Chisato Nakada

Genome-wide microRNA expression profiling in renal cell carcinoma: significant down-regulation of miR-141 and miR-200c.

We investigated expression profiles of microRNA (miRNA) in renal cell carcinoma [clear cell carcinomas (CCC) and chromophobe renal cell carcinomas (ChCC)] and in normal kidneys by using a miRNA microarray platform which covers a total of 470 human miRNAs (Sanger miRBase release 9.1). Unsupervised hierarchical cluster analysis revealed that CCC and ChCC were separable and that no subgroups were identified in CCCs. We found that 43 miRNAs were differentially expressed between CCC and normal kidney, of which 37 were significantly down‐regulated in CCC and the other 6 were up‐regulated. We also found that 57 miRNAs were differentially expressed between ChCC and normal kidney, of which 51 were significantly down‐regulated in ChCC and the other 6 were up‐regulated. Together, these observations indicate that expression of miRNAs tends to be down‐regulated in both CCC and ChCC compared with normal kidney. We observed that miR‐141 and miR‐200c were the most significantly down‐regulated miRNAs in CCCs. Indeed, in all cases of CCC analysed, both miR‐141 and miR‐200c were down‐regulated in comparison with normal kidney. Microarray data and quantitative RT–PCR showed that these two miRNAs were expressed concordantly. TargetScan algorithm revealed that ZFHX1B mRNA is a hypothetical target of both miR‐141 and ‐200c. We established by quantitative RT–PCR that, in CCCs in which miR‐141 and miR‐200c were down‐regulated, ZFHX1B, a transcriptional repressor for CDH1/E‐cadherin, tended to be up‐regulated. Furthermore, we found that overexpression of miR‐141 and miR‐200c caused down‐regulation of ZFHX1B and up‐regulation of E‐cadherin in two renal carcinoma cell lines, ACHN and 786‐O. On the basis of these findings, we suggest that down‐regulation of miR‐141 and miR‐200c in CCCs might be involved in suppression of CDH1/E‐cadherin transcription via up‐regulation of ZFHX1B. Copyright

Genome-wide analysis of DNA copy number alterations and gene expression in gastric cancer.

Genomic copy number aberrations (CNAs) are believed to play a major role in the development and progression of human cancers. Although many CNAs have been reported in gastric cancer, their genome‐wide transcriptional consequences are poorly understood. In this study, to reveal the impact of CNAs on genome‐wide expression in gastric cancer, we analysed 30 cases of gastric cancers for their CNAs by array comparative genomic hybridization (array CGH) and 24 of these 30 cases for their expression profiles by oligonucleotide‐expression microarray. We found that with the application of laser microdissection, most CNAs were detected at higher frequency than in previous studies. Notably, gain at 20q13 was detected in almost all cases (97%), suggesting that this may play an important role in the pathogenesis of gastric cancer. By comparing the array CGH data with expression profiles of the same samples, we showed that both genomic amplification and deletion strongly influence the expression of genes in altered genomic regions. Furthermore, we identified 125 candidate genes, consisting of 114 up‐regulated genes located in recurrent regions (>10%) of amplification and 11 down‐regulated genes located in recurrent regions of deletion. Up‐regulation of several candidate genes, such as CDC6, SEC61G, ANP32E, BYSL and FDFT1, was confirmed by immunohistochemistry. Interestingly, some candidate genes were localized at genomic loci adjacent to well‐known genes such as EGFR, ERBB2 and SMAD4, and concordantly deregulated by genomic alterations. Based on these results, we propose that our list of candidate genes may contain novel genes involved in the pathogenesis of advanced gastric cancer. Copyright

Overexpression of miR‐210, a downstream target of HIF1α, causes centrosome amplification in renal carcinoma cells

MiR‐210 is significantly up‐regulated in clear cell renal cell carcinoma (CCC), but the mechanism and biological consequences of miR‐210 up‐regulation are poorly understood. Here, we show that miR‐210 is highly expressed in renal carcinoma cell lines and that its expression is clearly correlated with accumulation of hypoxia‐inducible factor 1α (HIF1α) under normoxia as well as hypoxia, suggesting that miR‐210 up‐regulation in renal carcinoma cells is most likely due to accumulation of HIF1α. To reveal the effects of miR‐210 up‐regulation, the miR‐210 precursor was transfected into renal carcinoma cells. After transfection, the cells accumulated at the G2/M phase of the cell cycle and their viability was decreased, suggesting that miR‐210 overexpression may trigger an event that hinders normal cell division. Immunocytochemistry demonstrated a multipolar spindle accompanied by centrosome amplification in cells overexpressing miR‐210. It has been reported that centrosome amplification induces chromosome mis‐segregation, finally leading to chromosome instability and aneuploidy. Indeed, the proportion of aneuploid cells (>4n) was increased in miR‐210 overexpressed cells. By using the TargetScan and PicTar algorithms, E2F3 was identified as one of the possible targets of miR‐210 and was suppressed at the protein level by miR‐210. Moreover, the proportion of aneuploid cells was increased in E2F3 siRNA transfected cells. On the basis of these results, we propose that miR‐210 up‐regulation due to HIF1α accumulation may induce aneuploidy via E2F3 down‐regulation at least in part, and may play a role in tumourigenesis and/or progression of CCC. Copyright

Arpp, a New Homolog of Carp, Is Preferentially Expressed in Type 1 Skeletal Muscle Fibers and Is Markedly Induced by Denervation

In this study, we isolated and characterized a murine counterpart of the human Arpp (hArpp) gene. Sequence analysis revealed that the murine Arpp (mArpp) gene is almost identical to the Ankrd2 gene, which has recently been isolated as a mouse gene induced in stretched skeletal muscle. The mArpp gene encodes a protein of 332 amino acids that contains four well-conserved ankyrin-repeat domains in the central portion of the protein. The amino acid sequence of mArpp protein (mArpp) is highly homologous to that of mouse cardiac-restricted ankyrin-repeat protein (Carp), which is proposed to be a putative genetic marker for cardiac hypertrophy. Immunohistochemical analysis revealed that mArpp is preferentially expressed in type 1 skeletal muscle fibers, and that mArpp is localized in both the nucleus and the sarcomeric I-band of muscle fibers, suggesting that Arpp may function as a nuclear and sarcomeric protein. Furthermore, mArpp was also expressed in neurons of the cerebellum and cerebrum, the islets of Langerhans in the pancreas, and the esophageal epithelium, suggesting that mArpp may play a functional physiologic role in brain, pancreas, and esophagus as well as in type 1 muscle fibers. Interestingly, although mArpp was localized in both nucleus and cytoplasm in neurons, its localization was restricted to nucleus in pancreas and esophagus, suggesting that intracellular localization of mArpp is regulated in a tissue-specific manner. Furthermore, we found that mArpp- and Carp-expression in skeletal muscle were markedly up-regulated after denervation. Although the elevated expression level of Carp was kept only for two weeks after denervation, that of Arpp was kept at least for 4 weeks, suggesting that mArpp and Carp may play distinct functional roles in denervated skeletal muscle.

High-resolution analysis of DNA copy number alterations and gene expression in renal clear cell carcinoma†

We analysed chromosomal copy number aberrations (CNAs) in renal cell carcinomas by array‐based comparative genomic hybridization, using a genome‐wide scanning array with 2304 BAC and PAC clones covering the whole human genome at a resolution of roughly 1.3 Mb. A total of 30 samples of renal cell carcinoma were analysed, including 26 cases of clear cell carcinoma (CCC) and four cases of chromophobe renal cell carcinoma (ChCC). In CCCs, gains of chromosomes 5q33.1‐qter (58%), 7q11.22‐q35 (35%) and 16p12.3‐p13.12 (19%), and losses of chromosomes 3p25.1‐p25.3 (77%), 3p21.31‐p22.3 (81%), 3p14.1‐p14.2 (77%), 8p23.3 (31%), 9q21.13‐qter (19%) and 14q32.32‐qter (38%) were detected. On the other hand, the patterns of CNAs differed markedly between CCCs and ChCCs. Next, we examined the correlation of CNAs with expression profiles in the same tumour samples in 22/26 cases of CCC, using oligonucleotide microarray. We extracted genes that were differentially expressed between cases with and without CNAs, and found that significantly more up‐regulated genes were localized on chromosomes 5 and 7, where recurrent genomic gains have been detected. Conversely, significantly more down‐regulated genes were localized on chromosomes 14 and 3, where recurrent genomic losses have been detected. These results revealed that CNAs were correlated with deregulation of gene expression in CCCs. Furthermore, we compared the patterns of genomic imbalance with histopathological features, and found that loss of 14q appeared to be a specific and additional genetic abnormality in high‐grade CCC. When we compared the expression profiles of low‐grade CCCs with those of high‐grade CCCs, differentially down‐regulated genes tended to be localized on chromosomes 14 and 9. Thus, it is suggested that copy number loss at 14q in high‐grade CCC may be involved in the down‐regulation of genes located in this region. Copyright

Arpp/Ankrd2, a member of the muscle ankyrin repeat proteins (MARPs), translocates from the I-band to the nucleus after muscle injury

Ankyrin-repeat protein with a PEST motif and a proline-rich region (Arpp), also designated as Ankrd2, is a member of the muscle ankyrin repeat proteins (MARPs), which have been proposed to be involved in muscle stress response pathways. Arpp/Ankrd2 is localized mainly in the I-band of striated muscle. However, it has recently been reported that Arpp/Ankrd2 can interact with nuclear proteins, such as premyelocytic leukemia protein (PML), p53 and YB-1 in vitro. In this study, to determine whether nuclear accumulation of Arpp/Ankrd2 actually occurs, we performed an immunohistochemical investigation of gastrocnemius muscles that had been injured by injection of cardiotoxin or contact with dry ice. We found that Arpp/Ankrd2 accumulated in the nuclei of myofibers located adjacent to severely damaged myofibers after muscle injury. Double-labeled immunohistochemistry revealed that Arpp/Ankrd2 accumulated in the nuclei of sarcomere-damaged myofibers. Furthermore, we found that Arpp/Ankrd2 tended to be localized in euchromatin where genes are transcriptionally activated. Based on these findings, we suggest that Arpp/Ankrd2 may translocate from the I-band to the nucleus in response to muscle damage and may participate in the regulation of gene expression.

Cardiac ankyrin repeat protein is preferentially induced in atrophic myofibers of congenital myopathy and spinal muscular atrophy

Cardiac ankyrin repeat protein (CARP), which is structurally characterized by the presence of four ankyrin repeat motifs in its central region, is believed to be localized in the nucleus and to participate in the regulation of cardiac‐specific gene expression in cardiomyocytes. However, we recently found that CARP was induced in skeletal muscle by denervation, leading us to speculate that CARP may be induced under some pathological conditions. In the present study, we immunohistochemically analyzed the expression of CARP in 11 cases of spinal muscular atrophy (SMA) and 14 cases of congenital myopathy. In SMA, CARP was expressed selectively in severely atrophic myofibers, suggesting that CARP expression may reflect the status of muscle atrophy. Furthermore, in the congenital myopathies, the expression patterns of CARP were distinct among the subtypes, which included nemaline myopathy, myotubular myopathy, central core disease, and congenital fiber type disproportion. Although CARP was preferentially expressed in severely damaged myofibers in nemaline myopathy, it was not detected in central core disease. These findings suggest that immunohistochemical evaluation of CARP may be helpful in the diagnosis of SMA and the congenital myopathies.

Analysis of p53 Mutations and the Expression of p53 and p21WAF1/CIP1 Protein in 15 Cases of Sebaceous Carcinoma of the Eyelid

PURPOSE The purpose of this study was to detect mutation of the p53 gene, to assess its relationship with p53 or p21(WAF1/CIP1) expression, and to evaluate the correlation between p53 mutation or p21(WAF1/CIP1) expression and clinicopathologic findings in sebaceous carcinoma of the eyelid. METHODS Fifteen conventional paraffin-embedded samples of sebaceous carcinoma of the eyelid were analyzed. Using the single-strand conformation polymorphism technique, the authors sequenced coding exons 5-8 of the p53 gene. The expression of p53 and p21(WAF1/CIP1) protein was analyzed by immunohistochemistry. RESULTS In 10 of the 15 cases (66.7%), point mutations were detected in the p53 gene. CC to TT double-base changes (tandem mutations), which are known to be induced only by UV, were not detected in any of the mutations. Correlations between p53 mutation and expression were found to be statistically significant (P = 0.007). There was no significant correlation between p53 mutation and clinicopathologic findings or p21(WAF1/CIP1) expression. However, there was a significant inverse correlation between p21(WAF1/CIP1) expression and presence of lymph node metastasis (P = 0.007). CONCLUSIONS Among human cancers, sebaceous carcinoma of the eyelid may be one of those showing most frequent mutation of the p53 gene, which may not be caused by exposure to UV. p21(WAF1/CIP1) downregulation may be associated with lymph node metastasis.

Cardiac-Restricted Ankyrin-Repeated Protein Is Differentially Induced in Duchenne and Congenital Muscular Dystrophy

Cardiac ankyrin-repeated protein (CARP) has been shown to associate with a transcription factor, YB-1, that may activate expression of the ventricular myosin light chain-2 gene during cardiogenesis. CARP is induced in the adult hypertrophic heart subjected to pressure overload, suggesting that CARP may play important functional roles in both embryonic and adult hearts. Although CARP expression was initially believed to be restricted to the heart, we found recently that CARP is induced strongly in human fetal skeletal muscle and in experimentally denervated skeletal muscle, leading us to speculate that CARP may also play important roles in skeletal muscle. In the present study, we found that in rats initially damaged by a single injection of bupivacaine, CARP expression was induced strongly in regenerating muscles with a peak 3 days after the injection, followed by down-regulation to undetectable levels after 28 days. Although CARP was coexpressed with embryonic myosin heavy chain (MHC) in regenerating myofibers, CARP expression persisted even after down-regulation of embryonic MHC expression, whereas it began to decrease before the onset of slow or fast MHC expression, suggesting that CARP is expressed at a specific differentiation stage during muscle regeneration. We analyzed the expression of CARP in muscle biopsy specimens from 14 patients with muscular dystrophy (MD) and detected high expression of CARP in 13 of the 14 cases. CARP-positive myofibers were detected more often in congenital muscular dystrophy (CMD) than in Duchenne muscular dystrophy (DMD). We found that CARP was expressed exclusively, and at a high level, in small regenerating myofibers that express embryonic MHC in DMD, which suggested that CARP could be used as a marker of muscle regeneration in DMD. On the other hand, in CMD, expression of CARP was not limited to regenerating fibers, being detectable in myofibers expressing embryonic MHC and those expressing mature-type MHC. These findings suggest that the differentiation stage of CARP-positive myofibers in DMD and CMD may differ.

Role of leptin signaling in the pathogenesis of angiotensin II-mediated atrial fibrosis and fibrillation.

Background—We examined the hypothesis that leptin signaling contributes to the atrial fibrosis and atrial fibrillation (AF) evoked by angiotensin II (AngII). Methods and Results—Eight-week-old male CL57/B6 (CNT) and leptin-deficient ob/ob mice (Ob) were subcutaneously infused with AngII (2.0 mg/kg per day). Two weeks later, transesophageal burst pacing and an electrophysiological study using isolated perfused hearts were performed. Left-atrial tissues were collected to determine interstitial fibrosis by Masson trichrome staining, and the expressions of mRNAs related to inflammatory profibrotic signals were assessed. Left-atrial fibroblasts were isolated from adult Sprague-Dawley and Zucker rats. The effects of leptin (100 ng/mL) or AngII (100 nmol/L) treatment were evaluated. In CNT-AngII mice, leptin expression in the left atrium was upregulated (P<0.01). Transesophageal burst pacing induced atrial fibrillation in 88% (7/8) of CNT-AngII mice, but not in Ob-AngII mice (0/8; P<0.01). In isolated perfused hearts, atrial fibrillation was induced only in CNT-AngII mice (4/6; 67%). Interatrial conduction time was prolonged in CNT-AngII mice (P<0.01), but not in Ob-AngII mice. The upregulation of collagen 1, collagen 3, transforming growth factor-&bgr;1, &agr;-smooth muscle actin, MCP-1, F4/80, and RANTES mRNA, which was seen in CNT-AngII mice, was attenuated in Ob-AngII mice. In cultured Sprague-Dawley rat atrial fibroblasts, AngII treatment increased leptin expression (P<0.01). Addition of leptin increased transforming growth factor-&bgr;1, &agr;-smooth muscle actin, MCP-1, and RANTES expressions in Sprague-Dawley rat atrial fibroblasts, but not in Zucker rat atrial fibroblasts. Conclusions—Our results demonstrate for the first time that leptin signaling is essential for the development of atrial fibrosis and atrial fibrillation evoked by AngII.