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Mitsuo Oshimura

Publications and source records attributed to Mitsuo Oshimura.

At least 19 recordsLinked to original sources
PubMed2003

Proteomics-based identification of differentially expressed genes in human gliomas: down-regulation of SIRT2 gene.

A number of chromosomal abnormalities including 19q deletions have been associated with the formation of human gliomas. In this study, we employed a proteomics-based approach to identify possible genes involved in glioma tumorigenesis which may serve as potential diagnostic molecular markers for this type of cancer. By comparing protein spots from gliomas and non-tumor tissues using two-dimensional (2D) gel electrophoresis, we identified 11 up-regulated proteins and four down-regulated proteins in gliomas. Interestingly, we also discovered that a group of cytoskeleton-related proteins are differentially regulated in gliomas, suggesting the involvement of cytoskeleton modulation in glioma pathogenesis. We then focused on the cytoskeleton-related protein, SIRT2 (sirtuin homologue 2) tubulin deacetylase, which was down-regulated in gliomas. SIRT2 is located at 19q13.2, a region known to be frequently deleted in human gliomas. Subsequent Northern blot analysis revealed that RNA expression of SIRT2 was dramatically diminished in 12 out of 17 gliomas and glioma cell lines, in agreement with proteomic data. Furthermore, ectopic expression of SIRT2 in glioma cell lines led to the perturbation of the microtubule network and caused a remarkable reduction in the number of stable clones expressing SIRT2 as compared to that of a control vector in colony formation assays. These results suggest that SIRT2 may act as a tumor suppressor gene in human gliomas possibly through the regulation of microtubule network and may serve as a novel molecular marker for gliomas. Additional proteins were also identified, whose function in gliomas was previously unsuspected.

Blotting, Northern↗
PubMed2003

Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome.

To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.

Base Sequence↗
PubMed2003

Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation.

ARHI has been identified as a maternally imprinted tumor suppressor gene that maps to chromosome 1p31 and whose expression is markedly down-regulated in breast cancer. To explore possible mechanisms that could silence ARHI expression, we have tested the importance of DNA methylation, histone acetylation and histone methylation in regulating ARHI expression. We found that treatment with CpG demethylating agents and/or histone deacetylase inhibitors could reactivate both the silenced and the imprinted alleles of this tumor suppressor gene. Reactivation of ARHI expression by these reagents is related to the methylation status of the CpG islands in the ARHI promoter, especially CpG island II. Chromatin immunoprecipitation assays revealed that histone H3 lysine 9/18 acetylation levels associated with ARHI in normal cells were significantly higher than those in breast cancer cell lines that lacked ARHI expression. Treatment with a CpG demethylating agent and/or histone deacetylase inhibitor could increase ARHI expression in breast cancer cells, with a corresponding increase in histone H3 lysine 9/18 acetylation and decrease in histone H3 lysine 9 methylation.

Acetylation↗
PubMed2003

Aberrant methylation and silencing of ARHI, an imprinted tumor suppressor gene in which the function is lost in breast cancers.

ARHI is a maternally imprinted tumor suppressor gene that maps to a site on chromosome 1p31 where loss of heterozygosity has been observed in 40% of human breast and ovarian cancers. ARHI is expressed in normal ovarian and breast epithelial cells, but ARHI expression is lost in a majority of ovarian and breast cancers. Expression of ARHI from the paternal allele can be down-regulated by multiple mechanisms in addition to loss of heterozygosity. This article explores the role of DNA methylation in silencing ARHI expression. There are three CpG islands in the ARHI gene. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region. Consistent with imprinting, we have found that all three CpG islands were partially methylated in normal human breast epithelial cells. Additional confirmation of imprinting has been obtained by studying DNA methylation and ARHI expression in murine A9 cells that carry either the maternal or the paternal copy of human chromosome 1. All three CpG islands were methylated, and ARHI was not expressed in A9 cells that contained the maternal allele. Conversely, CpG islands were not methylated and ARHI was expressed in A9 cells that contained the paternal allele of human chromosome 1. Aberrant methylation was found in several breast cancer cell lines that exhibited decreased ARHI expression. Hypermethylation was detected in 67% (6 of 9) of breast cancer cell lines at CpG island I, 33% (3 of 9) at CpG island II, and 56% (5 of 9) at CpG island III. Hypomethylation was observed in 44% (4 of 9) of breast cancer cell lines at CpG island II. When methylation of CpG islands was studied in 20 surgical specimens, hypermethylation was not observed in CpG island I, but 3 of 20 cases exhibited hypermethylation in CpG island II (15%), and 4 of 20 cases had hypermethylation in CpG island III (20%). Treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, could reverse aberrant hypermethylation of CpG island I, II and III and partially restore ARHI expression in some, but not all of the cell lines. Treatment with 5-aza-2'-deoxycytidine partially reactivated ARHI expression in cell lines with hypermethylation of CpG islands I and II but not in cell lines with partial methylation or hypomethylation of these CpG islands. To test the impact of CpG island methylation on ARHI promoter activity more directly, constructs were prepared with the ARHI promoter linked to a luciferase reporter and transfected into SKBr3 and human embryo kidney 293 cells. Methylation of the entire construct destroyed promoter activity. Selective methylation of CpG island II alone or in combination with CpG island I also abolished ARHI promoter activity. Methylation of CpG I alone partially inhibited promoter activity of ARHI. Thus, hypermethylation of CpG island II in the promoter region of ARHI is associated with the complete loss of ARHI expression in breast cancer cells. Other epigenetic modifications such as hypermethylation in CpG island III may also contribute to the loss of ARHI expression.

Alleles↗
PubMed2003

Calcr, a brain-specific imprinted mouse calcitonin receptor gene in the imprinted cluster of the proximal region of chromosome 6.

Expressed sequence tags (ESTs) in the human chromosome 7q21-q31 region were recently used to screen for allelic expression bias in monochromosomal hybrids retaining a paternal or maternal human chromosome 7. Six candidate imprinted genes were identified. In this study, we investigated parent-of-origin-specific expression profiles of their mouse homologues in the proximal region of chromosome 6. An imprinting analysis, using F1 mice from reciprocal crosses between the B6 and JF strains, demonstrated that the mouse calcitonin receptor gene ( Calcr) was expressed preferentially from the maternal allele in brain, whereas no allelic bias was detected in other tissues. Our results indicate that Calcr is imprinted in a tissue-specific manner, with a predominant expression from the maternal allele in the brain.

Animals↗
PubMed2003

Predominant maternal expression of the mouse Atp10c in hippocampus and olfactory bulb.

The human chromosome 15q11-q13 region is one of the most intriguing imprinted domains, and the abnormalities inherited are associated with neurological disorders including Prader-Willi syndrome (PWS), Angelman syndrome (AS) and autism. Recently we have identified a novel maternally expressed gene, ATP10C, that encodes a putative aminophospholipid translocase within this critical region, 200 kb distal to UBE3A in an imprinted domain on human chromosome 15. ATP10C, with UBE3A, displayed tissue-specific imprinting with predominant expression of the maternal allele in the brain. In this study, we demonstrated that the mouse homologue, Atp10c/pfatp, showed tissue-specific maternal expression in the hippocampus and olfactory bulb, which overlapped the region of imprinted Ube3a expression. These data suggest that the imprinted transcript of Atp10c in the specific region of CNS may be associated with neurological disorders including AS and autism.

Adenosine Triphosphatases↗
PubMed2003

Identification of a </= 600-kb region on human chromosome 1q42.3 inducing cellular senescence.

The introduction of a human chromosome 1 via microcell-mediated chromosome transfer (MMCT) induces the cellular senescence in mouse melanoma B16-F10 cells. The senescent cells maintained still the telomerase activity, which is frequently associated with immortal growth of human cells, suggesting that a telomerase-independent mechanism is involved in the senescence observed in this mouse cell line. To map the senescence-inducing gene to a specific chromosomal region, we took two experimental approaches: identification of a minimal region with the senescence-inducing activity via MMCT of a series of subchromosomal transferrable fragments (STFs), each consisting of a different profile of human chromosome 1-derived regions, and identification of a region commonly deleted from the transferred chromosome 1 in the revertant clones that escaped cellular senescence. These approaches identified a 2.7-3.0 Mb of senescence-inducing region shared among the active STFs and a 2.4-3.0 Mb of commonly deleted region in the revertant clones. These two regions overlapped each other to map the responsible gene at the 450 to 600-kb interval between UniSTS93710 and D1S3542 on chromosome 1q42.3. This study provides essential information and materials for cloning and characterization of a novel senescence-inducing gene that functions in a telomerase-independent pathway, which is likely to be conserved between mice and humans.

Animals↗
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

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↗