Epigenetics as a mechanism driving polygenic clinical drug resistance

Aberrant methylation of CpG islands located at or near gene promoters is associated with inactivation of gene expression during tumour development. It is increasingly recognised that such epimutations may occur at a much higher frequency than gene mutation and therefore have a greater impact on selection of subpopulations of cells during tumour progression or acquisition of resistance to anticancer drugs. Although laboratory-based models of acquired resistance to anticancer agents tend to focus on specific genes or biochemical pathways, such 'one gene : one outcome' models may be an oversimplification of acquired resistance to treatment of cancer patients. Instead, clinical drug resistance may be due to changes in expression of a large number of genes that have a cumulative impact on chemosensitivity. Aberrant CpG island methylation of multiple genes occurring in a nonrandom manner during tumour development and during the acquisition of drug resistance provides a mechanism whereby expression of multiple genes could be affected simultaneously resulting in polygenic clinical drug resistance. If simultaneous epigenetic regulation of multiple genes is indeed a major driving force behind acquired resistance of patients' tumour to anticancer agents, this has important implications for biomarker studies of clinical outcome following chemotherapy and for clinical approaches designed to circumvent or modulate drug resistance

The atm-1 gene is required for genome stability in Caenorhabditis elegans

The Ataxia-telangiectasia-mutated (ATM) gene in humans was identified as the basis of a rare autosomal disorder leading to cancer susceptibility and is now well known as an important signal transducer in response to DNA damage. An approach to understanding the conserved functions of this gene is provided by the model system, Caenorhabditis elegans. In this paper we describe the structure and loss of function phenotype of the ortholog atm-1. Using bioinformatic and molecular analysis we show that the atm-1 gene was previously misannotated. We find that the transcript is in fact a product of three gene predictions, Y48G1BL.2 (atm-1), K10E9.1, and F56C11.4 that together make up the complete coding region of ATM-1. We also characterize animals that are mutant for two available knockout alleles, gk186 and tm5027. As expected, atm-1 mutant animals are sensitive to ionizing radiation. In addition, however, atm-1 mutants also display phenotypes associated with genomic instability, including low brood size, reduced viability and sterility. We document several chromosomal fusions arising from atm-1 mutant animals. This is the first time a mutator phenotype has been described for atm-1 in C. elegans. Finally we demonstrate the use of a balancer system to screen for and capture atm-1-derived mutational events. Our study establishes C. elegans as a model for the study of ATM as a mutator potentially leading to the development of screens to identify therapeutic targets in humans

Chemically induced DNA hypomethylation in breast carcinoma cells detected by the amplification of intermethylated sites

INTRODUCTION: Compromised patterns of gene expression result in genomic instability, altered patterns of gene expression and tumour formation. Specifically, aberrant DNA hypermethylation in gene promoter regions leads to gene silencing, whereas global hypomethylation events can result in chromosomal instability and oncogene activation. Potential links exist between environmental agents and DNA methylation, but the destabilizing effects of environmental exposures on the DNA methylation machinery are not understood within the context of breast cancer aetiology. METHODS: We assessed genome-wide changes in methylation patterns using a unique methylation profiling technique called amplification of intermethylated sites (AIMS). This method generates easily readable fingerprints that represent the investigated cell line's methylation profile, based on the differential cleavage of DNA with methylation-specific isoschisomeric restriction endonucleases. RESULTS: We validated this approach by demonstrating both unique and reoccurring sites of genomic hypomethylation in four breast carcinoma cell lines treated with the cytosine analogue 5-azacytidine. Comparison of treated with control samples revealed individual bands that exhibited methylation changes, and these bands were excized and cloned, and the precise genomic location individually identified. In most cases, these regions of hypomethylation coincided with susceptible target regions previously associated with chromosome breakage, rearrangement and gene amplification. Similarly, we observed that acute benzopyrene exposure is associated with altered methylation patterns in these cell lines. CONCLUSION: These results reinforce the link between environmental exposures, DNA methylation and breast cancer, and support a role for AIMS as a rapid, affordable screening method to identify environmentally induced DNA methylation changes that occur in tumourigenesis

Negative Feedback Regulation following Administration of Chronic Exogenous Corticosterone

Administration of exogenous glucocorticoids is known to suppress the HPA axis and has been reported to occupy brain glucocorticoid receptors, eventually leading to down-regulation. To determine the effects of chronic corticosterone administration on HPA axis function, corticosterone was administered as both 25% and 50% corticosteronekholesterol pellets. Rats were sacrificed 6 days after corticosterone pellet implantation. The 25% corticosterone pellets produced a small increase in morning corticosterone concentrations but no change in evening ACTH or corticosterone secretion. The 50% corticosterone pellets produced constant corticosterone concentrations of 5–6 pg/dl, with no circadian variation in corticosterone, indicating inhibition of evening ACTH and corticosterone secretion. The 25% corticosterone pellets produced no significant decrease in thymus weight or in adrenal weight; 50% corticosterone pellets produced significant decreases in thymus weight and adrenal weight. Neither 25% nor 50% corticosterone pellets produced significant decreases in GR in hippocampus and cortex. The 50% corticosterone pellets treatment resulted in a decrease in anterior pituitary POMC mRNA levels, a decrease in baseline and oCRH stimulated ACTH release from the anterior pituitary, and a near complete inhibition of the AM and PM response to restraint stress. These results suggest that: 1) the HPA axis was able to adjust to the small increase in glucocorticoids produced by the 25% cort pellets with minimal disturbances in function and 2) 50% corticosterone pellets exert a significant inhibitory effect on stress and diurnal ACTH secretion which appears to be exerted at the pituitary as well as possible inhibitory effects on brain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72571/1/j.1365-2826.1995.tb00665.x.pd

Frequency of KCNC3 DNA Variants as Causes of Spinocerebellar Ataxia 13 (SCA13)

Gain-of function or dominant-negative mutations in the voltage-gated potassium channel KCNC3 (Kv3.3) were recently identified as a cause of autosomal dominant spinocerebellar ataxia. Our objective was to describe the frequency of mutations associated with KCNC3 in a large cohort of index patients with sporadic or familial ataxia presenting to three US ataxia clinics at academic medical centers.DNA sequence analysis of the coding region of the KCNC3 gene was performed in 327 index cases with ataxia. Analysis of channel function was performed by expression of DNA variants in Xenopus oocytes.Sequence analysis revealed two non-synonymous substitutions in exon 2 and five intronic changes, which were not predicted to alter splicing. We identified another pedigree with the p.Arg423His mutation in the highly conserved S4 domain of this channel. This family had an early-onset of disease and associated seizures in one individual. The second coding change, p.Gly263Asp, subtly altered biophysical properties of the channel, but was unlikely to be disease-associated as it occurred in an individual with an expansion of the CAG repeat in the CACNA1A calcium channel.Mutations in KCNC3 are a rare cause of spinocerebellar ataxia with a frequency of less than 1%. The p.Arg423His mutation is recurrent in different populations and associated with early onset. In contrast to previous p.Arg423His mutation carriers, we now observed seizures and mild mental retardation in one individual. This study confirms the wide phenotypic spectrum in SCA13

Microcin H47 System: An Escherichia coli Small Genomic Island with Novel Features

Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria

Narrow band imaging for the detection of gastric intestinal metaplasia and dysplasia during surveillance endoscopy

Background: Surveillance of premalignant gastric lesions relies mainly on random biopsy sampling. Narrow band imaging (NBI) may enhance the accuracy of endoscopic surveillance of intestinal metaplasia (IM) and dysplasia. We aimed to compare the yield of NBI to white light endoscopy (WLE) in the surveillance of patients with IM and dysplasia. Methods: Patients with previously identified gastric IM or dysplasia underwent a surveillance endoscopy. Both WLE and NBI were performed in all patients during a single procedure. The sensitivity of WLE and NBI for the detection of premalignant lesions was calculated by correlating endoscopic findings to histological diagnosis. Results: Forty-three patients (28 males and 15 females, mean age 59 years) were included. IM was diagnosed in 27 patients; 20 were detected by NBI and WLE, four solely by NBI and three by random biopsies only. Dysplasia was detected in seven patients by WLE and NBI and in two patients by random biopsies only. Sixty-eight endoscopically detected lesions contained IM: 47 were detected by WLE and NBI, 21 by NBI only. Nine endoscopically detected lesions demonstrated dysplasia: eight were detected by WLE and NBI, one was detected by NBI only. The sensitivity, specificity, positive and negative predictive values for detection of premalignant lesions were 71, 58, 65 and 65% for NBI and 51, 67, 62 and 55% for WLE, respectively. Conclusions: NBI increases the diagnostic yield for detection of advanced premalignant gastric lesions compared to routine WLE

Chasing Migration Genes: A Brain Expressed Sequence Tag Resource for Summer and Migratory Monarch Butterflies (Danaus plexippus)

North American monarch butterflies (Danaus plexippus) undergo a spectacular fall migration. In contrast to summer butterflies, migrants are juvenile hormone (JH) deficient, which leads to reproductive diapause and increased longevity. Migrants also utilize time-compensated sun compass orientation to help them navigate to their overwintering grounds. Here, we describe a brain expressed sequence tag (EST) resource to identify genes involved in migratory behaviors. A brain EST library was constructed from summer and migrating butterflies. Of 9,484 unique sequences, 6068 had positive hits with the non-redundant protein database; the EST database likely represents ∼52% of the gene-encoding potential of the monarch genome. The brain transcriptome was cataloged using Gene Ontology and compared to Drosophila. Monarch genes were well represented, including those implicated in behavior. Three genes involved in increased JH activity (allatotropin, juvenile hormone acid methyltransfersase, and takeout) were upregulated in summer butterflies, compared to migrants. The locomotion-relevant turtle gene was marginally upregulated in migrants, while the foraging and single-minded genes were not differentially regulated. Many of the genes important for the monarch circadian clock mechanism (involved in sun compass orientation) were in the EST resource, including the newly identified cryptochrome 2. The EST database also revealed a novel Na+/K+ ATPase allele predicted to be more resistant to the toxic effects of milkweed than that reported previously. Potential genetic markers were identified from 3,486 EST contigs and included 1599 double-hit single nucleotide polymorphisms (SNPs) and 98 microsatellite polymorphisms. These data provide a template of the brain transcriptome for the monarch butterfly. Our “snap-shot” analysis of the differential regulation of candidate genes between summer and migratory butterflies suggests that unbiased, comprehensive transcriptional profiling will inform the molecular basis of migration. The identified SNPs and microsatellite polymorphisms can be used as genetic markers to address questions of population and subspecies structure

CpG island methylation is a common finding in colorectal cancer cell lines

Tumour cell lines are commonly used in colorectal cancer (CRC) research, including studies designed to assess methylation defects. Although many of the known genetic aberrations in CRC cell lines have been comprehensively described, no studies have been performed on their methylation status. In this study, 30 commonly used CRC cell lines as well as seven primary tumours from individuals with hereditary nonpolyposis colorectal cancer (HNPCC) were assessed for methylation at six CpG islands known to be hypermethylated in colorectal cancer: hMLH1, p16, methylated in tumour (MINT-)-1, -2, -12 and -31. The cell lines were also assessed for microsatellite instability (MSI), ploidy status, hMLH1 expression, and mutations in APC and Ki-ras. Methylation was frequently observed at all examined loci in most cell lines, and no differences were observed between germline-derived and sporadic cell lines. Methylation was found at MINT 1 in 63%, MINT 2 in 57%, MINT 12 in 71%, MINT 31 in 53%, p16 in 71%, and hMLH1 in 30% of cell lines. Overall only one cell line, SW1417, did not show methylation at any locus. Methylation was found with equal frequency in MSI and chromosomally unstable lines. MSI was over-represented in the cell lines relative to sporadic CRC, being detected in 47% of cell lines. The rate of codon 13 Ki-ras mutations was also over three times that expected from in vivo studies. We conclude that CpG island hypermethylation, whether acquired in vivo or in culture, is a ubiquitous phenomenon in CRC cell lines. We suggest that CRC cell lines may be only representative of a small subset of real tumours, and this should be taken into account in the use of CRC cell lines for epigenetic studies