関 直彦

Bcl6, a sequence-specific transcriptional repressor, is important for generation and maintenance of memory CD8+ T cells. Although memory CD8+ T cells are generated from effector CD8+ T cells, a role for Bcl6 in effector CD8+ T cells is largely unknown. We show here that Bcl6 expression was transiently induced in activated CD8+ T cells and continuously up-regulated in effector CD8+ T cells. The amount of granzyme B mRNA among effector molecules produced by effector CD8+ T cells inversely correlated with the amount of Bcl6 mRNA in CD8+ T cells. Overexpression of Bcl6 in CD8+ T cells resulted in lower killing activity at their effector phase, supporting the reduction of granzyme B expression in effector CD8+ T cells by Bcl6. We identified a putative Bcl6-binding DNA sequence in the promoter region of the granzyme B gene. Binding of Bcl6 to the Bcl6-binding sequence was detected in naive CD8+ T cells but not in activated CD8+ T cells by chromatin immunoprecipitation assay. Furthermore, the Bcl6-binding sequence was required for Bcl6 to repress the lucfferase reporter gene expression controlled by the granzyme B promoter. Thus, the granzyme B gene is a molecular target of Bcl6 in effector CD8+ T cells. © 2006 Wiley-VCH Verlag GmbH &amp Co. KGaA, Weinheim.

Diabetic retinopathy is one of the most frequent complications of diabetes and is a leading cause of vision loss in adulthood. To better understand the molecular pathophysiology of diabetic retinopathy, we performed comprehensive gene expression analysis of the mouse retina under diabetic conditions with an in-house cDNA microarray system that was designed to be suitable for the small amount of RNA available from a single mouse retina. Diabetes was induced in male C57BL/6 mice by an intraperitoneal injection of streptozotocin, and the changes in retinal mRNA levels were examined in three pairs of diabetic and age-matched control mice at 1 and 3 months after the injection of streptozotocin. Northern blot analysis with amplified total cRNA confirmed the increase in mRNA levels of several selected genes. Most of the significantly up-regulated genes could be classified into two functional categories: oxidative phosphorylation and protein turnover. All mitochondrial DNA-encoded and most of the nuclear DNA-encoded genes for oxidative phosphorylation were up-regulated in the diabetic retina. This was in sharp contrast with a previous report of a down-regulation of these genes in skeletal muscles of streptozotocin-induced diabetic mice and type 2 diabetic humans. Genes for protein synthesis and ubiquitin were also up-regulated in the diabetic retina, suggesting the increase in turnover rates for at least a part of the protein population. Taken together, the diabetic retina appears to be in a state activated for intermediary metabolism, presumably because of an increase in insulin-independent glucose influx. These results provide insights into possible preventive and therapeutic intervention of diabetic retinopathy. © 2006 Elsevier Ltd. All rights reserved.

OBJECTIVES: The extremely unfavorable prognosis of intrahepatic cholangiocarcinoma (ICC), even after surgical resection, is mainly attributed to a high rate of recurrence. The aim of this study was to identify the molecules associated with early recurrence of ICC following resection. METHODS: Between December 1984 and July 2003, 46 patients with ICC underwent surgical resection. The clinical outcome of these patients was evaluated in view of the time of recurrence. Consequently, we categorized ICC patients into subgroups, based on the clinical results, and screened differentially expressed genes by DNA microarray analysis. Furthermore, the obtained results were validated in an independent sample set by quantitative real-time reverse transcription- polymerase chain reaction (RT-PCR). Immunohistochemistry was performed to assess the expressed genes at the protein level. RESULTS: The survival of patients with early recurrence, occurring within a year after surgical resection, was significantly poor after surgery and even after recurrence, as compared to that of patients whose recurrence occurred beyond a year after surgery. By the DNA microarray analysis, 13 differentially expressed genes were picked up, and quantitative RT-PCR reaction identified the pancreatic secretory trypsin inhibitor (PSTI) as a candidate gene associated with early recurrence of ICC after resection. This observation was confirmed through examination of an independent set of samples, in which the patients with higher levels of PSTI mRNA expression had significantly shorter recurrence-free survival. Immunohistochemically, PSTI was expressed in the cytoplasm of cancer cells. CONCLUSIONS: PSTI might be a potential marker for identifying ICC patients with an increased risk of early recurrence after surgical resection. © 2006 by Am. Coll. of Gastroenterology. Published by Blackwell Publishing.

The mouse embryonal carcinoma cell line ATDC5 provides an excellent model system for chondrogenesis in vitro. To understand better the molecular mechanisms of endochondral bone formation, we investigated gene expression profiles during the differentiation course of ATDC5 cells, using an in-house microarray harboring full-length-enriched cDNAs. For 28 days following chondrogenic induction, 507 genes were up- or down-regulated at least 1.5-fold. These genes were classified into five clusters based on their expression patterns. Genes for growth factor and cytokine pathways were significantly enriched in the cluster characterized by increases in expression during late stages of chondrocyte differentiation. mRNAs for decorin and osteoglycin, which have been shown to bind to transforming growth factors-β and bone morphogenetic proteins, respectively, were found in this cluster and were detected in hypertrophic chondrocytes of developing mouse bones by in situ hybridization analysis. Taken together with assigned functions of individual genes in the cluster, interdigitated interaction between a number of intercellular signaling molecules is likely to take place in the late chondrogenic stage for autocrine and paracrine regulation among chondrocytes, as well as for chemoattraction and stimulation of progenitor cells of other lineages. © 2006 Elsevier Inc. All rights reserved.

BACKGROUND. 5-Aza-2'-deoxycytidine (5-AzaC) is known well for its demethylation effect and is a promising anticancer agent. However, to the authors' knowledge, serial changes in gene expression over time after 5-AzaC treatment have not been studied to date. To clarify the categories of genes that are up-regulated or downregulated after 5-AzaC treatment, the authors surveyed the genes that had expression levels changed by 5-AzaC treatment in 6 hepatoma cell lines (Hep3B, HLE, Huh7, HepG2, PLC/PRF/5, and Huh6). METHODS. Cell lines were grown in medium that contained 1 mu M of 5-AzaC. Changes in messenger RNA levels were monitored from 24 hours up to 120 hours after 5-AzaC treatment using an in-house microarray that consisted of 4608 combinational DNAs. Using clustering analysis to identify the genes that had gradually changed expression levels and to exclude the substantial experimental noise by microarray analysis, the authors focused on 206 up-regulated genes and 248 down-regulated genes. RESULTS. According to their functional characterization, genes that were involved in the cytoskeleton and the extracellular matrix were enriched significantly in the up-regulated genes. Conversely, genes that were involved in metabolism were enriched significantly in the down-regulated genes. CONCLUSIONS. The current results demonstrated that 5-AzaC can regulate the expression of groups of genes with characteristic functions.

Blood pressure and genetic factors are important factors for diabetic nephropathy. We investigated the relationship between the efficacy of renin angiotensin system (RAS) inhibitors and angiotensin-converting enzyme (ACE) genotypes. Patients with type 2 diabetes without proteinuria, were treated with RAS inhibitors, the first being an ACE inhibitor (ACEI) and the second, an angiotensin II (ATH) receptor blocker (ARB) for 8 weeks each. There was no significant difference (except serum ACE activity) between the two treatments. However, by analysis segregated with ACE gene polymorphism, ARB significantly decreased transforming growth factor-beta 1 (TGF-beta) compared to ACEI in patients with the I/I genotype but not in patients with the D/I + D/D genotype. Delta ATII and Delta TGF-beta have a negative correlation with the I/I genotype and a positive correlation with the D/I + D/D genotypes. These correlation coefficients are significantly different. We suggest that in I/I patients, TGF-beta was reduced by ARB via effects on (ATII) type 2 receptors (AT2). In our experiments, the effect of ARB on TGF-beta reduction was only detected by segregation of ACE genotypes. This indicates that the selection of medicine in light of a patient's genotype is important in treating diabetic nephropathy. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Cathepsin D is an aspartyl protease involved in protein catabolism and tissue remodeling which can be secreted from cancer cells. To identify a potential serum marker for gliomas, we investigated the gene expression levels of cathepsin D in 87 tissue samples and measured the protein concentrations in sera of glioma patients. The tissue samples consisted of 43 glioblastomas, 13 anaplastic astrocytomas, 22 astrocytomas, and 9 normal brain tissues. The results of real-time quantitative reverse transcription-PCR analysis showed that cathepsin D transcript levels became significantly higher as the glioma grade advanced (P = 0.0466, glioblastoma and anaplastic astrocytoma; P = 0.0008, glioblastoma and astrocytoma; P = 0.0271, glioblastoma and normal brain tissue, unpaired t test). Immunohistochemical analysis with anticathepsin D antibody revealed dense and spotty staining in the tumor cells with high transcript levels. The low expression of cathepsin D significantly correlated with long survival of the glioma patients. Furthermore, the glioblastoma patients with high gene expression of cathepsin D lived significantly shorter than those with low expression (P = 0.0104, Cox-Mantel log-rank test) and frequently had leptomeningeal dissemination (P = 0.0016, chi(2) test). The multivariate analysis confirmed that the cathepsin D expression level was an independent predictor for short survival (P = 0.0102, Cox proportional hazard regression model). Measurement of the serum cathepsin D concentrations by ELISA showed a significant increase in the patients with high-grade gliomas as compared with the low-grade tumors (p = 0.0081, chi(2) test). These results collectively suggest that cathepsin D could be a potential serum marker for the prediction of aggressive nature of human gliomas.