DNA methylation changes following DNA damage in prostate cancer cells.

Many cancer therapies operate by inducing double-strand breaks (DSBs) in cancer cells, however treatment-resistant cells rapidly initiate mechanisms to repair damage enabling survival. While the DNA repair mechanisms responsible for cancer cell survival following DNA damaging treatments are becoming better understood, less is known about the role of the epigenome in this process. Using prostate cancer cell lines with differing sensitivities to radiation treatment, we analysed the DNA methylation profiles prior to and following a single dose of radiotherapy (RT) using the Illumina Infinium HumanMethylation450 BeadChip platform. DSB formation and repair, in the absence and presence of the DNA hypomethylating agent, 5-azacytidine (5-AzaC), were also investigated using γH2A.X immunofluorescence staining. Here we demonstrate that DNA methylation is generally stable following a single dose of RT; however, a small number of CpG sites are stably altered up to 14 d following exposure. While the radioresistant and radiosensitive cells displayed distinct basal DNA methylation profiles, their susceptibility to DNA damage appeared similar demonstrating that basal DNA methylation has a limited influence on DSB induction at the regions examined. Recovery from DSB induction was also similar between these cells. Treatment with 5-AzaC did not sensitize resistant cells to DNA damage, but rather delayed recruitment of phosphorylated BRCA1 (S1423) and repair of DSBs. These results highlight that stable epigenetic changes are possible following a single dose of RT and may have significant clinical implications for cancer treatment involving recurrent or fractionated dosing regimens.

Optimized extraction of anthocyanins from Reid Fruits' Prunus avium 'Lapins' cherries.

The influence of process parameters on the extraction of anthocyanins from the edible portion of fresh, sweet cherry were investigated. The optimal extraction time and temperature were determined as 90 min and 37 °C, respectively. A solvent/solid ratio of 10 mL/g using 100% acidified solvent resulted in the greatest anthocyanin yield. No significant difference was observed between the use of methanol or ethanol as the extraction solvent. Ultra Performance Liquid Chromatography-MS analysis of the extract identified four anthocyanins, with cyanidin-3-rutinoside and peonidin-3-rutinoside accounting for over 95% of the anthocyanin content, while cyanidin-3-glucoside and pelargonidin-3-rutinoside accounted for the remaining 5%. 244 mg/100 g fresh weight total anthocyanins were determined in the fresh cherries using the optimal extraction conditions.

RNA-seq profiling of a radiation resistant and radiation sensitive prostate cancer cell line highlights opposing regulation of DNA repair and targets for radiosensitization.

BACKGROUND: Radiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy. METHODS: Using an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide. RESULTS: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02). CONCLUSIONS: These findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.

Elimination of background in film-based applications.

Erase-It Background Eliminator is a solution used directly on processed film to remove background or improve data resolution. Traditional methods, such as optimization of the scientific protocol or better estimation of exposure time, are tedious and uncertain. Nevertheless, autoradiography continues to be a simple, effective method to visualize data. Therefore, we evaluated the ability of Erase-It Working Solution to help solve background and resolution issues. To demonstrate the efficiency of the Background Eliminator, we analyzed the product's ability to remove signal evenly, performance on several brands of film, and usefulness with various detection methods. Even reduction of signal was demonstrated by performing densitometric analysis on film generated from a dot blot with serial dilutions of analyte. In addition, overexposed films from various suppliers were effectively treated to remove background and visualize data. Autoradiographs, generated with 32P-labeled probes, and chemiluminescent substrate were also processed resulting in clearer images. Our results demonstrate that film data can be treated quickly and conveniently without fear of artificial enhancement. We show the Background Eliminator to be a universal and timesaving tool to visualize results that otherwise may be difficult to interpret.