The role of TET-mediated DNA hydroxymethylation in prostate cancer.

Ten-eleven translocation (TET) proteins are recently characterized dioxygenases that regulate demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further derivatives. The recent finding that 5hmC is also a stable and independent epigenetic modification indicates that these proteins play an important role in diverse physiological and pathological processes such as neural and tumor development. Both the genomic distribution of (hydroxy)methylation and the expression and activity of TET proteins are dysregulated in a wide range of cancers including prostate cancer. Up to now it is still unknown how changes in TET and 5(h)mC profiles are related to the pathogenesis of prostate cancer. In this review, we explore recent advances in the current understanding of how TET expression and function are regulated in development and cancer. Furthermore, we look at the impact on 5hmC in prostate cancer and the potential underlying mechanisms. Finally, we tried to summarize the latest techniques for detecting and quantifying global and locus-specific 5hmC levels of genomic DNA.

Integration of copy number and transcriptomics provides risk stratification in prostate cancer: A discovery and validation cohort study.

BACKGROUND: Understanding the heterogeneous genotypes and phenotypes of prostate cancer is fundamental to improving the way we treat this disease. As yet, there are no validated descriptions of prostate cancer subgroups derived from integrated genomics linked with clinical outcome. METHODS: In a study of 482 tumour, benign and germline samples from 259 men with primary prostate cancer, we used integrative analysis of copy number alterations (CNA) and array transcriptomics to identify genomic loci that affect expression levels of mRNA in an expression quantitative trait loci (eQTL) approach, to stratify patients into subgroups that we then associated with future clinical behaviour, and compared with either CNA or transcriptomics alone. FINDINGS: We identified five separate patient subgroups with distinct genomic alterations and expression profiles based on 100 discriminating genes in our separate discovery and validation sets of 125 and 103 men. These subgroups were able to consistently predict biochemical relapse (p = 0.0017 and p = 0.016 respectively) and were further validated in a third cohort with long-term follow-up (p = 0.027). We show the relative contributions of gene expression and copy number data on phenotype, and demonstrate the improved power gained from integrative analyses. We confirm alterations in six genes previously associated with prostate cancer (MAP3K7, MELK, RCBTB2, ELAC2, TPD52, ZBTB4), and also identify 94 genes not previously linked to prostate cancer progression that would not have been detected using either transcript or copy number data alone. We confirm a number of previously published molecular changes associated with high risk disease, including MYC amplification, and NKX3-1, RB1 and PTEN deletions, as well as over-expression of PCA3 and AMACR, and loss of MSMB in tumour tissue. A subset of the 100 genes outperforms established clinical predictors of poor prognosis (PSA, Gleason score), as well as previously published gene signatures (p = 0.0001). We further show how our molecular profiles can be used for the early detection of aggressive cases in a clinical setting, and inform treatment decisions. INTERPRETATION: For the first time in prostate cancer this study demonstrates the importance of integrated genomic analyses incorporating both benign and tumour tissue data in identifying molecular alterations leading to the generation of robust gene sets that are predictive of clinical outcome in independent patient cohorts.

Peroxiredoxin-3 is overexpressed in prostate cancer and promotes cancer cell survival by protecting cells from oxidative stress.

OBJECTIVE: We have previously identified peroxiredoxin-3 (PRDX-3) as a cell-surface protein that is androgen regulated in the LNCaP prostate cancer (PCa) cell line. PRDX-3 is a member of the peroxiredoxin family that are responsible for neutralising reactive oxygen species. EXPERIMENTAL DESIGN: PRDX-3 expression was examined in tissue from 32 patients using immunohistochemistry. Subcellular distribution was determined using confocal microscopy. PRDX-3 expression was determined in antiandrogen-resistant cell lines by western blotting and quantitative RT-PCR. The pathways of PRDX-3 overexpression and knockdown on apoptosis and response to oxidative stress were investigated using protein arrays. RESULTS: PRDX-3 is upregulated in a number of endocrine-regulated tumours; in particular in PCa and prostatic intraepithelial neoplasia. Although the majority of PRDX-3 is localised to the mitochondria, we have confirmed that PRDX-3 at the cell membrane is androgen regulated. In antiandrogen-resistant LNCaP cell lines, PRDX-3 is upregulated at the protein but not RNA level. Resistant cells also possess an upregulation of the tricarboxylic acid (TCA) pathway and resistance to H2O2-induced apoptosis through a failure to activate pro-apoptotic pathways. Knockdown of PRDX-3 restored H2O2 sensitivity. CONCLUSION: Our results suggest that PRDX-3 has an essential role in regulating oxidation-induced apoptosis in antiandrogen-resistant cells. PRDX-3 may have potential as a therapeutic target in castrate-independent PCa.

Considerations for the processing and analysis of GoldenGate-based two-colour Illumina platforms.

Illumina's GoldenGate technology is a two-channel microarray platform that allows for the simultaneous interrogation of about 1,500 locations in the genome. GoldenGate has proved a flexible platform not only in the choice of those 1,500 locations, but also in the choice of the property being measured at them. It retains the desirable properties of Illumina's BeadArrays in that the probes (in this case 'beads') are randomly arranged across the microarray, there are multiple instances of each probe and many samples can be processed simultaneously. As for other Illumina technologies, however, these properties are not exploited as they might be. Here we review the various common adaptations of the GoldenGate platform, review the analysis methods that are associated with each adaptation and then, with the aid of a number of example data sets we illustrate some of the improvements that can be made over the default analysis.

BASH: a tool for managing BeadArray spatial artefacts.

SUMMARY: With their many replicates and their random layouts, Illumina BeadArrays provide greater scope fordetecting spatial artefacts than do other microarray technologies. They are also robust to artefact exclusion, yet there is a lack of tools that can perform these tasks for Illumina. We present BASH, a tool for this purpose. BASH adopts the concepts of Harshlight, but implements them in a manner that utilizes the unique characteristics of the Illumina technology. Using bead-level data, spatial artefacts of various kinds can thus be identified and excluded from further analyses. AVAILABILITY: The beadarray Bioconductor package (version 1.10 onwards), www.bioconductor.org

Early radiotherapy dose response and lack of hypersensitivity effect in normal brain tissue: a sequential dynamic susceptibility imaging study of cerebral perfusion.

AIMS: To determine if magnetic resonance perfusion markers can be used as an analytical marker of subclinical normal brain injury after radiotherapy, by looking for a dose-effect relationship. MATERIALS AND METHODS: Four patients undergoing conformal radiotherapy to 54Gy in 30 fractions for low-grade gliomas were imaged with conventional T(2)-weighted and fluid attenuated inversion recovery imaging as well as dynamic contrast susceptibility perfusion imaging. Forty regions of interest were determined from the periventricular white matter. All conventional sequences were examined for evidence of radiation-induced changes. Patients were imaged before radiotherapy, after one fraction, at the end of treatment and then at 1 and 3 months from the end of radiotherapy. For each region the relative cerebral blood volume (rCBV), relative cerebral blood flow (rCBF) and mean transit time (MTT) expressed as a ratio of the baseline value, and radiotherapy dose were determined. RESULTS: Of the 40 regions, seven occurred within the gross tumour volume and a further four occurred in regions later infiltrated by tumour, and were thus excluded. Regions within the 80% isodose showed a reduction in rCBV and rCBF over the 3 month period. There was no significant alteration in rCBV or rCBF in regions outside the 60% isodose (i.e. <32Gy). MTT did not alter in any region. There seemed to be a threshold effect at 132 days from the end of radiotherapy of 47% (standard error of the mean 11.5, about 25.4Gy) for rCBV and 59% (standard error of the mean 14.2, about 31.9Gy) for rCBF. CONCLUSIONS: There was a dose-related reduction in rCBV and rCBF in normal brain after radiotherapy at higher dose levels. Although this study used a limited number of patients, it suggests that magnetic resonance perfusion imaging seems to act as a marker of subclinical response of normal brain and that there is an absence of an early hypersensitivity effect with small doses per fraction. Further studies are required with larger groups of patients to show that these results are statistically robust.

Overnight intensive recovery: elective open aortic surgery without a routine ICU bed.

OBJECTIVES: Most patients are managed on the intensive care unit (ICU) after elective open aortic surgery. We preoperatively identify patients suitable for extubation in theatre with overnight management in theatre recovery before discharge back to the ward (overnight intensive recovery (OIR)). The safety of this was investigated. DESIGN: Retrospective case note analysis of all patients who underwent EOAS from 1998 to 2002, recording in-hospital morbidity and mortality. Physiological and operative severity score for the enUmeration of mortality and morbidity (POSSUM) data were collected prospectively. METHODS: Patients were divided into those selected for OIR and those booked for elective ICU admission. Observed morbidity and mortality data were compared with predicted outcomes generated by Portsmouth-POSSUM and POSSUM equations. RESULTS: Hundred and fifty-two out of 178 patients used OIR; 155 patients had abdominal aortic aneurysm (AAA) repair. The elective ICU group had significantly higher anaesthetic risk scores (ASA grade), larger AAA, greater intraoperative blood loss and longer operations. In the OIR group, ten patients (7%) needed ICU admission within 48h postoperatively. Complications occurred in 85/152, with two deaths. There was no excess morbidity or mortality in the OIR group (predicted 95% CI 83-105 and 5-17, respectively). CONCLUSION: Most patients having elective open aortic surgery can be managed safely using OIR.

Single nucleotide polymorphisms of the protoporphyrinogen oxidase gene: inter-population heterogeneity of allelic variation.

Five single nucleotide polymorphisms (SNPs) in the protoporphyrinogen oxidase gene (PPOX) were used for inter-population comparisons of six South African populations and two non-South African Caucasian populations. Novel polymorphisms identified in the promoter region and exon 11 of the PPOX gene, as well as three known variants in exon 1 and intron 2, were analysed using single-strand conformation polymorphism (SSCP) and restriction enzyme analyses. Significant population differences were found for four of the five polymorphisms analysed. A G-to-A transition was found at nucleotide position -1081 and is the first polymorphism to be identified in the 5' promoter region of the gene. A novel A-to-C substitution at nucleotide position 3880 in exon 11 was not detected in subjects of European descent. This study represents the first inter-population comparison of allelic variation at the PPOX locus. The significant differences observed between populations demonstrate the importance of population considerations when marker association studies are performed at this locus.

Polysome activity in relation to growth and protein starvation in brains and hearts of cultured early chick embryos.

In previous studies, brains but not hearts of intact early chick embryos were found to be sensitive to protein starvation. In this study, the in vitro protein synthetic activity of polysomes isolated from brains was found to be greater than those isolated from hearts. Starvation reduced the protein synthetic activity of polysomes in vitro but the extent of the reduction was approximately the same for both brains and hearts. A reduction in the amount of ribosomes as polysomes may have contributed to the lower synthetic activity of polysomes from tissues of starved embryos but not to the differences in synthetic activities between brains and hearts. In addition, neither the stability of isolated polysomes nor ribosome-associated ribonuclease activity appeared responsible for the differences observed in polysome synthetic activities. In direct relationship to the differential sensitivity of brains and hearts to starvation observed in the intact embryo, ribosomes isolated from brains of both growing and starved embryos were more readily degraded during in vitro incubation than those from hearts.

FOOD TEXTURE MEASUREMENTS IN CANADA.

A survey of the food industry, government agencies and educational institutions dealing with foods has been conducted to establish the nature and extent to which instrumental texture measurements are employed in Canada. Two hundred and fifteen questionnaires were sent out and 123 (57%) returned. Of those replying, 52% were using texture evaluation instruments. Within the food industry the use of texture measuring instruments was as follows: meat 36%; fish 20%; canner/freezer 79%; dairy 42%; confectionery 73%; baking 50%; fats and oils 78%; multi product 77%; beverage 0%. Most widely used instruments in the quality control area were rotational viscometers and penetrometers. The more complex instruments, such as the Universal Testing Machine and the Shear Press, were used to a larger extent in the research area. The use of some instruments appeared to be restricted to particular commodity groups. The questionnaire dealt among other things with the use of taste panels, statistical evaluation of results, and the need for expanded use of instruments. There was general agreement that more standardization of methods is desirable.