombination (HR) in the repair of ICL. In the drug resistant derivative cell lines, overall there was a significant increase in the number and persistence of both gamma-H2AX and Rad51 foci in the nuclei of cells over a 72 hr period, when compared to the non-resistant parental cell lines (ANOVA P < 0.0001). In a cisplatin resistant ovarian cancer cell line (A2780cisR) a similar enhancement of DNA repair was observed when compared to the non-drug resistant wild type ovarian cancer cells (A2780) following exposure to nitrogen mustard. Our data suggest that using DNA repair biomarkers to evaluate mechanisms of resistance in cancer cell lines and human tumours may be of experimental and clinical benefit. We concede however, that examination of a larger population of cell lines and tumours is required to fully evaluate the validity of this approach.The Barts Charity, East London, U