1.Molecular Culprits Generating Brain Tumor Stem Cells.
Brain Tumor Research and Treatment 2013;1(1):9-15
Despite current advances in multimodality therapies, such as surgery, radiotherapy, and chemotherapy, the outcome for patients with high-grade glioma remains fatal. Understanding how glioma cells resist various therapies may provide opportunities for developing new therapies. Accumulating evidence suggests that the main obstacle for successfully treating high-grade glioma is the existence of brain tumor stem cells (BTSCs), which share a number of cellular properties with adult stem cells, such as self-renewal and multipotent differentiation capabilities. Owing to their resistance to standard therapy coupled with their infiltrative nature, BTSCs are a primary cause of tumor recurrence post-therapy. Therefore, BTSCs are thought to be the main glioma cells representing a novel therapeutic target and should be eliminated to obtain successful treatment outcomes.
Adult Stem Cells
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Brain Neoplasms*
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Brain*
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Drug Therapy
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Glioma
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Humans
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Radiotherapy
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Recurrence
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Stem Cells*
2.Cellular characteristics of primary and immortal canine embryonic fibroblast cells.
Seungkwon YOU ; Jai Hee MOON ; Tae Kyung KIM ; Sung Chan KIM ; Jai Woo KIM ; Du Hak YOON ; Sungwook KWAK ; Ki Chang HONG ; Yun Jaie CHOI ; Hyunggee KIM
Experimental & Molecular Medicine 2004;36(4):325-335
Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.
Animals
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Catalytic Domain/genetics
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*Cell Aging/genetics
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Cell Line, Transformed
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Cell Transformation, Neoplastic
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Dogs
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Embryo/cytology
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Fibroblasts/*cytology/metabolism
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Gene Expression
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Protein p16/genetics
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Protein p53/genetics
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RNA, Messenger/analysis/metabolism
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Research Support, Non-U.S. Gov't
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Telomerase/genetics/metabolism
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ras Proteins/genetics/metabolism
3.Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death.
Young Ki CHOI ; Tae Kyung KIM ; Chul Joong KIM ; Joong Seob LEE ; Se Young OH ; Han Soo JOO ; Douglas N FOSTER ; Ki Chang HONG ; Seungkwon YOU ; Hyunggee KIM
Experimental & Molecular Medicine 2006;38(1):11-17
The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK-stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.
Animals
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Annexin A5/metabolism
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*Apoptosis
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Blotting, Western
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Cell Fractionation
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Cell Line
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Comparative Study
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Cytochrome c Group/metabolism
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Cytosol/chemistry
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DNA Fragmentation
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Enzyme Activation
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Gene Expression Regulation, Viral
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Hela Cells
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Humans
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Influenza A virus/*physiology
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Kinetics
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Mitochondria/metabolism/*physiology
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Precipitin Tests
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Proto-Oncogene Proteins c-bcl-2/genetics/metabolism
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Research Support, Non-U.S. Gov't
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Swine
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bcl-2-Associated X Protein/genetics/metabolism
4.Production of transgenic pigs using a pGFAP-CreER(T2)/EGFP(LoxP) inducible system for central nervous system disease models
Seon Ung HWANG ; Kiyoung EUN ; Junchul David YOON ; Hyunggee KIM ; Sang Hwan HYUN
Journal of Veterinary Science 2018;19(3):434-445
Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreER(T2)/LCMV-EGFP(LoxP) and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreER(T2)) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreER(T2)-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreER(T2)/EGFP(LoxP) recombination system via SCNT.
Animals, Genetically Modified
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Astrocytes
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Central Nervous System
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Cerebellum
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Cerebrum
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DNA
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Embryo Transfer
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Embryonic Development
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Estrogens
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Female
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Fibroblasts
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Fluorescent Antibody Technique
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Glial Fibrillary Acidic Protein
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Humans
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Nuclear Transfer Techniques
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Ovulation
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Polymerase Chain Reaction
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Pregnancy
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Real-Time Polymerase Chain Reaction
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Recombination, Genetic
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Regenerative Medicine
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RNA, Messenger
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Skin
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Swine
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Tissue Donors
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Toxicity Tests
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Transgenes
5.Human telomerase catalytic subunit (hTERT) suppresses p53-mediated anti-apoptotic response via induction of basic fibroblast growth factor.
Xun JIN ; Samuel BECK ; Young Woo SOHN ; Jun Kyum KIM ; Sung Hak KIM ; Jinlong YIN ; Xumin PIAN ; Sung Chan KIM ; Yun Jaie CHOI ; Hyunggee KIM
Experimental & Molecular Medicine 2010;42(8):574-582
Although human telomerase catalytic subunit (TERT) has several cellular functions including telomere homeostasis, genomic stability, cell proliferation, and tumorigenesis, the molecular mechanism underlying anti-apoptosis regulated by TERT remains to be elucidated. Here, we show that ectopic expression of TERT in spontaneously immortalized human fetal fibroblast (HFFS) cells, which are a telomerase- and p53-positive, leads to increases of cell proliferation and transformation, as well as a resistance to DNA damage response and inactivation of p53 function. We found that TERT and a mutant TERT (no telomerase activity) induce expression of basic fibroblast growth factor (bFGF), and ectopic expression of bFGF also allows cells to be resistant to DNA-damaging response and to suppress activation of p53 function under DNA-damaging induction. Furthermore, loss of TERT or bFGF markedly increases a p53 activity and DNA-damage sensitivity in HFFS, HeLa and U87MG cells. Therefore, our findings indicate that a novel TERT-bFGF axis accelerates the inactivation of p53 and consequent increase of resistance to DNA-damage response.
*Apoptosis
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*Catalytic Domain
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Cell Line, Transformed
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Cell Proliferation
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DNA Damage
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Fetus/cytology
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Fibroblast Growth Factor 2/*genetics/metabolism
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Fibroblasts/cytology/metabolism
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Gene Expression Regulation, Neoplastic
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Hela Cells
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Humans
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RNA, Messenger/genetics/metabolism
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Telomerase/deficiency/*metabolism
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Tumor Suppressor Protein p53/*metabolism