Yoshiyuki Tsukamoto

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

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

Aberrant DNA methylation status of endometriosis: epigenetics as the pathogenesis, biomarker and therapeutic target.

Endometriosis, a common, benign, estrogen‐dependent disease affecting 3–10% of women of reproductive age, is characterized by the ectopic growth of endometrial tissue that is found primarily in the peritoneum, ovaries and rectovaginal septum. Recently, endometriosis has been alternatively described as an immune disease, a genetic disease and a disease caused by exposure to environmental factors, in addition to its usual description as a hormonal disease. In addition, accumulating evidence suggests that various epigenetic aberrations play definite roles in the pathogenesis of endometriosis. Epigenetic alterations reported to date in endometriosis include the genomic DNA methylation of progesterone receptor‐B, E‐cadherin, homeobox A10, estrogen receptor‐β, steroidogenic factor‐1 and aromatase. Aberrant expression of DNA methyltransferases, which attach a methyl group to the 5‐carbon position of cytosine bases in the CpG island of the promoter region and silence the corresponding gene expression, has also been demonstrated in endometriosis. This review summarizes the recent studies on the aberrant DNA methylation status and aberrant expression of DNA methyltransferases, which regulate DNA methylation, in endometriosis. We also discuss the recent information on the diagnostic and therapeutic implications of epigenetic alterations occurring in endometriosis.

Immunohistochemical diagnosis of the cagA‐gene genotype of Helicobacter pylori with anti‐East Asian CagA‐specific antibody

Cytotoxin‐associated antigen A (CagA) protein produced by Helicobacter pylori is proposed to be associated with the pathogenesis of gastric cancer as well as gastritis and gastroduodenal ulcer. It has been reported that the CagA of H. pylori widespread in East Asian countries, where the mortality rate due to gastric cancer is high, is structurally different from that in Western countries, where the gastric cancer mortality rate is relatively low. In this study, we generated an antibody, East Asian CagA‐specific antibody (α‐EAS Ab), which is specifically immunoreactive with East Asian CagA but not with Western CagA. The CagA was immunohistochemically detected at the surface of the gastric mucosa. Interestingly, positive immunoreactivity was also detected in the nucleus and cytoplasm of the infected gastric epithelium, suggesting that CagA may play some pathogenic role in both the nucleus and cytoplasm. Immunohistochemistry of 47 gastric biopsy specimens detected East Asian CagA‐positive H. pylori in 43 cases. In 46 of the 47 cases examined, the data obtained by immunohistochemistry were completely consistent with those obtained by sequencing of the cagA gene of the isolated strain, suggesting that our immunohistochemical method is reliable and useful for diagnosis of the cagA genotype. (Cancer Sci 2007; 98: 521–528)

Helicobacter pylori dupA gene is not associated with clinical outcomes in the Japanese population.

The dupA gene of Helicobacter pylori was suggested to be a risk factor for duodenal ulcer but protective against gastric cancer. The present study aimed to re-examine the role of dupA in H. pylori-infected Japanese patients. We found that dupA status was not associated with any gastroduodenal disease, histological score of chronic gastritis or with the extent of interleukin-8 production from gastric cell lines. These results indicate that dupA is unlikely to be a virulence factor of H. pylori in the Japanese population.

Helicobacter pylori infection and gastroduodenal diseases in Vietnam: a cross-sectional, hospital-based study

BackgroundThe rate of H. pylori infection in Vietnam is reportedly high, but the spectrum of H. pylori-associated gastroduodenal diseases has not been systematically investigated. Moreover, despite the similarities of ethnicity and diet, the age-standardized incidence rate of gastric cancer in the northern city of Hanoi is higher than that in the southern city of Ho Chi Minh, but the reason for this phenomenon is unknown. The virulence of Vietnamese H. pylori has also not been investigated in detail.MethodsIndividuals undergoing esophagogastroduodenoscopy were randomly recruited. H. pylori infection status was determined based on the combined results of culture, histology, immunohistochemistry, rapid urine test and serum ELISA. Peptic ulcer (PU) and gastroesophageal reflux disease was diagnosed by endoscopy, and chronic gastritis was determined histologically. H. pylori virulence factors were investigated by PCR and sequencing.ResultsAmong the examined patients, 65.6% were infected with H. pylori. The prevalence of infection was significantly higher in those over 40 years of age than in those aged ≤40. Chronic gastritis was present in all H. pylori-infected individuals, 83.1% of whom had active gastritis, and 85.3% and 14.7% had atrophy and intestinal metaplasia, respectively. PU was present in 21% of infected patients, whereas its incidence was very low in non-infected individuals. The prevalence of PU was significantly higher in Hanoi than in Ho Chi Minh. The prevalence of vacA m1, which has been identified as an independent risk factor for PU in Vietnam, was significantly higher among H. pylori isolates from Hanoi than among those from Ho Chi Minh.ConclusionsH. pylori infection is common in Vietnam and is strongly associated with PU, active gastritis, atrophy and intestinal metaplasia. vacA m1 is associated with an increased risk for PU and might contribute to the difference in the prevalence of PU and gastric cancer between Hanoi and Ho Chi Minh.

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.

MiR-29c is downregulated in gastric carcinomas and regulates cell proliferation by targeting RCC2

BackgroundPreviously, using miRNA microarray, we have found that miR-29c is significantly downregulated in advanced gastric carcinoma. In the present study, we investigated whether miR-29c functions as a tumor-suppressor miRNA in gastric carcinoma cells. For this purpose, we verified the downregulation of miR-29c in gastric carcinoma tissues, and assessed the biological effect of miR-29c on gastric carcinoma cells.ResultsIn miR-29c-transfected cells, both proliferation and colony formation ability on soft agar were significantly decreased. Although apoptosis was not induced, BrdU incorporation and the proportion of cells positive for phospho-histone H3 (S10) were significantly decreased in miR-29c-transfected cells, indicating that miR-29c may be involved in the regulation of cell proliferation. To explain the mechanism of growth suppression by miR-29c, we explored differentially expressed genes (>2-fold) in miR-29c-transfected cells in comparison with negative control transfected cells using microarray. RCC2, PPIC and CDK6 were commonly downregulated in miR-29c-transfected MKN45, MKN7 and MKN74 cells, and all of the genes harbored miR-29c target sequences in the 3’-UTR of their mRNA. RCC2 and PPIC were actually upregulated in gastric carcinoma tissues, and therefore both were identified as possible targets of miR-29c in gastric carcinoma. To ascertain whether downregulation of RCC2 and/or PPIC is involved in the growth suppression by miR-29c, we transfected siRNAs against RCC2 and PPIC into MKN45 and determined cell viability, the rate of BrdU incorporation, and caspase activity. We found that RCC2-knockdown decreased both cell viability and BrdU incorporation without any increase of caspase activity, while PPIC-knockdown did not, indicating that downregulation of RCC2 may be at least partly responsible for the growth suppression by miR-29c.ConclusionsOur findings indicate that miR-29c may have tumor-suppressive functions in gastric carcinoma cells, and that its decreased expression may confer a growth advantage on tumor cells via aberrant expression of RCC2.