Characterizing key nucleotide polymorphisms of hepatitis C virus-disease associations via mass-spectrometric genotyping.

As more than 80% of hepatocellular carcinoma patients in Japan also suffer from hepatitis C virus infections some time in their medical history, identifying genetic aberrations associated to hepatitis C virulence in these individuals remains a high priority in the diagnosis and treatment of hepatocellular carcinoma. From the BioBank Japan Project, we acquired 480 subjects of hepatocellular carcinoma, chronic hepatitis and liver cirrhosis, and genotyped 131 clinically relevant host single nucleotide polymorphisms to survey the potential association between certain risk alleles and genes to a patient's predisposition to hepatitis C and liver cancer. Among those polymorphisms, we found 12 candidates with statistical significance to support association with hepatitis C virus susceptibility and genetic predisposition to hepatocellular carcinoma. SNPs in genes such as XPC, FANCA, KDR and BRCA2 also suggested likely connections between hepatitis C virus susceptibility and the contraction of liver diseases. Single nucleotide polymorphisms reported here provided suggestions for genes as biomarkers and elucidated insights briefing the linkage of hepatitis C virulence to the alteration of healthy liver genomic landscape as well as liver disease progression.

Identification of frequent differentially methylated region in sporadic bladder cancers.

INTRODUCTION: Aberrant methylation levels in the cytosine-phosphate-guanine island (CpGi) region from exon 1 to intron 1 of the zygote arrest 1 (ZAR1) gene have been reported in several types of human cancers, including melanoma, brain tumor, and hepatocellular carcinoma. In the present study, methylation levels at the CpGi of ZAR1 exon 1/intron 1 in bladder cancer specimens were analyzed using mass spectrometry. MATERIALS AND METHODS: Genomic DNA was extracted from 20 sporadic bladder cancers, and the methylation levels at ZAR1 CpGi were quantitatively examined by the MassARRAY EpiTYPER method. RESULT: The methylation levels at specific CpG sites of the ZAR1 CpGi were significantly lower in high-grade bladder cancers than in low-grade tumors. CONCLUSIONS: The results of the present study indicated a decreased methylation level at CpG sites of ZAR1 exon 1/intron 1. CpGi could serve as a biomarker for invasive bladder cancer.

Nr4a3, a possibile oncogenic factor for neuroblastoma associated with CpGi methylation within the third exon.

Aberrant methylation of Nr4a3 exon 3 CpG island (CpGi) was initially identified during multistep mouse skin carcinogenesis. Nr4a3 is also known as a critical gene for neuronal development. Thus, we examined the Nr4a3 exon 3 CpGi methylation in mouse brain tissues from 15-day embryos, newborns and 12-week-old adults and found significant increase of its methylation and Nr4a3 expression during mouse brain development after birth. In addition, homologous region in human genome was frequently and aberrantly methylated in neuroblastoma specimens. A quantitative analysis of DNA methylation revealed that hypomethylation of CpG islands on Nr4a3 exon 3, but not on exon 1 was identified in three neuroblastomas compared with matched adrenal glands. Additional analysis for 20 neuroblastoma patients was performed and 8 of 20 showed hypomethylation of the CpGi on Nr4a3 exon 3. The survival rate of those 8 patients was significantly lower compared with those in patients with hypermethylation. Immunohistochemical Nr4a3 expression was generally faint in neuroblastoma tissues compared with normal tissues. Moreover, the MYCN amplified NB9 cell line showed hypomethylation and low expression of Nr4a3, while the non-MYCN amplified NB69 cell line showed hypermethylation and high expression. These results indicate that DNA hypomethylation of the CpGi at Nr4a3 exon 3 is associated with low Nr4a3 expression, and correlates with poor prognosis of neuroblastoma. Since Nr4a3 upregulation associated with the hypermethylation and neuronal differentiation in mice, poor prognosis of neuroblastoma associated with Nr4a3 low expression may be partly explained by dysregulation of its differentiation.

Non-promoter DNA hypermethylation of Zygote Arrest 1 (ZAR1) in neuroblastomas.

BACKGROUND: The comprehensive methylation analysis of tumor-specific differently methylated regions in malignant melanomas and brain tumors has led to the identification of non-promoter hypermethylation of zygote arrest 1 (ZAR1). To search the non-promoter ZAR1 hypermethylation in neuroblastomas, we analyzed the levels of the methylation and transcript expression of ZAR1. METHODS: The MassARRAY® EpiTYPER (Sequenom Inc., San Diego, CA, USA) system was optimized to determine the quantitative methylation levels of ZAR1 for 12 neuroblastoma cell lines, 23 neuroblastoma samples and four adrenal samples. ZAR1 expression levels were evaluated through a quantitative, real-time reverse transcription-polymerase chain reaction. The quantitative methylation levels of ZAR1 were subjected to correlation studies with the established markers of progressive disease and outcome. RESULTS: Strikingly, the hypermethylation of ZAR1 regions and ZAR1 expression levels was observed in the neuroblastoma cell lines and neuroblastoma samples, compared to the adrenal samples. Somatic changes in ZAR1 methylation and ZAR1 expression were found in all three neuroblastoma patients. In the ZAR1 regions, poor-outcome tumors that were MYCN-amplified and/or Stage 3 or 4 and/or the age at diagnosis was≥18months, and/or showed an unfavorable histology were frequently hypermethylated. CONCLUSION: Our results indicate that the hypermethylation of ZAR1 regions is extremely frequent in neuroblastomas and correlates with established markers of progressive disease and outcome.

DNA hypomethylation at the ZNF206-exon 5 CpG island associated with neuronal differentiation in mice and development of neuroblastoma in humans.

Differentiation of human neuroblastoma recapitulates neural crest development. In our whole genome DNA methylation screening of tissue-specific differentially methylated regions (T-DMRs) and developmental stage specific differentially methylated regions (DS-DMRs) we reported that the exon 5 CpG island (CpGi) of Zfp206 (human: ZNF206), which was required to maintain embryonic stem cells in a pluripotent state, was one of potent brain and testis-specific T-DMRs in mice. In this study methylation level of the CpG sites at Zfp206-exon 5 CpGi in mouse brain samples at three different developmental stages (15-day-old embryo; E15, new born; NB, 12-week adult; AD) were quantitatively analyzed and it was identified that Zfp206-exon 5 CpGi was the DS-DMRs in mouse brain. In AD brains, Zfp206-exon 5 CpGi was significantly hypomethylated and Zfp206 expression was repressed, compared with E15 and NB brains. Hence, methylation level of human 5'-end of CpGi at ZNF206-exon 5, which is homologous CpGi to mice, was analyzed in neuroblastomas. Although all four adrenal samples showed complete methylation at the homologous region, we found the hypomethylation in 7 out of 26 neuroblastomas and a significant association between the hypomethylation and poor prognosis. In neuroblastoma cell lines and specimens, the hypomethylation was also associated with ZNF206 expression. These data indicated that the changes in DNA methylation levels at the Zfp206-exon 5 might be one of the important factors during neuronal development in mice and that the hypomethylation of the homologous region induced ZNF206 expression in humans and was associated with human neuroblastomagenesis. Even though the function of ZNF206 and its expression regulation in neuroblastoma remain elusive, ZNF206 might be a candidate differentiation suppressor and prognosis marker in neuroblastoma.

Non-promoter hypermethylation of zygote arrest 1 (ZAR1) in human brain tumors.

Zygote arrest 1 (ZAR1) is a novel maternal-effect gene of crucial importance during the oocyte-to-embryo transition. Comprehensive methylation analysis of tumor-specific differently methylated regions in human malignant melanomas has recently led to the identification of non-promoter hypermethylation of the ZAR1 gene that had never been identified as an aberrant methylated region in any human tumor. Notably, ZAR1 hypermethylation was frequently observed in melanomas but was absent in benign nevi, and ZAR1 expression was found to be up-regulated in methylated tumors. These findings prompted us to screen for ZAR1 non-promoter methylation in various types of human brain tumors using MassARRAY EpiTYPER. Strikingly, hypermethylation of ZAR1 was observed frequently in diffuse astrocytomas (100%), anaplastic astrocytomas (94%), glioblastomas (93%), oligodendrogliomas (100%), anaplastic oligodendrogliomas (100%), and pituitary adenomas (90%), but not at all in pilocytic astrocytomas. For other tumor types ZAR1 hypermethylation was infrequent: 17% of vestibular schwannomas and 33% of meningothelial meningiomas. Detectable ZAR1 transcript was not found in any of hypermethylated glioma cell lines. Our results indicate that hypermethylation of the ZAR1 non-promoter is extremely frequent in diffuse gliomas and pituitary adenomas, although ZAR1 expression is unlikely to play a tumorigenic role.

Identification and analysis of an early diagnostic marker for malignant melanoma: ZAR1 intra-genic differential methylation.

BACKGROUND: Epigenetic changes such as aberrant DNA methylation and histone modification have been shown to play an important role in the tumorigenesis of malignant melanoma. OBJECTIVE: To identify novel tumor-specific differentially methylated regions (DMRs) in human malignant melanoma. METHODS: The aberrant methylation at 14 candidate human genomic regions identified through a mouse model study with quantitative DNA methylation analysis using the Sequenom MassARRAY system was performed. RESULTS: The CpG island Exon 1 region of the Zygote arrest 1 (ZAR1) gene, which is responsible for oocyte-to-embryo transition, showed frequent aberrant methylation of 28 out of 30 (93%) melanoma surgical specimens, 16 of 17 (94%) melanoma cell lines, 0% of 4 normal human epidermal melanocyte (NHEM) cell lines, 0% of 10 melanocytic nevi and 100% of 51 various cancer cell lines. According to the real-time RT-PCR, the ZAR1 gene was overexpressed in part of the hypermethylated cell lines, while its low expression with bivalent histone methylation status was seen in unmethylated cell lines. CONCLUSION: Our findings suggest that the ZAR1 intra-genic differentially methylated region would be a useful tumor marker for malignant melanoma and may be other type of cancers. The involvement of ZAR1 in the carcinogenesis of melanoma, still remains unclear, although we have examined tumorigenic capacities by exogenous full-length ZAR1 over-expression and siRNA knock-down experiments.

Inhibition of MMP-9 transcription and suppression of tumor metastasis by pyrrole-imidazole polyamide.

Matrix metalloproteinase (MMP)-9, the 92-kDa type IV collagenase, contributes to tumor invasion and metastases, and strategies to down-regulate its expression could ultimately be of clinical utility. A pyrrole-imidazole (PI) polyamide that targets the activator protein-1 (AP-1)-binding site of the MMP-9 promoter was designed and synthesized as a gene-silencing agent for tumor metastases. The synthesized product showed selective DNA binding ability. The MMP-9 PI polyamide significantly inhibited MMP-9's mRNA expression, protein level, and enzymatic activity in human breast adenocarcinoma cells (MDA-MB-231). Furthermore, the MMP-9 PI polyamide inhibited migration and invasion by in vitro wound-healing and matrigel-invasion assay. The FITC-labeled PI polyamide was localized in nuclei in 45 min of incubation with an MDA-MB-231 cell and remained in the nuclei for up to 96 h after incubation in vitro. It was also quickly localized in the mouse cellular nuclei of many tissues, including liver, kidney, and spleen, after intravenous injection without using any drug-delivery system. Moreover, the polyamide treatment significantly decreased metastasis in a mouse model of liver metastasis. Our results suggest that this PI polyamide, which targets the MMP-9 gene promoter, can be a novel MMP-9 down-regulating molecule for antimetastasis.

Aberrant hypermethylation of non-promoter zygote arrest 1 (ZAR1) in human brain tumors.

Zygote arrest 1 (ZAR1) is a novel maternal-effect gene which is extremely important during the oocyte-to-embryo transition. Comprehensive methylation analysis of tumor-specific differentially methylated regions in human malignant melanoma has recently led to the identification of non-promoter hypermethylation of the ZAR1 gene that had never been previously linked to aberrant methylation. Strikingly, ZAR1 hypermethylation was frequently observed in melanomas but was absent in benign nevi, and ZAR1 expression was found to be up-regulated in methylated tumors. The present study searched for non-promoter ZAR1 hypermethylation in 90 primary human brain tumor samples, normal brain tissue from one autopsy case, and 7 glioma cell lines, employing Sequenom MassARRAY, in which bisulfite-treated fragments are quantitatively detected using time-of-flight mass spectroscopy. ZAR1 transcript expression levels were also evaluated by quantitative real-time reverse transcription-polymerase chain reaction in the 7 glioma cell lines. Hypermethylation of ZAR1 was frequently found in diffuse astrocytomas (7/7, 100%), anaplastic astrocytomas (16/17, 94%), glioblastomas (27/29, 93%), oligodendrogliomas (3/3, 100%), anaplastic oligodendrogliomas (3/3, 100%), and pituitary adenomas (9/10, 90%), but not in pilocytic astrocytomas (0/3). Other tumor types showed infrequent ZAR1 hypermethylation: 1 (17%) of 6 vestibular schwannomas and 4 (33%) of 12 meningothelial meningiomas. The normal brain tissue revealed no evidence of ZAR1 methylation. All 7 glioma cell lines displayed aberrant hypermethylation of ZAR1, but none had detectable ZAR1 transcript. Our findings indicate that non-promoter hypermethylation of ZAR1 is extremely frequent in diffuse gliomas and pituitary adenomas, but methylation-related aberrant ZAR1 expression is far less likely to be related to glioma tumorigenesis.

Quantitative analysis of human tissue-specific differences in methylation.

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.

Development of pyrrole-imidazole polyamide for specific regulation of human aurora kinase-A and -B gene expression.

Pyrrole-imidazole polyamide (PIP) is a nuclease-resistant novel compound that inhibits gene expression through binding to the minor groove of DNA. Human aurora kinase-A (AURKA) and -B (AURKB) are important regulators in mitosis during the cell cycle. In this study, two specific PIPs (PIP-A and PIP-B) targeting AURKA and AURKB promoter regions were designed and synthesized, and their biological effects were investigated by several in vitro assays. PIP-A and PIP-B significantly inhibited the promoter activities, mRNA expression, and protein levels of AURKA and AURKB, respectively, in a concentration-dependent manner. Moreover, 1:1 combination treatment with both PIPs demonstrated prominent antiproliferative synergy (CI value [ED(50)] = 0.256) to HeLa cells as a result of inducing apoptosis-mediated severe catastrophe of cell-cycle progression. The novel synthesized PIP-A and PIP-B are potent and specific gene-silencing agents for AURKA and AURKB.