Biomedical subjects

M Oshimura

Publications and source records attributed to M Oshimura.

At least 37 records · Page 2Linked to original sources

PubMed2001

14-3-3 is involved in p75 neurotrophin receptor-mediated signal transduction.

The low affinity neurotrophin receptor (p75NTR) has been shown to mediate the apoptosis signaling to neural cells. However, the specific mechanisms of intracellular signal transduction of this process are largely unknown. To understand p75NTR-mediated signal transduction, we previously identified a protein that interacts with the intracellular domain of p75NTR, and we named it p75NTR-associated cell death executor (NADE). To elucidate further the signaling mechanisms utilized by p75NTR and NADE, we screened for NADE-binding protein(s) with the yeast two-hybrid method, and we identified 14-3-3epsilon as a NADE-binding protein in vivo. To examine whether 14-3-3epsilon affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3epsilon were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interestingly, transient expression of the mutant form of 14-3-3epsilon lacking the 208-255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated apoptosis, although this mutant form of 14-3-3epsilon continued to associate with NADE. These results suggest that 14-3-3epsilon plays an important role in the modulation of nerve growth factor-dependent p75NTR/NADE-mediated apoptosis.

14-3-3 Proteins↗

PubMed2001

Functional evidence for a telomerase repressor gene on human chromosome 10p15.1.

Based on the sites of frequent allelic loss in hepatocellular carcinoma, five normal human chromosomes (2, 4, 5, 10 and 16) were transferred individually into a telomerase-positive human hepatocellular carcinoma cell line, Li7HM, by microcell-mediated chromosome transfer (MMCT). Chromosome 10, but not the others, repressed telomerase activity immediately and stopped cell growth after 50 population doublings (PDs). Loss of the transferred 10p loci resulted in the emergence of revertant cells that continued to proliferate and expressed telomerase activity, suggesting the presence of a telomerase repressor gene on this chromosomal arm. Transfer of a series of defined fragments from chromosome 10p successfully narrowed down the responsible region: a 28.9-cM region on 10p15 (between WI-4752 and D10S249), but not a 26.2-cM region (between D10S1728 and D10S249), caused repression of telomerase activity and progressive telomere shortening. A strong correlation between the expression level of telomerase catalytic subunit gene (hTERT) and telomerase activity was observed. These findings suggest that a novel telomerase repressor gene which controls the expression of hTERT is located on the 2.7-cM region (between WI-4752 and D10S1728) on chromosome 10p15.1.

Carcinoma, Hepatocellular↗

PubMed2001

Large-scale evaluation of imprinting status in the Prader-Willi syndrome region: an imprinted direct repeat cluster resembling small nucleolar RNA genes.

Loss of paternal gene expression at the imprinted domain on proximal human chromosome 15 causes Prader-Willi syndrome (PWS), a complex multiple-anomaly disorder involving variable mental retardation, hyperphasia leading to obesity and infantile hypotonia with failure to thrive. Although numerous paternally expressed transcripts have been identified that reside in the candidate region, the individual contributions to the development of PWS have not been firmly established. Recent studies of mouse models carrying a cytogenetic deletion suggest that paternal deficiency of the SNRPN-IPW interval is critical for perinatal lethality of potential relevance to PWS. Here we determined the allelic expression profiles of a total of 118 cDNA clones using monochromosomal hybrids retaining either a paternal or maternal human chromosome 15. Our results demonstrated a preponderance of unusual transcripts lacking protein-coding potential that were expressed exclusively from the paternal copy of the critical interval. This interval was also found to encompass a large direct repeat (DR) cluster displaying a potentially active chromatin conformation of paternal origin, as suggested by enhanced sensitivity to nuclease digestion. Database searches revealed an unexpected organization of tandemly repeated consensus elements, all of which possessed well-defined box C and D sequences characteristic of small nucleolar RNAs (snoRNAs). Southern blot analysis further demonstrated a considerable degree of phylogenetic conservation of the DR locus in the genomes of all mammalian species tested, but not in chicken, Xenopus and Drosophila. These findings imply a potential direct contribution of the DR locus, representing a cluster of multiple snoRNA genes, to certain phenotypic features of PWS.

Base Sequence↗

PubMed2001

Functional evidence for the presence of tumor suppressor gene on chromosome 10p15 in human prostate cancers.

Loss of heterozygosity on chromosome 10p was observed frequently in human prostate cancers. Studies have demonstrated that the introduction of the short arm of human chromosome 10 into a human prostate cancer cell line, PPC-1, by microcell-mediated chromosome transfer (MMCT), suppressed the malignant phenotype, suggesting the presence of a prostate tumor suppressor gene(s) within a region of 17 cM at distal 10p. To narrow down the candidate region harboring the tumor suppressor gene, a series of 10p fragments were transferred into PPC-1 cells by MMCT using a panel of hamster-human hybrid cells containing various portions of 10p. Four of the six hybrid cells obtained showed decreased tumorigenicity when injected subcutaneously into athymic nude mice. Tumors developed only at six of 40 injection sites for these four hybrid cells. In contrast, the other two hybrid cells, as well as parental PPC-1 cells, were judged to be fully tumorigenic because tumors appeared at a total 26 of 32 sites for the two hybrid cells and 15 of 16 sites for PPC-1. Allelotyping of 10p combined with fluorescence in situ hybridization in these hybrid cells suggested that a prostate tumor suppressor gene was located within a fragment of approximately 1.2 Mb flanked by D10S1172 and D10S226 on 10p15.1.

Adenocarcinoma↗

PubMed2001

Epigenetic silencing of PEG3 gene expression in human glioma cell lines.

Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele-specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele-specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high-level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.

Animals↗

PubMed2001

Frequent loss of imprinting of IGF2 and MEST in lung adenocarcinoma.

Genomic imprinting is a parental origin-specific chromosomal modification that causes differential expression of maternal and paternal alleles of a gene. Accumulating evidence suggests that deregulation of imprinted genes, including loss of imprinting (LOI), plays a role in oncogenesis. In the present study, we investigated allelic expression of six imprinted genes in human lung adenocarcinomas as well as in matched normal lung tissue. Informative cases showing heterozygosity for the gene of interest were selected from 35 patients. LOI of the insulin-like growth factor 2 gene (IGF2) and mesoderm-specific transcript (MEST, also known as paternally expressed gene 1) was noted in 47% (seven of 15) and 85% (11 of 13) of informative cases, respectively. Monoallelic expression was maintained in all the matched normal tissues examined. LOI of IGF2 was seen more frequently in moderately to poorly differentiated adenocarcinomas. In contrast, H19, small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN), necdin gene (NDN), and long QT intronic transcript 1 (LIT1) exhibited consistent monoallelic expression in all the informative samples. These findings indicated that independent deregulation took place in imprinted genes and suggested that aberrant imprinting of IGF2 and MEST was involved in the development of lung adenocarcinoma.

Adenocarcinoma↗

PubMed2001

A novel in vitro system for analyzing parental allele-specific histone acetylation in genomic imprinting.

One of the obstacles in studying human genomic imprinting is distinguishing the parental origin of alleles in diploid cells. To solve this problem, we have constructed a library of mouse A9 hybrids in which individual clones contain a single human chromosome of known parental origin. Here we extend this in vitro system to the analysis of the role of histone acetylation in the allelic expression of human imprinted genes. The levels of histone H4 acetylation of the imprinted human LIT1, H19, and SNRPN genes were examined by a chromatin immunoprecipitation (ChIP) assay in mouse A9 hybrids with a single human chromosome of known parental origin. We demonstrated that H4 histones associated with the actively expressed alleles of imprinted LIT1, H19, and SNRPN genes were highly acetylated, whereas they were hypoacetylated in the silent alleles. Furthermore, treatment of A9 hybrids with trichostatin A (TSA), an inhibitor of histone deacetylase, resulted in transcriptional reactivation of the silent alleles for LIT1 and SNRPN, suggesting that histone deacetylation is one of the key regulatory mechanisms in genomic imprinting. These results indicate that our monochromosomal hybrid system is a new technology for analyzing histone modifications between parental alleles in human imprinted genes.

Acetyltransferases↗

PubMed2001

Germline transmission of a transferred human chromosome 21 fragment in transchromosomal mice.

We generated transchromosomal (Tc) mice containing a human chromosome 21 fragment (hCF21) using mouse embryonic stem (ES) cells with the transferred hCF21. Here we report breeding analyses that test the maintenance rate of the hCF21 in Tc mice of two different genetic backgrounds, MCH (ICR) and C57BL/6. Fluorescence in situ hybridization and polymerase chain reaction-based DNA analyses revealed that the structure of the hCF21 fragment including the CBR1, SIM2, HLCS, and D21S268 markers, was approximately 5 Mb in size, and was transmitted at least to the F3 generation. Though the retention rate of the hCF21 was variable among individual mice, for example, 21%-92% in brain and 10%-92% in tail fibroblasts, the C57BL/6 background yielded a higher retention rate than did the MCH (ICR). These results suggest that the hCF21 could be maintained stably in Tc mice, depending on the genetic background. The panel of Tc mice will be a useful model to investigate the function of genes on the hCF21 fragment in various tissues through germinal transmission.

Animals↗

PubMed2001

Construction of 700 human/mouse A9 monochromosomal hybrids and analysis of imprinted genes on human chromosome 6.

As an in vitro assay system for the identification of human imprinted genes, a library of human/mouse A9 monochromosomal hybrids containing a single, intact bsr-tagged human chromosome of known parental origin, derived from normal human fibroblasts, has been previously generated by microcell-mediated chromosome transfer (MMCT). To supplement this assay system, we constructed additional 700 A9 monochromosomal hybrids, using a pSTneo or pPGKneo selection marker. To validate the A9 hybrids, we screened them with chromosome-specific polymorphic markers, and identified the hybrids containing either human chromosome 6, 7, 14, 18, or 21 of known parental origin. Matching paternal and maternal chromosome pairs of A9 hybrids were identified for chromosomes 6, 7, 14, and 18. The paternal-specific expression of ZAC (zinc finger protein, which regulates apoptosis and cell cycle arrest) and HYMAI (hydatidiform mole-associated and imprinted transcript), and the maternal-specific methylation of a CpG island within an imprinted domain on human chromosome 6q24, were maintained in A9 hybrids. For an example, we profiled the expression of expressed sequence tags (ESTs) and the methylation of CpG islands in the 300-kb imprinted domain around 6q24, which may be associated with cancers and transient neonatal diabetes mellitus (TNDM). Thus, the 700 A9 hybrids should be useful for various aspects of imprinting studies.

Animals↗

PubMed2001

A novel maternally expressed gene, ATP10C, encodes a putative aminophospholipid translocase associated with Angelman syndrome.

Lack of a maternal contribution to the genome at the imprinted domain on proximal chromosome 15 causes Angelman syndrome (AS) associated with neurobehavioral anomalies that include severe mental retardation, ataxia and epilepsy. Although AS patients have infrequent mutations in the gene encoding an E6-AP ubiquitin ligase required for long-term synaptic potentiation (LTP), most cases are attributed to de novo maternal deletions of 15q11-q13. We report here that a novel maternally expressed gene, ATP10C, maps within the most common interval of deletion and that ATP10C expression is virtually absent from AS patients with imprinting mutations, as well as from patients with maternal deletions of 15q11-q13.

Adenosine Triphosphatases↗

PubMed2001

Loss of imprinting of long QT intronic transcript 1 in colorectal cancer.

Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) and H19 genes on human chromosome 11 has been found not only in childhood tumors but also in common adult cancers including colorectal cancer. Recently, a transcript called LIT1 (long QT intronic transcript 1) has been identified within the KvLQT1 locus on chromosome 11. LIT1 is expressed preferentially from the paternal allele and is transcribed in most human tissues. LOI of LIT1 was found in a considerable number of Beckwith-Wiedemann syndrome (BWS) patients, suggesting that it is associated with the etiology of BWS. Since LOI of IGF2 was observed in association with overexpression of IGF2 in colorectal cancer in our previous study, we examined the status of genomic imprinting of LIT1 and H19 in comparison with IGF2 in colorectal cancer. We examined 44 surgically dissected colorectal cancer tissues. Ten of them represented informative cases for LIT1. None of these patients exhibited loss of heterozygosity (LOH) of LIT1, and LOI of LIT1 was observed in 4 of the 10 (40%) informative patients, but not in non-cancerous tissues. Neither LOH nor LOI of H19 was observed. LOI of IGF2 was observed in 4 of 18 (22%) informative patients. These results suggest that LOI of LIT1 is frequently observed in colorectal cancer and may be a useful marker for diagnosis of colorectal cancer.

Chromosomes, Human, Pair 11↗

PubMed2000

Identification of a mutated DNA ligase IV gene in the X-ray-hypersensitive mutant SX10 of mouse FM3A cells.

The mouse carcinoma cell line SX10 is a hypersensitive mutant to x-rays and bleomycin. An earlier complementation test suggests that SX10 would belong to x-ray-cross complementing group (XRCC) 4. However, in this study, a human XRCC4 expression vector failed to complement the SX10 phenotype. Consistent with the previous report, SX10 showed the same level of DNA-dependent protein kinase activity as the wild-type SR-1. We isolated and analyzed hybrids between SX10 and human diploid fibroblast cells and found that human chromosome 13 conferred the x-ray resistance to the hybrids, suggesting that a candidate gene would be located on this chromosome. Polymerase chain reaction analysis with these hybrids and x-ray-resistant transformants obtained by introducing human chromosomes into SX10 indicated that the mutant was likely to be defective in DNA ligase IV. Sequence analysis of the DNA ligase IV gene confirmed that a defect in SX10 was attributed to a transition of G to A at nucleotide position 1413 of the gene, leading to an amino acid substitution from Trp at residue 471 to a stop codon. Revertant clones (Rev1-3) derived from SX10 showed a restored x-ray resistance; Rev1 reverted to the original nucleotide G at position 1413, whereas Rev2 and Rev3 to C. Transfection of a mouse DNA ligase IV cDNA vector into SX10 restored the resistance to both x-rays and bleomycin. SX10 showed a reduced frequency of chromosomal integration of transfected DNA, but the revertants restored the frequency found in the wild-type cells. These results suggest a possible involvement of DNA ligase IV in the integration event of foreign DNA as well as a crucial role in DNA double-strand break repair.

Animals↗

PubMed2000

Correlation of telomere lengths in normal and cancers tissue in the large bowel.

The hypothesis that telomeres in colorectal cancer cells exhibit age-related shortening, as in normal cells of the colorectal epithelium, was tested with samples of non-cancerous mucosa and cancer tissue from 124 patients (aged 29-97 years). Shortening with aging could be demonstrated for both normal and cancer tissues; regression analysis showed rates for length reduction of 44 and 50 base pair/year, respectively. Straight, essentially parallel, lines were obtained for the two cases, normal tissue values being about 2 kilobase pairs (kbp) higher, with a significant correlation between data at the individual patient level.

Adult↗

PubMed2000

Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome.

Human chromosome 11p15.5 harbors an intriguing imprinted gene cluster of 1 Mb. This imprinted domain is implicated in a wide variety of malignancies and Beckwith-Wiedemann syndrome (BWS). Recently, several lines of evidence have suggested that the BWS-associated imprinting cluster consists of separate chromosomal domains. We have previously identified LIT1, a paternally expressed antisense RNA within the KvLQT1 locus through a positional screening approach using human monochromosomal hybrids. KvLQT1 encompasses the translocation breakpoint cluster in BWS and patients exhibit frequent loss of maternal methylation at the LIT1 CpG island, implying a regulatory role for the LIT1 locus in coordinate control of the imprinting cluster. Here we generated modified human chromosomes carrying a targeted deletion of the LIT1 CpG island using recombination-proficient chicken DT40 cells. Consistent with the prediction, this mutation abolished LIT1 expression on the paternal chromosome, accompanied by activation of the normally silent paternal alleles of multiple imprinted loci at the centromeric domain including KvLQT1 and p57(KIP2). The deletion had no effect on imprinting of H19 located at the telomeric end of the cluster. Our findings demonstrate that the LIT1 CpG island can act as a negative regulator in cis for coordinate imprinting at the centromeric domain, thereby suggesting a role for the LIT1 locus in a BWS pathway leading to functional inactivation of p57(KIP2). Thus, the targeting and precise modification of human chromosomal alleles using the DT40 cell shuttle system can be used to define regulatory elements that confer long-range control of gene activity within chromosomal domains.

Alleles↗

PubMed2000

A novel imprinted gene, HYMAI, is located within an imprinted domain on human chromosome 6 containing ZAC.

Transient neonatal diabetes mellitus (TNDM) is a rare disease characterized by intrauterine growth retardation, dehydration, and failure to thrive due to a lack of normal insulin secretion. This disease is associated with paternal uniparental disomy or paternal duplication of chromosome 6, suggesting that the causative gene(s) for TNDM is imprinted. Recently, Gardner et al. (1999, J. Med. Genet. 36: 192-196) proposed that a candidate gene for TNDM lies within chromosome 6q24.1-q24.3. To find human imprinted genes, we performed a database search for EST sequences that mapped to this region, followed by RT-PCR analysis using monochromosomal hybrid cells with a human chromosome 6 of defined parental origin. Here we report the identification of a novel imprinted gene, HYMAI. This gene exhibits differential DNA methylation between the two parental alleles at an adjacent CpG island and is expressed only from the paternal chromosome. A previously characterized imprinted gene, ZAC/LOT1, is located 70 kb downstream of HYMAI and is also expressed only from the paternal allele. In the pancreas, both genes are moderately expressed. HYMAI and ZAC/LOT1 are therefore candidate genes involved in TNDM. Furthermore, the human chromosome 6q24 region is syntenic to mouse chromosome 10 and represents a novel imprinted domain.

Alleles↗

PubMed2000

Specific impairment of cardiogenesis in mouse ES cells containing a human chromosome 21.

Down syndrome (DS) leads to cardiac defects which are common and significant in babies with DS. We recently generated chimeric mice carrying a human chromosome (hChr) 21. The contribution ratio of embryonic stem (ES) cells containing a hChr 21 was specifically low in the heart, compared to other organs, and cardiovascular malformations were observed, suggesting that an additional copy of hChr 21 also disrupts the normal development of heart in mice. Here we describe that the presence of hChr 21 in ES cells delays the appearance of beating cardiomyocyte during differentiation, whereas differentiation into other cell types is not disrupted. Furthermore, the defect in cardiogenesis was restored following the deletion of a specific region of hChr 21. Therefore, we conclude that the imbalance of specific gene(s) on hChr 21 may lead to the disturbance of cardiogenesis and that this may be a useful system to model and investigate the cardiac defects of human DS.

Animals↗

PubMed2000

Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies.

The use of a human chromosome or its fragment as a vector for animal transgenesis may facilitate functional studies of large human genomic regions. We describe here the generation and analysis of double trans-chromosomic (Tc) mice harboring two individual human chromosome fragments (hCFs). Two transmittable hCFs, one containing the Ig heavy chain locus (IgH, approximately 1.5 Mb) and the other the kappa light chain locus (Igkappa, approximately 2 Mb), were introduced into a mouse strain whose endogenous IgH and Igkappa loci were inactivated. In the resultant double-Tc/double-knockout mice, substantial proportion of the somatic cells retained both hCFs, and the rescue in the defect of Ig production was shown by high level expression of human Ig heavy and kappa chains in the absence of mouse heavy and kappa chains. In addition, serum expression profiles of four human Ig gamma subclasses resembled those seen in humans. They mounted an antigen-specific human antibody response upon immunization with human serum albumin, and human serum albumin-specific human monoclonal antibodies with various isotypes were obtained from them. These results represent a generation of mice with "humanized" loci by using the transmittable hCFs, which suggest that the Tc technology may allow for the humanization of over megabase-sized, complex loci in mice or other animals. Such animals may be useful not only for studying in vivo functions of the human genome but also for obtaining various therapeutic products.

Animals↗

PubMed2000

Cellular senescence of a human bladder carcinoma cell line (JTC-32) induced by a normal chromosome 11.

Human chromosome 11 is expected to carry tumor suppressor genes for a variety of human cancers, including bladder carcinoma. To examine the functional role of a putative tumor suppressor gene(s) on this chromosome in the development of bladder carcinoma, we performed microcell-mediated transfer of chromosome 11 into the bladder carcinoma cell line, JTC-32. Fifteen of 20 colonies formed by the transfer experiment showed a remarkable change in cell morphology. They flattened and ceased growing, or senesced, prior to 10 population doublings. The presence of transferred chromosome 11-derived fragments in the growth-arrested cells was confirmed by PCR-based polymorphism analyses. The remaining 5 microcell hybrid clones exhibited a parental cell-like morphology, and presumably escaped from senescence, which was accompanied by deletions and/or rearrangements of the transferred chromosome 11. On the other hand, a transferred normal chromosome 7 neither changed the cell morphology nor arrested the cell growth. These results support the hypothesis that chromosome 11 contains a gene or genes which restore the senescence program lost during the immortalization process of JTC-32 cells.

Aged↗