DNA methylome-wide alterations associated with estrogen receptor-dependent effects of bisphenols in breast cancer.

BACKGROUND: Bisphenol A (BPA), an estrogen-like endocrine disruptor used in plastics, has been associated with development and promotion of breast cancer, so plastic manufacturers shifted towards less-studied analogs, BPF and BPS. Studying the associated DNA methylome-wide mechanisms of these derivatives is timely, particularly in comparison with BPA. METHODS: We assessed proliferation, cell cycle, and migration of breast cancer cells (estrogen receptor (ER)-positive: MCF-7 and ER-negative: MDA-MB-231) treated with BPF and BPS ± estrogen receptor inhibitor (ERI) in comparison to BPA ± ERI. RNA expression and activity of DNA (de)methylation enzymes and LINE-1 methylation were quantified. DNA methylome-wide analysis was evaluated in bisphenol-exposed cells and compared to clinical breast cancer data. RESULTS: The three bisphenols caused ER-dependent increased proliferation and migration of MCF-7 but not MDA-MB-231 cells, with BPS being 10 times less potent than BPA and BPF. Although they have similar chemical structures, the three bisphenols induced differential DNA methylation alterations at several genomic clusters of or single CpG sites, with the majority of these being ER-dependent. At equipotent doses, BPA had the strongest effect on the methylome, followed by BPS then BPF. No pathways were enriched for BPF while BPA- and BPS-induced methylome alterations were enriched in focal adhesion, cGMP-PKG, and cancer pathways, which were also dysregulated in methylome-wide alterations comparing ER-positive breast cancer samples to adjacent normal tissues. CONCLUSIONS: The three bisphenols have important epigenetic effects in breast cell lines, with those of BPA and BPS overlapping with cancer-related pathways in clinical breast cancer models. Hence, further investigation of their safety is warranted.

Association of leukocyte DNA methylation changes with dietary folate and alcohol intake in the EPIC study.

BACKGROUND: There is increasing evidence that folate, an important component of one-carbon metabolism, modulates the epigenome. Alcohol, which can disrupt folate absorption, is also known to affect the epigenome. We investigated the association of dietary folate and alcohol intake on leukocyte DNA methylation levels in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Leukocyte genome-wide DNA methylation profiles on approximately 450,000 CpG sites were acquired with Illumina HumanMethylation 450K BeadChip measured among 450 women control participants of a case-control study on breast cancer nested within the EPIC cohort. After data preprocessing using surrogate variable analysis to reduce systematic variation, associations of DNA methylation with dietary folate and alcohol intake, assessed with dietary questionnaires, were investigated using CpG site-specific linear models. Specific regions of the methylome were explored using differentially methylated region (DMR) analysis and fused lasso (FL) regressions. The DMR analysis combined results from the feature-specific analysis for a specific chromosome and using distances between features as weights whereas FL regression combined two penalties to encourage sparsity of single features and the difference between two consecutive features. RESULTS: After correction for multiple testing, intake of dietary folate was not associated with methylation level at any DNA methylation site, while weak associations were observed between alcohol intake and methylation level at CpG sites cg03199996 and cg07382687, with qval = 0.029 and qval = 0.048, respectively. Interestingly, the DMR analysis revealed a total of 24 and 90 regions associated with dietary folate and alcohol, respectively. For alcohol intake, 6 of the 15 most significant DMRs were identified through FL. CONCLUSIONS: Alcohol intake was associated with methylation levels at two CpG sites. Evidence from DMR and FL analyses indicated that dietary folate and alcohol intake may be associated with genomic regions with tumor suppressor activity such as the GSDMD and HOXA5 genes. These results were in line with the hypothesis that epigenetic mechanisms play a role in the association between folate and alcohol, although further studies are warranted to clarify the importance of these mechanisms in cancer.

Epigenetics as a mechanism linking developmental exposures to long-term toxicity.

A variety of experimental and epidemiological studies lend support to the Developmental Origin of Health and Disease (DOHaD) concept. Yet, the actual mechanisms accounting for mid- and long-term effects of early-life exposures remain unclear. Epigenetic alterations such as changes in DNA methylation, histone modifications and the expression of certain RNAs have been suggested as possible mediators of long-term health effects of environmental stressors. This report captures discussions and conclusions debated during the last Prenatal Programming and Toxicity meeting held in Japan. Its first aim is to propose a number of criteria that are critical to support the primary contribution of epigenetics in DOHaD and intergenerational transmission of environmental stressors effects. The main criteria are the full characterization of the stressors, the actual window of exposure, the target tissue and function, the specificity of the epigenetic changes and the biological plausibility of the linkage between those changes and health outcomes. The second aim is to discuss long-term effects of a number of stressors such as smoking, air pollution and endocrine disruptors in order to identify the arguments supporting the involvement of an epigenetic mechanism. Based on the developed criteria, missing evidence and suggestions for future research will be identified. The third aim is to critically analyze the evidence supporting the involvement of epigenetic mechanisms in intergenerational and transgenerational effects of environmental exposure and to particularly discuss the role of placenta and sperm. While the article is not a systematic review and is not meant to be exhaustive, it critically assesses the contribution of epigenetics in the long-term effects of environmental exposures as well as provides insight for future research.

DNA methylation signal has a major role in the response of human breast cancer cells to the microenvironment.

Breast cancer-associated fibroblasts (CAFs) have a crucial role in tumor initiation, metastasis and therapeutic resistance by secreting various growth factors, cytokines, protease and extracellular matrix components. Soluble factors secreted by CAFs are involved in many pathways including inflammation, metabolism, proliferation and epigenetic modulation, suggesting that CAF-dependent reprograming of cancer cells affects a large set of genes. This paracrine signaling has an important role in tumor progression, thus deciphering some of these processes could lead to relevant discoveries with subsequent clinical implications. Here, we investigated the mechanisms underlying the changes in gene expression patterns associated with the cross-talk between breast cancer cells and the stroma. From RNAseq data obtained from breast cancer cell lines grown in presence of CAF-secreted factors, we identified 372 upregulated genes, exhibiting an expression level positively correlated with the stromal content of breast cancer specimens. Furthermore, we observed that gene expression changes were not mediated through significant DNA methylation changes. Nevertheless, CAF-secreted factors but also stromal content of the tumors remarkably activated specific genes characterized by a DNA methylation pattern: hypermethylation at transcription start site and shore regions. Experimental approaches (inhibition of DNA methylation, knockdown of methyl-CpG-binding domain protein 2 and chromatin immunoprecipitation assays) indicated that this set of genes was epigenetically controlled. These data elucidate the importance of epigenetics marks in the cancer cell reprogramming induced by stromal cell and indicated that the interpreters of the DNA methylation signal have a major role in the response of the cancer cells to the microenvironment.

Modeling cancer driver events in vitro using barrier bypass-clonal expansion assays and massively parallel sequencing.

The information on candidate cancer driver alterations available from public databases is often descriptive and of limited mechanistic insight, which poses difficulties for reliable distinction between true driver and passenger events. To address this challenge, we performed in-depth analysis of whole-exome sequencing data from cell lines generated by a barrier bypass-clonal expansion (BBCE) protocol. The employed strategy is based on carcinogen-driven immortalization of primary mouse embryonic fibroblasts and recapitulates early steps of cell transformation. Among the mutated genes were almost 200 COSMIC Cancer Gene Census genes, many of which were recurrently affected in the set of 25 immortalized cell lines. The alterations affected pathways regulating DNA damage response and repair, transcription and chromatin structure, cell cycle and cell death, as well as developmental pathways. The functional impact of the mutations was strongly supported by the manifestation of several known cancer hotspot mutations among the identified alterations. We identified a new set of genes encoding subunits of the BAF chromatin remodeling complex that exhibited Ras-mediated dependence on PRC2 histone methyltransferase activity, a finding that is similar to what has been observed for other BAF subunits in cancer cells. Among the affected BAF complex subunits, we determined Smarcd2 and Smarcc1 as putative driver candidates not yet fully identified by large-scale cancer genome sequencing projects. In addition, Ep400 displayed characteristics of a driver gene in that it showed a mutually exclusive mutation pattern when compared with mutations in the Trrap subunit of the TIP60 complex, both in the cell line panel and in a human tumor data set. We propose that the information generated by deep sequencing of the BBCE cell lines coupled with phenotypic analysis of the mutant cells can yield mechanistic insights into driver events relevant to human cancer development.

The exposome in practice: Design of the EXPOsOMICS project.

EXPOsOMICS is a European Union funded project that aims to develop a novel approach to the assessment of exposure to high priority environmental pollutants, by characterizing the external and the internal components of the exposome. It focuses on air and water contaminants during critical periods of life. To this end, the project centres on 1) exposure assessment at the personal and population levels within existing European short and long-term population studies, exploiting available tools and methods which have been developed for personal exposure monitoring (PEM); and 2) multiple "omic" technologies for the analysis of biological samples (internal markers of external exposures). The search for the relationships between external exposures and global profiles of molecular features in the same individuals constitutes a novel advancement towards the development of "next generation exposure assessment" for environmental chemicals and their mixtures. The linkage with disease risks opens the way to what are defined here as 'exposome-wide association studies' (EWAS).

The effect of high-power ultrasound and gas phase plasma treatment on Aspergillus spp. and Penicillium spp. count in pure culture.

AIMS: The aim of this study was to investigate and compare two nonthermal techniques in the inactivation of moulds. METHODS AND RESULTS: High power ultrasound (20 kHz) and nonthermal gas phase plasma treatments were studied in the inactivation of selected moulds. Aspergillus spp. and Penicillium spp. were chosen as the most common mould present in or on food. Experimental design was introduced to establish and optimize working variables. For high power ultrasound, the greatest reduction of moulds (indicated by the total removal of viable cells) was obtained after ultrasound treatments at 60°C (thermosonication) for 6 and 9 min (power applied, 20-39 W). For plasma treatment, the greatest inactivation of moulds was observed for the longest treatment time (5 min) and lowest sample volume (2 ml), (AP12, AP13, PP12 and PP13). CONCLUSIONS: The great amount of applied energy required for achieving a partial log reduction in viable cells is the limiting factor for using high-power ultrasound. However, both treatment methods could be combined in the future to produce beneficial outcomes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study deals with nonthermal food processing techniques and the results and findings present in this study are the root for further prospective studies. The food industry is looking for nonthermal methods that will enable food preservation, reduce deterioration of food compounds and structure and prolong food shelf life.

The histone acetyltransferase component TRRAP is targeted for destruction during the cell cycle.

Chromosomes are dynamic structures that must be reversibly condensed and unfolded to accommodate mitotic division and chromosome segregation. Histone modifications are involved in the striking chromatin reconfiguration taking place during mitosis. However, the mechanisms that regulate activity and function of histone-modifying factors as cells enter and exit mitosis are poorly understood. Here, we show that the anaphase-promoting complex or cyclosome (APC/C) is involved in the mitotic turnover of TRRAP (TRansformation/tRanscription domain-Associated Protein), a common component of histone acetyltransferase (HAT) complexes, and that the pre-mitotic degradation of TRRAP is mediated by the APC/C ubiquitin ligase activators Cdc20 and Cdh1. Ectopic expression of both Cdh1 and Cdc20 reduced the levels of coexpressed TRRAP protein and induced its ubiquitination. TRRAP overexpression or stabilization induces multiple mitotic defects, including lagging chromosomes, chromosome bridges and multipolar spindles. In addition, lack of sister chromatid cohesion and impaired chromosome condensation were found after TRRAP overexpression or stabilization. By using a truncated form of TRRAP, we show that mitotic delay is associated with a global histone H4 hyperacetylation induced by TRRAP overexpression. These results demonstrate that the chromatin modifier TRRAP is targeted for destruction in a cell cycle-dependent fashion. They also suggest that degradation of TRRAP by the APC/C is necessary for a proper condensation of chromatin and proper chromosome segregation. Chromatin compaction mediated by histone modifiers may represent a fundamental arm for APC/C orchestration of the mitotic machinery.

Gender-related differences in response to mutagens and carcinogens.

The incidences of many cancers can be very different in men and women. Besides differences in exposures to putative causative agents, it is plausible that both genetic and epigenetic effects play roles in these differences. In addition, gender-specific lifestyle and behavioural factors may modulate the effects of exposure to genotoxins. This commentary focuses on several aspects of gender-related differences in responses to mutagens and carcinogens, including sensitivity to chromosome damage, the contribution of genotypic variation and the role of DNA methylation. It is concluded that the reasons for gender differences in cancer susceptibility remain largely unknown in many cases, and the subject deserves more attention and study.

Orchestration of chromatin-based processes: mind the TRRAP.

Chromatin modifications at core histones including acetylation, methylation, phosphorylation and ubiquitination play an important role in diverse biological processes. Acetylation of specific lysine residues within the N terminus tails of core histones is arguably the most studied histone modification; however, its precise roles in different cellular processes and how it is disrupted in human diseases remain poorly understood. In the last decade, a number of histone acetyltransferases (HATs) enzymes responsible for histone acetylation, has been identified and functional studies have begun to unravel their biological functions. The activity of many HATs is dependent on HAT complexes, the multiprotein assemblies that contain one HAT catalytic subunit, adapter proteins, several other molecules of unknown function and a large protein called TRansformation/tRanscription domain-Associated Protein (TRRAP). As a common component of many HAT complexes, TRRAP appears to be responsible for the recruitment of these complexes to chromatin during transcription, replication and DNA repair. Recent studies have shed new light on the role of TRRAP in HAT complexes as well as mechanisms by which it mediates diverse cellular processes. Thus, TRRAP appears to be responsible for a concerted and context-dependent recruitment of HATs and coordination of distinct chromatin-based processes, suggesting that its deregulation may contribute to diseases. In this review, we summarize recent developments in our understanding of the function of TRRAP and TRRAP-containing HAT complexes in normal cellular processes and speculate on the mechanism underlying abnormal events that may lead to human diseases such as cancer.

Perception of ecological factors in asthmatic and coronary patients.

The aim of the study was to determine whether an examined group of asthmatic patients differ significantly from a control group of coronary patients with regard to perception of two groups of etiological factors and their interaction: a) ecological factors, and b) habits and behavior of the patients. The study included 100 patients with bronchial asthma and 102 with coronary disease. A questionnaire was used to obtain data on ecological factors in the living environment of the patients and information on habits and behavior. The questionnaire was structured according to the specific needs of the study, and as a starting point known, calibrated, psychometric scales were used. Asthmatic and coronary patients did not differ with regard to their place of residence, i.e. the same number lived in the town and village, in similar ecological environments, and they also did not differ with regard to life style and habits. The study indicated statistically significant differences between asthmatic and coronary patients in their perception of several ecological and other risk factors. The asthmatic patients significantly more frequently perceived harmful ecological factors in their environment and regarded them significant for the occurrence of their disease. The coronary patients perceived their unhealthy habits and behavior as the causal factors of their disease.

Disruption of Trrap causes early embryonic lethality and defects in cell cycle progression.

The transactivation/transformation-domain associated protein (TRRAP) belongs to the Ataxia-telangiectasia mutated (ATM) super-family and has been identified as a cofactor for c-MYC-mediated oncogenic transformation. TRRAP and its yeast homolog (Tra1p) are components of histone acetyltransferase (HAT) complexes, SAGA (refs. 2,4,5), PCAF (ref. 3) and NuA4 (ref. 6), which are important for the regulation of transcription and cell cycle progression and also have a role in cell viability. Yet the biological function of this molecule and how it controls proliferation are still unclear. Here we show that null mutation of Trrap in mice results in peri-implantation lethality due to a blocked proliferation of blastocysts. We use an inducible Cre-loxP system to show that loss of Trrap blocks cell proliferation because of aberrant mitotic exit accompanied by cytokinesis failure and endoreduplication. Trrap-deficient cells fail to sustain mitotic arrest despite chromosome missegregation and disrupted spindles, and display compromised cdk1 activity. Trrap is therefore essential for early development and required for the mitotic checkpoint and normal cell cycle progression.

Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death.

Poly(ADP-ribose) polymerase (PARP) is responsible for post-translational modification of proteins in the response to numerous endogenous and environmental genotoxic agents. PARP and poly(ADP-ribosyl)ation are proposed to be important for the regulation of many cellular processes such as DNA repair, cell death, chromatin functions and genomic stability. Activation of PARP is one of the early DNA damage responses, among other DNA sensing molecules, such as DNA-PK, ATM and p53. The generation and characterization of PARP deficient mouse models have been instrumental in defining the biological role of the molecule and its involvement in the pathogenesis of various diseases including diabetes, stroke, Parkinson disease, general inflammation as well as tumorigenesis, and have, therefore, provided information for the development of pharmaceutical strategies for the treatment of diseases.

Increased resistance to anticancer therapy of mouse cells lacking the poly(ADP-ribose) polymerase attributable to up-regulation of the multidrug resistance gene product P-glycoprotein.

Mouse embryo fibroblasts lacking poly(ADP-ribose) polymerase (PARP)-1 express a barely detectable level of wild-type (wt) p53 protein. Doxorubicin at concentrations activating wt p53 in normal mouse embryo fibroblasts failed to induce it in mutant cells. wt p53 was only activated in response to a 10-fold higher doxorubicin dose. Treatment with higher doxorubicin concentrations was cytotoxic for normal but not for PARP-1 -/- cells. The latter was also resistant to other anticancer agents. The increased resistance of mutant cells to drugs resembled a unique phenomenon known as multidrug resistance (MDR). Interestingly, the MDR gene product P-glycoprotein was clearly up-regulated in PARP-1-deficient cells as compared with normal counterparts. Pretreatment with verapamil reversed the MDR phenotype.

Doses in the vicinity of mobile X-ray equipment in a children's intensive care unit.

Most of the patients in the intensive care unit for children are newborns and infants having an infection of the central nervous system, with systemic septic and respiratory infections. Therefore, mobile X-ray equipment including mobile shields is routinely used for diagnosis of the respiratory tract, heart and endovascular catheterization. The aim of this work was to determine the radiation exposure to the children in the vicinity of the exposed patient in the same or next room. Three measurement runs were carried out with thermoluminescence dosimetry system. The results show that the homogeneity of the irradiation field is adequate, the exposure of children to radiation in the vicinity of the exposed patient in the same or next room is very low, practically in the range of the lowest detectable dose. The entrance dose on the breast of the patient was found to be 0.07 mSv. Therefore, there is no basis for the risk estimation of genetic, leukemogenic and cancerogenic detriment.

Hereditary hemorrhagic telangiectasia or Rendu-Osler-Weber syndrome in the same family.

The authors present the case of three patients from the same family in whom hereditary hemorrhagic telangiectasia (HHT) or Rendu-Osler-Weber syndrome was diagnosed. The disease is rare and occurs with multiple telangiectases of the skin and mucosa, and pulmonary arteriovenous fistulae. The clinical status of our patients included multiple telangiectases of the skin and mucosa, recurrent epistaxis, exertion dyspnea and cyanosis. Polycythemia and hypoxemia were observed in the blood. The clinical status and conventional radiological examination of the thoracic region, with the suspicion of arteriovenous (A-V) fistulae, pointed to HHT. A-V fistulae were confirmed by pulmonary angiography. The pulmonary A-V fistulae were operated in all three patients and diagnosis was confirmed by histopathological examination of the operated samples. Clinical improvement was observed after the operation and cyanosis, dyspnea, hypoxemia and polycythemia disappeared.

Differential susceptibility of normal and PARP knock-out mouse fibroblasts to proteasome inhibitors.

Recently we found a clearly reduced basal level of wt p53 protein in PARP-deficient cells. Interestingly, PARP deficiency affected only regularly spliced (RS) wt p53. No significant difference of the p53 transcription rate was observed between wt and PARP-lacking cells. To clarify whether the reduction of RS p53 protein is due to a lower translation rate or rather to its instability in the absence of functional PARP, we investigated the effect of the inhibition of proteasome activity and nuclear export on the p53 level. The p53 half-life was approximately eight-fold decreased in PARP-lacking cells. Surprisingly, treatment with three proteasome inhibitors increased RS p53 in normal but not in PARP-deficient cells. However, the inhibition of nuclear export resulted in a considerable accumulation of RS p53 in the latter. Therefore, we decided to increase concentrations of the inhibitors. Their higher concentrations strongly affected viability of normal, but not of PARP-deficient cells, about 70% of MEFs died. Interestingly, higher concentrations of proteasome inhibitors resulted in the appearance of RS p53 in PARP-lacking fibroblasts. Reconstitution of PARP-deficient cells with PARP restored the normal susceptibility to proteasome inhibitors thereby unequivocally demonstrating that the enhanced cytotoxicity of proteasome inhibitors and their action on p53 level depends on the presence of functional PARP.

Psychosocial characteristics of patients with bronchial asthma and coronary disease: similarities and differences.

The authors compare two groups of subjects: patients with bronchial asthma and those with coronary disease, with regard to some social characteristics, abilities and perception of factors which they conceive are important in the etiology of their disease. Data were obtained by means of a questionnaire based on a known calibrated scale. A group of 100 patients with bronchial asthma and a group of 102 patients with coronary disease were examined. The significance of the difference was tested by chi 2, t-test, Wilcoxon's test and multivariate discriminative analysis. The results showed statistically significant differences between the patients with bronchial asthma and those with coronary disease in some social and psychological characteristics and also with regard to perception of potential etiological factors of their disease. However, no difference was found in life style and habits between the coronary and asthmatic patients.

Failure of poly(ADP-ribose) polymerase cleavage by caspases leads to induction of necrosis and enhanced apoptosis.

Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD+ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD+ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD+ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD+ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.

p53 mutations in tumors derived from irradiated human thyroid epithelial cells.

A non-tumorigenic human thyroid epithelial cell line (HTori-3) has been transformed into tumorigenic cells by exposure in vitro to alpha particles or gamma-radiation. These transformants were tumorigenic in athymic nude mice and tumors were transplantable into other nude mice. To further characterize processes involved in neoplastic progression, the tumor cell lines derived from these radiation-induced primary tumors were screened for mutations in the p53 tumor suppressor gene. p53 mutation was detected by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 inclusive. Mutations detected by SSCP analysis were confirmed by sequencing. Mutations were detected in all four exons analysed, although there was no correlation between dose, LET or mutation position or frequency. Mutations in p53 exons 6 and 7 have been reported in the childhood papillary thyroid carcinomas in Belarus presumably as a result of radioiodine fall-out. Similarly here, p53 mutations are induced experimentally during the development of human thyroid tumors generated by irradiation of a human thyroid epithelial cell line in vitro.