Elevated 5hmC levels characterize DNA of the cerebellum in Parkinson’s disease

5-methylcytosine and the oxidation product 5-hydroxymethylcytosine are two prominent epigenetic variants of the cytosine base in nuclear DNA of mammalian brains. We measured levels of 5-methylcytosine and 5-hydroxymethylcytosine by enzyme-linked immunosorbent assay in DNA from post-mortem cerebella of individuals with Parkinson’s disease and age-matched controls. 5-methylcytosine levels showed no significant differences between Parkinson’s disease and control DNA sample sets. In contrast, median 5-hydroxymethylcytosine levels were almost twice as high (p < 0.001) in both male and female Parkinson’s disease individuals compared with controls. The distinct epigenetic profile identified in cerebellar DNA of Parkinson’s disease patients raises the question whether elevated 5-hydroxymethylcytosine levels are a driver or a consequence of Parkinson’s disease

Local iron homeostasis in the breast ductal carcinoma microenvironment

Abstract BACKGROUND: While the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored. METHODS: Immunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization. RESULTS: We confirm previous results by showing that breast cancer epithelial cells present an 'iron-utilization phenotype' with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an 'iron-donor' phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size. CONCLUSIONS: The present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context.info:eu-repo/semantics/publishedVersio

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics

Cerebellar Oxidative DNA Damage and Altered DNA Methylation in the BTBR T+tf/J Mouse Model of Autism and Similarities with Human Post Mortem Cerebellum

<div><p>The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (<i>Ogg1</i>) and increased expression of <i>de novo</i> DNA methyltransferases 3a and 3b (<i>Dnmt3a</i> and <i>Dnmt3b</i>). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression <i>1 (Tet1)</i> and <i>Tet2</i> genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of <i>OGG1</i> alterations in pathogenesis of autism.</p></div

Oxidative DNA damage in the cerebellum of BTBR mice.

<p>(<b>A</b>) Levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG) in genomic DNA isolated from the cerebellum of BTBR T+tf/J and C57BL/6J mice (mean ± SD, n = 5). (<b>B</b>) Levels of 8-oxodG in genomic DNA isolated from the cerebellum of autism individuals and unaffected control individuals (mean ± SD, n = 13). (<b>C</b>) The extent of mitochondrial DNA damage in the cerebellum of BTBR T+tf/J mice (mean ± SD, n = 5). (<b>D</b>) The expression of base excision DNA repair genes in the cerebellum of BTBR T+tf/J and C57BL/6J mice. The expression of <i>Ogg1, Ung, Apex1</i>, and <i>Polβ</i> genes was determined by qRT-PCR as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113712#s2" target="_blank">Materials and Methods</a>”. The results are presented as an average fold change in the expression of each gene in the cerebellum of BTBR T+tf/J mice relatively to that in C57BL/6J mice, which was assigned a value 1. * - Significantly different from C57BL/6J mice (mean ± SD, n = 5). (<b>E</b>) Correlation plots of the expression of <i>Ogg1</i> and the 8-oxodG level in mouse genomic DNA. Each symbol represents an individual animal.</p

Levels of 5mC and 5hmC in cerebellar genomic DNA.

<p>(<b>A</b>) Levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in genomic DNA isolated from the cerebellum of BTBR T+tf/J and C57BL/6J mice (mean ± SD, n = 5). (<b>B</b>) Levels of 5mC and 5hmC in genomic DNA isolated from the cerebellum of autism individuals and unaffected control individuals. Values were represented as open and closed circles as well as box plots. * - Significantly different from C57BL/6J mice or autism-free individuals. (<b>C</b>) Correlation plots of the 5hmC and the 8-oxo-7-hydrodeoxyguanosine (8-oxodG) levels in mouse and human cerebellar genomic DNA.</p

Western blot analysis of histone H3K4, H3K9, H3K27, and H4K20 trimethylation; H3K9, H4K20 dimethylation and H3K9, H3K27, and H4K16 acetylation in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>Densitometric analysis of the immunostaining results is shown as percent change in histone modification level in the cerebellum BTBR T+tf/J mice relative to the corresponding values in C57BL/6J mice, which was assigned a value of 100% (mean ± SD, n = 5).</p

Whole genome microarray analysis of gene expression in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>(<b>A</b>) Heat map illustrating significant differences in global gene expression between BTBR T+tf/J and C57BL/6J mice. The color bar identifies high-expressed (red) and low-expressed (green) genes. (<b>B</b>) Principal component analysis illustrating similarities and differences between BTBR T+tf/J and C57BL/6J mouse strains. (<b>C</b>) Venn diagram showing genes that were significantly different between BTBR T+tf/J and C57BL/6J mice. (<b>D</b>) Summary of molecular pathways that significantly differ between BTBR T+tf/J and C57BL/6J mice. The Ingenuity Pathway Analysis database (version 9.0) was used to determine and visualize molecular pathways enriched by the significant mRNA transcripts a P-value of <0.05 were considered “enriched”.</p

The expression of chromatin-modifying (A) and one carbon metabolism (B) genes in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>The gene expression was determined by qRT-PCR as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113712#s2" target="_blank">Materials and Methods</a>”. The results are presented as an average fold change in the expression of each gene in the cerebellum of BTBR T+tf/J mice relatively to that in C57BL/6J mice, which was assigned a value 1. * - Significantly different from C57BL/6J mice (mean ± SD, n = 5).</p