Precipitous Release of Methyl-CpG Binding Protein 2 and Histone Deacetylase 1 from the Methylated Human Multidrug Resistance Gene (MDR1) on Activation

Molecular and Cellular Biology. Mar, 2002  |  Pubmed ID: 11865062

Overexpression of the human multidrug resistance gene 1 (MDR1) is a negative prognostic factor in leukemia. Despite intense efforts to characterize the gene at the molecular level, little is known about the genetic events that switch on gene expression in P-glycoprotein-negative cells. Recent studies have shown that the transcriptional competence of MDR1 is often closely associated with DNA methylation. Chromatin remodeling and modification targeted by the recognition of methylated DNA provide a dominant mechanism for transcriptional repression. Consistent with this epigenetic model, interference with DNA methyltransferase and histone deacetylase activity alone or in combination can reactivate silent genes. In the present study, we used chromatin immunoprecipitation to monitor the molecular events involved in the activation and repression of MDR1. Inhibitors of DNA methyltransferase (5-azacytidine [5aC]) and histone deacetylase (trichostatin A [TSA]) were used to examine gene transcription, promoter methylation status, and the chromatin determinants associated with the MDR1 promoter. We have established that methyl-CpG binding protein 2 (MeCP2) is involved in methylation-dependent silencing of human MDR1 in cells that lack the known transcriptional repressors MBD2 and MBD3. In the repressed state the MDR1 promoter is methylated and assembled into chromatin enriched with MeCP2 and deacetylated histone. TSA induced significant acetylation of histones H3 and H4 but did not activate transcription. 5aC induced DNA demethylation, leading to the release of MeCP2, promoter acetylation, and partial relief of repression. MDR1 expression was significantly increased following combined 5aC and TSA treatments. Inhibition of histone deacetylase is not an overriding mechanism in the reactivation of methylated MDR1. Our results provide us with a clearer understanding of the molecular mechanism necessary for repression of MDR1.

FMR1 Silencing and the Signals to Chromatin: a Unified Model of Transcriptional Regulation

Biochemical and Biophysical Research Communications. Jul, 2002  |  Pubmed ID: 12099676

Understanding the effect of DNA methylation requires an appreciation of the determinants responsible for gene expression. To this end, we and others have demonstrated the exquisite silencing properties of methylation-dependent repressors that belong to the methyl-CpG binding domain (MBD) protein family. It is crucial, to establish a direct connection between the remodelling events observed with the fragile X mental retardation gene 1 (FMR1) promoter if we are to understand the mechanism of control. In this paper contrasting but not mutually exclusive models are discussed, which conceptually and experimentally reflects an appreciation of the surrounding chromatin environment. Examining general transcription systems controlled by DNA methylation, proteins that recognize the methyl-CpG moiety and the enzyme that maintains methylation, a unified model of FMR1 gene silencing are discussed.

On the Use of DNA Methylation Inhibitors and the Reversal of Transcriptional Silencing

Blood. Feb, 2003  |  Pubmed ID: 12560246

The Rise of DNA Methylation and the Importance of Chromatin on Multidrug Resistance in Cancer

Experimental Cell Research. Nov, 2003  |  Pubmed ID: 14567978

In recent years, the different classes of drugs and regimens used clinically have provided an improvement in tumour management. However, treatment is often palliative for the majority of cancer patients. Transformed cells respond poorly to chemotherapy mainly due to the development of the multidrug resistance (MDR) phenotype. Response to treatment does not generally result in complete remission and disease cure is uncommon for patients presenting with advanced stage cancer. Successful treatment of cancer requires a clearer understanding of chemotherapeutic resistance. Here, we examine what is known of one of the most extensively studied mechanisms of cellular drug resistance. The human multidrug resistance gene 1 (MDR1) is associated with expression of p-glycoprotein (Pgp). A transmembrane protein, Pgp acts as an efflux pump and reduces intracellular drug levels and thus its effectiveness as an antitumor agent. The precise mechanism of transcriptional regulation has been unclear due to the complex regulatory nature of the gene. It has become increasingly apparent that trans-activation or genetic amplification is by no means the only mechanism of activation. Consequently, alternative pathways have received more attention in the area of epigenetics to help explain transcriptional competence at a higher level of organization. The goal of this article is to highlight important findings in the field of methylation and explain how they impinge on MDR1 gene regulation. In this review, we cover the current information and postulate that epigenetic modification of MDR1 chromatin influences gene transcription in leukaemia. Finally, we explore transcriptional regulation and highlight recent progress with engineered ZFP's (zinc finger proteins).

DNMT Cooperativity--the Developing Links Between Methylation, Chromatin Structure and Cancer

BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Nov, 2003  |  Pubmed ID: 14579248

Controversy has reigned for some time over the biological connection between DNA methylation and cancer. For this reason, the methylation mechanism responsible for increased cancer risk has received greater attention in recent years. Tumor suppressor genes are often hypermethylated resulting in gene silencing. Although some have questioned this interpretation of the link between methylation and cancer, it appears that both hypermethylation and hypomethylation events can create epigenetic changes that can contribute to cancer development. Recent studies have shown that the methyltransferases DNMT1 and DNMT3b cooperatively maintain DNA methylation and gene silencing in human cancer cells. Disruption of the human DNMT3b only slightly reduces the overall global DNA methylation; however, demethylation was markedly potentiated when both DNMT1 and DNMT3b were simultaneously deleted. The results to these experiments provide compelling evidence towards a role for DNA methylation in cancer. This review discusses the current understanding of cancer-epigenetic information and highlights recent studies that connect the methylation machinery and chromatin remodelling with cancer susceptibility.

The Rise and Fall of Genomic Methylation in Cancer

Leukemia : Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K. Feb, 2004  |  Pubmed ID: 14628069

Changes in genomic methylation and its significance in carcinogenesis is in the spotlight once again, though the focus is not on the usual suspects, DNA hypermethylation and tumour suppressor gene (TSG) silencing. Several recent reports provide compelling evidence of the relevance of genomic hypomethylation in cancer. These findings provide the best evidence so far that links the loss of DNA methylation and chromosomal instability with cancer development. This review article discusses these recent findings and reflects on the antithetical association between DNA methylation and carcinogenesis and the re-examination of studies performed almost two decades ago.

Epigenetic Changes Activate Widespread Signals in Response to Double-strand Breaks

Cancer Biology & Therapy. Jul, 2004  |  Pubmed ID: 15136760

Double-strand breaks are one the most severe types of DNA damage with respect to cell survival and the preservation of genomic integrity. Therefore, cells have evolved complex mechanisms including cell cycle regulation, activation of repair pathways and in certain cases induction of apoptosis in response to these lesions. The molecular details of many of the cellular responses to double-strand breaks have been well characterized. Our understanding of these responses in the context of chromatin has also progressed recently. In this review, we focus our discussion on the significance of DNA damage-induced chromatin modifications in double-strand break signaling and repair pathways. In particular, findings from recent studies suggest mechanisms by which highly localized double-strand breaks may activate widespread signals throughout the cell by inducing alterations in chromatin structure.

Understanding the Consequences of Epigenetic Mechanisms and Its Effects on Transcription in Health and Disease

Cancer Biology & Therapy. Sep, 2004  |  Pubmed ID: 15326369

Over the course of thirty-five years of research into the chromosome we are confronted with a number of general conclusions central to chromatin biology, perhaps most pertinent of these is that organization of higher order nucleosome structures play a fundamental role in DNA behavior. Folding and packaging of DNA is linked with a number of processes ranging from transcriptional regulation, genome stability, chromosome segregation, including replication and repair. Intrinsic to nuclear function is chromatin structure and the relationship with epigenetic modification. While we are aware that genetic and epigenetic changes can influence tumorigenesis, it is still unclear what protein components are specified on chromatin or the molecular events that underlie the link between chromosomes and cancer. In recent years, our understanding of DNA methylation with cancer development has revealed some surprising findings with particular emphasis on the antithetical character of hypo- and hyper- methylation of DNA. The molecular mechanism by which methylation is responsible for increased cancer risk has received widespread attention and is relavent to our understanding of genetic stability. The biological significance of other epigenetic modifications such as histone tail changes and active chromatin remodeling are also a major driving force in chromosome research in cancer. In light of recent advances in the understanding of epigenetic changes in disease we have organized a focused review series and discuss the significance of Epigenetics in Normal Control and Deregulation in Cancer. Authors from divergent fields of study have contributed articles that examine chromatin dynamics in cancer, bringing together research interests from a wide variety of fields. In this vein, we discuss the consequences of deregulating epigenetic mechanisms with a viewpoint on transcriptional control, chromatin function and human disease.

MDR1, Chemotherapy and Chromatin Remodeling

Cancer Biology & Therapy. Sep, 2004  |  Pubmed ID: 15326379

The development of multidrug resistance (MDR) in cancer can severely impede the efficacy of chemotherapy treatment. P-glycoprotein (Pgp) overexpression, encoded by the MDR1 gene, is a well-established mediator of MDR. MDR1 expression is rapidly upregulated by chemotherapeutic drugs and a number of other exogenous stimuli, however the mechanisms underlying its transcriptional regulation remain unclear. In recent years, research has indicated that chromatin accessibility, or epigenetic modifications, will play a large role in controlling the endogenous MDR1 expression state, and its response to activation stimuli. This review examines some of these studies, and discusses how new developments from the greatly expanding epigenetics field may extend to MDR1 transcriptional research.

Expression Analysis of the Epigenetic Methyltransferases and Methyl-CpG Binding Protein Families in the Normal B-cell and B-cell Chronic Lymphocytic Leukemia (CLL)

Cancer Biology & Therapy. Oct, 2004  |  Pubmed ID: 15467427

The importance of epigenetic modifications in carcinogenesis has been a source of controversy for some time. There is little doubt that changes in genomic hypermethylation contribute to the silencing of tumor suppressor genes. Furthermore, recent studies have also identified the significance of genomic hypomethylation associated with chromosomal instability and tumorigenesis. One of the most perplexing questions regarding epigenetic modifications and leukemogenesis is the relationship with DNA methyltransferases (DNMT's). The primary function of the DNMT enzymes is to methylate genomic DNA, whereas the methyl-CpG binding domain proteins (MBD) interpret this methylation signal and regulate gene expression and chromatin behavior. In this study we analyse these gene families by quantitative real-time PCR to investigate whether expression levels and the B-cell chronic lymphocytic leukemia (B-CLL) phenotype are associated. Furthermore, given the epigenetic crosstalk between genome stability and the histone chromatin code we have analysed eukaryotic histone methyltransferase (Eu-HMTaseI). Surprisingly, we did not observe significant changes in DNMT1 expression in B-CLL cases when compared to normal lymphocytes, regardless of whether we normalise against GAPDH or PCNA as reference standards. Indeed, expression of the maintenance and de novo methylases were independently regulated. Of particular note was the significant down regulation of DNMT3b. Furthermore, we observed a positive correlation between HMTaseI expression levels and stage of leukemia suggesting that changes in the methylation patterns in B-CLL may represent deregulation of the epigenetic repertoire that also include the methylation dependent binding proteins, MBD2 and MeCP2. We envisage changes in the epigenetic program are multifactorial in nature and postulate that the prevalent genomic methylases just one component of a larger epigenetic repertoire.

SiRNAs: Mechanism of RNA Interference, in Vivo and Potential Clinical Applications

Cancer Biology & Therapy. Nov, 2004  |  Pubmed ID: 15539939

Small interfering RNAs are currently the most widely used nucleic acid-based sequence-specific gene silencing molecules. These molecules mediate RNA interference--a natural post-transcriptional gene-silencing pathway. Given the high reliability and higher efficiency of small interfering RNA-mediated RNA interference, compared to earlier reverse genetic technologies, this is now the preferred technique in functional genomics. Furthermore, the exquisite specificity and exceptional gene-silencing potency of small interfering RNAs has resulted in intense research related to potential target-specific therapeutic applications of these molecules. This review will discuss the mechanism of RNA interference and applications of the pathway in molecular biology including functional genomics will be overviewed. The article will outline in vivo and potential clinical applications of small interfering RNA molecules.

DNA Damage Detection and Repair, and the Involvement of Epigenetic States

Human Mutation. Feb, 2005  |  Pubmed ID: 15643607

Chromatin is a highly dynamic structure that acts alternately as a substrate and a template in a number of critical biological processes. Modification of chromatin is pertinent and is responsible for a number of nuclear processes, including DNA repair, replication, transcription, and recombination. The purpose of this review is to discuss specific interactions between chromatin remodeling, DNA repair, and transcription. These areas are demonstrated to share commonality, particularly with a number of key molecules that appear to have roles in a number of pathways. The implications of pathway cross-over and communication form a seamless continuation of genomic integrity and stability.

Brahma Links the SWI/SNF Chromatin-remodeling Complex with MeCP2-dependent Transcriptional Silencing

Nature Genetics. Mar, 2005  |  Pubmed ID: 15696166

Transcriptional repression of methylated genes can be mediated by the methyl-CpG binding protein MeCP2. Here we show that human Brahma (Brm), a catalytic component of the SWI/SNF-related chromatin-remodeling complex, associates with MeCP2 in vivo and is functionally linked with repression. We used a number of different molecular approaches and chromatin immunoprecipitation strategies to show a unique cooperation between Brm, BAF57 and MeCP2. We show that Brm and MeCP2 assembly on chromatin occurs on methylated genes in cancer and the gene FMR1 in fragile X syndrome. These experimental findings identify a new role for SWI/SNF in gene repression by MeCP2.

RNA Interference and Potential Therapeutic Applications of Short Interfering RNAs

Cancer Gene Therapy. Oct, 2005  |  Pubmed ID: 15891770

RNA interference is an endogenous gene-silencing mechanism that involves double-stranded RNA-mediated sequence-specific mRNA degradation. The discovery of this pathway together with the elucidation of the structure and function of short interfering RNAs--the effector molecules of RNA interference--has had an enormous impact on experimental biology. RNA interference technologies are currently the most widely utilized techniques in functional genomic studies. Furthermore, there is an intense research effort aimed at developing short interfering RNAs for therapeutic purposes. A number of proof-of-principle experiments have demonstrated the clinical potential of appropriately designed short interfering RNAs in various diseases including viral infections, cancer and neurodegenerative disorders. Already, in such a short time from their discovery, Acuity Pharmaceuticals (August 2004) and Sirna Therapeutics (September 2004) have filed Investigational New Drug applications with the US FDA to begin clinical trials with modified siRNA molecules in patients with age-related macular degeneration. This review will give a brief overview of the mechanism of RNA interference and applications of the pathway in experimental biology will be discussed. The article will focus on recent developments related to the use of RNA interference technologies in mammalian systems and on potential clinical applications of short interfering RNA-mediated RNA interference.

Modulation of Soluble Receptor for Advanced Glycation End Products by Angiotensin-converting Enzyme-1 Inhibition in Diabetic Nephropathy

Journal of the American Society of Nephrology : JASN. Aug, 2005  |  Pubmed ID: 15930093

Recent studies have identified that first-line renoprotective agents that interrupt the renin-angiotensin system not only reduce BP but also can attenuate advanced glycation end product (AGE) accumulation. This study used in vitro, preclinical, and human approaches to explore the potential effects of these agents on the modulation of the receptor for AGE (RAGE). Bovine aortic endothelial cells that were exposed to the angiotensin-converting enzyme inhibitor (ACEi) ramiprilat in the presence of high glucose demonstrated a significant increase in soluble RAGE (sRAGE) secreted into the medium. In streptozotocin-induced diabetic rats, ramipril treatment (ACEi) at 3 mg/L for 24 wk reduced the accumulation of skin collagen-linked carboxymethyllysine and pentosidine, as well as circulating and renal AGE. Renal gene upregulation of total RAGE (all three splice variants) was observed in ACEi-treated animals. There was a specific increase in the gene expression of the splice variant C-truncated RAGE (sRAGE). There were also increases in sRAGE protein identified within renal cells with ACEi treatment, which showed AGE-binding ability. This was associated with decreases in renal full-length RAGE protein from ACEi-treated rats. Decreases in plasma soluble RAGE that were significantly increased by ACEi treatment were also identified in diabetic rats. Similarly, there was a significant increase in plasma sRAGE in patients who had type 1 diabetes and were treated with the ACEi perindopril. Complexes between sRAGE and carboxymethyllysine were identified in human and rodent diabetic plasma. It is postulated that ACE inhibition reduces the accumulation of AGE in diabetes partly by increasing the production and secretion of sRAGE into plasma.

The Histone Deacetylase Inhibitor, Trichostatin A, Enhances Radiation Sensitivity and Accumulation of GammaH2A.X

Cancer Biology & Therapy. Jul, 2005  |  Pubmed ID: 16082178

Histone deacetylase inhibitors have been shown to induce numerous biologic effects including, altering cell cycle distribution, cytostasis and in certain cases apoptosis. Given their ability to disrupt critical biological processes in cancer cells, these agents are emerging as potential therapeutics for cancer. Recently, it has been identified that histone deacetylase inhibitors can also efficiently enhance the radiation sensitivity of cells, both in vitro and in vivo. In this study, we investigated whether the potent histone deacetylase inhibitor, Trichostatin A, modulates the radiation sensitivity of human erythroleukemic K562 cells. The endpoints we used were clonogenic survival, apoptosis and gammaH2AX immunoprecipitations of soluble chromatin. The findings from clonogenic survival assays indicated that incubation with Trichostatin A 24 hours prior to irradiation enhances the radiation sensitivity of K562 cells. The dose modification factors ranged from 1.1 when cells were incubated with 0.1 microM Trichostatin A to 2.3 at 1 microM Trichostatin A. Similarly, caspase-3 and caspase-7 assays indicated that Trichostatin A potentiates radiation-induced apoptosis in K562 cells, in a concentration dependent manner. Our results suggest the modulation of radiation effects observed at the lower Trichostatin A concentrations was associated with histone hyperacetylation and changes in phosphorylated gammaH2A.X formation on euchromatin. In contrast, at the higher Trichostatin A concentrations mechanisms such as drug-mediated cytotoxicity and G1 cell cycle arrest, contributed to the sensitization effect. More generally, our findings are consistent with those from recent studies and support the development of histone deacetylase inhibitors for use as radiation sensitizers, particularly for targeting radioresistant cancers.

Epigenetic Changes to the MDR1 Locus in Response to Chemotherapeutic Drugs

Oncogene. Dec, 2005  |  Pubmed ID: 16091741

The mechanism of action of chemotherapeutic drugs and their ability to induce multidrug resistance (MDR) are of relevance to cancer treatment. Overexpression of P-glycoprotein (Pgp) encoded by the MDR1 gene following chemotherapy can severely limit the efficacy of anticancer agents; however, the manner by which cells acquire high levels of Pgp has not been defined. Herein, we demonstrate that chemotherapeutic drugs induce specific epigenetic modifications at the MDR1 locus, concomitant with MDR1 upregulation mediated by transcriptional activation, and a potential post-transcriptional component. We have established that the mechanisms are not mutually exclusive and are dependent on the methylation state of the MDR1 promoter. MDR1 upregulation did not result in further changes to the CpG methylation profile. However, dramatic changes in the temporal and spatial patterning of histone modifications occurred within the 5' hypomethylated region of MDR1, directly correlating with MDR1 upregulation. Specifically, drug-induced upregulation of MDR1 was associated with increases in H3 acetylation and induction of methylated H3K4 within discrete regions of the MDR1 locus. Our results demonstrate that chemotherapeutic drugs can actively induce epigenetic changes within the MDR1 promoter, and enhance the MDR phenotype.

Demethylation Using the Epigenetic Modifier, 5-azacytidine, Increases the Efficiency of Transient Transfection of Macrophages

Journal of Lipid Research. Feb, 2005  |  Pubmed ID: 15520456

This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40, and EF-1alpha promoters and transient transfection of human HepG2 cells. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.

Mechanisms of Abnormal Gene Expression in Tumor Cells

EXS. 2006  |  Pubmed ID: 16383026

Epigenetic mechanisms are involved in critical nuclear processes such as transcriptional control, genome stability, replication and repair. Recent evidence suggests that changes in the epigenetic repertoire can drive tumorigenesis. This review examines the latest experimental evidence that questions the mechanisms underlying the consequence of epigenetic changes in gene regulation and cancer development.

The Paradox of Histone Deacetylase Inhibitor-mediated Modulation of Cellular Responses to Radiation

Cell Cycle (Georgetown, Tex.). Feb, 2006  |  Pubmed ID: 16418577

Given the widespread use of radiotherapy in cancer, there has been a longstanding interest in the development of chemical compounds that can modify cellular responses to ionizing radiation. Additionally, recent terrorism threats suggesting attacks with 'dirty bombs' containing combinations of radioactive isotopes with conventional explosives, has increased the interest in compounds that can protect from radiation injury. Histone deacetylase inhibitors represent a new class of compounds that can modulate the effects of radiation. Research with histone deacetylase inhibitors has largely focussed on the consequences of their ability to alter gene transcription via histone acetylation and on their properties as anti-cancer agents. They have been shown to cause cell cycle and growth arrest, differentiation and in certain cases apoptosis in cell cultures and in vivo. In addition to their intrinsic anti-cancer properties, numerous studies have demonstrated that histone deacetylase inhibitors can modulate cellular responses to other toxicity-inducing modalities including ionizing radiation. The consensus is that histone deacetylase inhibitors markedly enhance the sensitivity of cells to radiation by altering numerous molecular pathways. Intriguingly, a report has also shown that histone deacetylase inhibitors can reduce radiation induced acute and late skin damage using a well-established animal model of cutaneous radiation syndrome. Hence, there is an emerging interest in potential use of histone deacetylase inhibitors as radiation sensitizers or protectors. This review focuses on the different mechanisms by which histone deacetylase inhibitors modify cellular responses to ionizing radiation.

The Neuronal Noradrenaline Transporter, Anxiety and Cardiovascular Disease

Journal of Psychopharmacology (Oxford, England). Jul, 2006  |  Pubmed ID: 16785272

Panic disorder can serve as a clinical model for testing whether mental stress can cause heart disease. Potential neural mechanisms of cardiac risk are the sympathetic activation during panic attacks, continuing release of adrenaline as a co-transmitter in the cardiac sympathetic nerves, and impairment of noradrenaline neuronal reuptake, augmenting sympathetic neural respnses. The phenotype of impaired neuronal reuptake of noradrenaline: an epigenetic mechanism? We suspect that this phenotype, in sensitizing people to heart symptom development, is a cause of panic disorder, and by magnifying the sympathetic neural signal in the heart, underlies increased cardiac risk. No loss of function mutations of the coding region of the norepinephrine transporter (NET) are evident, but we do detect hypermethylation of CpG islands in the NET gene promoter region. Chromatin immunoprecipitation methodology demonstrates binding of the inhibitory transcription factor, MeCP2, to promoter region DNA in panic disorder patients. Cardiovascular illnesses co-morbid with panic disorder. Panic disorder commonly coexists with essential hypertension and the postural tachycardia syndrome. In both of these cardiovascular disorders the impaired neuronal noradrenaline reuptake phenotype is also present and, as with panic disorder, is associated with NET gene promoter region DNA hypermethylation. An epigenetic 'co-morbidity' perhaps underlies the clinical concordance. Brain neurotransmitters. Using internal jugular venous sampling, in the absence of a panic attack we find normal norepinephrine turnover, but based on measurements of the overflow of the serotonin metabolite, 5HIAA, a marked increase (six to sevenfold) in brain serotonin turnover in patients with panic disorder. This appears to represent the underlying neurotransmitter substrate for the disorder. Whether this brain serotonergic activation is a prime mover, or consequential on other primary causes of panic disorder, including cardiac sensitization by faulty neuronal noradrenaline reuptake leading to cardiac symptoms and the enhanced vigilance which accompanies them, is unclear at present.

Clinical Potential of Histone Deacetylase Inhibitors As Stand Alone Therapeutics and in Combination with Other Chemotherapeutics or Radiotherapy for Cancer

Epigenetics : Official Journal of the DNA Methylation Society. Jul-Sep, 2006  |  Pubmed ID: 17965606

Histone deacetylase inhibitors are emerging as a new class of cancer chemotherapeutics and already are being heralded as the first anti-cancer drugs targeting the epigenome. Through histone hyperacetylation-mediated changes in chromatin conformation and gene expression, histone deacetylase inhibitors induce differentiation, cell cycle arrest, apoptosis, growth inhibition and cell death, which are more pronounced in transformed cell-lines than in normal cells. Additional anti-cancer effects of HDAC inhibitors include inhibition of migration, invasion and angiogenesis in vivo. Indeed, clinical anti-cancer activity has been observed using HDAC inhibitors as single agents or in combination with conventional chemotherapeutics, in phase I and II trials. Furthermore, numerous preclinical studies are suggesting a potential clinical role for HDAC inhibitors in radiotherapy either as radiation sensitizers or protectors. In this article the molecular basis for the clinical potential of HDAC inhibitors, either as stand alone cancer therapeutics or in combination with other chemotherapy agents or ionizing radiation will be overviewed.

The Epigenetic Modifier, Valproic Acid, Enhances Radiation Sensitivity

Epigenetics : Official Journal of the DNA Methylation Society. Jul-Sep, 2006  |  Pubmed ID: 17965607

Valproic acid is an established therapeutic for a variety of seizure disorders and in certain cases for depression and anxiety. In addition, valproic acid has been shown to possess histone deacetylase inhibition activity and is currently being investigated as an anti-cancer agent, either alone or in combination with other conventional cancer therapies such as ionizing radiation. In this study, we investigated whether valproic acid modulates cellular responses to radiation in human erythroleukemic, K562 cells. Hyperacetylation of nuclear histones 3 and 4 was used to correlate the effects of valproic acid to inhibition of histone deacetylase activity, clonogenic survival, apoptosis and apoptosis. The findings from the clonogenic survival and caspase induction assays indicated that pretreatment of cells with valproic acid for 24 hours, markedly enhanced radiation induced cell-death and apoptosis in K562 cells, respectively. Mechanisms involving drug-mediated cytotoxicity and changes in cell cycle distribution were associated with the radiation sensitizing properties of valproic acid, particularly at the higher concentrations. Overall, our findings are consistent with the general consensus that HDAC inhibitors efficiently sensitize cancer cells to the effects of ionizing radiation and support the idea of developing clinically relevant combinations of HDAC inhibitors and radiotherapy.

FOXO1 Regulates the Expression of 4E-BP1 and Inhibits MTOR Signaling in Mammalian Skeletal Muscle

The Journal of Biological Chemistry. Jul, 2007  |  Pubmed ID: 17510058

The mammalian target of rapamycin (mTOR) is regulated by growth factors to promote protein synthesis. In mammalian skeletal muscle, the Forkhead-O1 transcription factor (FOXO1) promotes catabolism by activating ubiquitin-protein ligases. Using C2C12 mouse myoblasts that stably express inducible FOXO1-ER fusion proteins and transgenic mice that specifically overexpress constitutively active FOXO1 in skeletal muscle (FOXO(++/+)), we show that FOXO1 inhibits mTOR signaling and protein synthesis. Activation of constitutively active FOXO1 induced the expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) mRNA via binding to the promoter. This resulted in an increased total 4E-BP1 abundance and a reduced 4E-BP1 (Thr-37/46) phosphorylation. The reduction in 4E-BP1 phosphorylation was associated with a reduction in the abundance of Raptor and mTOR proteins, Raptor-associated mTOR, reduced phosphorylation of the downstream protein p70S6 kinase, and attenuated incorporation of [(14)C]phenylalanine into protein. The FOXO(++/+) mice, characterized by severe skeletal muscle atrophy, displayed similar patterns of mRNA expression and protein abundance to those observed in the constitutively active FOXO1 C2C12 myotubes. These data suggest that FOXO1 may be an important therapeutic target for human diseases where anabolism is impaired.

Human Sympathetic Nerve Biology: Parallel Influences of Stress and Epigenetics in Essential Hypertension and Panic Disorder

Annals of the New York Academy of Sciences. Dec, 2008  |  Pubmed ID: 19120127

Patients with panic disorder provide a clinical model of stress. On a "good day," free from a panic attack, they show persistent stress-related changes in sympathetic nerve biology, including abnormal sympathetic nerve single-fiber firing ("salvos" of multiple firing within a cardiac cycle) and release of epinephrine as a cotransmitter. The coreleased epinephrine perhaps originates from in situ synthesis by phenylethanolamine N-methyltransferase (PNMT). In searching for biological evidence that essential hypertension is caused by mental stress--a disputed proposition--we note parallels with panic disorder, which provides an explicit clinical model of stress: (1) There is clinical comorbidity; panic disorder prevalence is increased threefold in essential hypertension. (2) For both, epinephrine cotransmission is present in sympathetic nerves. (3) In panic disorder and essential hypertension, but not in health, single-fiber sympathetic nerve firing salvos occur. (4) Tissue nerve growth factor is increased in both conditions (nerve growth factor is a stress reactant). (5) There is induction of PNMT in sympathetic nerves. Essential hypertension exhibits a further manifestation of mental stress: there is activation of noradrenergic brain stem neurons projecting to the hypothalamus and amygdala. These pathophysiological findings strongly support the view that chronic mental stress is important in the pathogenesis of essential hypertension. A hypothesis now under test is whether in both disorders, under prevailing conditions of ongoing stress, PNMT induced in sympathetic nerves acts as a DNA methylase, causing the norepinephrine transporter (NET) gene silencing that is present in both conditions. PNMT can have an intranuclear distribution, binding to DNA. We have demonstrated that the reduced neuronal noradrenaline reuptake present in both disorders does have an epigenetic mechanism, with demonstrable reduction in the abundance of the transporter protein, the NET gene silencing being associated with DNA binding by the methylation-related inhibitory transcription factor MeCP2.

Effect of Valproic Acid on Radiation-induced DNA Damage in Euchromatic and Heterochromatic Compartments

Cell Cycle (Georgetown, Tex.). Feb, 2008  |  Pubmed ID: 18239454

The distinction between heterochromatin and euchromatin in the double-strand break (DSB) damage pathway is of interest, recent reports indicate that chromatin is not created equally nor is it acquiescent to DSBs. Using the classical histone deacetylase inhibitor, Trichostatin A, we have previously demonstrated that chromatin represents a heterogeneous substrate with respect to histone tail modification by histone deacetylase inhibitors and consequent responses to DNA damage and repair. Here, we extended the initial findings by investigating the radiation sensitizing properties of the widely used antiepileptic, valproic acid. Clonogenic survival assays confirm that valproic acid is an efficient sensitizer of radiation-induced cell death. The radiosensitizing effect is correlated with valproic acid-mediated histone hyperacetylation, chromatin decondensation and enhanced formation of radiation-induced gammaH2AX preferentially on euchromatic alleles. Heterochromatin was much more resistant to histone tail modification, changes in chromatin architecture and DNA damage. These findings are consolidated by studies with the structurally related analogue, valpromide, which does not inhibit histone deacetylase enzymes. At a relatively low concentration (1 mM) valpromide did not cause chromatin modifications and radiation sensitivity, providing further evidence that the radiation sensitizing properties of valproic acid are at least in part, due to histone modification-dependent effects on euchromatin. When higher concentrations (5 mM) were used, both compounds resulted in significant radiation sensitivity, albeit, with differing efficacy (dose modifying factors of 1.5 and 1.2 for valproic acid and valpromide, respectively). The findings imply that histone-modification independent mechanisms also contribute to the radiation sensitizing properties of valproic acid. Overall, our findings are consistent with the emerging interest in the use histone deacetylase inhibitors in combination with radiotherapy for the treatment of cancer.

Epigenetic Regulation and Fetal Programming

Best Practice & Research. Clinical Endocrinology & Metabolism. Feb, 2008  |  Pubmed ID: 18279777

Fetal programming encompasses the role of developmental plasticity in response to environmental and nutritional signals during early life and its potential adverse consequences (risk of cardiovascular, metabolic and behavioural diseases) in later life. The first studies in this field highlighted an association between poor fetal growth and chronic adult diseases. However, environmental signals during early life may lead to adverse long-term effects independently of obvious effects on fetal growth. Adverse long-term effects reflect a mismatch between early (fetal and neonatal) environmental conditions and the conditions that the individual will confront later in life. The mechanisms underlying this risk remain unclear. However, experimental data in rodents and recent observations in humans suggest that epigenetic changes in regulatory genes and growth-related genes play a significant role in fetal programming. Improvements in our understanding of the biochemical and molecular mechanisms at play in fetal programming would make it possible to identify biomarkers for detecting infants at high risk of adult-onset diseases. Such improvements should also lead to the development of preventive and therapeutic strategies.

Transient High Glucose Causes Persistent Epigenetic Changes and Altered Gene Expression During Subsequent Normoglycemia

The Journal of Experimental Medicine. Sep, 2008  |  Pubmed ID: 18809715

The current goal of diabetes therapy is to reduce time-averaged mean levels of glycemia, measured as HbA1c, to prevent diabetic complications. However, HbA1c only explains <25% of the variation in risk of developing complications. Because HbA1c does not correlate with glycemic variability when adjusted for mean blood glucose, we hypothesized that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications. We show that transient hyperglycemia induces long-lasting activating epigenetic changes in the promoter of the nuclear factor kappaB (NF-kappaB) subunit p65 in aortic endothelial cells both in vitro and in nondiabetic mice, which cause increased p65 gene expression. Both the epigenetic changes and the gene expression changes persist for at least 6 d of subsequent normal glycemia, as do NF-kappaB-induced increases in monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression. Hyperglycemia-induced epigenetic changes and increased p65 expression are prevented by reducing mitochondrial superoxide production or superoxide-induced alpha-oxoaldehydes. These results highlight the dramatic and long-lasting effects that short-term hyperglycemic spikes can have on vascular cells and suggest that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications.

Survival Motor Neuron Gene 2 Silencing by DNA Methylation Correlates with Spinal Muscular Atrophy Disease Severity and Can Be Bypassed by Histone Deacetylase Inhibition

Human Molecular Genetics. Jan, 2009  |  Pubmed ID: 18971205

Spinal muscular atrophy (SMA), a common neuromuscular disorder, is caused by homozygous absence of the survival motor neuron gene 1 (SMN1), while the disease severity is mainly influenced by the number of SMN2 gene copies. This correlation is not absolute, suggesting the existence of yet unknown factors modulating disease progression. We demonstrate that the SMN2 gene is subject to gene silencing by DNA methylation. SMN2 contains four CpG islands which present highly conserved methylation patterns and little interindividual variations in SMN1-deleted SMA patients. The comprehensive analysis of SMN2 methylation in patients suffering from severe versus mild SMA carrying identical SMN2 copy numbers revealed a correlation of CpG methylation at the positions -290 and -296 with the disease severity and the activity of the first transcriptional start site of SMN2 at position -296. These results provide first evidence that SMN2 alleles are functionally not equivalent due to differences in DNA methylation. We demonstrate that the methyl-CpG-binding protein 2, a transcriptional repressor, binds to the critical SMN2 promoter region in a methylation-dependent manner. However, inhibition of SMN2 gene silencing conferred by DNA methylation might represent a promising strategy for pharmacologic SMA therapy. We identified histone deacetylase (HDAC) inhibitors including vorinostat and romidepsin which are able to bypass SMN2 gene silencing by DNA methylation, while others such as valproic acid and phenylbutyrate do not, due to HDAC isoenzyme specificities. These findings indicate that DNA methylation is functionally important regarding SMA disease progression and pharmacological SMN2 gene activation which might have implications for future SMA therapy regimens.

Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated with Gene-activating Epigenetic Marks That Coexist on the Lysine Tail

Diabetes. May, 2009  |  Pubmed ID: 19208907

Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as "hyperglycemic memory." We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information.

The Emerging Role of Epigenetic Modifications and Chromatin Remodeling in Spinal Muscular Atrophy

Journal of Neurochemistry. Jun, 2009  |  Pubmed ID: 19383090

As the leading genetic cause for infantile death, Spinal Muscular Atrophy (SMA) has been extensively studied since its first description in the early 1890s. Though today much is known about the cause of the disease, a cure or effective treatment is not currently available. Recently the short chain fatty acid valproic acid, a drug used for decades in the management of epilepsy and migraine therapy, has been shown to elevate the levels of the essential survival motor neuron protein in cultured cells. In SMA mice, valproic acid diminished the severity of the disease phenotype. This effect was linked to the ability of the short chain fatty acid to suppress histone deacetylase activity and activate gene transcription. Since then, the study of different histone deacetylase inhibitors and their epigenetic modifying capabilities has been of high interest in an attempt to find potential candidates for effective treatment of SMA. In this review, we summarize the current knowledge about use of histone deacetylase inhibitors in SMA as well as their proposed effects on chromatin structure and discuss further implications for possible treatments of SMA arising from research examining epigenetic change.

Gamma-radiation-induced GammaH2AX Formation Occurs Preferentially in Actively Transcribing Euchromatic Loci

Cellular and Molecular Life Sciences : CMLS. Jan, 2010  |  Pubmed ID: 19859659

The central dogma in radiation biology is that nuclear DNA is the critical target with respect to radiosensitivity. In accordance with the theoretical expectations, and in the absence of a conclusive model, the general consensus in the field has been to view chromatin as a homogeneous template for DNA damage and repair. This paradigm has been called into question by recent findings indicating a disparity in gamma-irradiation-induced gammaH2AX foci formation in euchromatin and heterochromatin. Here, we have extended those studies and provide evidence that gammaH2AX foci form preferentially in actively transcribing euchromatin following gamma-irradiation.

Epigenetic Regulation of Multidrug Resistance 1 Gene Expression: Profiling CpG Methylation Status Using Bisulphite Sequencing

Methods in Molecular Biology (Clifton, N.J.). 2010  |  Pubmed ID: 19949925

Methylation of CpG dinucleotides is one of the major epigenetic processes involved in the regulation of gene expression. Catalyzed by DNA methyltransferases, hypermethylation of CpG islands in promoter regions is typically associated with gene silencing. DNA methylation plays an important role in normal differentiation, development, and maintenance of genomic stability, with aberrant CpG methylation being linked with a number of disease states. Three CpG islands within a 1.15- kb region characterize the chromatin landscape surrounding the transcriptional start site of the multidrug resistance 1 (MDR1) gene. We and others have demonstrated that hypermethylation of this region is correlated with MDR1 gene silencing and the inability of chemotherapeutic agents to activate MDR1 transcription. The bisulphite sequencing and cloning method allows a precise interpretation of the methylation status of each individual CpG dinucleotide in the MDR1 region.

Analysis of the IGF2/H19 Imprinting Control Region Uncovers New Genetic Defects, Including Mutations of OCT-binding Sequences, in Patients with 11p15 Fetal Growth Disorders

Human Molecular Genetics. Mar, 2010  |  Pubmed ID: 20007505

The imprinted expression of the IGF2 and H19 genes is controlled by the imprinting control region 1 (ICR1) located at chromosome 11p15.5. This methylation-sensitive chromatin insulator works by binding the zinc-finger protein CTCF in a parent-specific manner. DNA methylation defects involving the ICR1 H19/IGF2 domain result in two growth disorders with opposite phenotypes: an overgrowth disorder, the Beckwith-Wiedemann syndrome (maternal ICR1 gain of methylation in 10% of BWS cases) and a growth retardation disorder, the Silver-Russell syndrome (paternal ICR1 loss of methylation in 60% of SRS cases). Although a few deletions removing part of ICR1 have been described in some familial BWS cases, little information is available regarding the mechanism of ICR1 DNA methylation defects. We investigated the CTCF gene and the ICR1 domain in 21 BWS patients with ICR1 gain of methylation and 16 SRS patients with ICR1 loss of methylation. We identified four constitutional ICR1 genetic defects in BWS patients, including a familial case. Three of those defects are newly identified imprinting defects consisting of small deletions and a single mutation, which do not involve one of the CTCF binding sites. Moreover, two of those defects affect OCT-binding sequences which are suggested to maintain the unmethylated state of the maternal allele. A single-nucleotide variation was identified in a SRS patient. Our data extends the spectrum of constitutive genetic ICR1 abnormalities and suggests that extensive and accurate analysis of ICR1 is required for appropriate genetic counseling in BWS patients with ICR1 gain of methylation.

Cardiac Ventricular Chambers Are Epigenetically Distinguishable

Cell Cycle (Georgetown, Tex.). Feb, 2010  |  Pubmed ID: 20090419

The left and right ventricles are muscular chambers of the heart that differ significantly in the extent of pressure work-load. The regional and differential distribution of gene expression patterns is critical not only for heart development, but, also in the establishment of cardiac hypertrophy phenotypes. the cells of the myocardium employ elaborate regulatory mechanisms to establish changes in chromatin structure and function, yet, the role of epigenetic modifications and specific gene expression patterns in cardiac ventricles remains poorly understood. We have examined gene expression changes and studied histone H3 and H4 acetylation as well as dimethylation of lysine 4 on histone H3 on promoters of alpha-Myosin heavy chain gene (alpha-MHC), beta-Myosin heavy chain gene (beta-MHC), Atrial natriuretic peptide gene (ANp), B-type natriuretic peptide gene (BNP) and Sarcoplasmic reticulum Ca(2+) ATPase gene (SERCA2a). The recruitment of histone acetyltransferase (HAT) enzyme p300, which is a transcriptional coactivator, was also studied on the hyperacetylated promoters using immunopurification of soluble chromatin in the left and right ventricles of the mouse. We present evidence for the first time that the pattern of gene expression is closely linked with histone modifications and propose the left and right chambers of the heart are epigenetically distinguishable.

Alleviating Transcriptional Inhibition of the Norepinephrine Slc6a2 Transporter Gene in Depolarized Neurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2010  |  Pubmed ID: 20107077

Recent studies have brought to light additional experimental information, namely, that the MeCP2 protein complex is not only capable of associating with members of the ATPase-dependent bromodomain family, but also found on nonmethylated genomic sequences. These unexpected results are indicative of a multifunctional role for MeCP2, more importantly; our view of the molecular mechanisms that regulate gene activity may not be necessarily distinguishable. Depolarized mouse neuronal cortical cells were examined for increased Slc6a2 mRNA synthesis, changes in CpG methylation status using bisulfite sequencing, and binding of MeCP2 and Smarca2 on the Slc6a2 promoter sequence by chromatin immunopurification strategies. Increased Slc6a2 gene expression in response to membrane depolarization was strongly correlated with the dissociation of MeCP2 and Smarca2 complex on the unmethylated gene. We identified that gene expression in neuronal cortical cells involves increased histone hyperacetylation on the Slc6a2 promoter, which is commensurate with the recruitment of SP1 and RNA Polymerase II and is inversely correlated with H3K9 trimethylation. We hypothesize that the MeCP2 corepressor is capable of associating with multiple forms of SWI/SNF to remodel chromatin for important regulatory roles. The results of our experiments indicate that these proteins are asymmetrically bound to chromatin independent of DNA methylation and not inevitably diametrically opposed. These results now begin to offer a new perspective on the mechanism of Slc6a2 gene regulation.

GammaH2AX As a Molecular Marker of Aging and Disease

Epigenetics : Official Journal of the DNA Methylation Society. Feb, 2010  |  Pubmed ID: 20150765

Double-strand breaks are one of the most critical DNA lesions with respect to cell-death and preservation of genomic integrity. Rapid phosphorylation of the histone variant H2AX at Ser-139 to form gammaH2AX is an early cellular response to DNA double-strand breaks. Visualization of discrete gammaH2AX foci using immunofluorescence-based assays has provided a sensitive and effective method for detecting DSBs which may be implicated in various pathologies including cancer, age-related diseases, chronic inflammatory diseases and ischemia-reperfusion injury. In this review, the potential utility and significance of gammaH2AX as a molecular marker of aging and disease is analysed.

Downstream Targets of Methyl CpG Binding Protein 2 and Their Abnormal Expression in the Frontal Cortex of the Human Rett Syndrome Brain

BMC Neuroscience. 2010  |  Pubmed ID: 20420693

The Rett Syndrome (RTT) brain displays regional histopathology and volumetric reduction, with frontal cortex showing such abnormalities, whereas the occipital cortex is relatively less affected.

Metabolic Memory and Diabetic Nephropathy: Potential Role for Epigenetic Mechanisms

Nature Reviews. Nephrology. Jun, 2010  |  Pubmed ID: 20421885

Many clinical studies have shown that intensive glycemic control in patients with diabetes can reduce the incidence and progression of diabetic nephropathy and can also reduce the incidence of other complications. These beneficial effects persist after patients return to usual (often worse) glycemic control. The Diabetes Control and Complications Trial was the first to refer to this phenomenon as 'metabolic memory'. Many patients with diabetes, however, still develop diabetic nephropathy despite receiving intensified glycemic control. Preliminary work in endothelial cells has shown that transient episodes of hyperglycemia can induce changes in gene expression that are dependent on modifications to histone tails (for example, methylation), and that these changes persist after return to normoglycemia. The persistence of such modifications cannot yet be fully explained, but certain epigenetic changes, as well as biochemical mechanisms such as advanced glycation, may provide new and interesting clues towards explaining the pathogenesis of this phenomenon. Further elucidation of the molecular events that enable prior glycemic control to result in end-organ protection in diabetes may lead to the development of new approaches for reducing the burden of diabetic nephropathy.

Glycemic Memory Associated Epigenetic Changes

Biochemical Pharmacology. Dec, 2010  |  Pubmed ID: 20599797

It is evident that metabolic memory, whereby diabetic complications continue to develop and progress in individuals who returned to normal glycemic control after a period of transient hyperglycemia, can have long lasting effects. We have primary findings that transient hyperglycemia causes profound transcriptional changes in vascular endothelial cells. We hypothesized that ambient hyperglycemia triggers gene-activating events of the NFκB p65 promoter that are mediated by changes in epigenetic modifications. In a follow-up study we identified two histone-specific writing and erasing enzymes involved in the underlying regulation of gene expression during transient hyperglycemia and subsequent return to normoglycemia. Experimental evidence indicates that previous hyperglycemia is associated with persistent expression of the NFκB p65 gene, which activates NFκB-dependent proteins, such as MCP-1, which are implicated in diabetes-associated vascular injury. Increased gene transcription is correspondent with H3K4m1, but not H3K4m2 and H3K4m3, on the NFκB p65 gene. In vascular endothelial cells the histone methyltransferase Set7 can write the mono-methylation mark H3K4m1 and this methyl-writing enzyme is recruited as a gene co-activator in response to glucose. Furthermore, Set7 knockdown prevents glucose-induced p65 expression. We hypothesize that these molecular events represent an integrated response of the epigenome that lead to changes in the expression of genes and proteins that regulate the development and progression of diabetic vascular complications. Further characterisation of these glucose-induced epigenetic events and the identification of key enzymes involved will improve our understanding of the pathways implicated in diabetic vascular injury.

Trichostatin A Accentuates Doxorubicin-induced Hypertrophy in Cardiac Myocytes

Aging. Oct, 2010  |  Pubmed ID: 20930262

Histone deacetylase inhibitors represent a new class of anticancer therapeutics and the expectation is that they will be most effective when used in combination with conventional cancer therapies, such as the anthracycline, doxorubicin. The dose-limiting side effect of doxorubicin is severe cardiotoxicity and evaluation of the effects of combinations of the anthracycline with histone deacetylase inhibitors in relevant models is important. We used a well-established in vitro model of doxorubicin-induced hypertrophy to examine the effects of the prototypical histone deacetylase inhibitor, Trichostatin A. Our findings indicate that doxorubicin modulates the expression of the hypertrophy-associated genes, ventricular myosin light chain-2, the alpha isoform of myosin heavy chain and atrial natriuretic peptide, an effect which is augmented by Trichostatin A. Furthermore, we show that Trichostatin A amplifies doxorubicin-induced DNA double strand breaks, as assessed by γH2AX formation. More generally, our findings highlight the importance of investigating potential side effects that may be associated with emerging combination therapies for cancer.

Epigenetic Phenomena Linked to Diabetic Complications

Nature Reviews. Endocrinology. Dec, 2010  |  Pubmed ID: 21045787

Diabetes mellitus (type 1 and type 2) and the complications associated with this condition are an urgent public health problem, as the incidence of diabetes mellitus is steadily increasing. Environmental factors, such as diet and exposure to hyperglycemia, contribute to the etiology of diabetes mellitus and its associated microvascular and macrovascular complications. These vascular complications are the main cause of the morbidity and mortality burden of diabetes mellitus. The DCCT-EDIC and UKPDS epidemiological studies correlated poor glycemic control with the development of vascular complications in patients with type 1 or type 2 diabetes mellitus. The findings of these studies suggest that early exposure to hyperglycemia predisposes individuals to the development of diabetic complications, a phenomenon referred to as metabolic memory or the legacy effect. The first experimental evidence for metabolic memory was reported >20 years ago and the underlying molecular mechanisms are currently being characterized. Interestingly, transient exposure to hyperglycemia results in long-lasting epigenetic modifications that lead to changes in chromatin structure and gene expression, which mediate these persistent metabolic characteristics.

Histone Modifications Regulate the Norepinephrine Transporter Gene

Cell Cycle (Georgetown, Tex.). Nov, 2010  |  Pubmed ID: 21088495

Epigenetics: Mechanisms and Implications for Diabetic Complications

Circulation Research. Dec, 2010  |  Pubmed ID: 21148447

Epigenetic modifications regulate critical functions that underlie chromosome metabolism. Understanding the molecular changes to chromatin structure and the functional relationship with altered signaling pathways is now considered to represent an important conceptual challenge to explain diabetes and the phenomenon of metabolic or hyperglycemic memory. Although it remains unknown as to the specific molecular mechanisms whereby hyperglycemic memory leads to the development of diabetic vascular complications, emerging evidence now indicates that critical gene-activating epigenetic changes may confer future cell memories. Chemical modification of the H3 histone tail of lysine 4 and 9 has recently been identified with gene expression conferred by hyperglycemia. The persistence of these key epigenetic determinants in models of glycemic variability and the development of diabetic complications has been associated with these primary findings. Transient hyperglycemia promotes gene-activating epigenetic changes and signaling events critical in the development and progression of vascular complications. As for the role of specific epigenomic changes, it is postulated that further understanding enzymes involved in writing and erasing chemical changes could transform our understanding of the pathways implicated in diabetic vascular injury providing new therapeutic strategies.

Atherogenic Factors and Their Epigenetic Relationships

International Journal of Vascular Medicine. 2010  |  Pubmed ID: 21152193

Hypercholesterolemia, homocysteine, oxidative stress, and hyperglycemia have been recognized as the major risk factors for atherogenesis. Their impact on the physiology and biochemistry of vascular cells has been widely demonstrated for the last century. However, the recent discovery of the role of epigenetics in human disease has opened up a new field in the study of atherogenic factors. Thus, epigenetic tags in endothelial, smooth muscle, and immune cells seem to be differentially affected by similar atherogenic stimuli. This paper summarizes some recent works on expression of histone-modifying enzymes and DNA methylation directly linked to the presence of risk factors that could lead to the development or prevention of the atherosclerotic process.

Genetic and Epigenetic Events in Diabetic Wound Healing

International Wound Journal. Feb, 2011  |  Pubmed ID: 21159125

The prevalence of the chronic metabolic disorder, diabetes mellitus, is expected to increase in the coming years and worldwide pandemic levels are predicted. Inevitably, this will be accompanied by an increase in the prevalence of diabetic complications, including diabetic foot ulcers. At present, treatment options for diabetic foot ulcers are in many cases insufficient, and progression of the condition results in the requirement for limb amputation in a proportion of patients. To improve therapy, an increase in our understanding of the pathobiology of diabetic complications such as impaired wound healing is necessary. In this review, recent advances in molecular aspects of normal and impaired diabetic wound healing are discussed. Furthermore, investigations of the role of epigenetic processes in the pathogenesis of impaired diabetic wound healing are now emerging. Indeed, epigenetic changes have already been identified as key factors in diabetes and related complications and these are overviewed in this review.

Double-strand Breaks and the Concept of Short- and Long-term Epigenetic Memory

Chromosoma. Apr, 2011  |  Pubmed ID: 21174214

Double-strand breaks represent an extremely cytolethal form of DNA damage and thus pose a serious threat to the preservation of genetic and epigenetic information. Though it is well-known that double-strand breaks such as those generated by ionising radiation are among the principal causative factors behind mutations, chromosomal aberrations, genetic instability and carcinogenesis, significantly less is known about the epigenetic consequences of double-strand break formation and repair for carcinogenesis. Double-strand break repair is a highly coordinated process that requires the unravelling of the compacted chromatin structure to facilitate repair machinery access and then restoration of the original undamaged chromatin state. Recent experimental findings have pointed to a potential mechanism for double-strand break-induced epigenetic silencing. This review will discuss some of the key epigenetic regulatory processes involved in double-strand break (DSB) repair and how incomplete or incorrect restoration of chromatin structure can leave a DSB-induced epigenetic memory of damage with potentially pathological repercussions.

Phosphoinositide 3-kinase As a Novel Functional Target for the Regulation of the Insulin Signaling Pathway by SIRT1

Molecular and Cellular Endocrinology. Mar, 2011  |  Pubmed ID: 21241768

The protein deacetylase SIRT1, and its activator resveratrol, exert beneficial effects on glucose metabolism. Different SIRT1 targets have been identified, including PTP1B, AMPK, FOXO, PGC-1α and IRS2. The latter may underscore a tight link between SIRT1 and insulin signaling components. However, whether SIRT1 has a direct effect on insulin resistance and whether resveratrol acts directly or indirectly in this context is still a matter of controversy and this question has not been addressed in muscle cells. Here, we show that SIRT1 protein expression is decreased in muscle biopsies and primary myotubes derived from type 2 diabetic patients, suggesting a contribution of diminished SIRT1 in the determination of muscle insulin resistance. To investigate the functional impact of SIRT1 on the insulin pathway, the activation of insulin downstream effector PKB was evaluated after SIRT1 inactivation by RNAi, SIRT1 overexpression, or resveratrol treatments. In muscle cells and HEK293 cells, downregulation of SIRT1 reduced, while overexpression increased, insulin-induced PKB activatory phosphorylation. Further molecular characterisation revealed that SIRT1 interacts in an insulin-independent manner with the PI3K adapter subunit p85. We then investigated whether resveratrol may improve insulin signaling in muscle cells via SIRT1, or alternative targets. Incubation of muscle cells with resveratrol reverted the insulin-resistant state induced by prolonged TNFα or insulin treatment. Resveratrol-dependent improvement of insulin-resistance occurred through inhibition of serine phosphorylation of IRS1/2, implicating resveratrol as a serine kinase inhibitor. Finally, a functional interaction between PI3K and SIRT1 was demonstrated in C. elegans, where constitutively active PI3K - mimicking increased IIS signaling - lead to shortened lifespan, while removal of sir-2.1 abolished PI3K-induced lifespan shortening. Our data identify SIRT1 as a positive modulator of insulin signaling in muscle cells through PI3K, and this mechanism appears to be conserved from C. elegans through humans.

Evaluation of the Efficacy of Radiation-modifying Compounds Using γH2AX As a Molecular Marker of DNA Double-strand Breaks

Genome Integrity. 2011  |  Pubmed ID: 21261999

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds.

Utility of γH2AX As a Molecular Marker of DNA Double-Strand Breaks in Nuclear Medicine: Applications to Radionuclide Therapy Employing Auger Electron-Emitting Isotopes

Current Radiopharmaceuticals. Jan, 2011  |  Pubmed ID: 22191615

There is an intense interest in the development of radiopharmaceuticals for cancer therapy. In particular, radiopharmaceuticals which involve targeting radionuclides specifically to cancer cells with the use of monoclonal antibodies (radioimmunotherapy) or peptides (targeted radiotherapy) are being widely investigated. For example, the ultra-short range Auger electron-emitting isotopes, which are discussed in this review, are being considered in the context of DNAtargeted radiotherapy. The efficient quantitative evaluation of the levels of damage caused by such potential radiopharmaceuticals is required for assessment of therapeutic efficacy and determination of relevant doses for successful treatment. The DNA double-strand break surrogate marker, γH2AX, has emerged as a useful biomonitor of damage and thus effectiveness of treatment, offering a highly specific and sensitive means of assessment. This review will cover the potential applications of γH2AX in nuclear medicine, in particular radionuclide therapy.

Histone Deacetylase Inhibitors Augment Doxorubicin-induced DNA Damage in Cardiomyocytes

Cellular and Molecular Life Sciences : CMLS. Dec, 2011  |  Pubmed ID: 21584806

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutics with suberoylanilide hydroxamic acid (Vorinostat) and depsipeptide (Romidepsin) already being approved for clinical use. Numerous studies have identified that histone deacetylase inhibitors will be most effective in the clinic when used in combination with conventional cancer therapies such as ionizing radiation and chemotherapeutic agents. One promising combination, particularly for hematologic malignancies, involves the use of histone deacetylase inhibitors with the anthracycline, doxorubicin. However, we previously identified that trichostatin A can potentiate doxorubicin-induced hypertrophy, the dose-limiting side-effect of the anthracycline, in cardiac myocytes. Here we have the extended the earlier studies and evaluated the effects of combinations of the histone deacetylase inhibitors, trichostatin A, valproic acid and sodium butyrate on doxorubicin-induced DNA double-strand breaks in cardiomyocytes. Using γH2AX as a molecular marker for the DNA lesions, we identified that all of the broad-spectrum histone deacetylase inhibitors tested augment doxorubicin-induced DNA damage. Furthermore, it is evident from the fluorescence photomicrographs of stained nuclei that the histone deacetylase inhibitors also augment doxorubicin-induced hypertrophy. These observations highlight the importance of investigating potential side-effects, in relevant model systems, which may be associated with emerging combination therapies for cancer.

Cardiac Genes Show Contextual SWI/SNF Interactions with Distinguishable Gene Activities

Epigenetics : Official Journal of the DNA Methylation Society. Jun, 2011  |  Pubmed ID: 21586902

Recent experimental evidence indicates that cardiac and chromatin remodeling are associated with changes in gene expression mediated by Brahma-related gene 1 (Brg1), a member of the large group of SWI/SNF subunits. The second catalytic member of this family is Brahma (Brm), which shares close sequence homology to Brg1. Despite the sequence similarities, these determinants are found in distinct regulatory complexes; however, the precise nature and role of these remodeling enzymes in the failing heart remains unknown. Here we have hypothesized that Brg1 and Brm form distinct complexes in regulating gene expression in an animal model of cardiac hypertrophy. We have identified that the hypertrophic myocardium is characterized by profound morphological changes associated with increased expression of ANP (Nppa), BNP (Nppb) and β-MHC (Myh7) genes, correlating with reduced expression of the α-MHC (Myh6) and SERCA2A (Atp2a2) genes. Histone deacetylase inhibition prevented left ventricular hypertrophy indicating that the re-expression of gene activity can be associated with both contextual and distinct SWI/SNF interactions. We hypothesize that cardiac hypertrophy and the fetal gene expression program are associated with distinguishable binding of Brm and Brg1 on genes present in distinct complexes, suggesting possible independent-regulatory roles.

Remodeling is at the Heart of Chromatin: the Heartaches of Chromatin

Epigenetics : Official Journal of the DNA Methylation Society. Jul, 2011  |  Pubmed ID: 21646860

Chromatin modifications are integral elements of chromosome structure and its function and the vasculature depends on tissue-specific genome regulation for its development. A general concept for the de-regulation of chromatin modifications in cardiac and vascular disease is also emerging. The recognition that metabolic memory contributes to disease persistence highlights the benefit of early and aggressive treatment. As for the importance of memory, we do know that good metabolic control delays the onset of long-term diabetic complications. There are striking parallels between the timing of disease and the development of complications. Landmark multicenter clinical trials on diabetes patients have popularized the concept that glucose is also a demonstrable determinant for the development of complications, indicating the prolonged benefit of intensive therapy and the lasting damage of conventional therapy. Each cell type experiences thousands of modifications to the epigenome in response to environmental changes it is exposed to. Therefore, history is neither lost nor forgotten and previous experiences and exposure may form future memories. There is now a strong resurgence in research trying to understand gene-environment interactions and to determine what commits specific vascular cell types to specific memories. Recent insights show that cardiac gene expression is distinguished by specific chromatin remodeling events and histone modifications that are associated with heart disease.

Chromatin Modifying Agents - the Cutting Edge of Anticancer Therapy

Drug Discovery Today. Jul, 2011  |  Pubmed ID: 21664485

Chromatin modifying compounds are emerging as the next generation of anticancer therapies. By altering gene expression they could be able to correct uncontrolled proliferation and, in certain cases, aberrant apoptotic pathways, which are hallmarks of malignant cells. The modulation of gene expression is regulated via chromatin remodelling processes that include DNA methylation and chromatin modifications. The identification of aberrant methylation of genes and dysregulated histone acetylation status in cancer cells provides a basis for novel epigenetic therapies. Currently available chromatin modifying agents, a group that includes DNA methyltransferase and histone deacetylase inhibitors, exert anticancer effects by reactivating tumour suppressor genes, inhibiting proliferation and inducing apoptosis. It is anticipated that massive parallel sequencing will identify new epigenetic targets for drug development.

New Insights into the Pathogenesis of Beckwith-Wiedemann and Silver-Russell Syndromes: Contribution of Small Copy Number Variations to 11p15 Imprinting Defects

Human Mutation. Oct, 2011  |  Pubmed ID: 21780245

The imprinted 11p15 region is organized in two domains, each of them under the control of its own imprinting control region (ICR1 for the IGF2/H19 domain and ICR2 for the KCNQ1OT1/CDKN1C domain). Disruption of 11p15 imprinting results in two fetal growth disorders with opposite phenotypes: the Beckwith-Wiedemann (BWS) and the Silver-Russell (SRS) syndromes. Various 11p15 genetic and epigenetic defects have been demonstrated in BWS and SRS. Among them, isolated DNA methylation defects account for approximately 60% of patients. To investigate whether cryptic copy number variations (CNVs) involving only part of one of the two imprinted domains account for 11p15 isolated DNA methylation defects, we designed a single nucleotide polymorphism array covering the whole 11p15 imprinted region and genotyped 185 SRS or BWS cases with loss or gain of DNA methylation at either ICR1 or ICR2. We describe herein novel small gain and loss CNVs in six BWS or SRS patients, including maternally inherited cis-duplications involving only part of one of the two imprinted domains. We also show that ICR2 deletions do not account for BWS with ICR2 loss of methylation and that uniparental isodisomy involving only one of the two imprinted domains is not a mechanism for SRS or BWS.

Genome-wide Analysis Distinguishes Hyperglycemia Regulated Epigenetic Signatures of Primary Vascular Cells

Genome Research. Oct, 2011  |  Pubmed ID: 21890681

Emerging evidence suggests that poor glycemic control mediates post-translational modifications to the H3 histone tail. We are only beginning to understand the dynamic role of some of the diverse epigenetic changes mediated by hyperglycemia at single loci, yet elevated glucose levels are thought to regulate genome-wide changes, and this still remains poorly understood. In this article we describe genome-wide histone H3K9/K14 hyperacetylation and DNA methylation maps conferred by hyperglycemia in primary human vascular cells. Chromatin immunoprecipitation (ChIP) as well as CpG methylation (CpG) assays, followed by massive parallel sequencing (ChIP-seq and CpG-seq) identified unique hyperacetylation and CpG methylation signatures with proximal and distal patterns of regionalization associative with gene expression. Ingenuity knowledge-based pathway and gene ontology analyses indicate that hyperglycemia significantly affects human vascular chromatin with the transcriptional up-regulation of genes involved in metabolic and cardiovascular disease. We have generated the first installment of a reference collection of hyperglycemia-induced chromatin modifications using robust and reproducible platforms that allow parallel sequencing-by-synthesis of immunopurified content. We uncover that hyperglycemia-mediated induction of genes and pathways associated with endothelial dysfunction occur through modulation of acetylated H3K9/K14 inversely correlated with methyl-CpG content.

Genetic Examination of SETD7 and SUV39H1/H2 Methyltransferases and the Risk of Diabetes Complications in Patients with Type 1 Diabetes

Diabetes. Nov, 2011  |  Pubmed ID: 21896933

Hyperglycemia plays a pivotal role in the development and progression of vascular complications, which are the major sources of morbidity and mortality in diabetes. Furthermore, these vascular complications often persist and progress despite improved glucose control, possibly as a result of prior episodes of hyperglycemia. Epigenetic modifications mediated by histone methyltransferases are associated with gene-activating events that promote enhanced expression of key proinflammatory molecules implicated in vascular injury. In this study, we investigated genetic polymorphisms of the SETD7, SUV39H1, and SUV39H2 methyltransferases as predictors of risk for micro- and macrovascular complications in type 1 diabetes.

Investigation into the Biological Properties of the Olive Polyphenol, Hydroxytyrosol: Mechanistic Insights by Genome-wide MRNA-Seq Analysis

Genes & Nutrition. Sep, 2011  |  Pubmed ID: 21953375

The medicinal properties of the leaves and fruit of Olea Europaea (olive tree) have been known since antiquity. Numerous contemporary studies have linked the Mediterranean diet with increased health. In particular, consumption of olive oil has been associated with a decreased risk of cardiovascular disease and certain cancers. Increasingly, there has been an interest in the biological properties of polyphenols, which are minor constituents of olive oil. For example, hydroxytyrosol has been shown to be a potent antioxidant and has anti-atherogenic and anti-cancer properties. The overall aim of this study was to provide insights into the molecular mechanisms of action of hydroxytyrosol using genome-wide mRNA-Seq. Initial experiments were aimed at assessing cytotoxicity, apoptosis and cell cycle effects of hydroxytyrosol in various cell lines. The findings indicated a dose-dependent reduction in cell viability in human erythroleukemic K562 and human keratinocytes. When comparing the viability in parental CEM-CCRF and R100 cells (which overexpress the P-glycoprotein pump), it was determined that the R100 cells were more resistant to effects of hydroxytyrosol suggesting efflux by the multi-drug resistance pump. By comparing the uptake of Hoechst 33342 in the two cell lines that had been pretreated with hydroxytyrosol, it was determined that the polyphenol may have P-glycoprotein-modulating activity. Further, initial studies indicated modest radioprotective effects of relatively low doses of hydroxytyrosol in human keratinocytes. Analysis of mRNA sequencing data identified that treatment of keratinocytes with 20 μM hydroxytyrosol results in the upregulation of numerous antioxidant proteins and enzymes, including heme oxygenase-1 (15.46-fold upregulation), glutaredoxin (1.65) and glutathione peroxidase (1.53). This may account for the radioprotective activity of the compound, and reduction in oxidative stress suggests a mechanism for chemoprevention of cancer by hydroxytyrosol. Alteration in the expression of transcription factors may also contribute to the anti-cancer effects described in numerous studies. These include changes in the expression of STAT3, STAT6, SMAD7 and ETS-1. The telomerase subunit TERT was also found to be downregulated in K562 cells. Overall, our findings provide insights into the mechanisms of action of hydroxytyrosol, and more generally, we identify potential gene candidates for further exploration.

Protective Effects of Valproic Acid Against Airway Hyperresponsiveness and Airway Remodeling in a Mouse Model of Allergic Airways Disease

Epigenetics : Official Journal of the DNA Methylation Society. Dec, 2011  |  Pubmed ID: 22139576

Airway remodeling and airway hyperresponsiveness are major aspects of asthma pathology that are not targeted optimally by existing anti-inflammatory drugs. Histone deacetylase inhibitors have a wide range of effects that may potentially abrogate aspects of remodeling. One such histone deacetylase inhibitor is valproic acid (2-propylvaleric acid). Valproic acid is used clinically as an anti-epileptic drug and is a potent inhibitor of class I histone deacetylases but also inhibits class II histone deacetylases. We used valproic acid as a molecular model of histone deacetylase inhibition in vivo in chronic allergic airways disease mice with airway remodeling and airway hyperresponsiveness. Wild-type Balb/c mice with allergic airways disease were treated with valproic acid or vehicle control. Airway inflammation was assessed by bronchoalveolar lavage fluid cell counts and examination of lung tissue sections. Remodeling was assessed by morphometric analysis of histochemically stained slides and lung function was assessed by invasive plethysmography measurement of airway resistance. Valproic acid treatment did not affect inflammation parameters; however, valproic acid treatment resulted in reduced epithelial thickness as compared to vehicle treated mice (p < 0.01), reduced subepithelial collagen deposition (p < 0.05) and attenuated airway hyperresponsiveness (p < 0.05 and p < 0.01 for the two highest doses of methacholine, respectively). These findings show that treatment with valproic acid can reduce structural airway remodeling changes and hyperresponsiveness, providing further evidence for the potential use of histone deacetylase inhibitors for the treatment of asthma.

Glycemic Memory

Current Opinion in Lipidology. Feb, 2012  |  Pubmed ID: 22186662

The mistake of predicting the future is perhaps not tending to repressed or past memories. Hamlet's 17th-Century soliloquy 'the heartache and the thousand natural shocks, that flesh is heir to', (3.1. 7-8) is a tale that looks beyond the present by linking the past with the future. The present article examines the resurgence in the field to understand gene-regulating epigenetic changes conferring glycemic memory.

Influence of Natural and Synthetic Histone Deacetylase Inhibitors on Chromatin

Antioxidants & Redox Signaling. Feb, 2012  |  Pubmed ID: 22229817

Abstract Significance: Histone deacetylase inhibitors (HDACIs) have emerged as a new class of anticancer therapeutics. The hydroxamic acid, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™), and the cyclic peptide, depsipeptide (Romidepsin, Istodax™), were approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma in 2006 and 2009, respectively. At least 15 HDACIs are currently undergoing clinical trials either alone or in combination with other therapeutic modalities for the treatment of numerous hematological and solid malignancies. Recent Advances: The potential utility of HDACIs has been extended to nononcologic applications, including autoimmune disorders, inflammation, diseases of the central nervous system, and malaria. Critical Issues: Given the promise of HDACIs, there is growing interest in the potential of dietary compounds that possess HDAC inhibition activity. This review is focused on the identification of and recent findings with HDACIs from dietary, medicinal plant, and microbial sources. We discuss the mechanisms of action and clinical potential of natural HDACIs. Future Directions: Apart from identification of further HDACI compounds from dietary sources, further research will be aimed at understanding the effects on gene regulation on lifetime exposure to these compounds. Another important issue that requires clarification. Antioxid. Redox Signal. 00, 000-000.

Microparticles: Major Transport Vehicles for Distinct MicroRNAs in Circulation

Cardiovascular Research. Feb, 2012  |  Pubmed ID: 22258631

AIMS: Circulating microRNAs (miRNAs) have attracted major interest as biomarkers for cardiovascular diseases. Since RNases are abundant in circulating blood, there needs to be a mechanism protecting miRNAs from degradation. We hypothesized that microparticles (MP) represent protective transport vehicles for miRNAs and that these are specifically packaged by their maternal cells. METHODS AND RESULTS: Conventional plasma preparations, such as the ones used for biomarker detection, are shown to contain substantial numbers of platelet-, leucocyte-, and endothelial cell-derived MP. To analyse the widest spectrum of miRNAs, Next Generation Sequencing was used to assess miRNA profiles of MP and their corresponding stimulated and non-stimulated cells of origin. THP-1 (monocytic origin) and human umbilical vein endothelial cell (HUVEC) MP were used for representing circulating MP at a high purity. miRNA profiles of MP differed significantly from those of stimulated and non-stimulated maternal THP-1 cells and HUVECs, respectively. Quantitative reverse transcription-polymerase chain reaction of miRNAs which have been associated with cardiovascular diseases also demonstrated significant differences in miRNA profiles between platelets and their MP. Notably, the main fraction of miRNA in plasma was localized in MP. Furthermore, miRNA profiles of MP differed significantly between patients with stable and unstable coronary artery disease. CONCLUSION: Circulating MP represent transport vehicles for large numbers of specific miRNAs, which have been associated with cardiovascular diseases. miRNA profiles of MP are significantly different from their maternal cells, indicating an active mechanism of selective 'packaging' from cells into MP. These findings describe an interesting mechanism for transferring gene-regulatory function from MP-releasing cells to target cells via MP circulating in blood.

Molecular Model of Naphthalene-induced DNA Damage in the Murine Lung

Human & Experimental Toxicology. Jan, 2012  |  Pubmed ID: 21508073

Airway epithelial damage and repair represents a novel therapeutic target in asthma and chronic obstructive pulmonary disease. An established mouse model of airway epithelial damage involves the Clara cell cytotoxicity of parenterally administered naphthalene, an important environmental toxicant with genotoxic and carcinogenic potential. The objective of the current study was to investigate naphthalene-induced toxicity and to identify and quantify DNA double-strand breaks in a murine naphthalene model of airway epithelial damage. Male C57/BL6 mice were injected with 200 mg/kg naphthalene and culled at 12-, 24-, 48- and 72-h time points. Lung function and bronchoalveolar lavage was performed and the lungs were dissected for histological analysis and for quantitation of DNA double-strand breaks using γH2AX as a molecular marker. Mice injected with naphthalene had increased epithelial denudation, bronchoalveolar lavage fluid cellularity and reactivity to nebulized methacholine chloride as compared to corn oil vehicle controls. Histological changes were most pronounced at the 12- and 24-h time points. DNA double-strand breaks, quantitated as the number of γH2AX foci per cell, were highest at the 24- and 48-h time points. All parameters had decreased at the 72-h time point, consistent with airway re-epithelization and cellular repair. Our findings indicate a time-dependent accumulation of γH2AX foci in mouse airway epithelial cells following administration of naphthalene. Naphthalene airway epithelial injury constitutes a model of DNA double-strand breaks in mice, which can be adapted as a suitable model for further investigation of genotoxic damage for evaluating the efficacy of potential therapeutics.