Altered expression of the IGF2­H19 locus and mitochondrial respiratory complexes in adrenocortical carcinoma.

Abnormalities of the insulin­like growth factor 2 (IGF2)­H19 locus with the overexpression of IGF2 are frequent findings in adrenocortical carcinoma (ACC). The present study assessed the expression of RNAs and microRNAs (miRNAs/miRs) from the IGF2­H19 locus using PCR­based methods in ACC and adrenocortical adenoma (ACA). The results were associated with proteomics data. IGF2 was overexpressed in ACC, and its expression correlated with that of miR­483­3p and miR­483­5p hosted by IGF2. The downregulated expression of H19 in ACC compared to ACA correlated with miR­675 expression hosted by H19. Several proteins exhibited an inverse correlation in expression and were predicted as targets of miR­483­3p, miR­483­5p or miR­675. Subsets of these proteins were differentially expressed between ACC and ACA. These included several proteins involved in mitochondrial metabolism. Among the mitochondrial respiratory complexes, complex I and IV were significantly decreased in ACC compared to ACA. The protein expression of NADH:ubiquinone oxidoreductase subunit C1 (NDUFC1), a subunit of mitochondrial respiratory complex I, was further validated as being lower in ACC compared to ACA and normal adrenals. The silencing of miR­483­5p increased NDUFC1 protein expression and reduced both oxygen consumption and glycolysis rates. On the whole, the findings of the present study reveal the dysregulation of the IGF2­H19 locus and mitochondrial respiration in ACC. These findings may provide a basis for the further understanding of the pathogenesis of ACC and may have potential values for diagnostics and treatment.

Comprehensive longitudinal study of epigenetic mutations in aging.

BACKGROUND: The role of DNA methylation in aging has been widely studied. However, epigenetic mutations, here defined as aberrant methylation levels compared to the distribution in a population, are less understood. Hence, we investigated longitudinal accumulation of epigenetic mutations, using 994 blood samples collected at up to five time points from 375 individuals in old ages. RESULTS: We verified earlier cross-sectional evidence on the increase of epigenetic mutations with age, and identified important contributing factors including sex, CD19+ B cells, genetic background, cancer diagnosis, and technical artifacts. We further classified epigenetic mutations into High/Low Methylation Outliers (HMO/LMO) according to their changes in methylation, and specifically studied methylation sites (CpGs) that were prone to mutate (frequently mutated CpGs). We validated four epigenetically mutated CpGs using pyrosequencing in 93 samples. Furthermore, by using twins, we concluded that the age-related accumulation of epigenetic mutations was not related to genetic factors, hence driven by stochastic or environmental effects. CONCLUSIONS: Here we conducted a comprehensive study of epigenetic mutation and highlighted its important role in aging process and cancer development.

Tobacco smoking induces changes in true DNA methylation, hydroxymethylation and gene expression in bronchoalveolar lavage cells.

BACKGROUND: While smoking is known to associate with development of multiple diseases, the underlying mechanisms are still poorly understood. Tobacco smoking can modify the chemical integrity of DNA leading to changes in transcriptional activity, partly through an altered epigenetic state. We aimed to investigate the impact of smoking on lung cells collected from bronchoalveolar lavage (BAL). METHODS: We profiled changes in DNA methylation (5mC) and its oxidised form hydroxymethylation (5hmC) using conventional bisulphite (BS) treatment and oxidative bisulphite treatment with Illumina Infinium MethylationEPIC BeadChip, and examined gene expression by RNA-seq in healthy smokers. FINDINGS: We identified 1667 total 5mC + 5hmC, 1756 5mC and 67 5hmC differentially methylated positions (DMPs) between smokers and non-smokers (FDR-adjusted P <.05, absolute Δß >0.15). Both 5mC DMPs and to a lesser extent 5mC + 5hmC were predominantly hypomethylated. In contrast, almost all 5hmC DMPs were hypermethylated, supporting the hypothesis that smoking-associated oxidative stress can lead to DNA demethylation, via the established sequential oxidation of which 5hmC is the first step. While we confirmed differential methylation of previously reported smoking-associated 5mC + 5hmC CpGs using former generations of BeadChips in alveolar macrophages, the large majority of identified DMPs, 5mC + 5hmC (1639/1667), 5mC (1738/1756), and 5hmC (67/67), have not been previously reported. Most of these novel smoking-associating sites are specific to the EPIC BeadChip and, interestingly, many of them are associated to FANTOM5 enhancers. Transcriptional changes affecting 633 transcripts were consistent with DNA methylation profiles and converged to alteration of genes involved in migration, signalling and inflammatory response of immune cells. INTERPRETATION: Collectively, these findings suggest that tobacco smoke exposure epigenetically modifies BAL cells, possibly involving a continuous active demethylation and subsequent increased activity of inflammatory processes in the lungs. FUND: The study was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, the Stockholm County Council (ALF), the King Gustav's and Queen Victoria's Freemasons' Foundation, Knut and Alice Wallenberg Foundation, Neuro Sweden, and the Swedish MS foundation.

Author Correction: Smoking induces DNA methylation changes in Multiple Sclerosis patients with exposure-response relationship.

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Smoking induces DNA methylation changes in Multiple Sclerosis patients with exposure-response relationship.

Cigarette smoking is an established environmental risk factor for Multiple Sclerosis (MS), a chronic inflammatory and neurodegenerative disease, although a mechanistic basis remains largely unknown. We aimed at investigating how smoking affects blood DNA methylation in MS patients, by assaying genome-wide DNA methylation and comparing smokers, former smokers and never smokers in two Swedish cohorts, differing for known MS risk factors. Smoking affects DNA methylation genome-wide significantly, an exposure-response relationship exists and the time since smoking cessation affects methylation levels. The results also show that the changes were larger in the cohort bearing the major genetic risk factors for MS (female sex and HLA risk haplotypes). Furthermore, CpG sites mapping to genes with known genetic or functional role in the disease are differentially methylated by smoking. Modeling of the methylation levels for a CpG site in the AHRR gene indicates that MS modifies the effect of smoking on methylation changes, by significantly interacting with the effect of smoking load. Alongside, we report that the gene expression of AHRR increased in MS patients after smoking. Our results suggest that epigenetic modifications may reveal the link between a modifiable risk factor and the pathogenetic mechanisms.

Human Herpesvirus 6B Induces Hypomethylation on Chromosome 17p13.3, Correlating with Increased Gene Expression and Virus Integration.

Human herpesvirus 6B (HHV-6B) is a neurotropic betaherpesvirus that achieves latency by integrating its genome into host cell chromosomes. Several viruses can induce epigenetic modifications in their host cells, but no study has investigated the epigenetic modifications induced by HHV-6B. This study analyzed methylation with an Illumina 450K array, comparing HHV-6B-infected and uninfected Molt-3 T cells 3 days postinfection. Bisulfite pyrosequencing was used to validate the Illumina results and to investigate methylation over time in vitro Expression of genes was investigated using quantitative PCR (qPCR), and virus integration was investigated with PCR. A total of 406 CpG sites showed a significant HHV-6B-induced change in methylation in vitro Remarkably, 86% (351/406) of these CpGs were located <1 Mb from chromosomal ends and were all hypomethylated in virus-infected cells. This was most evident at chromosome 17p13.3, where HHV-6B had induced CpG hypomethylation after 2 days of infection, possibly through TET2, which was found to be upregulated by the virus. In addition, virus-induced cytosine hydroxymethylation was observed. Genes located in the hypomethylated region at 17p13.3 showed significantly upregulated expression in HHV-6B-infected cells. A temporal experiment revealed HHV-6B integration in Molt-3 cell DNA 3 days after infection. The telomere at 17p has repeatedly been described as an integration site for HHV-6B, and we show for the first time that HHV-6B induces hypomethylation in this region during acute infection, which may play a role in the integration process, possibly by making the DNA more accessible.IMPORTANCE The ability to establish latency in the host is a hallmark of herpesviruses, but the mechanisms differ. Human herpesvirus 6B (HHV-6B) is known to establish latency through integration of its genome into the telomeric regions of host cells, with the ability to reactivate. Our study is the first to show that HHV-6B specifically induces hypomethylated regions close to the telomeres and that integrating viruses may use the host methylation machinery to facilitate their integration process. The results from this study contribute to knowledge of HHV-6B biology and virus-host interaction. This in turn will lead to further progress in our understanding of the underlying mechanisms by which HHV-6B contributes to pathological processes and may have important implications in both disease prevention and treatment.

Integrin α9 gene promoter is hypermethylated and downregulated in nasopharyngeal carcinoma.

Epigenetic silencing of tumor suppressor genes (TSGs) by promoter methylation can be an early event in the multi-step process of carcinogenesis. Human chromosome 3 contains clusters of TSGs involved in many cancer types including nasopharyngeal carcinoma (NPC), the most common cancer in Southern China. Among ten candidate TSGs identified in chromosome 3 using NotI microarray, ITGA9 and WNT7A could be validated. 5'-aza-2' deoxycytidine treatment restored the expression of ITGA9 and WNT7A in two NPC cell lines. Immunostaining showed strong expression of these genes in the membrane and cytoplasm of adjacent control nasopharyngeal epithelium cells, while they were weakly expressed in NPC tumor cells. The ITGA9 promoter showed marked differentially methylation between tumor and control tissue, whereas no differentially methylation could be detected for the WNT7A promoter. The expression level of ITGA9 in NPC tumors was downregulated 4.9-fold, compared to the expression in control. ITGA9 methylation was detected by methylation specific PCR (MSP) in 56% of EBV positive NPC-cases with 100% specificity. Taken together, this suggests that ITGA9 might be a TSG in NPC that is involved in tumor cell biology. The possibility of using ITGA9 methylation as a marker for early detection of NPC should further be explored.

Cesarean delivery and hematopoietic stem cell epigenetics in the newborn infant: implications for future health?

OBJECTIVE: Cesarean section (CS) has been associated with a greater risk for asthma, diabetes, and cancer later in life. Although elective CS continues to rise, it is unclear whether and how it may contribute to compromised future health. Our aim was to investigate the influence of mode of delivery on the epigenetic state in neonatal hematopoietic stem cells. STUDY DESIGN: This was an observational study of 64 healthy, singleton, newborn infants (33 boys) born at term. Cord blood was sampled after elective CS (n = 27) and vaginal delivery. Global deoxyribonucleic acid (DNA) methylation in hematopoietic stem cells (CD34+) was determined by luminometric methylation assay, and genome-wide, locus-specific DNA methylation analysis was performed by Illumina Infinium 450K (Illumina, San Diego, CA), validated by bisulfite-pyrosequencing. RESULTS: CD34+ cells from infants delivered by CS were globally more DNA methylated (+2%) than DNA from infants delivered vaginally (P = .02). In relation to mode of delivery, a locus-specific analysis identified 343 loci with a difference in DNA methylation of 10% or greater (P < .01). A majority of the differentially methylated loci in neonatal CD34+ cells (76%) were found to be hypermethylated after vaginal delivery. In these infants, the degree of DNA methylation in 3 loci correlated to the duration of labor. The functional relevance of differentially methylated loci involved processes such as immunoglobulin biosynthetic process, regulation of glycolysis and ketone metabolism, and regulation of the response to food. CONCLUSION: A possible interpretation is that mode of delivery affects the epigenetic state of neonatal hematopoietic stem cells. Given the functional relevance indicated, our findings may have important implications for health and disease in later life.

Human adenovirus-36 is uncommon in type 2 diabetes and is associated with increased insulin sensitivity in adults in Sweden.

BACKGROUND: Human adenovirus-36 (Adv36) increases adiposity, but also upregulates distal insulin signaling in vitro in human adipose and muscle tissue and in vivo in the rodent independently of adiposity. Accordingly, healthy adults and children with antibodies against Adv36 had increased insulin sensitivity and reduced hepatic lipid accumulation. We hypothesized that Adv36 infection would be less frequent in individuals with type 2 diabetes or impaired glycemic control. METHODS: Presence of antibodies against Adv36 was analyzed for association to type 2 diabetes or impaired glycemic control in a two-wave population-based sample of well-characterized adults (n = 1734). Indices of impaired glycemic control included oral glucose tolerance, and circulating fasting levels of glucose, insulin, and insulin-like growth factor binding protein-1 (IGFBP-1). RESULTS: Adv36 seropositivity was more common in those with normal glucose tolerance (NGT) than in those with diabetes (females: OR 17.2, 95% CI 4.0-74.3; males: OR 3.5, 95% CI 1.8-6.7). Also, females with NGT had higher frequency of Adv36 seropositivity than females with prediabetes (impaired glucose tolerance and/or impaired fasting glucose; OR 1.8, 95% CI 1.1-3.1). Within the female prediabetes group Adv36 seropositivity was associated with higher insulin sensitivity reflected by reduced HOMA-IR and increased IGFBP-1. CONCLUSION: Adv36 infection is associated with lower occurrence of type 2 diabetes and better insulin sensitivity in adults, particularly among females.

Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor.

Gemcitabine is indicated in combination with cisplatin as first-line therapy for solid tumours including non-small cell lung cancer (NSCLC), bladder cancer and mesothelioma. Gemcitabine is an analogue of pyrimidine cytosine and functions as an anti-metabolite. Structurally, however, gemcitabine has similarities to 5-aza-2-deoxycytidine (decitabine/Dacogen®), a DNA methyltransferase inhibitor (DNMTi). NSCLC, mesothelioma and prostate cancer cell lines were treated with decitabine and gemcitabine. Reactivation of epigenetically silenced genes was examined by RT-PCR/qPCR. DNA methyltransferase activity in nuclear extracts and recombinant proteins was measured using a DNA methyl-transferase assay, and alterations in DNA methylation status were examined using methylation-specific PCR (MS-PCR) and pyrosequencing. We observe a reactivation of several epigenetically silenced genes including GSTP1, IGFBP3 and RASSF1A. Gemcitabine functionally inhibited DNA methyltransferase activity in both nuclear extracts and recombinant proteins. Gemcitabine dramatically destabilised DNMT1 protein. However, DNA CpG methylation was for the most part unaffected by gemcitabine. In conclusion, gemcitabine both inhibits and destabilises DNA methyltransferases and reactivates epigenetically silenced genes having activity equivalent to decitabine at concentrations significantly lower than those achieved in the treatment of patients with solid tumours. This property may contribute to the anticancer activity of gemcitabine.

Adenovirus-36 is associated with obesity in children and adults in Sweden as determined by rapid ELISA.

BACKGROUND: Experimental and natural human adenovirus-36 (Adv36) infection of multiple animal species results in obesity through increasing adipogenesis and lipid accumulation in adipocytes. Presence of Adv36 antibodies detected by serum neutralization assay has previously been associated with obesity in children and adults living in the USA, South Korea and Italy, whereas no association with adult obesity was detected in Belgium/The Netherlands nor among USA military personnel. Adv36 infection has also been shown to reduce blood lipid levels, increase glucose uptake by adipose tissue and skeletal muscle biopsies, and to associate with improved glycemic control in non-diabetic individuals. PRINCIPAL FINDINGS: Using a novel ELISA, 1946 clinically well-characterized individuals including 424 children and 1522 non-diabetic adults, and 89 anonymous blood donors, residing in central Sweden representing the population in Stockholm area, were studied for the presence of antibodies against Adv36 in serum. The prevalence of Adv36 positivity in lean individuals increased from ∼7% in 1992-1998 to 15-20% in 2002-2009, which paralleled the increase in obesity prevalence. We found that Adv36-positive serology was associated with pediatric obesity and with severe obesity in females compared to lean and overweight/mildly obese individuals, with a 1.5 to 2-fold Adv36 positivity increase in cases. Moreover, Adv36 positivity was less common among females and males on antilipid pharmacological treatment or with high blood triglyceride level. Insulin sensitivity, measured as lower HOMA-IR, showed a higher point estimate in Adv36-positive obese females and males, although it was not statistically significant (p = 0.08). CONCLUSION: Using a novel ELISA we show that Adv36 infection is associated with pediatric obesity, severe obesity in adult females and lower risk of high blood lipid levels in non-diabetic Swedish individuals.

Population-based study of antiepileptic drug exposure in utero--influence on head circumference in newborns.

PURPOSE: To study the effect of AED exposure on head circumference in the newborn. METHODS: Data on all Swedish singletons births between 1995 and 2005, over 900,000 births, were obtained from the Swedish Medical Birth Registry. The effects of AEDs on birth-weight-adjusted mean head circumference (bw-adj-HC) were estimated by comparison with data from all births in an analysis which was adjusted for year of birth, maternal age, parity, maternal smoking, and maternal body mass index. RESULTS: A significant reduction of mean bw-adj-HC was seen after both carbamazepine (CBZ) (standard deviation scores (SDS)=0.15, p<0.001) and valproic acid (VPA) (SDS=0.10, p=0.04) in monotherapy. No effect on mean bw-adj-HC was seen for phenytoin, clonazepam, lamotrigine and gabapentin. There was a significant increase in the occurrence of microcephaly (bw-adj-HC smaller than 2 SD below the mean) after any AED polytherapy (OR=2.85, 95% CI: 1.74-4.78) but not after AED monotherapy or monotherapy with CBZ or VPA. CBZ or VPA was taken by 71% of the pregnant mothers on AED, and the usage increased over time. CONCLUSIONS: CBZ and VPA in monotherapy during pregnancy reduce mean bw-adj-HC. AED polytherapy increases the rate of microcephaly but no significant effect is seen of AED monotherapy. The possible significance for the further development of the child is uncertain but should be explored.

Nucleoside diphosphate kinase A/nm23-H1 promotes metastasis of NB69-derived human neuroblastoma.

Nucleoside diphosphate kinase A (NDPK-A), encoded by the nm23-H1 gene, acts as a metastasis suppressor in certain human tumors such as breast carcinoma. However, evidence also points to NDPK-A functioning as a metastasis promoter in other human tumors including neuroblastoma. In fact, amplification and overexpression of nm23-H1 as well as S120G mutation of NDPK-A (NDPK-A(S120G)) have been detected in 14% to 30% of patients with advanced stages of neuroblastoma. To test whether NDPK-A promotes neuroblastoma metastasis, we established stable transfectants and an orthotopic xenograft animal model from the human neuroblastoma NB69 cell line. We demonstrate that overexpressed NDPK-A or NDPK-A(S120G) increased both incidence and colonization of neuroblastoma metastasis in animal lungs without significantly affecting primary tumor development. In vitro, these metastasis-associated NDPK-A aberrations abrogated retinoic acid-induced neuronal differentiation while increasing cloning efficiency, cell survival, and colony formation of NB69 derivatives. Furthermore, NDPK-A(S120G) reduced cell adhesion and increased cell migration. Compared with its wild-type, NDPK-A(S120G) appears more effective in promoting neuroblastoma metastasis. Our results provide the first evidence that NDPK-A behaves as a metastasis promoter at least in human neuroblastoma derived from NB69 cells. The findings not only suggest a prognostic value of NDPK-A in neuroblastoma patients but also caution NDPK-A-targeted treatment for patients with different tumor types.

A fluorescent orthotopic mouse model for reliable measurement and genetic modulation of human neuroblastoma metastasis.

Neuroblastoma is the most common extra-cranial solid tumor of infancy and childhood, and majority of patients die from the metastatic disease. Orthotopic xenograft mouse models are valuable tools for improving our understanding and control of neuroblastoma metastasis, because they readily represent genetic diversity and allow spontaneous metastasis. Intra-adrenal injection is commonly used for establishing the orthotopic animal models since human neuroblastoma frequently originates in the adrenal gland. However, it is unclear whether the metastatic potential of neuroblastoma can be reliably determined in adrenally-injected mice because their gland size is so small. In this study, we developed and characterized a fluorescent orthotopic xenograft animal model of NB69-derived human neuroblastoma. By comparing animals receiving adrenal injection and adrenal overlay, with the latter mimicking injection spillover, we found that the metastatic potential of neuroblastoma can be reliably determined in animal lungs. Furthermore, the lung metastasis can be genetically modulated in these animals. The results also show that the expression of Renilla green fluorescent protein (GFP) was exceptionally stable in NB69 cells, allowing rapid and sensitive detection of lung metastases at the macroscopic level. Additional features of our model include 100% tumor take, a 1-week tumor latency, resemblance to tumor behaviors in neuroblastoma patients, and the ability to monitor the expression of a gene of interest with GFP. This animal model of human neuroblastoma will be useful for studying genes involved in the metastatic process and for evaluating anti-metastasis agents in pre-clinical trials.