Basic Research and Clinical Examination of Tumor Virus

Tumor viruses cause cancer; thus, extensive studies are being conducted on them. In this article, we will review the basic medical research on the current clinical genetic tests for tumor viruses such as human papilloma virus, hepatitis B virus, and T-cell leukemia virus. Recently, clinical genetic tests have been developed for quick diagnosis of the tumor virus infection. Additionally, we will review the mutagenesis of murine leukemia retrovirus. In particular, we will focus on the insertional mutagenesis. This will help in deciding the direction of future virus research by combining clinical and basic research

Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5\u27-RCGTG-3\u27), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2\u27-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells

Hierarchical Cluster and Region of Interest Analyses Based on Mass Spectrometry Imaging of Human Brain Tumours

Imaging mass spectrometry (IMS) has been rarely used to examine specimens of human brain tumours. In the current study, high quality brain tumour samples were selected by tissue observation. Further, IMS analysis was combined with a new hierarchical cluster analysis (IMS-HCA) and region of interest analysis (IMS-ROI). IMS-HCA was successful in creating groups consisting of similar signal distribution images of glial fibrillary acidic protein (GFAP) and related multiple proteins in primary brain tumours. This clustering data suggested the relation of GFAP and these identified proteins in the brain tumorigenesis. Also, high levels of histone proteins, haemoglobin subunit α, tubulins, and GFAP were identified in a metastatic brain tumour using IMS-ROI. Our results show that IMS-HCA and IMS-ROI are promising techniques for identifying biomarkers using brain tumour samples

The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

<p>Abstract</p> <p>Background</p> <p>Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity.</p> <p>Methods</p> <p>Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices.</p> <p>Result</p> <p>The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted.</p> <p>Conclusions</p> <p>The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.</p

Evaluation of the morphological characteristics and culture performance of Cladosiphon okamuranus strains

This study aimed to determine whether morphological differences of Cladosiphon okamuranus strains at different aquaculture sites were inherent characteristics and to select useful forms for aquaculture production and processing. Three candidate strains with potentially excellent morphological characteristics, that is ON, SY and KT, were selected from six local strains collected from Okinawa Islands. Then, these three candidate strains were transplanted to four aquaculture sites to evaluate their characteristics. The thallus length was significantly larger (e.g. 1.6-2.1 times) in the SY strain than in the ON strain in each area and the density of the primary lateral branches of the latter was significantly (e.g. 2.4-5.9 times) higher than that of the other two strains. The SY and ON strain characteristics were also distinguished by a comprehensive evaluation of eight traits with principal component analysis. Conversely, the KT strain tended to have an intermediate length between ON and SY strains, the biomass yield of the SY and KT strains was higher than that of ON strain. These results, indicating that some morphological differences are intrinsic strain characteristics, will provide information to aquaculture producers using appropriate strains to maximize the unit yield and thalli quality

CCAAT/Enhancer-Binding Protein-α Suppresses Lung Tumor Development in Mice through the p38α MAP Kinase Pathway

<div><p>The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) is a basic leucine zipper transcription factor and is expressed in alveolar type II cells, alveolar macrophages and Clara cells in the lung. Although decrease or absence of C/EBPα expression in human non-small cell lung cancer suggests a possible role of C/EBPα as a lung tumor suppressor, there is no direct proof for this hypothesis. In this study, we investigated, for the first time, the role of C/EBPα in lung tumors in vivo using transgenic mice with lung epithelial specific conditional deletion of <i>Cebpa</i> (<i>Cebpα^Δ/Δ</i> mice) and a urethane-induced lung tumor model. C/EBPα expression in the lung was dispensable, and its deletion was not oncogenic under unstressed conditions. However, at 28 wk after urethane injection, the number and size of tumors and the tumor burden were significantly higher in <i>Cebpα^Δ/Δ</i> mice than in littermate control mice. Urethane-injected <i>Cebpα^Δ/Δ</i> mice showed highly proliferative adenomas and adenocarcinomas in the lung, and survival time after urethane-injection was significantly shorter than that in control mice. In control mice, C/EBPα was strongly induced in the tumor tissues at 28 weeks after urethane-injection, but became weakened or absent as tumors progressed after long-term observation for over 1 year. Using intraperitoneal injection of p38 inhibitor (SB203580), we demonstrated that the induction of C/EBPα is strongly regulated by the p38 MAP kinase in murine alveolar epithelial cells. A high correlation was demonstrated between the expression of C/EBPα and p38α MAP kinase in tumor cells, suggesting that C/EBPα silencing in tumor cells is caused by down-regulation of p38α MAP kinase. In conclusion, the role of C/EBPα as a lung tumor suppressor was demonstrated for the first time in the present study, and the extinguished C/EBPα expression through p38α inactivation leads tumor promotion and progression.</p> </div

Urethane Induced Tumors in Early <i>Cebpa</i> Deletion Study.

<p>(A): The tumor numbers, tumor size and tumor burden were all significantly higher in the lungs of <i>Cebpα^Δ/Δ</i> mice at 20 and 28 wk after urethane injection (10 wk: n = 10, 20 wk: n = 11, 28 wk: n = 17/group, *p<0.01). The bar indicates the average. (B): Appearance of the lung at 28 weeks after urethane injection. <i>Arrow</i>: surface tumors. Urethane injection induced lung tumors in both control and <i>Cebpα^Δ/Δ</i> mice. Small tumors were observed on the surface of the lung in control mice. In contrast, <i>Cebpα^Δ/Δ</i> mice developed multiple large tumors. Representative microphotographs of H&E stained sections showed the histology of the adenoma in control mice and adenocarcinomas with airway invasion in <i>Cebpα^Δ/Δ</i> mice. Immunohistochemistry showed strong C/EBPα expression in tumor of control mice, but no staining in <i>Cebpα^Δ/Δ</i> mice. Tu: Tumor. (C): <i>Cebpa</i> mRNA expression was evaluated by qRT-PCR. At 28 wk after urethane injection, <i>Cebpa</i> mRNA expression in control mice was significantly higher than saline injected mice (n = 4/group, p<0.05). The expression in <i>Cebpα^Δ/Δ</i> mice was not affected by urethane injection. In control mice, <i>Cebpa</i> mRNA expression was induced in tumor tissues and was significantly higher than that in non-tumor lung tissues (n = 4/group, p<0.05). (D): <i>Kaplan-Meier</i> survival curves of control and <i>Cebpα^Δ/Δ</i> mice after urethane injection (n = 20/group). <i>Cebpα^Δ/Δ</i> mice showed significantly shorter survival than did control mice (log rank test, p<0.00001, the average survival after urethane injection: control mice (63.5±2.7 wk), <i>Cebpα^Δ/Δ</i> mice (28.5±0.9 wk).</p

Cell Death and Proliferation in Urethane-Induced Tumors.

<p>(A): Immunohistochemistry for Ki-67 and pH 3 at 20 wk after urethane injection. (B): Mitotic index in the tumors of control and <i>Cebpα^Δ/Δ</i> mice (n = 7/group). <i>Cebpα^Δ/Δ</i> mice showed significantly higher numbers of Ki-67 positive cells in tumors (*p<0.01). The tumors in <i>Cebpα^Δ/Δ</i> mice were more proliferative than those in control mice. (C): Arrows indicate TUNEL stain and cleaved-caspase 3 positive cells in tumors. Apoptotic cells were not obvious in this model.</p

Natural C/EBPα Silencing and Promoter DNA Methylation in Urethane-Induced Tumors.

<p>(A): H&E staining and C/EBPα immunohistochemistry in urethane induced tumors in control mice evaluated 60 wk after urethane injection. <i>Left column</i>: C/EBPα-entirely positive tumor, <i>Middle column</i>: C/EBPα-partially positive tumor and <i>Right column</i>: C/EBPα-entirely negative tumor. The samples in each row were taken from serial sections. At least one partially positive tumor was observed in all mice (26/26). Entirely negative tumors were observed in 6 out of 26 mice. (B): Double immunofluorescence of C/EBPα and 5-mC in entirely-positive and entirely-negative tumor in long-observed model. C/EBPα-positive cells were weak or negative for 5-mC (upper row), while C/EBPα-negative cells stained positive for 5-mC (lower row), suggesting that C/EBPα-negative cells revealed DNA hypermethylation. (C): Bisulfite sequencing analysis for 3 entirely C/EBPα-negative tumors (M1, M2 and M3) and 2 C/EBPα entirely positive tumors (C1 and C2, 28 wk after urethane injection). <i>Arrow</i>, transcription start site; the diagrams of the core-promoter sites (−337 to +49) are drawn to scale. Promoter DNA methylation was weak in entirely C/EBPα-negative tumors. Each row represents an individual clone. <i>White circles, unmethylated CG dinucleotides; black circles, methylated CG dinucleotides</i>.</p