Biomedical subjects

Jun Nishikawa

Publications and source records attributed to Jun Nishikawa.

9 recordsLinked to original sources

PubMed2007

Relationship between molecular markers and endoscopic findings in laterally spreading tumors.

BACKGROUND AND AIM: Characteristic clinicopathological features of laterally spreading tumors (LSTs) have been reported by endoscopists; however, only a few studies have been conducted on the biological features. These studies were not fully associated with the endoscopic findings of LSTs. The aim of this study was to estimate the biological features of each type of endoscopic finding of LST using two molecular markers, matrix metalloproteinase-7 (MMP-7) and beta-catenin. METHODS: Expression of the molecular markers and the endoscopic findings were compared in 22 LSTs and 14 subpedunculated polyps. MMP-7 and beta-catenin were immunostained. Three types of representative endoscopic findings of LST were defined as segments in LSTs. They were 15 granular segments, seven large nodular segments, and seven flat segments that corresponded to the area composed of aggregates of similar size granules, large nodules of diameter more than 10 mm, and a flat surface with no granule, respectively. RESULTS: Expression of MMP-7 and coexpression of MMP-7 and beta-catenin were higher in large nodular segments than in granular segments (P < 0.0167). Among the three types of segments, flat segments showed the highest expression densities of beta-catenin accumulated in the nucleus. Large nodular segments and subpedunculated polyps showed similar expression patterns for the molecular markers. CONCLUSIONS: This study provides new and important information on the relationship between the molecular markers and endoscopic findings of LSTs.

Aged↗

PubMed2005

In vitro EBV-infected subline of KMH2, derived from Hodgkin lymphoma, expresses only EBNA-1, while CD40 ligand and IL-4 induce LMP-1 but not EBNA-2.

In about 50% of classical Hodgkin lymphomas, the Hodgkin/Reed Sternberg (H/RS) cells carry Epstein-Barr virus (EBV). The viral gene expression in these cells is restricted to EBNA-1, EBERs, LMP-1 and LMP-2 (type II latency). The origin of H/RS cells was defined as crippled germinal center B cells that escaped apoptosis. In spite of numerous attempts, only few typical Hodgkin lymphoma (HL) lines have been established. This suggests that the cells require survival factors that they receive in the in vivo microenvironment. If EBV is expected to drive the cells for growth in culture, the absence of EBNA-2 may explain the incapacity of H/RS cells for in vitro proliferation. In EBV carrying B lymphocytes, functional EBNA-2 and LMP-1 proteins are required for in vitro growth. For analysis of the interaction between EBV and the H/RS cells, we infected the CD21-positive HL line KMH2 with the B958 and Akata viral strains. Only EBNA-1 expression was detected in a few cells in spite of the fact that all cells could be infected. Using a neomycin-resistance-tagged recombinant EBV strain (Akata-Neo) we established an EBV-positive subline that was carried on selective medium. In contrast to the type II EBV expression pattern of H/RS cells in vivo, the KMH2 EBV cells did not express LMP-1. The EBV expression pattern could be modified in this type I subline. LMP-1 could be induced by the histone deacetylase inhibitors TSA and n-butyrate, by 5-AzaC, a demethylating agent, and by phorbol ester. None of these treatments induced EBNA-2. Importantly, exposure to CD40 ligand and IL-4 induced LMP-1 without EBNA-2 expression and lytic replication. The KMH2 EBV cells expressed LMP-2A, but not LMP-2B mRNAs. This result is highly relevant for the type II expression pattern of H/RS cells in vivo, since these stimuli can be provided by the surrounding activated T lymphocytes.

Bacterial Outer Membrane Proteins↗

PubMed2005

EBV infection induces expression of the transcription factors ATF-2/c-Jun in B lymphocytes but not in B-CLL cells.

B cell type chronic lymphocytic leukaemia (B-CLL) cells carry the Epstein-Barr virus (EBV) receptor CD21 and can be infected in vitro with the virus. The infected cells exhibit an unusual EBV program, they express the nuclear proteins but not latent membrane protein 1 (LMP-1). Similar cells were encountered in lymphoid tissues of infectious mononucleosis (IM) patients and in lymphoproliferations of immunosuppressed patients. EBV infected B-CLL cells can be regarded as model for this viral program. In B cells the regulation of LMP-1 is executed mainly by EBV encoded nuclear antigen 2 (EBNA-2), interacting with several cellular proteins and these complexes bind to specific sequences in the LMP-1 promoter. ATF2 and c-Jun were shown to be among the interacting partners of EBNA-2. These molecules can be detected in experimentally infected B lymphocytes. We found c-Jun and/or phosphorylated ATF-2 (p-ATF-2) expression in some B-CLL ex vivo samples. They disappeared or their expression declined promptly in explanted cells, even if they were infected with EBV in vitro. Activation of the infected B-CLL cells by exposure to CD40L was accompanied by p-ATF-2 and c-Jun but not by LMP-1 expression. In one of three clones tested, subsequent treatment with histone deacetylase inhibitors (HDACi), TSA or n-butyrate, could induce LMP-1. Treatment with phorbol-12, 13-dibutyrate (PDB) induced LMP-1 expression in three of four clones. Neither the HDACi nor the PDB treated cells survived.

Activating Transcription Factor 2↗

PubMed2004

Clinical impact of strip biopsy for early gastric cancer.

BACKGROUND: The impact of EMR (strip biopsy method) on the selection of subsequent treatment for early gastric cancer was analyzed retrospectively. METHODS: A total of 163 consecutive patients with gastric epithelial tumors (186 lesions) underwent strip biopsy. On the basis of pretherapeutic findings, the indications for strip biopsy were classified into 4 groups: benign-malignant borderline group (93 lesions), curative indication group (65), diagnostic indication group (22), and palliative indication group (6). The clinical impact of the strip biopsy result on the subsequent treatment strategy was assessed. RESULTS: Of the lesions in the benign-malignant borderline group, 36.6% were intramucosal cancer. In the curative indication group, the results of strip biopsy differed from the pretherapeutic findings for 7.7% of the lesions. Strip biopsy was effective treatment for all lesions in the benign-malignant borderline group and for 92.3% of those in the curative indication group. Strip biopsy avoided unnecessary surgery in 50% of patients in the diagnostic indication group and 16.7% of those in the palliative indication group. After the strip biopsy results were explained, 50% of the patients in the palliative indication group reversed their initial decision and opted for surgery. Strip biopsy results reversed the decision for surgery, which had been based on inaccurate pretherapeutic information, in 20% of cases of early gastric cancer. CONCLUSIONS: Strip biopsy has a major clinical impact, because it provides an accurate diagnosis, aids in the selection of an appropriate treatment strategy, and reduces unnecessary surgery.

Biopsy↗

PubMed2004

Upregulation of LMP1 expression by histone deacetylase inhibitors in an EBV carrying NPC cell line.

OBJECTIVES: In about 60% of Epstein-Barr virus (EBV) carrying nasopharyngeal carcinomas (NPC) LMP1 expressing cells can be detected. The frequency of LMP1 positive cells and the expression level varies from cell to cell in the different tumors. Cell lines derived from EBV positive NPCs loose the virus during in vitro culture. The in vitro infected NPC cell line TWO3-EBV used in our study carries the neomycin-resistance gene containing EBV and expresses low level of LMP1. With this cell line it was thus possible to study the regulation of LMP1 expression by modification of chromatin acetylation state. STUDY DESIGN: The TWO-EBV cell line was treated with n -butyrate (NB) or trichostatin A (TSA). RESULTS: Shown by immunoblotting, the LMP1 level was elevated in the treated samples. Already 2 h after TSA exposure LMP1 expression was higher and it increased up to 24 h. Immunofluorescence staining showed that nearly all cells were LMP1 positive. Neither EBNA2 nor BZLF1 were induced. Tested first 2 h after the treatment, acetylated histone H3 and H4 were already detectable, and their level increased up to 8 h. Chromatin immunoprecipitation (ChIP) verified that the LMP1-promoter (LMP1p) (ED-L1) was acetylated after TSA treatment. CONCLUSION: EBV carrying epithelial cells do not express EBNA-2. We showed that LMP1 expression was upregulated by histone deacetylase inhibitors in an in vitro infected, EBV carrier NPC cell line.

Acetylation↗

PubMed2003

Upregulation of the truncated basic hair keratin 1(hHb1-DeltaN) in carcinoma cells by Epstein-Barr virus (EBV).

To investigate the role of Epstein-Barr virus (EBV) in epithelial tumors, we compared the expression pattern of cellular genes in the EBV-infected gastric carcinoma cell line, NU-GC-3, and its uninfected control. Subtractive suppression hybridization (SSH) was combined with high-density DNA array screening to identify differentially expressed genes. We have discovered that EBV infection upregulated a truncated variant of human basic hair keratin 1 (hHb1-DeltaN), a gene that had previously been identified in metastatic breast carcinoma. We verified the differential expression of hHb1-DeltaN in 3 independent EBV-positive and -negative NU-GC-3 clones by Northern blotting. We further verified the EBV-dependent upregulation of hHb1-DeltaN in 3 other carcinoma cell lines (AGS, TWO3 and DLD1) by RT-PCR. Inhibition of CpG methylation by 5-Aza-CdR induced hHb1-DeltaN mRNA expression in the EBV-negative clones but did not alter the expression in the EBV-positive clones. The expression of hHb1-DeltaN protein was detectable by immunofluorescence and Western blotting in EBV-positive but not in EBV-negative NU-GC-3 clones after proteasome inhibitor (MG132) treatment. hHb1-DeltaN protein formed fibrous structures in the cytoplasm and accumulated in distinct nuclear bodies in the euchromatic areas of the cell nucleus. We suggest that the unstable hHb1-DeltaN protein may inhibit some of the functions of the keratin cytoskeleton and/or interfere with transcription regulation. It also may establish a link between EBV and the low differentiated or anaplastic status of the carcinomas that carry the virus.

Azacitidine↗

PubMed2003

T cell leukemia I oncogene expression depends on the presence of Epstein-Barr virus in the virus-carrying Burkitt lymphoma lines.

We used a modified subtractive suppression hybridization to identify cellular genes that show altered expression in Burkitt lymphomas (BLs) in the presence of Epstein-Barr virus (EBV). Comparison of the gene expression patterns of an EBV-negative clone of the originally EBV-positive BL line Akata, with its Neo(R)-EBV derivative, revealed a significant difference in the expression of the T cell leukemia 1 oncogene (TCL-1). Subsequent expression studies showed that the original EBV-positive Akata line and the EBV-reconstituted derivative expressed high levels of TCL-1, whereas the EBV-negative variant showed only a low level of expression. Two other independently established EBV-positive BLs (Mutu and OMA) that have also thrown off EBV showed a similar decrease in TCL-1 expression after virus loss. Reinfection with Neo(R)-EBV restored the TCL-1 expression levels in the EBV loss variants to as high a level as the originally EBV-positive lines. High-resolution immunostaining showed that TCL-1 was localized in both the cytoplasm and the nucleus. Our findings suggest that high expression of TCL-1 is necessary for the development of the BL phenotype. In view of the fact that germinal center B cells, regarded as the progenitors of BL, do not express TCL-1, we suggest that constitutive expression of this oncogene occurs by genetic or epigenetic changes in the EBV-negative BLs. In the originally EBV-positive BLs, the ability of the virus to switch on TCL-1 expression would obviate this need.

Base Sequence↗

PubMed2003

Improved subtractive suppression hybridization combined with high density cDNA array screening identifies differentially expressed viral and cellular genes.

Suppression subtractive hybridization (SSH) combines normalization and suppression PCR effect step in a single cycle to isolate differentially expressed genes in two cDNA samples. The PCR suppression effect is mediated by long inverted terminal repeats. The efficiency of the restriction enzyme digestion and the adapter ligation are crucial in the success of the SSH. We modified the original SSH protocol in order to improve the efficiency of the subtraction. A magnetic bead based separation step has been included after the ligation step, to purify the successfully ligated fraction of the tester. EBV(NEO) infected Akata(-) Burkitt's lymphoma cell line was compared with the EBV(-) Akata(-) cell line to isolate differentially expressed genes with the improved SSH protocol. Some 44 cDNA clones that showed the greatest differences in expression have been sequenced. Of them, 20 showed more than 3-fold difference in expression. Seven of the 20 genes were EBV genes. To quantitate the expression levels, high density nylon cDNA array hybridization was optimized. Statistical analysis of the data revealed that the spotting of the arrayer is exceptionally reproducible, which makes the comparison of the hybridization of parallel filters possible.

Cell Line, Transformed↗

PubMed2002

SH2D1A expression in Burkitt lymphoma cells is restricted to EBV positive group I lines and is downregulated in parallel with immunoblastic transformation.

The SH2 domain containing SH2D1A protein has been characterized in relation to the X-linked lymphoproliferative disease (XLP), a primary immunodeficiency that leads to serious clinical conditions after Epstein-Barr virus (EBV) infection. The SH2D1A gene is mutated in the majority of XLP patients. We previously detected SH2D1A in activated T and NK cells, but not in B lymphocytes. We have found SH2D1A protein in Burkitt lymphoma (BL) lines, but only in those that carried EBV and had a Group I (germinal center) phenotype. All the EBV-carrying Group III (immunoblastic) and the EBV-negative BL lines tested were SH2D1A-negative. Motivated by these differences, we studied the impact of EBV and the cellular phenotype on SH2D1A expression. We approached the former question with BL sublines after both the loss of the virus and subsequent reinfection. We also tested original EBV-negative BL lines carrying transfected EBV genes, such as EBNA1, EBNA2, EBNA6, EBER1, 2 and LMP1, respectively. In our experiments, no direct relationship could be seen between EBV and SH2D1A expression. We modified the phenotype of the Group I BL cells by LMP1 transfection or CD40 ligation. The phenotypic changes, indicated by expression of immunoblastic markers, e.g., SLAM, were accompanied by downregulation of SH2D1A. It seems, therefore, that the presence of EBV and the phenotype of the cell together regulate SH2D1A expression in the BL cells. It is possible that SH2D1A is expressed in a narrow window of B cell development represented by germinal center cells.

Animals↗