Long Term Transcriptional Reactivation of Epigenetically Silenced Genes in Colorectal Cancer Cells Requires DNA Hypomethylation and Histone Acetylation

Epigenetic regulation of genes involves the coordination of DNA methylation and histone modifications to maintain transcriptional status. These two features are frequently disrupted in malignancy such that critical genes succumb to inactivation. 5-aza-2′-deoxycytidine (5-aza-dC) is an agent which inhibits DNA methyltransferase, and holds great potential as a treatment for cancer, yet the extent of its effectiveness varies greatly between tumour types. Previous evidence suggests expression status after 5-aza-dC exposure cannot be explained by the DNA methylation status alone. Aim: We sought to identify chromatin changes involved with short and long term gene reactivation following 5-aza-dC exposure. Two colorectal cancer cell lines, HCT116 and SW480, were treated with 5-aza-dC and then grown in drug-free media to allow DNA re-methylation. DNA methylation and chromatin modifications were assessed with bisulfite sequencing and Chromatin Immuno-Precipitation analysis. Results: Increased H3 acetylation, H3K4 tri-methylation and loss of H3K27 tri-methylation were associated with reactivation. Hypermethylated genes that did not show increased acetylation were transiently expressed with 5-aza-dC treatment before reverting to an inactive state. Three reactivated genes, CDO1, HSPC105 and MAGEA3, were still expressed 10 days post 5-aza-dC treatment and displayed localised hypomethylation at the transcriptional start site, and also an increased enrichment of histone H3 acetylation. Conclusions: These observations suggest that hypomethylation alone is insufficient to reactivate silenced genes and that increased Histone H3 acetylation in unison with localised hypomethylation allows long term reversion of these epigenetically silenced genes. This study suggests that combined DNA methyltransferase and histone deacetylase inhibitors may aid long term reactivation of silenced genes

Psychosocial impact of undergoing prostate cancer screening for men with BRCA1 or BRCA2 mutations.

OBJECTIVES: To report the baseline results of a longitudinal psychosocial study that forms part of the IMPACT study, a multi-national investigation of targeted prostate cancer (PCa) screening among men with a known pathogenic germline mutation in the BRCA1 or BRCA2 genes. PARTICPANTS AND METHODS: Men enrolled in the IMPACT study were invited to complete a questionnaire at collaborating sites prior to each annual screening visit. The questionnaire included sociodemographic characteristics and the following measures: the Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), 36-item short-form health survey (SF-36), Memorial Anxiety Scale for Prostate Cancer, Cancer Worry Scale-Revised, risk perception and knowledge. The results of the baseline questionnaire are presented. RESULTS: A total of 432 men completed questionnaires: 98 and 160 had mutations in BRCA1 and BRCA2 genes, respectively, and 174 were controls (familial mutation negative). Participants' perception of PCa risk was influenced by genetic status. Knowledge levels were high and unrelated to genetic status. Mean scores for the HADS and SF-36 were within reported general population norms and mean IES scores were within normal range. IES mean intrusion and avoidance scores were significantly higher in BRCA1/BRCA2 carriers than in controls and were higher in men with increased PCa risk perception. At the multivariate level, risk perception contributed more significantly to variance in IES scores than genetic status. CONCLUSION: This is the first study to report the psychosocial profile of men with BRCA1/BRCA2 mutations undergoing PCa screening. No clinically concerning levels of general or cancer-specific distress or poor quality of life were detected in the cohort as a whole. A small subset of participants reported higher levels of distress, suggesting the need for healthcare professionals offering PCa screening to identify these risk factors and offer additional information and support to men seeking PCa screening

John Watt : TV Blong Vanuatu

Werden discusses the impact of television on North America and the medium's ability to "contextualize a culture" in an introduction to Watt's video simulation of a day of community programming from Vanuatu, a culture sheltered from the global reaches of the medium

Drying of vegetation: how fast does the moisture go?

Live and dead vegetation both need to be dried out prior to combustion in a bushfire. This article presents a simple model that accounts for the heating that occurs inside a cylindrical vegetation sample which has a prescribed initial moisture content and is subjected to a heating regime characteristic of a passing bushfire. The model allows us to predict the death or survival of live vegetation samples of any given diameter. It can also be extended to include any thermal properties and any shape for the vegetation sample

Skeletal muscle total creatine content and creatine transporter gene expression in vegetarians prior to and following creatine supplementation

This study examined the effect of vegetarianism on skeletal muscle total creatine (TCr) content and creatine transporter (CreaT) gene expression, prior to and during 5 d of Cr supplementation (CrS). In a double-blind, crossover design, 7 vegetarians (VEG) and nonvegetarians (NVEG) were assigned Cr or placebo supplements for 5 d and after 5 wk, received the alternative treatment. Muscle sampling occurred before, and after 1 and 5 d of treatment ingestion. Basal muscle TCr content was lower (P &lt; 0.05) in VEG compared with NVEG. Muscle TCr increased (P &lt; 0.05) throughout the Cr trial in both groups but was greater (P &lt; 0.05) in VEG compared with NVEG, at days 1 and 5. CreaT gene expression was not different between VEG and NVEG. The results indicate that VEG have a lower muscle TCr content and an increased capacity to load Cr into muscle following CrS. Muscle CreaT gene expression does not appear to be affected by vegetarianism.<br /

Effect of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (CT) values were established for &szlig;-actin, &szlig;2-microglobulin (&szlig;2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between CT values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw CT values and the linear value of 2-CT, respectively. Interassay variability was 2.3% for raw CT values and 34% for the linear value of 2-CT. We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P &lt; 0.05) muscle total creatine content above placebo levels; however, there were no changes (P &gt; 0.05) in CT values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas &szlig;-actin, &szlig;2M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for &szlig;2M with more stable expressions for both &szlig;-actin and CYC. We conclude that, using real-time RT-PCR, &szlig;-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise.<br /