[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Previously, 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. RESULTS: In 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. CONCLUSIONS: Our 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.
Molecular Cancer 02/2013; 12(1):15. · 5.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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/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 (SD) and Zucker rats. The effects of leptin (100 ng/ml) or AngII (100 nM) treatment were evaluated. In CNT-AngII mice, leptin expression in the left atrium was upregulated (p<0.01). Transesophageal burst pacing induced AF in 88% (7/8) of CNT-AngII mice, but not in Ob-AngII mice (0/8, p<0.01). In isolated perfused hearts, AF was induced only in CNT-AngII mice (4/6, 67%). Inter-atrial conduction time was prolonged in CNT-AngII mice (p<0.01), but not in Ob-AngII mice. The upregulation of collagen 1, collagen 3, TGF-ß1, α-SMA, MCP-1, F4/80 and RANTES mRNA that was seen in CNT-AngII mice, was attenuated in Ob-AngII mice. In cultured SD rat atrial fibroblasts, AngII treatment increased leptin expression (p<0.01). Addition of leptin increased TGF-ß1, α-SMA, MCP-1 and RANTES expressions in SD 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 AF evoked by AngII.
Circulation Arrhythmia and Electrophysiology 02/2013; · 5.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: STUDY QUESTION: What is the global expression pattern of microRNAs (miRNAs) in endometriotic stromal cells and is miR-196b involved in the pathogenesis of endometriosis? SUMMARY ANSWER: Several miRNAs are aberrantly expressed in endometriotic cyst stromal cells (ECSCs), including miR-196b whose expression is repressed in endometriotic stromal cells. WHAT IS KNOWN ALREADY: Although, histologically, endometriotic tissues and normal proliferative endometrium are similar, a number of distinct molecular differences have been reported to date. The anti-apoptotic and excessive proliferative properties of endometriotic cells are considered to be involved in the development and progression of endometriosis. STUDY DESIGN AND SIZE DURATION: ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometriotic tissues and eutopic endometrial tissues, respectively and compared. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Aberrantly expressed miRNAs in ECSCs were identified by a global miRNA microarray technique. The roles of miR-196b in ECSC proliferation, apoptosis, and c-myc and B-cell lymphoma/leukemia (Bcl)-2 mRNA expression were investigated with precursor hsa-miR-196b transfection. The methylation status of the miR-196b gene in ECSCs and the effect of a DNA demethylating agent on miR-196b expression were also examined. MAIN RESULTS AND THE ROLE OF CHANCE: miRNA microarray analysis identified eight down-regulated miRNAs (including miR-196b) and four up-regulated miRNAs in ECSCs. Compulsory expression of miR-196b directed the inhibition of proliferation and the induction of apoptosis in ECSCs. miR-196b was found to suppress c-myc and Bcl-2 mRNA expression in ECSCs, and there was a significant correlation between miR-196b and HOXA10 expression in ECSCs and NESCs. The miR-196b gene was hypermethylated in ECSCs when compared with NESCs, and the treatment with a DNA demethylating agent restored the expression of miR-196b in ECSCs. LIMITATIONS AND REASONS FOR CAUTION: miRNA expression profiles were investigated only in the stromal component of ectopic and eutopic endometrium samples. In addition to miR-196b, the roles of other miRNAs aberrantly expressed in ECSCs should be examined. WIDER IMPLICATIONS OF THE FINDINGS: The present findings suggest that aberrant miRNA expression plays an important role in the pathogenesis of endometriosis as a part of epigenetic mechanisms, that expression of miR-196b in ECSCs is repressed by DNA hypermethylation of the miR-196b gene and this repression may be involved in the development of proliferative and anti-apoptotic characteristics of endometriosis. STUDY FUNDING: This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 20591920 to K.N. and no. 23592407 to H.N.) and The Uehara Memorial Foundation (to K.N.).
[Show abstract][Hide abstract] ABSTRACT: We designed a study to investigate genetic relationships between primary tumors of oral squamous cell carcinoma (OSCC) and their lymph node metastases, and to identify genomic copy number aberrations (CNAs) related to lymph node metastasis. For this purpose, we collected a total of 42 tumor samples from 25 patients and analyzed their genomic profiles by array-based comparative genomic hybridization. We then compared the genetic profiles of metastatic primary tumors (MPTs) with their paired lymph node metastases (LNMs), and also those of LNMs with non-metastatic primary tumors (NMPTs). Firstly, we found that although there were some distinctive differences in the patterns of genomic profiles between MPTs and their paired LNMs, the paired samples shared similar genomic aberration patterns in each case. Unsupervised hierarchical clustering analysis grouped together 12 of the 15 MPT-LNM pairs. Furthermore, similarity scores between paired samples were significantly higher than those between non-paired samples. These results suggested that MPTs and their paired LNMs are composed predominantly of genetically clonal tumor cells, while minor populations with different CNAs may also exist in metastatic OSCCs. Secondly, to identify CNAs related to lymph node metastasis, we compared CNAs between grouped samples of MPTs and LNMs, but were unable to find any CNAs that were more common in LNMs. Finally, we hypothesized that subpopulations carrying metastasis-related CNAs might be present in both the MPT and LNM. Accordingly, we compared CNAs between NMPTs and LNMs, and found that gains of 7p, 8q and 17q were more common in the latter than in the former, suggesting that these CNAs may be involved in lymph node metastasis of OSCC. In conclusion, our data suggest that in OSCCs showing metastasis, the primary and metastatic tumors share similar genomic profiles, and that cells in the primary tumor may tend to metastasize after acquiring metastasis-associated CNAs.
PLoS ONE 01/2013; 8(2):e56165. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clinical outcome of patients with high-grade ccRCC (clear cell renal cell carcinoma) remains still poor despite recent advances in treatment strategies. Molecular mechanism of pathogenesis in developing high-grade ccRCC must be clarified. In the present study, we found that SAV1 was significantly downregulated with copy number loss in high-grade ccRCCs. Therefore, we investigated the SAV1 function on cell proliferation and apoptosis in vitro. Furthermore, we attempted to clarify the downstream signaling which is regulated by SAV1.
We performed array CGH and gene expression analysis of 8 RCC cell lines (786-O, 769-P, KMRC-1, KMRC-2, KMRC-3, KMRC-20, TUHR4TKB, and Caki-2), and expression level of mRNA was confirmed by quantitative RT-PCR (qRT-PCR) analysis. We next re-expressed SAV1 in 786-O cells, and analyzed its colony-forming activity. Then, we transfected siRNAs of SAV1 into the kidney epithelial cell line HK2 and renal proximal tubule epithelial cells (RPTECs), and analyzed their proliferation and apoptosis. Furthermore, the activity of YAP1, which is a downstream molecule of SAV1, was evaluated by western blot analysis, reporter assay and immunohistochemical analysis.
We found that SAV1, a component of the Hippo pathway, is frequently downregulated in high-grade ccRCC. SAV1 is located on chromosome 14q22.1, where copy number loss had been observed in 7 of 12 high-grade ccRCCs in our previous study, suggesting that gene copy number loss is responsible for the downregulation of SAV1. Colony-forming activity by 786-O cells, which show homozygous loss of SAV1, was significantly reduced when SAV1 was re-introduced exogenously. Knockdown of SAV1 promoted proliferation of HK2 and RPTEC. Although the phosphorylation level of YAP1 was low in 786-O cells, it was elevated in SAV1-transduced 786-O cells. Furthermore, the transcriptional activity of the YAP1 and TEAD3 complex was inhibited in SAV1-transduced 786-O cells. Immunohistochemistry frequently demonstrated nuclear localization of YAP1 in ccRCC cases with SAV1 downregulation, and it was preferentially detected in high-grade ccRCC.
Taken together, downregulation of SAV1 and the consequent YAP1 activation are involved in the pathogenesis of high-grade ccRCC. It is an attractive hypothesis that Hippo signaling could be candidates for new therapeutic target.
[Show abstract][Hide abstract] ABSTRACT: The purpose of the present study was to determine the genomic profile of renal cell carcinoma (RCC) in end-stage renal disease (ESRD) by analyzing genomic copy number aberrations. Seventy-nine tumor samples from 63 patients with RCC-ESRD were analyzed by array comparative genomic hybridization using the Agilent Whole Human Genome 4 × 44K Oligo Micro Array (Agilent Technologies Inc., Palo Alto, CA, USA). Unsupervised hierarchical clustering analysis revealed that the 63 cases could be divided into two groups, Clusters A and B. Cluster A was comprised mainly of clear cell RCC (CCRCC), whereas Cluster B was comprised mainly of papillary RCC (PRCC), acquired cystic disease (ACD)-associated RCC, and clear cell papillary RCC. Analysis of the averaged frequencies revealed that the genomic profiles of Clusters A and B resembled those of sporadic CCRCC and sporadic PRCC, respectively. Although it has been proposed on the basis of histopathology that ACD-associated RCC, clear cell papillary RCC and PRCC-ESRD are distinct subtypes, the present data reveal that the genomic profiles of these types, categorized as Cluster B, resemble one another. Furthermore, the genomic profiles of PRCC, ACD-associated RCC and clear cell papillary RCC admixed in one tissue tended to resemble one another. On the basis of genomic profiling of RCC-ESRD, we conclude that the molecular pathogenesis of CCRCC-ESRD resembles that of sporadic CCRCC. Although various histologic subtypes of non-clear cell RCC-ESRD have been proposed, their genomic profiles resemble those of sporadic PRCC, suggesting that the molecular pathogenesis of non-CCRCC-ESRD may be related to that of sporadic PRCC.
Cancer Science 12/2011; 103(3):569-76. · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Pathology 01/2011; 224(2):280-8. · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genomic copy number aberrations (CNAs) in gastric cancer have already been extensively characterized by array comparative genomic hybridization (array CGH) analysis. However, involvement of genomic CNAs in the process of submucosal invasion and lymph node metastasis in early gastric cancer is still poorly understood. In this study, to address this issue, we collected a total of 59 tumor samples from 27 patients with submucosal-invasive gastric cancers (SMGC), analyzed their genomic profiles by array CGH, and compared them between paired samples of mucosal (MU) and submucosal (SM) invasion (23 pairs), and SM invasion and lymph node (LN) metastasis (9 pairs). Initially, we hypothesized that acquisition of specific CNA(s) is important for these processes. However, we observed no significant difference in the number of genomic CNAs between paired MU and SM, and between paired SM and LN. Furthermore, we were unable to find any CNAs specifically associated with SM invasion or LN metastasis. Among the 23 cases analyzed, 15 had some similar pattern of genomic profiling between SM and MU. Interestingly, 13 of the 15 cases also showed some differences in genomic profiles. These results suggest that the majority of SMGCs are composed of heterogeneous subpopulations derived from the same clonal origin. Comparison of genomic CNAs between SMGCs with and without LN metastasis revealed that gain of 11q13, 11q14, 11q22, 14q32 and amplification of 17q21 were more frequent in metastatic SMGCs, suggesting that these CNAs are related to LN metastasis of early gastric cancer. In conclusion, our data suggest that generation of genetically distinct subclones, rather than acquisition of specific CNA at MU, is integral to the process of submucosal invasion, and that subclones that acquire gain of 11q13, 11q14, 11q22, 14q32 or amplification of 17q21 are likely to become metastatic.
PLoS ONE 01/2011; 6(7):e22313. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated expression profiles of microRNA (miRNA) in gastric carcinomas by use of a miRNA microarray platform covering a total of 470 human miRNAs. We identified 39 differentially expressed miRNAs in gastric carcinoma, of which six were significantly downregulated and the other 33 were upregulated. We found that miRNA-375 (miR-375) was the most downregulated and that its ectopic expression in gastric carcinoma cells markedly reduced cell viability via the caspase-mediated apoptosis pathway. Interestingly, we found that expression of miR-375 inhibited expression of PDK1, which is a direct target of miR-375, followed by suppression of Akt phosphorylation. Further analysis by gene expression microarray revealed that 14-3-3zeta, a potent antiapoptotic gene, was significantly downregulated at both the mRNA and protein levels in cells transfected with miR-375. The activity of a luciferase reporter containing the miR-375 binding sequence at the 3' untranslated region (UTR) of 14-3-3zeta mRNA was repressed by the ectopic expression of miR-375, suggesting that miR-375 targets the 3' UTR of 14-3-3zeta. In addition, knockdown of either PDK1 or 14-3-3zeta in gastric carcinoma cells induced caspase activation, which was also observed in miR-375-transfected cells, suggesting that miR-375 may exert its proapoptotic function, at least in part, through the downregulation of PDK1 and 14-3-3zeta. Taken together, we propose that miR-375 is a candidate tumor suppressor miRNA in gastric carcinoma.
Cancer Research 03/2010; 70(6):2339-49. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although genomic copy number aberrations (CNAs) of gastric carcinoma at the advanced stage have already been extensively characterized by array comparative genomic hybridization (array CGH) analysis, those of gastric carcinoma in situ (CIS) are still poorly understood. Furthermore, no reports have demonstrated correlations between CNAs and histopathological features of gastric adenoma. In this study, we investigated CNAs of 20 gastric CISs (Vienna category 4.2) and 20 adenomas including seven low-grade adenomas (LGA; Vienna category 3) and 13 high-grade adenomas (HGA; Vienna category 4.1), using oligonucleotide-based array CGH. The most frequent aberrations in CIS were gains at 8q (85%) and 20q (50%), and losses at 5q (50%) and 17p (50%), suggesting that these CNAs are involved in the development of CIS. We found that the pattern of CNAs in HGA was quite different from that in LGA. The most frequent CNAs in HGA were gains at 8q (62%) and 7pq (54%), whereas those in LGA were gain at 7q21.3-q22.1 (57%) and loss at 5q (43%). Interestingly, gains at 8q and 7pq, both of which occurred most frequently in HGA, were not detected in any cases of LGA. Of note, 8q gain was detected most frequently in both HGA and CIS but was undetected in LGA. Since HGA is believed to have a higher risk of progression to invasive carcinoma than LGA, these data suggest that 8q gain is important for the malignant transformation of gastric adenoma.
The Journal of Pathology 01/2010; 221(1):96-105. · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
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.
[Show abstract][Hide abstract] ABSTRACT: This study aimed to determine whether epidermal growth factor receptor (EGFR), which has been reported to be frequently overexpressed in esophageal carcinoma cells, is actually activated in the cells. Paraffin-embedded specimens of 39 cases of esophageal carcinoma were analyzed immunohistochemically with anti-EGFR polyclonal antibody (alpha-EGFR Ab) and also an anti-phospho-EGFR-specific polyclonal antibody (alpha-p-EGFR(Tyr992) Ab) that specifically recognizes phosphorylated tyrosine 992 of EGFR. All of the 39 cases were found to express EGFR, but the expression levels were not significantly higher than those in basal cells of the normal esophageal epithelium. In 38 of the 39 cases, alpha-p-EGFR(Tyr992) immunoreactivity was evident. Interestingly, the positively stained carcinoma cells were not distributed diffusely, and strongly immunostained cells tended to be localized in areas of severe dysplasia and in microinvasive foci just adjacent to the main invasive carcinoma. However, the deeply invasive front never exhibited positive immunoreactivity. The present findings suggest that phosphorylation of EGFR( Tyr992) may play some specific functional role in esophageal carcinomas besides promotion of cell proliferation.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Pathology 08/2008; 216(4):418-27. · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Pathology 08/2008; 216(4):471-82. · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human runt-related transcription factor 3 gene (RUNX3) is considered to be a candidate tumor suppressor gene in gastric carcinoma. However, the role of RUNX3 in the regulation of cell proliferation remains unclear. In the present study, we constructed an adenoviral vector encoding human RUNX3 cDNA under the control of a Tet-responsive promoter (Ad-Tet-FLAG-RUNX3), which regulates the expression of RUNX3 in the presence or absence of doxycycline. A recombinant adenoviral expression vector encoding LacZ (Ad-Tet-LacZ) was used as a negative control. The effect of the transduction of RUNX3 on cell growth was examined using the Tet-On system in a human gastric carcinoma cell line, MKN-1. Exogenous RUNX3 expression was induced successfully by Ad-Tet-FLAG-RUNX3, but not Ad-Tet-LacZ, in the presence of doxycycline in the MKN-1 cells. At 72 h after infection, the proliferative activity in RUNX3-expressing cells was 55% or less of that of the control cells. Flow cytometry revealed that the sub-G(1) peak was increased in cells expressing RUNX3 (34.11%), indicating that the inhibition of cell growth was due to apoptosis, which was confirmed based on Hoechst 33258 staining, the release of cytochrome c from mitochondria into the cytosol, and detection of cleaved caspase-3 by western blotting in MKN-1 cells. Comprehensive analysis using a cDNA microarray showed that RUNX3 upregulated 17 apoptosis-related genes (including FADD, TRAF6, caspase-2, ING1, ING4, Calpain 10, and DNase1) and downregulated 135 apoptosis-related genes (including FLIP, PEA15, TXN2, HSPD1, IKK, and TIAL1) in MKN-1 cells. Pathway analyses to generate functional networks of the genes suggested that promotion of the formation of the death-inducing signaling complex and activation of the mitochondria-mediated pathway were associated with RUNX3-induced apoptosis. In conclusion, our findings suggest that exogenous RUNX3 expression suppressed cell proliferation by inducing apoptosis via the death-receptor mitochondria-mediated pathway in MKN-1 cells.
Cancer Science 02/2008; 99(1):23-30. · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Pathology 01/2008; 213(4):392-401. · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human runt-related transcription factor gene 3 (RUNX3) is considered as a possible candidate of tumour suppressor gene in human gastric carcinoma.
To investigate the RUNX3 protein expression in human gastric mucosa and carcinoma, we generated a polyclonal antibody, AS251, which recognized amino acid sequences from 251 to 266 of human RUNX3. The AS251 antibody was immunoreactive with only RUNX3 protein, but not with RUNX1 and RUNX2. The AS251-antibody was available for Western blotting and immunohistochemistry using paraffin-embedded specimens.
Western-blot analysis revealed that three (MKN-1, -7 and -45) of six human gastric carcinoma cell lines variably expressed RUNX3 protein, consistent with the expression pattern of RUNX3 mRNA reported previously by Li et al. (Cell 2002;109:113-24). Immunohistochemistry disclosed RUNX3 protein in most chief cells and a few gastrin-containing G cells in normal mucosa, but not in intestinal metaplasia and carcinoma cells.
These data suggest that RUNX3 may play a physiologic role in chief cells and G cells in gastric mucosa, and that suppression of RUNX3 expression in intestinal metaplasia and carcinoma of human stomach may be implicated in gastric carcinogenesis.
European Journal of Clinical Investigation 10/2004; 34(9):605-12. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ankyrin-repeated protein with PEST and a proline-rich region (ARPP) is a recently identified protein with 4 ankyrin-repeated motifs in its central portion. Type 1 myofibers of skeletal muscle express high levels of ARPP. Recently, we have found that ARPP expression was induced in mouse denervated skeletal muscle. This led us to hypothesize that ARPP expression might be induced in skeletal muscle under some pathological conditions.
In this study, we performed immunohistochemical analysis of ARPP expression in biopsy specimens of muscle tissue from 15 patients with muscular dystrophies (MDs), 13 with congenital myopathies and 11 with spinal muscular atrophies (SMAs).
The ARPP expression levels of all the specimens from MD patients appeared to be lower than control muscle levels. In contrast, the specimens from the 13 patients with congenital myopathies were all ARPP positive. We also found increased numbers of ARPP-positive myofibers in patients with congenital myopathies, and these myofibers co-expressed the slow myosin heavy chain. Indeed, it has been reported that type 1 myofibers are predominant in patients with congenital myopathies, suggesting that increased numbers of ARPP-positive myofibers in such patients may be associated with increased numbers of type 1 fibers. In patients with SMAs, we found that ARPP-positive myofibers tended to be distributed in groups. As grouped myofibers have been reported to result from the process of denervation, innervation and subsequent denervation of re-innervated myofibers, the grouped ARPP-positive myofibers in SMA patients may result from denervation of the motor units.
These findings suggest that evaluation of ARPP may be helpful for the histological diagnosis of muscle diseases.