Publications by authors named "Candice G T Tahimic"

18 Publications

  • Page 1 of 1

Circulating miRNA Spaceflight Signature Reveals Targets for Countermeasure Development.

Cell Rep 2020 Dec 25;33(10):108448. Epub 2020 Nov 25.

KBR, Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA 94035, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

We have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated, short-duration and long-duration spaceflight. Previous studies have identified miRNAs that regulate rodent responses to spaceflight in low-Earth orbit, and we have confirmed the expression of these proposed spaceflight-associated miRNAs in rodents reacting to simulated spaceflight conditions. Moreover, astronaut samples from the NASA Twins Study confirmed these expression signatures in miRNA sequencing, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase accessible chromatin (scATAC-seq) data. Additionally, a subset of these miRNAs (miR-125, miR-16, and let-7a) was found to regulate vascular damage caused by simulated deep space radiation. To demonstrate the physiological relevance of key spaceflight-associated miRNAs, we utilized antagomirs to inhibit their expression and successfully rescue simulated deep-space-radiation-mediated damage in human 3D vascular constructs.
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http://dx.doi.org/10.1016/j.celrep.2020.108448DOI Listing
December 2020

Neutrophil-to-Lymphocyte Ratio: A Biomarker to Monitor the Immune Status of Astronauts.

Front Immunol 2020 2;11:564950. Epub 2020 Nov 2.

Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA, United States.

A comprehensive understanding of spaceflight factors involved in immune dysfunction and the evaluation of biomarkers to assess in-flight astronaut health are essential goals for NASA. An elevated neutrophil-to-lymphocyte ratio (NLR) is a potential biomarker candidate, as leukocyte differentials are altered during spaceflight. In the reduced gravity environment of space, rodents and astronauts displayed elevated NLR and granulocyte-to-lymphocyte ratios (GLR), respectively. To simulate microgravity using two well-established ground-based models, we cultured human whole blood-leukocytes in high-aspect rotating wall vessels (HARV-RWV) and used hindlimb unloaded (HU) mice. Both HARV-RWV simulation of leukocytes and HU-exposed mice showed elevated NLR profiles comparable to spaceflight exposed samples. To assess mechanisms involved, we found the simulated microgravity HARV-RWV model resulted in an imbalance of redox processes and activation of myeloperoxidase-producing inflammatory neutrophils, while antioxidant treatment reversed these effects. In the simulated microgravity HU model, mitochondrial catalase-transgenic mice that have reduced oxidative stress responses showed reduced neutrophil counts, NLR, and a dampened release of selective inflammatory cytokines compared to wildtype HU mice, suggesting simulated microgravity induced oxidative stress responses that triggered inflammation. In brief, both spaceflight and simulated microgravity models caused elevated NLR, indicating this as a potential biomarker for future in-flight immune health monitoring.
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http://dx.doi.org/10.3389/fimmu.2020.564950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667275PMC
November 2020

Skeletal tissue regulation by catalase overexpression in mitochondria.

Am J Physiol Cell Physiol 2020 10 12;319(4):C734-C745. Epub 2020 Aug 12.

Space Biosciences Division, NASA Ames Research Center, Moffett Field, California.

Accumulation of oxidative damage from excess reactive oxygen species (ROS) may contribute to skeletal aging and mediate adverse responses to physiological challenges. Wild-type (WT) mice and transgenic mice (male, 16 wk of age) with human catalase targeted to the mitochondria (mCAT) were analyzed for skeletal responses to the remodeling stimuli of combined hind-limb unloading and exposure to ionizing radiation (Cs, 2 Gy). Treatment for 2 wk caused lipid peroxidation in the bones WT but not mCAT mice, showing that transgene expression mitigated oxidative stress. Ex vivo osteoblast colony growth rate was 95% greater in mCAT than WT mice and correlated with catalase activity levels ( < 0.005, r = 0.67), although terminal osteoblast and osteoclast differentiation were unaffected. mCAT mice had lower cancellous bone volume and cortical size than WT mice. Ambulatory control mCAT animals also displayed reduced cancellous and cortical structural properties compared with control WT mice. In mCAT but not WT mice, treatment caused an unexpectedly rapid radial expansion (+8% cortical area, +22% moment of inertia), reminiscent of compensatory bone growth during advancing age. In contrast, treatment caused similar structural deficits in cancellous tissue of mCAT and WT mice. In sum, mitochondrial ROS signaling via HO was important for the acquisition of adult bone structure and catalase overexpression failed to protect cancellous tissue from treatment. In contrast, catabolic stimuli caused radial expansion in mCAT not WT mice, suggesting that mitochondrial ROS in skeletal cells act to suppress tissue turnover in response to remodeling challenges.
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http://dx.doi.org/10.1152/ajpcell.00068.2020DOI Listing
October 2020

Dietary countermeasure mitigates simulated spaceflight-induced osteopenia in mice.

Sci Rep 2020 04 16;10(1):6484. Epub 2020 Apr 16.

Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA, 94035, USA.

Spaceflight is a unique environment that includes at least two factors which can negatively impact skeletal health: microgravity and ionizing radiation. We have previously shown that a diet supplemented with dried plum powder (DP) prevented radiation-induced bone loss in mice. In this study, we investigated the capacity of the DP diet to prevent bone loss in mice following exposure to simulated spaceflight, combining microgravity (by hindlimb unloading) and radiation exposure. The DP diet was effective at preventing most decrements in bone micro-architectural and mechanical properties due to hindlimb unloading alone and simulated spaceflight. Furthermore, we show that the DP diet can protect osteoprogenitors from impairments resulting from simulated microgravity. Based on our findings, a dietary supplementation with DP could be an effective countermeasure against the skeletal deficits observed in astronauts during spaceflight.
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http://dx.doi.org/10.1038/s41598-020-63404-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162976PMC
April 2020

Influence of Social Isolation During Prolonged Simulated Weightlessness by Hindlimb Unloading.

Front Physiol 2019 13;10:1147. Epub 2019 Sep 13.

Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA, United States.

The hindlimb unloading (HU) model has been used extensively to simulate the cephalad fluid shift and musculoskeletal disuse observed in spaceflight with its application expanding to study immune, cardiovascular and central nervous system responses, among others. Most HU studies are performed with singly housed animals, although social isolation also can substantially impact behavior and physiology, and therefore may confound HU experimental results. Other HU variants that allow for paired housing have been developed although no systematic assessment has been made to understand the effects of social isolation on HU outcomes. Hence, we aimed to determine the contribution of social isolation to tissue responses to HU. To accomplish this, we developed a refinement to the traditional NASA Ames single housing HU system to accommodate social housing in pairs, retaining desirable features of the original design. We conducted a 30-day HU experiment with adult, female mice that were either singly or socially housed. HU animals in both single and social housing displayed expected musculoskeletal deficits versus housing matched, normally loaded (NL) controls. However, select immune and hypothalamic-pituitary-adrenal (HPA) axis responses were differentially impacted by the HU social environment relative to matched NL controls. HU led to a reduction in % CD4 T cells in singly housed, but not in socially housed mice. Unexpectedly, HU increased adrenal gland mass in socially housed but not singly housed mice, while social isolation increased adrenal gland mass in NL controls. HU also led to elevated plasma corticosterone levels at day 30 in both singly and socially housed mice. Thus, musculoskeletal responses to simulated weightlessness are similar regardless of social environment with a few differences in adrenal and immune responses. Our findings show that combined stressors can mask, not only exacerbate, select responses to HU. These findings further expand the utility of the HU model for studying possible combined effects of spaceflight stressors.
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http://dx.doi.org/10.3389/fphys.2019.01147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753329PMC
September 2019

Mice Exposed to Combined Chronic Low-Dose Irradiation and Modeled Microgravity Develop Long-Term Neurological Sequelae.

Int J Mol Sci 2019 Aug 22;20(17). Epub 2019 Aug 22.

Division of Biomedical Engineering Sciences (BMES), Department of Basic Sciences, Loma Linda University, Loma Linda, CA 92354, USA.

Spaceflight poses many challenges for humans. Ground-based analogs typically focus on single parameters of spaceflight and their associated acute effects. This study assesses the long-term transcriptional effects following single and combination spaceflight analog conditions using the mouse model: simulated microgravity via hindlimb unloading (HLU) and/or low-dose γ-ray irradiation (LDR) for 21 days, followed by 4 months of readaptation. Changes in gene expression and epigenetic modifications in brain samples during readaptation were analyzed by whole transcriptome shotgun sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). The results showed minimal gene expression and cytosine methylation alterations at 4 months readaptation within single treatment conditions of HLU or LDR. In contrast, following combined HLU+LDR, gene expression and promoter methylation analyses showed multiple altered pathways involved in neurogenesis and neuroplasticity, the regulation of neuropeptides, and cellular signaling. In brief, neurological readaptation following combined chronic LDR and HLU is a dynamic process that involves pathways that regulate neuronal function and structure and may lead to late onset neurological sequelae.
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http://dx.doi.org/10.3390/ijms20174094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747492PMC
August 2019

Redox Signaling and Its Impact on Skeletal and Vascular Responses to Spaceflight.

Int J Mol Sci 2017 Oct 16;18(10). Epub 2017 Oct 16.

Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA 94035, USA.

Spaceflight entails exposure to numerous environmental challenges with the potential to contribute to both musculoskeletal and vascular dysfunction. The purpose of this review is to describe current understanding of microgravity and radiation impacts on the mammalian skeleton and associated vasculature at the level of the whole organism. Recent experiments from spaceflight and ground-based models have provided fresh insights into how these environmental stresses influence mechanisms that are related to redox signaling, oxidative stress, and tissue dysfunction. Emerging mechanistic knowledge on cellular defenses to radiation and other environmental stressors, including microgravity, are useful for both screening and developing interventions against spaceflight-induced deficits in bone and vascular function.
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http://dx.doi.org/10.3390/ijms18102153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666834PMC
October 2017

Dose- and Ion-Dependent Effects in the Oxidative Stress Response to Space-Like Radiation Exposure in the Skeletal System.

Int J Mol Sci 2017 Oct 10;18(10). Epub 2017 Oct 10.

Bone and Signaling Laboratory, Space BioSciences Division, NASA Ames Research Center, Mail-Stop 236-7, Moffett Field, CA 94035, USA.

Space radiation may pose a risk to skeletal health during subsequent aging. Irradiation acutely stimulates bone remodeling in mice, although the long-term influence of space radiation on bone-forming potential (osteoblastogenesis) and possible adaptive mechanisms are not well understood. We hypothesized that ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-weeks old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 MeV/n) or high-LET Fe ions (600 MeV/n) using either low (5 or 10 cGy) or high (50 or 200 cGy) doses at NASA's Space Radiation Lab. Five weeks or one year after irradiation, tissues were harvested and analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed by RT-PCR during the proliferative or mineralizing phase of growth, and differentiation was analyzed by imaging mineralized nodules. As expected, a high dose (200 cGy), but not lower doses, of either Fe or protons caused a loss of cancellous bone volume/total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; Fe (200 cGy) inhibited osteoblastogenesis by more than 90% (5 weeks and 1 year post-IR). After 5 weeks, irradiation (protons or Fe) caused few changes in gene expression levels during osteoblastogenesis, although a high dose Fe (200 cGy) increased and . The addition of exogenous superoxide dismutase (SOD) protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET (Cs γ) when irradiated in vitro, but had limited protective effects on high-LET Fe-exposed cells. In sum, either protons or Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET Fe increased redox-related gene expression, albeit to a limited extent, and inhibited osteoblastogenesis. Doses below 50 cGy did not elicit widespread responses in any parameter measured. We conclude that high-LET irradiation at 200 cGy impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss.
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http://dx.doi.org/10.3390/ijms18102117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666799PMC
October 2017

Regulation of Ligand and Shear Stress-induced Insulin-like Growth Factor 1 (IGF1) Signaling by the Integrin Pathway.

J Biol Chem 2016 Apr 10;291(15):8140-9. Epub 2016 Feb 10.

From the Endocrine Research Unit, Veterans Affairs Medical Center, San Francisco, California 94121, University of California, San Francisco, California 94158,

Mechanical loading of the skeleton, as achieved during daily movement and exercise, preserves bone mass and stimulates bone formation, whereas skeletal unloading from prolonged immobilization leads to bone loss. A functional interplay between the insulin-like growth factor 1 receptor (IGF1R), a major player in skeletal development, and integrins, mechanosensors, is thought to regulate the anabolic response of osteogenic cells to mechanical load. The mechanistic basis for this cross-talk is unclear. Here we report that integrin signaling regulates activation of IGF1R and downstream targets in response to both IGF1 and a mechanical stimulus. In addition, integrins potentiate responsiveness of IGF1R to IGF1 and mechanical forces. We demonstrate that integrin-associated kinases, Rous sarcoma oncogene (SRC) and focal adhesion kinase (FAK), display distinct actions on IGF1 signaling; FAK regulates IGF1R activation and its downstream effectors, AKT and ERK, whereas SRC controls signaling downstream of IGF1R. These findings linked to our observation that IGF1 assembles the formation of a heterocomplex between IGF1R and integrin β3 subunit indicate that the regulation of IGF1 signaling by integrins proceeds by direct receptor-receptor interaction as a possible means to translate biomechanical forces into osteoanabolic signals.
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http://dx.doi.org/10.1074/jbc.M115.693598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825016PMC
April 2016

Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone Formation During Fracture Healing.

J Bone Miner Res 2015 Sep;30(9):1572-84

Endocrine Unit, VA Medical Center and University of California, San Francisco, CA, USA.

Insulin-like growth factors (IGFs) are important local regulators during fracture healing. Although IGF1 deficiency is known to increase the risk of delayed union or non-union fractures in the elderly population, the underlying mechanisms that contribute to this defect remains unclear. In this study, IGF1 signaling during fracture healing was investigated in an osteoblast-specific IGF1 receptor (IGF1R) conditional knockout (KO) mouse model. A closed tibial fracture was induced in IGF1R(flox/flox) /2.3-kb α1(1)-collagen-Cre (KO) and IGF1R(flox/flox) (control) mice aged 12 weeks. Fracture callus samples and nonfractured tibial diaphysis were collected and analyzed by μCT, histology, immunohistochemistry, histomorphometry, and gene expression analysis at 10, 15, 21, and 28 days after fracture. A smaller size callus, lower bone volume accompanied by a defect in mineralization, bone microarchitectural abnormalities, and a higher cartilage volume were observed in the callus of these KO mice. The levels of osteoblast differentiation markers (osteocalcin, alkaline phosphatase, collagen 1α1) were significantly reduced, but the early osteoblast transcription factor runx2, as well as chondrocyte differentiation markers (collagen 2α1 and collagen 10α1) were significantly increased in the KO callus. Moreover, increased numbers of osteoclasts and impaired angiogenesis were observed during the first 15 days of fracture repair, but decreased numbers of osteoclasts were found in the later stages of fracture repair in the KO mice. Although baseline nonfractured tibias of KO mice had decreased trabecular and cortical bone compared to control mice, subsequent studies with mice expressing the 2.3-kb α1(1)-collagen-Cre ERT2 construct and given tamoxifen at the time of fracture and so starting with comparable bone levels showed similar impairment in fracture repair at least initially. Our data indicate that not only is the IGF1R in osteoblasts involved in osteoblast differentiation during fracture repair, but it plays an important role in coordinating chondrocyte, osteoclast, and endothelial responses that all contribute to the endochondral bone formation required for normal fracture repair.
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http://dx.doi.org/10.1002/jbmr.2510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690481PMC
September 2015

Anabolic effects of IGF-1 signaling on the skeleton.

Front Endocrinol (Lausanne) 2013 4;4. Epub 2013 Feb 4.

Endocrine Research Unit, Veterans Affairs Medical Center, Department of Medicine, University of California San Francisco, CA, USA.

This review focuses on the anabolic effects of IGF-1 signaling on the skeleton, emphasizing the requirement for IGF-1 signaling in normal bone formation and remodeling. We first discuss the genomic context, splicing variants, and species conservation of the IGF-1 locus. The modulation of IGF-1 action by growth hormone (GH) is then reviewed while also discussing the current model which takes into account the GH-independent actions of IGF-1. Next, the skeletal phenotypes of IGF-1-deficient animals are described in both embryonic and postnatal stages of development, which include severe dwarfism and an undermineralized skeleton. We then highlight two mechanisms by which IGF-1 exerts its anabolic action on the skeleton. Firstly, the role of IGF-1 signaling in the modulation of anabolic effects of parathyroid hormone (PTH) on bone will be discussed, presenting in vitro and in vivo studies that establish this concept and the proposed underlying molecular mechanisms involving Indian hedgehog (Ihh) and the ephrins. Secondly, the crosstalk of IGF-1 signaling with mechanosensing pathways will be discussed, beginning with the observation that animals subjected to skeletal unloading by hindlimb elevation are unable to mitigate cessation of bone growth despite infusion with IGF-1 and the failure of IGF-1 to activate its receptor in bone marrow stromal cell cultures from unloaded bone. Disrupted crosstalk between IGF-1 signaling and the integrin mechanotransduction pathways is discussed as one of the potential mechanisms for this IGF-1 resistance. Next, emerging paradigms on bone-muscle crosstalk are examined, focusing on the potential role of IGF-1 signaling in modulating such interactions. Finally, we present a future outlook on IGF research.
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http://dx.doi.org/10.3389/fendo.2013.00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563099PMC
February 2013

Methyl CpG-binding protein isoform MeCP2_e2 is dispensable for Rett syndrome phenotypes but essential for embryo viability and placenta development.

J Biol Chem 2012 Apr 28;287(17):13859-67. Epub 2012 Feb 28.

Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira 187-8502, Japan.

Methyl CpG-binding protein 2 gene (MeCP2) mutations are implicated in Rett syndrome (RTT), one of the common causes of female mental retardation. Two MeCP2 isoforms have been reported: MeCP2_e2 (splicing of all four exons) and MeCP2_e1 (alternative splicing of exons 1, 3, and 4). Their relative expression levels vary among tissues, with MeCP2_e1 being more dominant in adult brain, whereas MeCP2_e2 is expressed more abundantly in placenta, liver, and skeletal muscle. In this study, we performed specific disruption of the MeCP2_e2-defining exon 2 using the Cre-loxP system and examined the consequences of selective loss of MeCP2_e2 function in vivo. We performed behavior evaluation, gene expression analysis, using RT-PCR and real-time quantitative PCR, and histological analysis. We demonstrate that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin. In addition, we reveal a specific requirement for MeCP2_e2 function in extraembryonic tissue, where selective loss of MeCP2_e2 results in placenta defects and up-regulation of peg-1, as determined by the parental origin of the mutant allele. Taken together, our findings suggest a novel role for MeCP2 in normal placenta development and illustrate how paternal X chromosome inactivation in extraembryonic tissues confers a survival disadvantage for carriers of a mutant maternal MeCP2_e2 allele. Moreover, our findings provide an explanation for the absence of reports on MeCP2_e2-specific exon 2 mutations in RTT. MeCP2_e2 mutations in humans may result in a phenotype that evades a diagnosis of RTT.
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http://dx.doi.org/10.1074/jbc.M111.309864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340147PMC
April 2012

Efficient integration of transgenes into a defined locus in human embryonic stem cells.

Nucleic Acids Res 2010 Apr 13;38(7):e96. Epub 2010 Jan 13.

Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.

Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency.
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http://dx.doi.org/10.1093/nar/gkp1234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853137PMC
April 2010

Ku70/80 modulates ATM and ATR signaling pathways in response to DNA double strand breaks.

J Biol Chem 2007 Apr 1;282(14):10138-45. Epub 2007 Feb 1.

Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishimachi, Yonago, Tottori 683-8503, Japan.

Double strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ataxia telangiectasia-mutated (ATM) and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATM- and Rad3-related (ATR) kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53(Ser18) phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2 h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53(Ser18) in Ku-deficient cells at 12 h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response.
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http://dx.doi.org/10.1074/jbc.M611880200DOI Listing
April 2007

Exogenous gene expression and growth regulation of hematopoietic cells via a novel human artificial chromosome.

J Hum Genet 2006 7;51(2):147-150. Epub 2005 Dec 7.

Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan.

A number of gene delivery systems are currently being developed for potential use in gene therapy. Here, we demonstrate the feasibility of 21deltaqHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. We first introduced a 21deltaqHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21deltaqHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21deltaqHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21deltaqHAC could still be detected at two weeks post-transplantation in immunodeficient mice. We also showed effective expansion of hematopoietic cells by introducing the 21deltaqHAC containing ScFvg, a gp130-based chimeric receptor that transmits growth signals in response to specific-antigen of this receptor. All of these results demonstrate the usefulness of HAC in gene therapy.
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http://dx.doi.org/10.1007/s10038-005-0334-9DOI Listing
October 2006

Construction of a novel expression system on a human artificial chromosome.

Biochem Biophys Res Commun 2005 Apr;329(3):1018-25

Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medical Sciences, Tottori University, 86 Nishicho, Yonago, Tottori 683-8503, Japan.

Efficient regulation of transgene would greatly facilitate the analysis of gene function in biological systems for basic research and clinical applications. The tetracycline-regulatable system (TRS) has proven to be a promising tool for such purposes. Despite their widespread application, a number of challenges are still associated with the use of TRS, including clonal variability in the regulation and copy number. We have recently constructed a novel human artificial chromosome (HAC) called 21DeltaqHAC. By housing a TRS-based DNA-PKcs expression cassette in this HAC, we were able to circumvent the problems associated with conventional TRS-based vectors. We achieved tight control of DNA-PKcs expression and rescued the radiosensitive phenotype of DNA-PKcs-deficient CHO cells. The combined use of HAC and the TRS serves as a model for controllable and fixed copy number expression vectors. Our study also demonstrates the suitability of the HAC to accommodate multi-subunit constructs such as that of the TRS.
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http://dx.doi.org/10.1016/j.bbrc.2005.02.079DOI Listing
April 2005

Coordinate downregulation of a novel imprinted transcript ITUP1 with PEG3 in glioma cell lines.

DNA Res 2004 Feb;11(1):37-49

Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, 86 Nishimachi, Yonago City, Tottori 683-8503, Japan.

The human paternally expressed gene 3 (PEG3) on chromosome 19q13.4 is one of the candidate tumor suppressor genes for glioma. We have previously reported that the epigenetic silencing of PEG3 expression in glioma cell lines is dependent on aberrant DNA methylation of an exonic CpG island. Here, we have identified three expressed sequence tags (ESTs), H80201, H78825 and AW197312, that exhibit paternal allele-specific expression, using human monochromosomal hybrids containing the paternal or maternal origin of PEG3 locus. The EST H80201 was shown to be expressed only from the paternal allele in normal human lymphoblasts by utilizing a single nucleotide polymorphism (SNP). Monoallelic expression of EST H80201 was also detected in non-tumor adult human brain tissues of gliomas. These ESTs were located directly adjacent to PEG3 in a head-to-head orientation. We have named this new transcript, imprinted transcript 1, which is located upstream but oppositely oriented to PEG3 (ITUP1). The ITUP1 showed a similar expression profile with PEG3 in glioma cell lines. Bisulfite genomic sequencing and reverse transcription (RT)-PCR analysis indicated that hypermethylation of the promoter region correlated with the absence of these transcripts. This suggests that ITUP1 and PEG3 are coordinately regulated, and that downregulation of the both genes may be important in the development of glioma.
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http://dx.doi.org/10.1093/dnares/11.1.37DOI Listing
February 2004

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

Biochem Biophys Res Commun 2003 Sep;309(3):558-66

Department of Molecular and Cell Genetics, Graduate School of Medical Science, Tottori University, Nishimachi 86, Yonago, Tottori 683-8503, Japan.

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.
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http://dx.doi.org/10.1016/j.bbrc.2003.08.029DOI Listing
September 2003