Biomedical subjects

Junko Oshima

Publications and source records attributed to Junko Oshima.

17 recordsLinked to original sources

PubMed2006

Collagen expression in fibroblasts with a novel LMNA mutation.

Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.

Adolescent↗

PubMed2006

WRN's tenth anniversary.

Werner syndrome (WS) is a segmental progeroid syndrome in which patients display pleiotropic features of aging seen in the normal population. The advent of positional cloning in the 1990s markedly accelerated the identification of human disease-causing genes. In 1996, mutations in WRN, which was shown to encode a new, putative member of the family of RecQ DNA helicases, were identified in four patients as the cause of WS. Ten years after the identification of WRN, what have we learned about its role in WS, and its contribution to normal aging?

Adult↗

PubMed2006

The spectrum of WRN mutations in Werner syndrome patients.

The International Registry of Werner syndrome (www.wernersyndrome.org) has been providing molecular diagnosis of the Werner syndrome (WS) for the past decade. The present communication summarizes, from among 99 WS subjects, the spectrum of 50 distinct mutations discovered by our group and by others since the WRN gene (also called RECQL2 or REQ3) was first cloned in 1996; 25 of these have not previously been published. All WRN mutations reported thus far have resulted in the elimination of the nuclear localization signal at the C-terminus of the protein, precluding functional interactions in the nucleus; thus, all could be classified as null mutations. We now report two new mutations in the N-terminus that result in instability of the WRN protein. Clinical data confirm that the most penetrant phenotype is bilateral ocular cataracts. Other cardinal signs were seen in more than 95% of the cases. The median age of death, previously reported to be in the range of 46-48 years, is 54 years. Lymphoblastoid cell lines (LCLs) have been cryopreserved from the majority of our index cases, including material from nuclear pedigrees. These, as well as inducible and complemented hTERT (catalytic subunit of human telomerase) immortalized skin fibroblast cell lines are available to qualified investigators.

Adult↗

PubMed2005

Correction of cellular phenotypes of Hutchinson-Gilford Progeria cells by RNA interference.

The great majority of cases of the Hutchinson-Gilford progeroid syndrome (HGPS) ("Progeria of Childhood'') are caused by a single nucleotide mutation (1824 C->T) in the LMNA gene which encodes lamin A and C, nuclear intermediate filaments that are important components of the nuclear lamina. The resultant mutant protein (Delta50 lamin A) is thought to act in a dominant fashion. We exploited RNA interference technology to suppress Delta50 lamin A expression, with the long range goal of intervening in the pathogenesis of the coronary artery atherosclerosis that typically leads to the death of HGPS patients. Short hairpin RNA (shRNA) constructs were designed to target the mutated pre-spliced or mature LMNA mRNAs, and were expressed in HGPS fibroblasts carrying the 1824 C->T mutations using lentiviruses. One of the shRNAs targeted to the mutated mRNA reduced the expression levels of Delta50 lamin A to 26% or lower. The reduced expression was associated with amelioration of abnormal nuclear morphology, improvement of proliferative potential, and reduction in the numbers of senescent cells. These findings provide a rationale for potential gene therapy.

Cell Culture Techniques↗

PubMed2005

Phenotypic heterogeneity in body fat distribution in patients with atypical Werner's syndrome due to heterozygous Arg133Leu lamin A/C mutation.

CONTEXT: A heterozygous missense mutation substituting arginine at position 133 to leucine in the lamin A/C protein has been reported in two young women with clinical features of short stature, bird-like faces, and early onset of aging processes. OBJECTIVE: The objective of the study was to carry out detailed phenotyping of these two women by evaluating the pattern of fat loss using anthropometry, dual-energy x-ray absorptiometry (DEXA), and magnetic resonance imaging (MRI) and study metabolic abnormalities in glucose and lipid metabolism. DESIGN: The study consisted of descriptive case reports. SETTING: The study was conducted at a referral center. PATIENTS: Patient 1 was a 23-yr-old African-American female with progeroid features. Patient 2 was a 24-yr-old Caucasian female with generalized lipodystrophy, hypertriglyceridemia, and severe insulin resistance diabetes who required more than 200 U of insulin daily. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Body fat distribution to characterize pattern of lipodystrophy and nuclear morphology abnormalities in skin fibroblasts were studied. RESULTS: Patient 1 had normal body fat (27%) by DEXA. However, MRI revealed relative paucity of sc fat in the distal extremities, with preservation of sc truncal fat. She had impaired glucose tolerance and elevated postprandial serum insulin levels. Patient 2, in contrast, had only 11.6% body fat as determined by DEXA and had generalized loss of sc and intraabdominal fat on MRI. Skin fibroblasts from patient 2 showed marked abnormal nuclear morphology, compared with those from patient 1. Despite the deranged nuclear morphology, the lamin A/C remained localized to the nuclear envelope, and the nuclear DNA remained within the nucleus. CONCLUSIONS: Atypical Werner's syndrome associated with Arg133Leu mutation in the LMNA gene presents with a phenotypically heterogeneous disorder. Furthermore, the severity of metabolic complications seems to correlate with the extent of lipodystrophy.

Adipose Tissue↗

PubMed2005

Accumulation of Werner protein at DNA double-strand breaks in human cells.

Werner syndrome is an autosomal recessive accelerated-aging disorder caused by a defect in the WRN gene, which encodes a member of the RecQ family of DNA helicases with an exonuclease activity. In vitro experiments have suggested that WRN functions in several DNA repair processes, but the actual functions of WRN in living cells remain unknown. Here, we analyzed the kinetics of the intranuclear mobilization of WRN protein in response to a variety of types of DNA damage produced locally in the nucleus of human cells. A striking accumulation of WRN was observed at laser-induced double-strand breaks, but not at single-strand breaks or oxidative base damage. The accumulation of WRN at double-strand breaks was rapid, persisted for many hours, and occurred in the absence of several known interacting proteins including polymerase beta, poly(ADP-ribose) polymerase 1 (PARP1), Ku80, DNA-dependent protein kinase (DNA-PKcs), NBS1 and histone H2AX. Abolition of helicase activity or deletion of the exonuclease domain had no effect on accumulation, whereas the presence of the HRDC (helicase and RNaseD C-terminal) domain was necessary and sufficient for the accumulation. Our data suggest that WRN functions mainly at DNA double-strand breaks and structures resembling double-strand breaks in living cells, and that an autonomous accumulation through the HRDC domain is the initial response of WRN to the double-strand breaks.

Animals↗

PubMed2005

LMNA mutations in progeroid syndromes.

Segmental progeroid syndromes are disorders in which affected individuals. present various features that suggest accelerated ageing. The two best-known examples are Hutchinson-Gilford progeria syndrome (HGPS, 'Progeria of childhood') and Werner syndrome (WS, 'Progeria of the adult'). A novel, recurrent de novo mutation in the LMNA gene, responsible for the majority of HGPS cases, results in an in-frame deletion of 50 amino acids, including endoproteolytic sites required for processing of prelamin A to mature lamin A protein. Another mutation results in a 35 amino acid in-frame deletion with a milder HGPS phenotype. WRN, the gene responsible for the majority of WS cases, encodes a multifunctional nuclear protein with exonuclease and helicase activities and may participate in optimizing DNA repair/recombination. A subset of WS patients do not show mutations at the WRN locus (atypical WS), but show heterozygous amino acid substitutions in the heptad repeat region of lamin A. Structural analysis suggests that mutations in atypical WS may interfere with protein-protein interactions. When compared to WRN-mutant WS, LMNA-mutant atypical WS patients appear to show earlier onset and possibly more severe ageing-related symptoms.

Animals↗

PubMed2004

The genetics of human longevity.

Many of the genes that affect aging and longevity in model organisms, such as mice, fruit flies, and worms, have human homologs. This article reviews several genetic pathways that may extend lifespan through effects on aging, rather than through effects on diseases such as atherosclerosis or cancer. These include some of the genes involved in the regulation of DNA repair and nuclear structure, which cause the progeroid syndromes when mutated, as well as those that may affect telomere length, since shorter telomeres have been associated with shorter survival. Other potential longevity genes, such as sirtuins, are involved in regulating the response to cellular stress, including caloric restriction. The best-studied pathway involves insulin and insulin-like growth factor 1 signaling; mutations in homologs of these genes have extended lifespan up to sixfold in model organisms. Other potential candidates include mitochondrial DNA and the genes that regulate the inflammatory response. Despite the challenges in study design and analysis that face investigators in this area, the identification of genetic pathways that regulate longevity may suggest potential targets for therapy.

Aging↗

PubMed2003

LMNA mutations in atypical Werner's syndrome.

BACKGROUND: Werner's syndrome is a progeroid syndrome caused by mutations at the WRN helicase locus. Some features of this disorder are also present in laminopathies caused by mutant LMNA encoding nuclear lamin A/C. Because of this similarity, we sequenced LMNA in individuals with atypical Werner's syndrome (wild-type WRN). METHODS: Of 129 index patients referred to our international registry for molecular diagnosis of Werner's syndrome, 26 (20%) had wildtype WRN coding regions and were categorised as having atypical Werner's syndrome on the basis of molecular criteria. We sequenced all exons of LMNA in these individuals. Mutations were confirmed at the mRNA level by RT-PCR sequencing. In one patient in whom an LMNA mutation was detected and fibroblasts were available, we established nuclear morphology and subnuclear localisation. FINDINGS: In four (15%) of 26 patients with atypical Werner's syndrome, we noted heterozygosity for novel missense mutations in LMNA, specifically A57P, R133L (in two people), and L140R. The mutations altered relatively conserved residues within lamin A/C. Fibroblasts from the patient with the L140R mutation had a substantially enhanced proportion of nuclei with altered morphology and mislocalised lamins. Individuals with atypical Werner's syndrome with mutations in LMNA had a more severe phenotype than did those with the disorder due to mutant WRN. INTERPRETATION: Our findings indicate that Werner's syndrome is molecularly heterogeneous, and a subset of the disorder can be judged a laminopathy.

Adolescent↗

PubMed2003

Werner syndrome protein limits MYC-induced cellular senescence.

The MYC oncoprotein is a transcription factor that coordinates cell growth and division. MYC overexpression exacerbates genomic instability and sensitizes cells to apoptotic stimuli. Here we demonstrate that MYC directly stimulates transcription of the human Werner syndrome gene, WRN, which encodes a conserved RecQ helicase. Loss-of-function mutations in WRN lead to genomic instability, an elevated cancer risk, and premature cellular senescence. The overexpression of MYC in WRN syndrome fibroblasts or after WRN depletion from control fibroblasts led to rapid cellular senescence that could not be suppressed by hTERT expression. We propose that WRN up-regulation by MYC may promote MYC-driven tumorigenesis by preventing cellular senescence.

Cell Division↗

PubMed2003

The G2-phase decatenation checkpoint is defective in Werner syndrome cells.

It has been proposed that cells monitor chromatid catenation status after DNA replication and inhibit progression into mitosis until chromatids are correctly decatenated by topoisomerase II (TopoII). Studies in yeast have suggested that TopoII may interact with RecQ helicases during this process. Using ICRF187, a TopoII catalytic inhibitor that prevents chromatid decatenation without producing DNA strand breaks, we demonstrated that cells deficient of WRN, a human RecQ helicase, displayed a defect in decatenation checkpoint activation, which was corrected by ectopic expression of wild-type WRN. We also provide evidence that BRCA1 is phosphorylated in an ATR-dependent manner in response to decatenation checkpoint activation and that this phosphorylation is not detectable in Werner syndrome cells. Furthermore, ICRF187 treatment resulted in coimmunoprecipitation of WRN and TopoII. Finally, we demonstrated that override of the decatenation checkpoint resulted in enhanced chromosomal damage and apoptosis only in the absence of WRN, but not in normal cells. Our findings suggest that WRN plays a role in the activation of G(2) decatenation checkpoint and that the abortive function of this pathway itself does not appear to be sufficient to cause genomic instability but rather predisposes to genomic instability and apoptotic cell death in the absence of other "caretaker" genes, such as WRN.

Apoptosis↗

PubMed2003

WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair.

Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ-like helicase and is thought to participate in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non-homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild-type (wt) WRN. WRN possesses both 3' --> 5' exonuclease and 3' --> 5' helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN-/-) complemented with either wtWRN, exonuclease-defective WRN (E-), helicase-defective WRN (H-) or exonuclease/helicase-defective WRN (E-H-). The single E-and H- mutants each partially complemented the NHEJ abnormality of WRN-/- cells. Strikingly, the E-H- double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E- and H- single mutants increased HR to levels higher than those restored by either E-H- or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events.

Adenosine Triphosphatases↗

PubMed2002

Functional evidence for a metastasis suppressor gene for rat prostate cancer within a 60-kilobase region on human chromosome 8p21-p12.

We recently demonstrated that the human chromosome 8p21-p12 region encodes a metastasis suppressor gene for rat prostate cancer. The presence of this region suppresses the metastatic ability of rat prostate cancer cells (N. Nihei et al., Genes Chromosomes Cancer, 17: 260-268, 1996). To define further the region harboring the metastasis suppressor gene, a truncated human chromosome 8 containing this region was transferred into highly metastatic AT6.3 rat prostate cancer cells by microcell-mediated chromosome transfer. The region of human chromosome 8 retained in each microcell hybrid was determined by a PCR analysis with sequence-tagged site markers, and this analysis placed the metastasis suppressor gene in the interval between D8S2249 and D8S2244 on human chromosome 8p21-p12. One of the metastasis-suppressed microcell hybrids was used for construction of representative yeast/bacterial artificial chromosome (YAC/BAC) library covering the candidate region using a transformation-associated recombination technology (N. Kouprina et al., Genomics, 53: 21-28, 1998). The final contig corresponding to the candidate region was assembled by four YAC/BAC clones. Each clone was transfected into the AT6.3 cells, and the resultant transfectants were tested for their metastatic ability in athymic nude mice. Introduction of a 60-kb YAC/BAC clone resulted in significant suppression of the metastatic ability without suppression of the tumorigenicity. In contrast, other YAC/BAC clones in the contig had neither metastasis nor tumor suppressor activity. This demonstrates that the 60-kb fragment from the human chromosome 8p21-p12 region contains the metastasis suppressor gene for the AT6.3 cells. Frequent loss of heterozygosity of this region is detected in human prostate cancer, which suggests that our target metastasis suppressor gene may also play an important role in the progression of human prostate cancer.

Animals↗

PubMed2002

Lack of WRN results in extensive deletion at nonhomologous joining ends.

Loss of WRN causes the genomic instability progeroid syndrome, Werner syndrome. WRN encodes a multifunctional nuclear protein with 3'-->5' exonuclease and 3'-->5' helicase activities. Linear plasmids with noncompatible ends introduced to Werner syndrome cells underwent extensive deletions at nonhomologous joining ends, particularly at the 3' protruding single-stranded end. This extensive deletion phenotype was complemented by wild-type WRN. These results suggest that WRN can out-compete other exonucleases that participate in double-strand break repair or stabilize the broken DNA end.

DNA↗

PubMed2002

Alterations of chaperone protein expression in presenilin mutant neurons in response to glutamate excitotoxicity.

Mutations in the presenilin-1 (PS1) gene underlie the most common form of familial dementia of the Alzheimer type (DAT). We demonstrated previously that the expression of PS1 with a M146V mutation in transgenic mice potentiates glutamate toxicity to neurons, due to an altered calcium homeostasis. Here, using a subtractive cDNA library approach, we report the identification of several genes, the altered expression of which may be associated with this unique PS1-related vulnerability to glutamate. The identified genes, including chaperonin subunit 2 and nucleophosmin 1/B23, are involved in the intracellular trafficking of proteins and ions. Northern blot analysis revealed that the effect of glutamate on calcium-binding proteins was augmented in neurons from PS1 mutation mice, compared with neurons from mice lacking other genes relevant to the pathogenesis of DAT (FE65 and APOE) or neurons from control wild-type mice. Interestingly, mRNA for two chaperone proteins were expressed at lower levels specifically in neurons from PS1 mutant mice. These findings suggest that PS1 mutations may, in part, contribute to the development of DAT via altered expression of chaperone proteins.

Alzheimer Disease↗

PubMed2002

Werner Syndrome.

Werner syndrome is a premature aging disease caused by the mutation in the WRN gene. The cloning and characterization of the WRN gene and its product allows investigators to study the disease and the human aging process at molecular level. This review summarizes the recent progresses on various aspects of the WRN research including functional analysis of the protein, interactive cloning, complexes formation, mouse models, and SNPs (single nucleotide polymorphisms). These in depth investigations have greatly advanced our understanding of the disease and elucidated future research direction for Werner syndrome and the human aging process.

Journal Article↗

PubMedDate not supplied

Transfection of neuroprogenitor cells with iron nanoparticles for magnetic resonance imaging tracking: cell viability, differentiation, and intracellular localization.

PURPOSE: Magnetic resonance imaging (MRI) can track labeled cells in the brain. The use of hemagglutinating virus of Japan envelopes (HVJ-Es) to effectively introduce the contrast agent to neural progenitor cells (NPCs) is limited to date despite their high NPC affinity. PROCEDURES: HVJ-Es and Lipofectamine 2000 were compared as transfection vehicles of superparamagnetic iron oxide (SPIO). Labeled NPCs were examined for iron content, MRI signal change, and fundamental cell characteristics. Prussian Blue staining was used after differentiation to determine SPIO localization. RESULTS: HVJ-Es transfected up to 12.5 +/- 8.8 times more SPIO into NPCs. HVJ-Es do not affect cell viability or differentiation capability. Superparamagnetic iron oxide was disseminated in both the soma and neurites. CONCLUSIONS: These findings indicate that HVJ-Es are an effective vehicle for SPIO transfection of NPCs. The intracellular localization after differentiation raises the question as to the capability of MRI to distinguish cell migration from axonal or dendritic growth in vivo.

Animals↗