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Issei Imoto

Publications and source records attributed to Issei Imoto.

At least 37 records · Page 2Linked to original sources
PubMed2005

Overexpressed Skp2 within 5p amplification detected by array-based comparative genomic hybridization is associated with poor prognosis of glioblastomas.

To better understand the pathogenesis of glioblastoma multiforme (GBM) and to increase the accuracy of predicting outcomes for patients with this disease, we performed genome-wide screening for DNA copy-number aberrations in 22 glioma-derived cell lines using a custom-made comparative genomic hybridization-array. Copy-number gains were frequently detected at 3q, 7p, 7q, 20q, Xp and Xq, and losses at 4q, 9p, 10p, 10q, 11q, 13q, 14q, 18q, and 22q. Among several non-random chromosomal aberrations, the gain/amplification of DNA at 5p, which has never been reported before in GBM, was detected with a relatively high ratio (log2 ratio = 0.41-1.19) in four cell lines. Amplification and subsequent overexpression of SKP2, a possible target of amplification within 5p, were detected in four of the 22 cell lines. We also investigated the expression of the gene product in primary GBM by immunohistochemistry, which revealed increased levels of Skp2 in 11 of the 35 tumors examined (31.4%). Heightened expression of Skp2 was associated with shorter overall survival (P = 0.001, logrank test), especially in patients younger than 65 years. These results suggest that overexpression of Skp2 through gene amplification may contribute to the pathogenesis of GBM, and that overabundance of the protein might be a useful prognostic tool in patients with this disease.

Biomarkers, Tumor↗
PubMed2005

[Exploration of novel molecular targets for cancer therapy based on genomic aberrations].

Tumors develop through the combined processes of genetic instability and selection, resulting in clonal expansion of cells that have accumulated the most advantageous set of genetic aberrations. Many types of instability may occur, resulting in point mutations, chromosomal rearrangements, DNA dosage abnormalities, and epigenetic changes such as methylation. In order to identify novel molecular targets for cancer therapy, we have focused on genomic copy-number aberrations as landmarks. Recent advances in array-based CGH technology (CGH-array) have enabled examination of chromosomal regions in unprecedented detail, prompting us to screen cancer-related genes including candidates of molecular targets. Indeed, applying our in-house CGH-array, we have identified several amplification target oncogenes as well as tumor suppressor genes. Useful molecular targets for cancer therapy will be identified more rapidly through exploring genomic and/or epigenomic abnormalities using genomic arrays including CGH-array system.

Chromosome Aberrations↗
PubMed2004

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines.

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.

Blotting, Western↗
PubMed2004

Translocation (1;22)(p36;q11.2) with concurrent del(22)(q11.2) resulted in homozygous deletion of SNF5/INI1 in a newly established cell line derived from extrarenal rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a highly malignant pediatric cancer, which arises in various sites such as the kidney, brain, and soft tissues. Cytogenetic studies have revealed alterations of 22q11 in MRT. Recently, deletions and mutations of the SNF5/INI1 locus in 22q11.2 have been reported in MRT, suggesting that SNF5/INI1 is a tumor suppressor gene for MRT. Here we report our molecular cytogenetic study for a newly established cell line from extrarenal MRT with t(1;22)(p36;q11.2). Consequently, the reciprocal translocation was associated with the interstitial deletion of a small segment including SNF5/INI1, and another, chromosome 22, showed terminal deletion, the breakpoint of which was located 70-80 kb centromeric to SNF5/INI1, resulting in homozygous deletion of SNF5/INI1 in this cell line.

Base Sequence↗
PubMed2004

Frequent silencing of low density lipoprotein receptor-related protein 1B (LRP1B) expression by genetic and epigenetic mechanisms in esophageal squamous cell carcinoma.

Low-density lipoprotein receptor-related protein 1B (LRP1B) is frequently deleted in tumors of various types, but its status and expression in esophageal squamous cell carcinomas (ESCs) have never been reported. In the course of a program to screen ESC cell lines for copy-number aberrations using array-based comparative genomic hybridization, we identified a homozygous deletion of LRP1B. Genomic PCR experiments revealed homozygous deletions of LRP1B in additional ESC cell lines (total, 6 of 43; 14.0%) and in primary esophageal tumors (30 of 70; 42.9%). Moreover, expression of LRP1B mRNA was frequently silenced in ESC lines without homozygous deletions (14 of 37; 37.8%). Using bisulfite-PCR analysis and sequencing, we found that LRP1B-nonexpressing cells without homozygous deletions were highly methylated at a CpG island of LRP1B, a sequence possessing promoter activity. Treatment with 5-aza-2'-deoxycytidine restored expression of LRP1B in those ESC lines. Histone acetylation status correlated directly with expression of LRP1B and inversely with the methylation status of the CpG island. Methylation of LRP1B was also detected in primary esophageal tumors. Restoration of LRP1B expression in ESC cells reduced colony formation. These results suggest that loss of LRP1B function in esophageal carcinogenesis most often occurs either by homozygous deletion or by transcriptional silencing through hypermethylation of its CpG island.

Acetylation↗
PubMed2004

Alteration in copy numbers of genes as a mechanism for acquired drug resistance.

Chemoresistance is a major obstacle for successful treatment of cancer. To identify regions of the genome associated with acquired resistance to therapeutic drugs, we conducted molecular cytogenetic analyses of 23 cancer-cell lines, each resistant to either camptothecin, cisplatin, etoposide (VP-16), Adriamycin, or 1-beta-D-arabinofuranosylcytosine, although the parental tumor lines were not. Subtractive comparative genomic hybridization studies revealed regions of gain or loss in DNA-copy numbers that were characteristic of drug-resistant cell lines; i.e., differences from their drug-sensitive parental cell lines. Thirteen ATP-binding cassette (ABC) transporter genes [ABCA3, ABCB1 (MDR1), ABCB6, ABCB8, ABCB10, ABCB11, ABCC1 (MRP1), ABCC4, ABCC9, ABCD3, ABCD4, ABCE1, and ABCF2] were amplified among 19 of the resistant cell lines examined. Three genes encoding antiapoptotic BCL-2 proteins (BCL2L2, MCL1, and BCL2L10) were also amplified and consequently overexpressed in three of the derivative lines. Down-regulation of BCL2L2 with an antisense oligonucleotide sensitized a VP-16 resistant ovarian-cancer cell line (SKOV3/VP) to VP-16. A decrease in copy numbers of genes encoding deoxycytidine kinase, DNA topoisomerase I, and DNA topoisomerase II alpha reduced their expression levels in one cytosine arabinoside-resistant line, two of three camptothecin-resistant lines, and two of five VP-16-resistant cell lines, respectively. Our results indicated that changes in DNA-copy numbers of the genes mentioned can activate or down-regulate them in drug-resistant cell lines, and that such genomic alterations might be implicated in acquired chemoresistance.

ATP-Binding Cassette Transporters↗
PubMed2004

Overexpression of PDZK1 within the 1q12-q22 amplicon is likely to be associated with drug-resistance phenotype in multiple myeloma.

We investigated DNA copy number aberrations in 37 cell lines derived from multiple myelomas (MMs) using comparative genomic hybridization, and 11 (29.7%) showed high-level gain indicative of gene amplification at 1q12-q22. A corresponding transcriptional mapping using oligonucleotide arrays extracted three up-regulated genes (IRTA2, PDZK1, and S100A6) within the smallest region of overlapping in amplifications. Among them PDZK1 showed amplification and consequent overexpression in the MM cell lines. Amplification of PDZK1 was observed in primary cases of MM as well. MM cell lines with amplification of PDZK1 exhibited the resistance to melphalan-, cis-platin-, and vincristin-induced cell death compared with MM cell lines without its amplifications. Furthermore, down-regulation of PDZK1 with an anti-sense oligonucleotide sensitized a cell line KMS-11 to melphalan, cis-platin, and vincristin. Taken together, our results indicate that PDZK1 is likely to be one of targets for 1q12-q22 amplification in MM and may be associated with the malignant phenotype, including drug resistance, in this type of tumor.

Antineoplastic Agents↗
PubMed2004

Comparative genomic hybridization (CGH)-arrays pave the way for identification of novel cancer-related genes.

Comparative genomic hybridization (CGH) has already made a significant impact on cancer cytogenetics. However, CGH to metaphase chromosomes can provide only limited resolution at the 5-10 Mb level. To circumvent this limitation, array-based CGH has been devised. Since spotted DNAs in a CGH-array contain sequence information directly connected with the genome database, we can easily note particular biological aspects of genes that lie within regions involved in copy-number aberrations. High-density, sub-megabase arrays can reveal nonrandom chromosome copy-number aberrations responsible for neoplastic transformation that have been masked under complex karyotypes in epithelial solid tumors. High-density CGH-array therefore paves the way for identification of disease-related genetic aberrations that have not yet been detected by existing technologies, and array-based CGH technology should soon be practical for diagnosis of cancer or genetic diseases in the clinical setting.

Chromosome Aberrations↗
PubMed2004

Skp2 overexpression is a p27Kip1-independent predictor of poor prognosis in patients with biliary tract cancers.

To better understand the pathogenesis of biliary tract carcinoma (BTC) and to increase the accuracy of predicting outcomes for patients with this disease, we performed molecular cytogenetic analyses of BTC cell lines and tumors to identify non-random amplification(s) and target gene(s) within the amplicons. Among several non random chromosomal aberrations detected in BTC cell lines by comparative genomic hybridization, gain/ampli-fication of DNA at 5p was the most frequently observed alteration. We assessed the copy number and expression status of the possible target gene SKP2 for 5p amplification in cell lines and 33 primary stage II or III tumors of BTC. SKP2 was amplified, and subsequently overexpressed in both cell lines and primary tumors of BTC. However, levels of Skp2 and p27Kip1 proteins were not correlated inversely. Heightened expression of Skp2 and reduced expression of p27Kip1 were both associated with a shorter disease-free and/or overall survival in univariate analyses. In multivariate regression analyses, Skp2 and p27Kip1 were independent predictive factors. Those results suggest that (a) overexpression of Skp2 through an amplification mechanism may contribute to the progression of BTC, (b) not only each molecule, but also the combination of Skp2 and p27Kip1, might be a useful predictor of the prognosis of BTC, and (c) molecular targets of Skp2 other than p27Kip1 may also be important factors in the pathogenesis of this disease.

Aged↗
PubMed2004

Identification of a novel fusion gene in a pre-B acute lymphoblastic leukemia with t(1;19)(q23;p13).

The most common nonrandom translocation found among childhood pre-B acute lymphoblastic leukemias (ALL) is t(1;19)(q23;p13), which frequently results in fusion of E2A with PBX1. However, rare cases of childhood ALL and various other hematological diseases with t(1;19) lack the E2A-PBX1 fusion. Analyzing a cell line with pre-B-cell phenotype, TS-2, that carries t(1;19)(q23;p13) but lacks the E2A-PBX1 fusion, we successfully cloned the breakpoints, which fell within introns of MEF2D and DAZAP1. Both chimeric transcripts, MEF2D-DAZAP1 and DAZAP1-MEF2D, whose sequences indicated in-frame fusions between MEF2D and DAZAP1, were expressed in TS-2 cells and in bone-marrow cells of the patient from whom the TS-2 was established. MEF2D-DAZAP1 and DAZAP1-MEF2D proteins were both located in the nucleus, and MEF2D-DAZAP1 was able to form dimers with MEF2D and HDAC4. In addition, exogenous expression of MEF2D-DAZAP1 and DAZAP1-MEF2D promoted the growth of HeLa cells. Given the frequency of t(1;19) without the E2A-PBX1 fusion in hematological malignancies, we suggest that MEF2D / DAZAP1 rearrangements might be involved in the pathogenesis of those diseases.

Child, Preschool↗
PubMed2003

Unfavorable prognostic factors associated with high frequency of microsatellite instability and comparative genomic hybridization analysis in endometrial cancer.

PURPOSE: Although many articles have been published regarding chromosomal instability (CI) and microsatellite instability (MI) in endometrial adenocarcinoma, the relationship between prognostic factors and the biological mechanisms accounting for genetic instability in these tumors has not yet been precisely defined. To do that, it will be necessary to clarify the molecular mechanisms involved in endometrial carcinogenesis. EXPERIMENTAL DESIGN: Tissue samples from 43 human primary endometrioid endometrial adenocarcinomas (EACs) were analyzed for CI and MI status using comparative genomic hybridization and 11 microsatellite loci, respectively. Methylation status of the promoter of MLH1 was also determined. We analyzed all three of these parameters in relation to each other and to clinicopathological factors. RESULTS: Sixty-five percent of the EACs we examined had detectable CI. Frequent copy number gains were seen at 1q25-41 (23%), 8q11.1-q21.1 (23%), 8q21.3-qter (21%); 28% of these tumors exhibited high-frequency MI (MSI-H); Methylation of the MLH1 promoter was observed in 92% of EACs with MSI-H. Southern blotting showed amplification of MYCN in one tumor, which has been documented for the first time in a primary human EAC. CONCLUSIONS: MSI-H was correlated with histological grade, International Federation of Gynecologists and Obstetricians (FIGO) stage, myometrial invasion, and lymphonode metastasis. Our comparative genomic hybridization results demonstrated that the number of chromosomes involved in genomic alterations in EACs was distinctively fewer than those in other types of tumor. The carcinogenetic process leading to EAC appears to be highly complex; for example, MI and CI may act synergistically, whereas CI and/or MI are likely to be linked with tumor heterogeneity.

Adaptor Proteins, Signal Transducing↗
PubMed2003

TERC identified as a probable target within the 3q26 amplicon that is detected frequently in non-small cell lung cancers.

PURPOSE: Cell lines derived from non-small cell lung cancers (NSCLCs) revealed frequent high-level gains of chromosomal DNA at 3q23-q29 when examined by comparative genomic hybridization (CGH). Within this amplicon, a minimal common region of amplification in lung tumors had been mapped to 3q26 by earlier studies. The aim of the present work was to identify specific targets of the 3q26 amplification in NSCLCs. EXPERIMENTAL DESIGN: We examined genomic alterations in 19 NSCLC cell lines (10 derived from squamous cell carcinomas and 9 from adenocarcinomas) by using CGH and fluorescence in situ hybridization. We determined amplification and expression levels of four candidates (EVI1, TERC, SNO, and PIK3CA) in those cell lines and also in 25 primary NSCLC tumors. Because the TERC gene encodes the RNA component of human telomerase, we examined telomerase activity by the telomeric repeat amplification protocol (TRAP) assay. RESULTS: Copy numbers of EVI1 and TERC increased more than those of SNO and PIK3CA in our panel of NSCLC cell lines. Significant correlation between amplification and expression levels was observed only for TERC (P = 0.006), however, among these four candidate genes. Expression of TERC was also up-regulated in 24 (96%) of 25 primary NSCLC tumors compared with their nontumorous counterparts (P = 0.0001), and elevated expression of TERC was associated with high levels of telomerase activity (P = 0.048). CONCLUSIONS: TERC is a likely target of the 3q26 amplification, and, therefore, may be a useful biomarker for diagnosis and an attractive, novel target for molecular therapy of NSCLC.

Blotting, Northern↗
PubMed2003

Identification of ZASC1 encoding a Krüppel-like zinc finger protein as a novel target for 3q26 amplification in esophageal squamous cell carcinomas.

Among the transcription factors that direct proliferation, differentiation, and death of cells, several Krüppel-like zinc finger molecules such as GLI1 and ZNF147 can become oncogenic when the genes encoding them are overexpressed by the DNA amplification mechanism. Here, we report the discovery of a novel member of this class, ZASC1, from a recently reported critical region of 3q26 amplification frequently observed in various squamous cell carcinomas, including esophageal squamous cell carcinomas (ESCs). The deduced 485-amino acid protein product of ZASC1 contained a putative nuclear localization signal; the exogenously transfected ZASC1 was translocated into cell nuclei. ZASC1 was frequently coamplified and subsequently overexpressed with PIK3CA in our panel of ESC cell lines and primary tumors. In patients with ESC, a higher mRNA expression level of ZASC1 appeared to be associated with shorter overall survival, and a multivariate analysis demonstrated that ZASC1 mRNA expression was an independent prognosticator. In addition, exogenous expression of ZASC1 promoted the growth of ESC cells, whereas down-regulation of ZASC1 expression by means of an antisense oligonucleotide suppressed the growth of ESC cells. Taken together, our results suggest that ZASC1 might be involved in the pathogenesis of ESC as one of the targets for 3q26 amplification, alone or in concert with other targets.

Aged↗
PubMed2003

GPC5 is a possible target for the 13q31-q32 amplification detected in lymphoma cell lines.

Comparative genomic hybridization (CGH) analyses have detected gains of copy number on 13q, especially at 13q31-q32, in cell lines and primary cases of various types of lymphoma. Since amplification of chromosomal DNA is one of the mechanisms that can activate tumor-associated genes, and because 13q amplification had been reported in various other types of tumors as well, we attempted to define by fluorescence in situ hybridization (FISH) a common region at 13q31-q32 in which to explore genes that might be targets for the amplification events. Although the commonly amplified region we defined was relatively large (approximately 4 Mb), only one true gene, GPC5, was found there. GPC5 was over-expressed in lymphoma cell lines that had shown amplification, in comparison with those that had not. Our findings suggest that GPC5 is a likely target for amplification, and that over-expression of this gene may contribute to development and/or progression of lymphomas and other tumors.

Cell Line, Tumor↗
PubMed2003

PPM1D is a potential target for 17q gain in neuroblastoma.

Neuroblastomas (NBs) show complex patterns of genetic abnormalities, which may include amplification of the MYCN gene, deletion of 1p, or a gain of DNA at 17q, the last being the most frequent observation in NB tumors. However, the specific genes and the molecular mechanisms responsible for development and progression of NB remain poorly understood. We investigated aberrations of DNA copy number in 25 NB cell lines using comparative genomic hybridization and identified a minimal common region of gain at 17q23. Although gain of distal 17q is the most powerful genetic predictor of adverse outcome currently available for patients with NB, thus far, no potential target genes have been reported for that region. Therefore, we defined the 17q23 amplicon in detail and determined expression levels of 15 genes located within the smallest region of overlap observed among our NB cell lines to identify the most likely target gene(s). Among them, seven (CLTC, VMP1, delta-tubulin, RPS6KB1, FLJ22087, APPBP2, and PPM1D) were consistently overexpressed through increases in regional copy number. Analysis of expression levels of those seven genes in 32 primary NB tumors revealed a significant correlation between higher expression and poorer clinical outcome only with respect to PPM1D. Moreover, down-regulation of PPM1D by transfection of an antisense oligonucleotide suppressed the growth of NB cell lines to a remarkable degree, at least partly by participating in a process leading to apoptotic cell death. Taken together, our results indicate that PPM1D is the most likely target of the 17q23 gain/amplification in NB tumors and may have an important role in the pathogenesis of this disease.

Cell Death↗
PubMed2003

Physical map and haplotype analysis of 16q-linked autosomal dominant cerebellar ataxia (ADCA) type III in Japan.

Autosomal dominant cerebellar ataxia (ADCA) is a group of heterogeneous neurodegenerative disorders. We previously mapped a gene locus for ADCA with pure cerebellar syndrome (ADCA type III) to a 3-cM region in chromosome 16q, and found a common haplotype among affected individuals. This region was exactly within the locus for another ADCA, spinocerebellar ataxia type 4 (SCA4). To identify the gene causing 16q-linked ADCA type III, we constructed a contig with 38 bacterial artificial chromosome clones between D16S3043 and D16S3095. The size of this contig was estimated to be 4.8 Mb. We found more than 500 nucleotide tandem repeats, including 9 CAG/CTG repeats in this candidate region, although none of the 94 tandem repeats analyzed were expanded in affected individuals. However, we found 11 new polymorphic markers, giving 22 markers spanning the candidate region. By typing these markers on eight Japanese families with ADCA type III, including two new families, we found that a common "founder" haplotype is seen in a more restricted 3.8-Mb region, spanning markers GGAA05 and D16S3095. We present here a newly refined critical interval of 16q-ADCA type III/SCA4. Data of 11 new DNA markers on 16q22.1 would also be useful for other research of genes mapped to this region.

Cerebellar Ataxia↗
PubMed2003

Cell culture in esophageal squamous cell carcinoma and the association with molecular markers.

PURPOSE: We reported previously that the patients in whom cancer cells could be cultured as continuous cell lines had a poor prognosis of esophageal squamous cell carcinoma (ESCC) patients. In this study, to evaluate additional evidence of prognostic significance and the genetic background of cell culture, we analyzed 203 ESCC patients. EXPERIMENTAL DESIGN: Culture samples were obtained from resected 203 primary ESCC (from 1986 to 1998; R0 resection). The expression of six molecular markers was evaluated retrospectively in resected primary esophageal tumors by immunohistochemical analysis, and the capability of establishing cell lines was compared. RESULTS: Thirty-five cell lines (17.2%) were established from 203 ESCC patients: group 1 (n = 35), from whom cancer cells could be cultured as continuous cell lines, and group 2 (n = 168), from whom cell lines could not be established. The cumulative survival rate of patients in group 1 was significantly lower than that of those in group 2 (P = 0.0006). Cox's proportional hazard model revealed that cell culture capability was an independent prognostic factor (risk ratio, 1.98; P = 0.007). Univariate logistic regression analysis revealed that cell culture capability had associations with the following molecular biological factors: cyclin D1, p53, murine double minute 2, p27, and fragile histidine triad gene (P < 0.05). However, multivariate logistic regression analysis revealed that p53 protein accumulation and MDM2 protein expression predict establishment of cell line in ESCC (odds ratio, 7.72 and 8.62, respectively). CONCLUSIONS: Cell culture capability is a significant prognostic factor in ESCC. p53 and MDM2 may have a crucial role in the establishment of ESCC cell lines.

Aged↗