Transgenerational changes in the genome stability and methylation in pathogen-infected plants: (Virus-induced plant genome instability)

Previously, we reported the generation of a virus-induced systemic signal that increased the somatic and meiotic recombination rates in tobacco mosaic virus (TMV)-infected tobacco plants. Here, we analyzed the progeny of plants that received the signal and found that these plants also have a higher frequency of rearrangements in the loci carrying the homology to LRR region of the gene of resistance to TMV (N-gene). Analysis of the stability of repetitive elements from Nicotiana tabacum loci and 5.8S ribosomal RNA loci did not show any changes. Further analysis of the changes in the progeny of infected plants revealed that they had substantially hypermethylated genomes. At the same time, loci-specific methylation analysis showed: (1) profound hypomethylation in several LRR-containing loci; (2) substantial hypermethylation of actin loci and (3) no change in methylation in the loci of repetitive elements from N. tabacum or 5.8S ribosomal RNA. Global genome hypermethylation of the progeny is believed to be part of a general protection mechanism against stress, whereas locus-specific hypomethylation is associated with a higher frequency of rearrangements. Increased recombination events combined with the specific methylation pattern induced by pathogen attack could be a sign of an adaptive response by plants

Role of epigenetic aberrations in the development and progression of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers in humans. The molecular mechanisms leading to the development of HCC are extremely complicated and consist of prominent genetic, genomic, and epigenetic alterations. This review summarizes the current knowledge of the role of epigenetic aberrations, including changes in DNA methylation, histone modifications, and expression of microRNAs in the pathogenesis of HCC. It also emphasizes that identification of the underlying epigenetic alterations that drive cell transformation and promote development and progression of HCC is crucially important for understanding mechanisms of hepatocarcinogenesis, its detection, therapeutic intervention, and prevention

Characterization of copy number alterations in a mouse model of fibrosis-associated hepatocellular carcinoma reveals concordance with human disease

AbstractHepatocellular carcinoma (HCC) is a prevalent human cancer with rising incidence worldwide. Human HCC is frequently associated with chronic liver inflammation and cirrhosis, pathophysiological processes that are a consequence of chronic viral infection, disturbances in metabolism, or exposure to chemical toxicants. To better characterize the pathogenesis of HCC, we used a human disease‐relevant mouse model of fibrosis‐associated hepatocarcinogenesis. In this model, marked liver tumor response caused by the promutagenic chemical N‐nitrosodiethylamine in the presence of liver fibrosis was associated with epigenetic events indicative of genomic instability. Therefore, we hypothesized that DNA copy number alterations (CNAs), a feature of genomic instability and a common characteristic of cancer, are concordant between human HCC and mouse models of fibrosis‐associated hepatocarcinogenesis. We evaluated DNA CNAs and changes in gene expression in the mouse liver (normal, tumor, and nontumor fibrotic tissues). Additionally, we compared our findings to DNA CNAs in human HCC cases (tumor and nontumor cirrhotic/fibrotic tissues) using publicly available data from The Cancer Genome Atlas (TCGA). We observed that while fibrotic liver tissue is largely devoid of DNA CNAs, highly frequently occurring DNA CNAs are found in mouse tumors, which is indicative of a profound increase in chromosomal instability in HCC. The cross‐species gene‐level comparison of CNAs identified shared regions of CNAs between human fibrosis‐ and cirrhosis‐associated liver tumors and mouse fibrosis‐associated HCC. Our results suggest that CNAs most commonly arise in neoplastic tissue rather than in fibrotic or cirrhotic liver, and demonstrate the utility of this mouse model in replicating the molecular features of human HCC

Epigenetic aspects of genotoxic and non-genotoxic hepatocarcinogenesis: Studies in rodents

Hepatocellular carcinoma, which is one of the most prevalent life-threatening human cancers, is showing an increased incidence worldwide. Recent evidence indicates that the development of hepatocellular carcinoma is associated with not only genetic alterations, but also with profound epigenetic changes. This review summarizes the current knowledge about epigenetic alterations during rodent hepatocarcinogenesis, considers the similarities and differences in epigenetic effects of genotoxic and non-genotoxic rodent liver carcinogens, and discusses the possible role of these effects in the causality of liver tumor development

Chronic administration of ethanol leads to an increased incidence of hepatocellular adenoma by promoting H-ras-mutated cells

This study used tissue samples from male B6C3F1 mice treated with ethanol in drinking water (0, 2.5, or 5%) for 4 or 104 weeks. We tested whether chronic alcohol drinking promotes oxidative stress in the liver and characterized the mutation profile of spontaneous and ethanol-induced tumors. We show that ethanol does not cause detectable oxidative stress in the liver at any time point and acts by promoting H-ras mutated cells

Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor α

Peroxisome proliferators are potent rodent liver carcinogens that act via a non-genotoxic mechanism. The mode of action of these agents in rodent liver includes increased cell proliferation, decreased apoptosis, secondary oxidative stress and other events; however, it is not well understood how peroxisome proliferators are triggering the plethora of the molecular signals leading to cancer. Epigenetic changes have been implicated in the mechanism of liver carcinogenesis by a number of environmental agents. Short-term treatment with peroxisome proliferators and other non-genotoxic carcinogens leads to global and locus-specific DNA hypomethylation in mouse liver, events that were suggested to correlate with a burst of cell proliferation. In the current study, we investigated the effects of long-term exposure to a model peroxisome proliferator WY-14,643 on DNA and histone methylation. Male SV129 mice were fed a control or WY-14,643-containing (1000 ppm) diet for 1 wk, 5 wks or 5 months. Treatment with WY-14,643 led to progressive global hypomethylation of liver DNA as determined by an HpaII-based cytosine extension assay with the maximum effect reaching over 200% at 5 months. Likewise, trimethylation of histone H4 lysine 20 and H3 lysine 9 was significantly decreased at all time points. The majority of cytosine methylation in mammals resides in repetitive DNA sequences. In view of this, we measured the effect of WY-14,643 on the methylation status of major and minor satellites, as well as in IAP, LINE1 and LINE2 elements in liver DNA. Exposure to WY-14,643 resulted in a gradual loss of cytosine methylation in major and minor satellites, IAP, LINE1 and LINE2 elements. The epigenetic changes correlated with the temporal effects of WY-14,643 on cell proliferation rates in liver, but no sustained effect on c-Myc promoter methylation was observed. Finally, WY-14,643 had no effect on DNA and histone methylation status in Pparα-null mice at any of the time points considered in this study. These data indicate the importance of epigenetic alterations in the mechanism of action of peroxisome proliferators and the key role of PPARα

Comparative analysis of promoter methylation and gene expression endpoints between tumorous and non-tumorous tissues from HCV-positive patients with hepatocellular carcinoma

Transcriptional silencing of tumor suppressor genes and other cancer-related genes induced by promoter CpG island hypermethylation is an important epigenetic mechanism of hepatocarcinogenesis. Previous studies have established methylation profiles of hepatocellular carcinomas (HCCs) and demonstrated that methylation of several candidate genes in resected tissues may be associated with time to recurrence. The goals of our study were to test whether specific promoter methylation and mRNA levels of candidate genes, as well as global changes in DNA methylation, can be linked with time to recurrence and clinicopathological variables in a homogenous study group of HCC patients. Forty-three tumorous and 45 non-tumorous liver tissue samples from the surgical margin were obtained from HCV-positive, HBV-negative HCC patients who underwent tumor resection surgery and who were monitored for tumor recurrence thereafter (median follow-up time: 16 months (range, 0 – 79 months)). Methylation-specific PCR was used to assess the promoter methylation status of P16(INK4a), SOCS-1, RASSF1A, APC, GSTP1, RIZ1, and MGMT genes, while the level of LINE-1 methylation was used as marker of global DNA methylation levels. Methylation frequencies in P16(INK4a), RASSF1A, APC, GSTP1, and RIZ1 genes were significantly greater in tumorous versus non-tumorous tissues. Methylation of RIZ1 in non-tumorous tissues was significantly associated with time to recurrence. Additionally, genomic DNA was significantly more hypomethylated in tumorous tissues, and this change was associated with shorter recurrence, but not with clinicopathological features. In conclusion, this study supports the role of aberrant methylation in the pathobiology of HCV-positive HCCs. The finding that RIZ1 methylation and increased levels of LINE-1 hypomethylation in non-tumorous tissues are associated with time to recurrence underscores the importance of assessing the epigenetic state of the liver remnant

Gene Expression and DNA Methylation Alterations During Non-alcoholic Steatohepatitis-Associated Liver Carcinogenesis

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers. HCC is characterized by an acquisition of multiple abnormal phenotypes driven by genetic and epigenetic alterations, especially abnormal DNA methylation. Most of the existing clinical and experimental reports provide only a snapshot of abnormal DNA methylation patterns in HCC rather than their dynamic changes. This makes it difficult to elucidate the significance of these changes in the development of HCC. In the present study, we investigated hepatic gene expression and gene-specific DNA methylation alterations in mice using the Stelic Animal Model (STAM) of non-alcoholic steatohepatitis (NASH)-derived liver carcinogenesis. Analysis of the DNA methylation status in aberrantly expressed epigenetically regulated genes showed the accumulation of DNA methylation abnormalities during the development of HCC, with the greatest number of aberrantly methylated genes being found in full-fledged HCC. Among these genes, only one gene, tubulin, beta 2B class IIB (Tubb2b), was increasingly hypomethylated and over-expressed during the progression of the carcinogenic process. Furthermore, the TUBB2B gene was also over-expressed and hypomethylated in poorly differentiated human HepG2 cells as compared to well-differentiated HepaRG cells. The results of this study indicate that unique gene-expression alterations mediated by aberrant DNA methylation of selective genes may contribute to the development of HCC and may have diagnostic value as the disease-specific indicator

Epigenetic Alterations in Liver of C57BL/6J Mice after Short-Term Inhalational Exposure to 1,3-Butadiene

Background1,3-Butadiene (BD) is a high-volume industrial chemical and a known human carcinogen. The main mode of BD carcinogenicity is thought to involve formation of genotoxic epoxides.ObjectivesIn this study we tested the hypothesis that BD may be epigenotoxic (i.e., cause changes in DNA and histone methylation) and explored the possible molecular mechanisms for the epigenetic changes.Methods and ResultsWe administered BD (6.25 and 625 ppm) to C57BL/6J male mice by inhalation for 2 weeks (6 hr/day, 5 days a week) and then examined liver tissue from these mice for signs of toxicity using histopathology and gene expression analyses. We observed no changes in mice exposed to 6.25 ppm BD, but glycogen depletion and dysregulation of hepatotoxicity biomarker genes were observed in mice exposed to 625 ppm BD. We detected N-7-(2,3,4-trihydroxybut-1-yl)guanine (THB-Gua) adducts in liver DNA of exposed mice in a dose-responsive manner, and also observed extensive alterations in the cellular epigenome in the liver, including demethylation of global DNA and repetitive elements and a decrease in histone H3 and H4 lysine methylation. In addition, we observed down-regulation of DNA methyltransferase 1 (Dnmt1) and suppressor of variegation 3–9 homolog 1, a histone lysine methyltransferase (Suv39h1), and up-regulation of the histone demethylase Jumonji domain 2 (Jmjd2a), proteins responsible for the accurate maintenance of the epigenetic marks. Although the epigenetic effects were most pronounced in the 625-ppm exposure group, some effects were evident in mice exposed to 6.25 ppm BD.ConclusionsThis study demonstrates that exposure to BD leads to epigenetic alterations in the liver, which may be important contributors to the mode of BD carcinogenicity

Mechanisms of peroxisome proliferator-induced DNA hypomethylation in rat liver☆

Genomic hypomethylation is a consistent finding in both human and animal tumors and mounting experimental evidence suggests a key role for epigenetic events in tumorigenesis. Furthermore, it has been suggested that early changes in DNA methylation and histone modifications may serve as sensitive predictive markers in animal testing for carcinogenic potency of environmental agents. Alterations in metabolism of methyl donors, disturbances in activity and/or expression of DNA methyltransferases, and presence of DNA single-strand breaks could contribute to the loss of cytosine methylation during carcinogenesis; however, the precise mechanisms of genomic hypomethylation induced by chemical carcinogens remain largely unknown. This study examined the mechanism of DNA hypomethylation during hepatocarcinogenesis induced by peroxisome proliferators WY-14,643 (4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid) and DEHP (di-(2-ethylhexyl)phthalate), agents acting through non-genotoxic mode of action. In the liver of male Fisher 344 rats exposed to WY-14,643 (0.1% (w/w), 5 months), the level of genomic hypomethylation increased by ~2-fold, as compared to age-matched controls, while in the DEHP group (1.2% (w/w), 5 months) DNA methylation did not change. Global DNA hypomethylation in livers from WY-14,643 group was accompanied by the accumulation of DNA single-strand breaks, increased cell proliferation, and diminished expression of DNA methyltransferase 1, while the metabolism of methyl donors was not affected. In contrast, none of these parameters changed significantly in rats fed DEHP. Since WY-14,643 is much more potent carcinogen than DEHP, we conclude that the extent of loss of DNA methylation may be related to the carcinogenic potential of the chemical agent, and that accumulation of DNA single-strand breaks coupled to the increase in cell proliferation and altered DNA methyltransferase expression may explain genomic hypomethylation during peroxisome proliferator-induced carcinogenesis