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Mitsuo Oshimura

Publications and source records attributed to Mitsuo Oshimura.

At least 73 records · Page 4Linked to original sources
PubMed2003

Human chromosome 5 carries a putative telomerase repressor gene.

Telomerase, the ribonucleoprotein enzyme that maintains the telomere, is active in human germ and stem cells and in a majority of tumor tissues and immortalized cell lines. In contrast, telomerase activity is not detected in most somatic cells, suggesting that normal human cells contain a regulatory factor(s) to repress this activity. To identify which human chromosomes carry a gene or genes that function as telomerase repressors, we investigated telomerase activity in hybrids of the B16-F10 cell line, which contain individual human chromosomes transferred previously by microcell fusion and therefore represent a hybrid panel for the entire genome except for the Y chromosome. Microcell hybrids with an introduced normal human chromosome 5 showed inhibition of telomerase activity, but clones at a late passage exhibited reactivation of telomerase activity. Reactivation of telomerase activity was accompanied by deletion and/or rearrangement of the transferred human chromosome 5. The introduction of other human chromosomes did not significantly affect the telomerase activity of B16-F10 cells. The effect of suppression of telomerase activity in microcell hybrids containing chromosome 5 was accompanied by a reduction in the level of mTERT mRNA, which encodes a component of the telomerase complex. The putative telomerase repressor gene was mapped to human chromosome bands 5p11-p13 by a combination of functional analysis using transfer of subchromosomal transferable fragments of chromosome 5 into B16-F10 cells and deletion mapping of revertant clones with reactivated telomerase activity. Thus, these results suggest that loss of a gene(s) on this chromosome was responsible for telomerase reactivation, indicating that human chromosome 5 contains a gene or genes that can regulate the expression of mTERT in B16-F10 cells.

Animals↗
PubMed2003

A new imprinted cluster on the human chromosome 7q21-q31, identified by human-mouse monochromosomal hybrids.

We have previously established a series of human monochromosomal hybrids containing a single human chromosome of defined parental origin as an in vitro resource for the investigation of human imprinted loci. Using the hybrids with a paternal or maternal human chromosome 7, we determined the allelic expression profiles of 76 ESTs mapped to the human chromosome 7q21-q31. Seven genes/transcripts, including PEG10 which has previously been reported to be imprinted, showed parent-of-origin-specific expression in monochromosomal hybrids. One of the 6 candidate genes/transcripts, i.e., DLX5 was confirmed to be imprinted in normal human lymphoblasts and brain tissues by a polymorphic analysis. Thus, an imprinted domain has been newly defined in the region of human chromosome 7q21-q31 using human-mouse monochromosomal hybrids.

Animals↗
PubMed2003

Discovery of imprinted transcripts in the mouse transcriptome using large-scale expression profiling.

Candidate imprinted transcriptional units in the mouse genome were identified systematically from 27,663 FANTOM2 full-length mouse cDNA clones by expression profiling. Large-scale cDNA microarrays were used to detect differential expression dependent upon chromosomal parent of origin by comparing the mRNA levels in the total tissue of 9.5 dpc parthenogenote and androgenote mouse embryos. Of the FANTOM2 transcripts, 2114 were identified as candidates on the basis of the array data. Of these, 39 mapped to known imprinted regions of the mouse genome, 56 were considered as nonprotein-coding RNAs, and 159 were natural antisense transcripts. The imprinted expression of two transcripts located in the mouse chromosomal region syntenic to the human Prader-Willi syndrome region was confirmed experimentally. We further mapped all candidate imprinted transcripts to the mouse and human genome and were shown in correlation with the imprinting disease loci. These data provide a major resource for understanding the role of imprinting in mammalian inherited traits.

Animals↗
PubMed2003

Epigenetic regulation of ARHI in breast and ovarian cancer cells.

ARHI (Ras homologue member I) encodes a 26-kDa GTPase with 50-60% amino acid homology to Ras and Rap. ARHI and Ras share similar GTP/GDP binding domains, but exert opposite functions. ARHI is one of the first reported tumor suppressors in the ras superfamily. ARHI is expressed consistently in normal breast and ovarian epithelial cells, but not in breast or ovarian cancers. The loss of ARHI can be related to tumor progression. Reexpression of ARHI induces apoptosis of breast and ovarian cancer cells by a caspase-independent, calpain-dependent pathway. ARHI is consistently expressed in normal breast and ovarian epithelial cells but is dramatically downregulated in more then 70% of breast and ovarian cancers. ARHI is maternally imprinted with methylation of the three CpG islands in the maternal allele of normal cells. ARHI is expressed only from the paternal allele whose three CpG islands are not methylated. Loss of ARHI expression can occur through a genetic event, with loss of heterozygosity observed in 40% of breast, ovarian, and pancreatic cancers; but it can also occur through epigenetic mechanisms, including DNA methylation, histone deacetylation, histone methylation, and transcriptional regulation. Our data suggest that acetylation and methylation of chromatin associated with the ARHI promoter leads to loss of both ARHI expression and the ability to suppress tumor growth. Changes in chromatin that silence ARHI may be driven by methylation-dependent and -independent pathways. Reactivation of both the silenced paternal and imprinted maternal alleles can be achieved by demethylation and inhibition of histone deacetylation.

Acetylation↗
PubMed2003

Searching for genes involved in arteriosclerosis: proteomic analysis of cultured human umbilical vein endothelial cells undergoing replicative senescence.

It is known that replicative senescence of endothelium in vivo contributes at least partially to age-related vascular disorders such as arteriosclerosis. However, the genes involved in this process remain to be identified. In this study, we employed a proteomics-based approach to identify candidate genes using in vitro cultured human umbilical vein endothelial cells (HUVECs) as an experimental model for replicative senescence. By comparing protein spots from young and senescent HUVECs using two-dimensional electrophoresis, we identified three up-regulated proteins and five down-regulated proteins in senescent HUVECs as compared to young HUVECs, whose alteration was not observed during replicative senescence of primary human fibroblasts. Consistent results were obtained in Western blotting analysis using specific antibodies raised against some of these proteins, whereas there were no significant changes in the mRNA levels of these genes during senescence of HUVECs. Among them, cathepsin B, a protease participating in both intracellular proteolysis and extracellular matrix remodeling was observed to be dramatically up-regulated in senescent HUVECs and whose activity is known to be up-regulated in atherosclerotic lesions with senescence-associated phenotypes in vivo. Additional proteins, including cytoskeletal proteins and proteins involved in the processes of synthesis, turnover and modification of protein, were identified, whose function in endothelium was previously unsuspected. These proteins identified by a proteomics-based approach using cultured HUVECs may be involved not only in replicative senescence but also in functional alterations in vascular endothelial cells with senescence-associated phenotypes and may serve as molecular markers for these processes.

Arteriosclerosis↗
PubMed2003

Use of real-time RT-PCR for the detection of allelic expression of an imprinted gene.

Measurement of the relative amounts of transcripts from two alleles is important in the study of imprinted genes, since quantitative differences that vary among tissues or individuals, and subtle differences in the ratio of allelic expression can have pathobiological significance. Discrimination of alleles is commonly based on PCR, followed by restriction endonuclease digestion to recognize a polymorphic site. However, the use of restriction enzymes misses most of the available single nucleotide polymorphisms. Practically, it requires substantial post-PCR analyses including the restriction enzyme digestion and gel electrophoresis, all of which increase turn around time. Taking advantage of our previous study identifying lung adenocarcinomas displaying biallelic expression of the imprinted gene MEST, we investigated the validity of a method of allelic discrimination in a real-time PCR assay using allele-specific probes. Allelic expression of the MEST gene in the range of 4-fold differences was detected. This new method should enhance our ability to rapidly and accurately assess allelic expression of imprinted genes in a number of samples.

Adenocarcinoma↗
PubMed2002

Elevated apoptosis in pre-mature neurons differentiated from mouse ES cells containing a single human chromosome 21.

A decrease in the number and density of neurons is the most common phenotype in the brains of Down syndrome (DS) patients, causing mental retardation. Studies using primary cultured neurons from DS patients or from model mice have suggested that a defect in metabolism of reactive oxygen species, or diminished levels of glutathione, causes mitochondrial and caspase-mediated neuronal apoptosis in vitro. However, it is not well documented whether neuronal apoptosis also occurs in immature DS neurons, owing to the difficulty in isolating or identifying neuronal stem cells in human or mouse fetuses. Here we utilized an in vitro model system for neuronal differentiation, with mouse embryonic stem cells containing human chromosome 21 (TT2F/hChr.21) to examine the effect of an additional hChr.21 on the early phases of neurogenesis. The differentiation profile of TT2F/hChr.21 cells was essentially the same as those of parental TT2F ES cells. In differentiations of both TT2F and TT2F/hChr.21 cells, high level of apoptosis was observed in neuronal stem cells, but the rate of apoptosis in TT2F/hChr.21 cells was significantly higher than that of parental cells. These results suggest that quantitative changes in the level of apoptosis in DS neuronal stem cells may account for the reduction of neuronal number and density in the DS brain.

Animals↗
PubMed2002

Identification and characterization of an imprinted antisense RNA (MESTIT1) in the human MEST locus on chromosome 7q32.

Imprinted gene(s) on human chromosome 7 are thought to be involved in Russell-Silver syndrome (RSS), based on the fact that approximately 10% of patients have maternal uniparental disomy of chromosome 7. However, involvement of the known imprinted genes (GRB10 at 7p12, PEG10 at 7q21.3 and MEST at 7q32) in RSS has yet to be established. To screen for new imprinted genes, we are initially using somatic cell hybrids containing a paternal or maternal human chromosome 7. Transcripts located between D7S530 and D7S649 (a 1.5 Mb interval encompassing MEST ) were subjected to RT-PCR analysis using somatic cell hybrids. One transcript named MESTIT1 (for MEST intronic transcript 1) reproducibly showed paternal-specific expression. Upon further analysis, we found MESTIT1 to be (1) paternally (and not maternally) expressed in all fetal tissues and fibroblasts examined, (2) to be located in an intron of one of the two isoforms of MEST but transcribed in the opposite direction, (3) to be composed of at least two exons without any significant open reading frame, and (4) to exist as a 4.2 kb transcript in many fetal and adult tissues. We could also identify two isoforms of the mouse Mest gene as observed in humans, but it is still unknown if a murine ortholog of MESTIT1 exists. We also examined the imprinting status of MEST isoforms as a first step in assessing whether MESTIT1 might influence the allelic expression pattern of the sense transcript. MEST isoform 1 was determined to be exclusively expressed from the paternal allele in all fetal tissues and cell lines examined, whereas MEST isoform 2 was only preferentially expressed from the paternal allele in a tissue/cell-type-specific manner. Our results suggest that MESTIT1 is a paternally expressed non-coding RNA that may be involved in the regulation of MEST expression during development. MESTIT1 (also known as PEG1-AS) is now the third independent transcript (with MEST and COPG2IT1) identified at human chromosome 7q32 demonstrating paternal chromosome-specific expression.

Blotting, Northern↗
PubMed2002

An extra human chromosome 21 reduces mlc-2a expression in chimeric mice and Down syndrome.

An extra copy of human chromosome 21 (Chr 21) causes Down syndrome (DS), which is characterized by mental retardation and congenital heart disease (CHD). Chimeric mice containing Chr 21 also exhibit phenotypic traits of DS including CHD. In this study, to identify genes contributing to DS phenotypes, we compared the overall protein expression patterns in hearts of Chr 21 chimeras and wild type mice by two-dimensional electrophoresis. The endogenous mouse atrial specific isoform of myosin light chain-2 (mlc-2a) protein was remarkably downregulated in the hearts of chimeric mice. We also confirmed that the human MLC-2A protein level was significantly lower in a human DS neonate heart, as compared to that of a normal control. Since mouse mlc-2a is involved in heart morphogenesis, our data suggest that the downregulation of this gene plays a crucial role in the CHD observed in DS. The dosage imbalance of Chr 21 has a trans-acting effect which lowers the expression of other genes encoded elsewhere in the genome.

Animals↗
PubMed2002

Overexpression of Icat induces G(2) arrest and cell death in tumor cell mutants for adenomatous polyposis coli, beta-catenin, or Axin.

Aberrant activation of Wnt signaling caused by mutations in adenomatous polyposis coli (APC) or beta-catenin is a critical event in the development of human colorectal tumors. Wnt signaling stabilizes beta-catenin, which in turn associates with TCF/LEF family transcription factors, ultimately altering the expression of Wnt target genes. We have recently identified ICAT, a beta-catenin-interacting protein that interferes with the interaction between beta-catenin and TCF-4, thereby negatively regulating Wnt signaling. In the present study, we generated a recombinant adenovirus encoding ICAT and examined its effect on the growth of tumor cells. We found that Icat inhibits proliferation of colorectal tumor cells mutated in APC or beta-catenin and hepatocellular carcinoma cells mutated in AXIN: By contrast, Icat did not inhibit growth of either normal or tumor cells containing the wild-type APC, beta-catenin, and Axin genes. Icat also inhibited the anchorage-independent growth of colorectal tumor cells and tumorigenic growth of colorectal tumor xenografts. Furthermore, we found that Icat inhibits both dephosphorylation of Cdc2 and nuclear translocation of cyclin B1 and induces G(2) arrest followed by cell death in colorectal tumor cells. These results suggest that Wnt signaling is critical for the growth of colorectal tumors and some hepatocellular carcinomas and that expression of ICAT or drugs which mimic its effects may be useful in the treatment of these tumors.

Adaptor Proteins, Signal Transducing↗
PubMed2002

A dyad oct-binding sequence functions as a maintenance sequence for the unmethylated state within the H19/Igf2-imprinted control region.

DNA methylation of an imprinted control region (ICR) directs the allele-specific and reciprocal expression of the mouse H19 and the insulin-like growth factor 2 (Igf2) genes, mediated by controlling enhancer access. The ICR shows enhancer blocking activity through CTCF binding to an unmethylated sequence. The unmethylated state of the maternal ICR is maintained throughout development after establishment in the germ line; however, little is known of the molecular mechanisms that regulate DNA methylation. Hence, in this study we show that a dyad Oct-binding sequence (DOS) in the ICR mediates the demethylation of low-density methylation but not hypermethylation and is required to maintain the unmethylated state against the tendency for de novo methylation within the ICR in the embryonic carcinoma cell line P19. Furthermore, we also reveal that the unmethylated state of at least one CTCF-binding site within the ICR is under the control of DOS. Our results suggest that the ICR, as a CTCF-dependent insulator, requires DOS as well as CTCF-binding sites and that DOS maintains the maternal specific unmethylated state of the ICR at postimplantation stages.

Animals↗
PubMed2002

Asb4, Ata3, and Dcn are novel imprinted genes identified by high-throughput screening using RIKEN cDNA microarray.

Genes differentially expressed between parthenogenetic and androgenetic embryos are candidates for the identification of imprinted genes, which are expressed specifically from the maternal or paternal allele. To search for genes differentially expressed between parthenogenetic and androgenetic embryos, we used the RIKEN full-length enriched mouse cDNA microarray. The 25 candidates obtained included 8 known imprinted genes (such as IgfII, Snrpn, and Neuronatin) and 3 new ones--Asb4 (ankyrin repeat and SOCS box-containing protein 4), Ata3 (amino acid transport system A3), and Decorin--which were confirmed by using normal diploid embryos from the reciprocal F1 crosses of B6 and JF1 mice. The 25 candidates also included genes that showed no imprinting-associated expression in normal diploid embryos. We describe a feasible high-throughput method of screening for novel imprinted genes by using the RIKEN cDNA microarray.

Amino Acid Transport System A↗
PubMed2002

Suppression of the tumorigenic phenotype by chromosome 18 transfer into pancreatic cancer cell lines.

A number of lines of evidence have suggested that the long arm of chromosome 18 apart from SMAD4 may carry a tumor-suppressor gene(s) that plays a role in the early stage of pancreatic ductal carcinogenesis. Thus, adenovirus-mediated introduction of SMAD4 does not suppress in vitro growth in cells with completely inactivated SMAD4, and frequent loss of 18q at the SMAD4 locus is observed in pancreatic cancers but no abnormalities of the normal SMAD4 homolog have been detected. In this study, we introduced a normal copy of chromosome 18 into some pancreatic ductal carcinoma cells with and without a complete inactivation of SMAD4. Both anchorage-dependent and -independent proliferation as well as invasiveness were significantly suppressed in the hybrid clones compared with that of their parental cells. Moreover, significant suppression of tumorigenesis was observed after inoculation in nude mice, irrespective of the SMAD4 status. Our present study provides the first functional evidence of the existence of an additional tumor-suppressor gene(s), other than SMAD4 and DCC, that is responsible for the pathogenesis in the early stage of pancreatic ductal carcinogenesis.

Animals↗
PubMed2002

Multiple human chromosomes carrying tumor-suppressor functions for the mouse melanoma cell line B16-F10, identified by microcell-mediated chromosome transfer.

Many tumor-suppressor genes are involved in the development and progression of cellular malignancy. To understand the functional role of tumor-suppressor genes in melanoma and to identify the human chromosome that carries these genes, we transferred individually each normal human chromosome, except for the Y chromosome, into the mouse melanoma cell line B16-F10, by microcell fusion. We examined the tumorigenicity of hybrid cells in nude mice and their in vitro growth properties. The introduction of human chromosomes 1 and 2 elicited a remarkable change in cell morphologic features, and cellular senescence was induced at seven to 10 population doublings. The growth rates of tumors derived from microcell hybrid clones containing introduced human chromosome 5, 7, 9, 10, 11, 13, 14, 15, 16, 19, 20, 21, 22, or X were significantly slower than that of the parental B16-F10 cells, whereas the introduction of other human chromosomes had no effect on the tumorigenicity of these cells. The majority of microcell hybrid clones that exhibited suppressed tumorigenicity also showed a moderate reduction in doubling time compared with B16-F10 cells. Microcell hybrid clones with an introduced human chromosome 5 showed complete suppression of in vitro-transformed phenotypes, including cell growth, saturation density, and colony-forming efficiency in soft agar. Thus, these results indicated the presence of many cell senescence-related genes and putative tumor-suppressor genes for the mouse melanoma cell line B16-F10 and showed in vitro that many tumor-suppressor genes control the phenotypes of transformed cells in the multistep process of neoplastic development.

Animals↗
PubMed2002

Molecular genetic studies of human chromosome 7 in Russell-Silver syndrome.

Russell-Silver syndrome (RSS) is a form of congenital short stature characterized by severe growth retardation and variable dysmorphic features. In some RSS individuals, alterations in imprinted genes may be involved because approximately 7% of sporadic patients have been observed to have maternal uniparental disomy (mUPD) of chromosome 7. RSS patients with structural abnormalities of chromosome 7 have also been described. In these individuals the chromosome rearrangement could disrupt the balance of imprinted genes, contribute to a recessive form of RSS, or lead to haploinsufficiency of a crucial developmental gene product. Because the mechanism and molecular defects on chromosome 7 causing RSS are still unknown, we tested our collection of 77 RSS families for mUPD7 and were able to identify three new cases. We also characterized two RSS patients with de novo cytogenetic abnormalities involving the short arm of chromosome 7. One had a partial duplication [46, XX, dup(7)(p12 p14)] and the second contained a paracentric inversion [46, XY, inv(7)(p14 p21)]. Fluorescence in situ hybridization (FISH) mapping revealed that the breakpoints on 7p14 were localized to the same novel gene, C7orf10, which encompasses >700 kb of DNA. We also identified other transcription units from this immediate region, but all seem to be biallelically expressed when using a somatic cell hybrid assay.

Abnormalities, Multiple↗
PubMed2002

Telomere lengths are characteristic in each human individual.

BACKGROUND: A great deal of attention has been focused on telomeres in relation to cellular aging, immortality, and cancer. However, there is no simple link between telomeres and tissue turnover. We recently proposed a hypothesis that telomere shortening with aging and telomere lengths in different organs are characteristic for human individuals. METHODS: To test this, telomere lengths were measured using DNA from cerebral cortex, myocardium, liver, renal cortex and spleen tissues obtained from human subjects ranging in age from neonates to centenarians. RESULTS: Regression analyses demonstrated telomere reduction rates of 29-60 base pair (bp) per year in the liver, renal cortex and spleen, but no such decrease in the cerebral cortex and myocardium. Significant correlation was found between tissues within individuals, such as cerebral cortex versus (vs) myocardium, cerebral cortex vs liver, cerebral cortex vs renal cortex, myocardium vs liver, myocardium vs renal cortex, and liver vs renal cortex. In most cases, the longest telomeres were observed in the myocardium and the shortest in the liver or renal cortex. CONCLUSIONS: Telomere lengths did not show clear correlation with tissue renewal times in vivo, but rather were characteristic for individuals.

Adolescent↗
PubMed2002

Comparative analysis of telomere lengths and erosion with age in human epidermis and lingual epithelium.

We investigated progressive telomere shortening in normal human epidermis and lingual epithelium during aging, and attempted, in particular, to ascertain whether the telomere shortening that accompanies aging occurs at the same rate in different tissues. We studied telomeric DNA integrity, and estimated annual telomere loss, in 52 specimens of epidermis and 48 specimens of lingual epithelium collected at autopsy from subjects who had died at ages between 0 and 101 y. Most of the DNA samples were measured twice by southern blot hybridization. In addition, the correlation between telomere lengths in the two types of tissues was examined. The telomere reduction rates in epidermis and lingual epithelium were 36 bp and 30 bp per y, respectively, and these were significantly different. The rates obtained by the second measurements in epidermis and lingual epithelium were 39 and 32 bp per y, respectively, and these were also significantly different. The mean telomere lengths in the epidermis of eight neonates and the lingual epithelium of seven neonates were 13.2+/-1.0 and 13.8+/-1.0 kb, respectively. Comparison of telomere lengths in the two tissues for 41 paired samples showed that the mean telomere length in the epidermis (10.7+/-2.3 kb) was less than that in the lingual epithelium (12.4+/-2.5 kb); however, statistical analysis revealed a very significant relationship between epidermal and lingual epithelial telomere length (r=0.842, p<0.0001). These results indicate that the telomeres in epidermis and lingual epithelium are characterized by tissue-specific loss rates.

Adolescent↗