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author:("Sato, kiko")
1.  Genome-Wide Analysis of DNA Methylation Dynamics during Early Human Development 
PLoS Genetics  2014;10(12):e1004868.
DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5–10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.
Author Summary
DNA methylation reprogramming after fertilization is critical for normal mammalian development. Early embryos are sensitive to environmental stresses and a number of reports have pointed out the increased risk of DNA methylation errors associated with assisted reproduction technologies. Therefore, it is very important to understand normal DNA methylation patterns during early human development. Recent reduced representation bisulfite sequencing studies reported partial methylomes of human gametes and early embryos. To provide a more comprehensive view of DNA methylation dynamics during early human development, we report on whole genome bisulfite sequencing of human gametes and blastocysts. We show that the paternal genome is globally demethylated in blastocysts whereas the maternal genome is demethylated to a much lesser extent. We also reveal unique regulation of imprinted differentially methylated regions, gene bodies and repeat sequences during early human development. Our high-resolution methylome maps are essential to understand epigenetic reprogramming by human oocytes and will aid in the preimplantation epigenetic diagnosis of human embryos.
doi:10.1371/journal.pgen.1004868
PMCID: PMC4263407  PMID: 25501653
2.  Imprinting methylation errors in ART 
Reproductive Medicine and Biology  2014;13(4):193-202.
There has been an increase in incidence reports of rare imprinting disorders associated with assisted reproductive technology (ART). ART, including in vitro fertilization and intracytoplasmic sperm injections, is an important treatment for infertile people of reproductive age and increasingly produces children. The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that ART techniques themselves may predispose embryos to acquire imprinting errors and diseases. In this review, we note that the particular steps of ART may be prone to induction of imprinting methylation errors during gametogenesis, fertilization and early embryonic development. In addition, we explain imprint-associated diseases and their causes. Moreover, from a Japanese nationwide epidemiological study of imprint-associated diseases, we determine their associations with ART. Epigenetic studies will be required to understand the pathogenesis, ART-related risk factor(s) and what precautions can be taken to prevent the occurrence of input methylation errors. We hope that the constitution of children born after each ART procedure will reveal the safest and most ethical approach to use, which will be invaluable for the future development of standard ART.
doi:10.1007/s12522-014-0183-3
PMCID: PMC4182590  PMID: 25298744
Assisted reproductive technologies (ART); DNA methylation; Genomic imprinting; Intracytoplasmic sperm injection (ICSI); In vitro fertilization (IVF)
3.  Magnetic nanoparticles of Fe3O4 enhance docetaxel-induced prostate cancer cell death 
Docetaxel (DTX) is one of the most important anticancer drugs; however, the severity of its adverse effects detracts from its practical use in the clinic. Magnetic nanoparticles of Fe3O4 (MgNPs-Fe3O4) can enhance the delivery and efficacy of anticancer drugs. We investigated the effects of MgNPs-Fe3O4 or DTX alone, and in combination with prostate cancer cell growth in vitro, as well as with the mechanism underlying the cytotoxic effects. MgNPs-Fe3O4 caused dose-dependent increases in reactive oxygen species levels in DU145, PC-3, and LNCaP cells; 8-hydroxydeoxyguanosine levels were also elevated. MgNPs-Fe3O4 alone reduced the viability of LNCaP and PC-3 cells; however, MgNPs-Fe3O4 enhanced the cytotoxic effect of a low dose of DTX in all three cell lines. MgNPs-Fe3O4 also augmented the percentage of DU145 cells undergoing apoptosis following treatment with low dose DTX. Expression of nuclear transcription factor κB in DU145 was not affected by MgNPs-Fe3O4 or DTX alone; however, combined treatment suppressed nuclear transcription factor κB expression. These findings offer the possibility that MgNPs-Fe3O4–low dose DTX combination therapy may be effective in treating prostate cancer with limited adverse effects.
doi:10.2147/IJN.S40766
PMCID: PMC3753150  PMID: 23990723
prostate cancer; magnetic nanoparticles; docetaxel; reactive oxidative species
4.  Aberrant methylation of microRNA-34b/c is a predictive marker of metachronous gastric cancer risk 
Journal of Gastroenterology  2013;49(7):1135-1144.
Background
Metachronous gastric cancer (GC) can develop after endoscopic resection of GC and cannot be predicted based on clinical signature. Aberrant DNA methylation in noncancerous gastric mucosa is strongly implicated in gastric carcinogenesis and could be a useful biomarker of GC risk. We evaluated the clinical utility of DNA methylation as a biomarker of metachronous GC risk.
Method
We carried out scheduled follow-up endoscopy in 129 patients after curative endoscopic resection of GC. Biopsy specimens were collected from noncancerous mucosa in the gastric antrum and body, after which quantitative methylation analysis of miR-34b/c, SFRP1, SFRP2, SFRP5, DKK2 and DKK3 was carried out using bisulfite pyrosequencing. The utility of the methylation for predicting the risk of metachronous GC development was assessed using Kaplan–Meier and Cox proportional hazards model analyses.
Results
During the follow-up period, 17 patients (13 %) developed metachronous GCs. The cumulative incidence of metachronous GC was significantly higher among patients with elevated miR-34b/c, SFRP2 and DKK2 methylation in their gastric body. MiR-34b/c showed the strongest association with the risk of metachronous GC, and the cumulative incidence of metachronous GC was much higher in the high-miR-34b/c-methylation group than the low-methylation group. Multivariate analysis adjusted for age, sex, H. pylori status and pathological findings showed miR-34b/c methylation in gastric body to be an independent predictor of metachronous GC risk.
Conclusion
Our results suggest that methylation of miR-34b/c in the mucosa of the noncancerous gastric body may be a useful biomarker for predicting the risk of metachronous GC.
Electronic supplementary material
The online version of this article (doi:10.1007/s00535-013-0861-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s00535-013-0861-7
PMCID: PMC4083150  PMID: 23942619
Metachronous gastric cancer; DNA methylation; Biomarker; Risk assessment
5.  Effects of Fe3O4 Magnetic Nanoparticles on A549 Cells 
Fe3O4 magnetic nanoparticles (MgNPs-Fe3O4) are widely used in medical applications, including magnetic resonance imaging, drug delivery, and in hyperthermia. However, the same properties that aid their utility in the clinic may potentially induce toxicity. Therefore, the purpose of this study was to investigate the cytotoxicity and genotoxicity of MgNPs-Fe3O4 in A549 human lung epithelial cells. MgNPs-Fe3O4 caused cell membrane damage, as assessed by the release of lactate dehydrogenase (LDH), only at a high concentration (100 μg/mL); a lower concentration (10 μg/mL) increased the production of reactive oxygen species, increased oxidative damage to DNA, and decreased the level of reduced glutathione. MgNPs-Fe3O4 caused a dose-dependent increase in the CD44+ fraction of A549 cells. MgNPs-Fe3O4 induced the expression of heme oxygenase-1 at a concentration of 1 μg/mL, and in a dose-dependent manner. Despite these effects, MgNPs-Fe3O4 had minimal effect on cell viability and elicited only a small increase in the number of cells undergoing apoptosis. Together, these data suggest that MgNPs-Fe3O4 exert little or no cytotoxicity until a high exposure level (100 μg/mL) is reached. This dissociation between elevated indices of cell damage and a small effect on cell viability warrants further study.
doi:10.3390/ijms140815546
PMCID: PMC3759872  PMID: 23892599
magnetic nanoparticles; cytotoxicity; genotoxicity; A549; CD44
6.  Stability of genomic imprinting in human induced pluripotent stem cells 
BMC Genetics  2013;14:32.
Background
hiPSCs are generated through epigenetic reprogramming of somatic tissue. Genomic imprinting is an epigenetic phenomenon through which monoallelic gene expression is regulated in a parent-of-origin-specific manner. Reprogramming relies on the successful erasure of marks of differentiation while maintaining those required for genomic imprinting. Loss of imprinting (LOI), which occurs in many types of malignant tumors, would hinder the clinical application of hiPSCs.
Results
We examined the imprinting status, expression levels and DNA methylation status of eight imprinted genes in five independently generated hiPSCs. We found a low frequency of LOI in some lines. Where LOI was identified in an early passage cell line, we found that this was maintained through subsequent passages of the cells. Just as normal imprints are maintained in long-term culture, this work suggests that abnormal imprints are also stable in culture.
Conclusions
Analysis of genomic imprints in hiPSCs is a necessary safety step in regenerative medicine, with relevance both to the differentiation potential of these stem cells and also their potential tumorigenic properties.
doi:10.1186/1471-2156-14-32
PMCID: PMC3751563  PMID: 23631808
Genomic imprinting; Loss of imprinting (LOI); DNA methylation; Histone modification; Human induced pluripotent cells
7.  High-throughput detection of aberrant imprint methylation in the ovarian cancer by the bisulphite PCR-Luminex method 
Background
Aberrant DNA methylation leads to loss of heterozygosity (LOH) or loss of imprinting (LOI) as the first hit during human carcinogenesis. Recently we developed a new high-throughput, high-resolution DNA methylation analysis method, bisulphite PCR-Luminex (BPL), using sperm DNA and demonstrated the effectiveness of this novel approach in rapidly identifying methylation errors.
Results
In the current study, we applied the BPL method to the analysis of DNA methylation for identification of prognostic panels of DNA methylation cancer biomarkers of imprinted genes. We found that the BPL method precisely quantified the methylation status of specific DNA regions in somatic cells. We found a higher frequency of LOI than LOH. LOI at IGF2, PEG1 and H19 were frequent alterations, with a tendency to show a more hypermethylated state. We detected changes in DNA methylation as an early event in ovarian cancer. The degree of LOI (LOH) was associated with altered DNA methylation at IGF2/H19 and PEG1.
Conclusions
The relative ease of BPL method provides a practical method for use within a clinical setting. We suggest that DNA methylation of H19 and PEG1 differentially methylated regions (DMRs) may provide novel biomarkers useful for screening, diagnosis and, potentially, for improving the clinical management of women with human ovarian cancer.
doi:10.1186/1755-8794-5-8
PMCID: PMC3342152  PMID: 22443985
Genomic imprinting; Ovarian cancer; DNA methylation; Bisulphite PCR-Luminex(BPL)method; LOI (loss of imprinting)
8.  DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm 
European Journal of Human Genetics   2009;17(12):1582-1591.
There is an increased prevalence of imprinting disorders, such as Beckwith–Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.
doi:10.1038/ejhg.2009.68
PMCID: PMC2845511  PMID: 19471309
genomic imprinting; assisted reproductive technologies; DNA methylation; oligospermic patients; DNA methyltransferase 3-like protein
9.  DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm 
European Journal of Human Genetics  2009;17(12):1582-1591.
There is an increased prevalence of imprinting disorders, such as Beckwith–Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.
doi:10.1038/ejhg.2009.68
PMCID: PMC2845511  PMID: 19471309
genomic imprinting; assisted reproductive technologies; DNA methylation; oligospermic patients; DNA methyltransferase 3-like protein
10.  Growth Inhibition of Re-Challenge B16 Melanoma Transplant by Conjugates of Melanogenesis Substrate and Magnetite Nanoparticles as the Basis for Developing Melanoma-Targeted Chemo-Thermo-Immunotherapy 
Melanogenesis substrate, N-propionyl-cysteaminylphenol (NPrCAP), is selectively incorporated into melanoma cells and inhibits their growth by producing cytotoxic free radicals. Magnetite nanoparticles also disintegrate cancer cells and generate heat shock protein (HSP) upon exposure to an alternating magnetic field (AMF). This study tested if a chemo-thermo-immunotherapy (CTI therapy) strategy can be developed for better management of melanoma by conjugating NPrCAP on the surface of magnetite nanoparticles (NPrCAP/M). We examined the feasibility of this approach in B16 mouse melanoma and evaluated the impact of exposure temperature, frequency, and interval on the inhibition of re-challenged melanoma growth. The therapeutic protocol against the primary transplanted tumor with or without AMF exposure once a day every other day for a total of three treatments not only inhibited the growth of the primary transplant but also prevented the growth of the secondary, re-challenge transplant. The heat-generated therapeutic effect was more significant at a temperature of 43°C than either 41°C or 46°C. NPrCAP/M with AMF exposure, instead of control magnetite alone or without AMF exposure, resulted in the most significant growth inhibition of the re-challenge tumor and increased the life span of the mice. HSP70 production was greatest at 43°C compared to that with 41°C or 46°C. CD8+T cells were infiltrated at the site of the re-challenge melanoma transplant.
doi:10.1155/2009/457936
PMCID: PMC2760320  PMID: 19830247

Results 1-10 (10)