Formation of elongated giant mitochondria in DFO-induced cellular senescence: Involvement of enhanced fusion process through modulation of Fis1

Yoon, Young-Sil,Yoon, Dong-Sun,Lim, In Kyoung,Yoon, Soo-Han,Chung, Hae-Young,Rojo, Manuel,Malka, Florence,Jou, Mei-Jie,Martinou, Jean-Claude,Yoon, Gyesoon Liss 2006 Journal of Cellular Physiology Vol.209 No.2

<P>Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H<SUB>2</SUB>O<SUB>2</SUB> (200 µM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF β1- or H<SUB>2</SUB>O<SUB>2</SUB>-induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-ΔTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence. J. Cell. Physiol. 209: 468–480, 2006. © 2006 Wiley-Liss, Inc.</P>

Cellular localization of the full-length isoform of the type 2 corticotropin releasing factor receptor in the postnatal mouse cerebellar cortex

Lee, Kyung-Hoon,Bishop, Georgia A.,Tian, Jin Bin,Jang, Yoon-Jin,Bui, Bao Chi,Nguyen, Truong Le Xuan,Ahn, Jee-Yin,King, James S. Liss 2007 Journal of neuroscience research Vol.85 No.9

<P>Corticotropin releasing factor (CRF) and its cognate receptors, defined as Type 1 and Type 2 have been localized within the cerebellum. The Type 2 CRF receptor (CRF-R2) is known to have both a full length (CRF-R2α) and a truncated (CRF-R2α-tr) isoform. A recent study documented CRF-R2α primarily in Bergann glia and astrocytes, as well as in populations of Purkinje cells in the adult cerebellum. The goal of the present study is to determine if CRF-R2α is present in the postnatal cerebellum, and if so to describe its cellular distribution. RT-PCR data showed that CRF-R2α is expressed in the mouse cerebellum from birth through postnatal day 21. Between birth and P14, CRF-R2α-immunoreactivity was localized within the somata of Purkinje cells, and migrating GABAergic interneurons. GFAP-immunoreactive astrocytes, including Bergmann glia, also expressed CRF-R2α-immunoreactivity from P3-P14. There is a change, however, in CRF-R2α immunolabeling within neurons as the cerebellum matures. Compared to its expression in the adult cerebellum, Purkinje cells, and GABAergic interneurons showed more extensive CRF-R2α immunolabeling during early postnatal development. We postulate that CRF-R2α could be involved in developmental events related to the survival and differentiation of Purkinje cells and GABAergic neurons, whereas in the adult, this isoform of the CRF receptor family is likely involved in modulating Bergmann glia that have been shown to play a role in regulating the synaptic environment around Purkinje neurons. © 2007 Wiley-Liss, Inc.</P>

Four novel RUNX2 mutations including a splice donor site result in the cleidocranial dysplasia phenotype

Kim, Hyo-Jin,Nam, Soon-Hyeun,Kim, Hyun-Jung,Park, Hyo-Sang,Ryoo, Hyun-Mo,Kim, Shin-Yoon,Cho, Tae-Joon,Kim, Seung-Gon,Bae, Suk-Chul,Kim, In-San,Stein, Janet L.,van Wijnen, Andre J.,Stein, Gary S.,Lian, Liss 2006 Journal of Cellular Physiology Vol.207 No.1

<P>Cleidocranial dysplasia (CCD) is an autosomal dominant disorder caused by haploinsufficiency of the RUNX2 gene. In this study, we analyzed by direct sequencing RUNX2 mutations from eleven CCD patients. Four of seven mutations were novel: two nonsense mutations resulted in a translational stop at codon 50 (Q50X) and 112 (E112X); a missense mutation converted arginine to glycine at codon 131 (R131G); and an exon 1 splice donor site mutation (donor splice site GT/AT, IVS1 + 1G > A) at exon 1–intron junction resulted in the deletion of QA stretch contained in exon 1 of RUNX2. We focused on the functional analysis of the IVS1 + 1G > A mutation. A full-length cDNA of this mutation was cloned (RUNX2Δe1) and expressed in Chinese hamster ovary (CHO) and HeLa cells. Functional analysis of RUNX2Δe1 was performed with respect to protein stability, nuclear localization, DNA binding, and transactivation activity of a downstream RUNX2 target gene. Protein stability of RUNX2Δe1 is similar to wild-type RUNX2 as determined by Western blot analysis. Subcellular localization of RUNX2Δe1, assessed by in situ immunofluorescent staining, was observed with partial retention in both the nucleus and cytoplasm. This finding is in contrast to RUNX2 wild-type, which is detected exclusively in the nucleus. DNA binding activity was also compromised by the RUNX2Δe1 in gel shift assay. Finally, RUNX2Δe1 blocked transactivation of the osteocalcin gene determined by transient transfection assay. Our findings demonstrate for the first time that the CCD phenotype can be caused by a splice site mutation, which results in the deletion of N-terminus amino acids containing the QA stretch in RUNX2 that contains a previously unidentified second nuclear localization signal (NLS). We postulate that the QA sequence unique to RUNX2 contributes to a competent structure of RUNX2 that is required for nuclear localization, DNA binding, and transactivation function. J. Cell. Physiol. 207: 114–122, 2006. © 2005 Wiley-Liss, Inc.</P>

Protein kinase C-ERK1/2 signal pathway switches glucose depletion-induced necrosis to apoptosis by regulating superoxide dismutases and suppressing reactive oxygen species production in A549 lung cancer cells

Kim, Cho Hee,Han, Song Iy,Lee, Su Yeon,Youk, Hyun Suk,Moon, Ji Young,Duong, Hong Quan,Park, Min Jung,Joo, Young Mi,Park, Hye Gyeong,Kim, Yung Jin,Yoo, Mi Ae,Lim, Sung-Chul,Kang, Ho Sung Liss 2007 Journal of Cellular Physiology Vol.211 No.2

<P>Cells typically die by either apoptosis or necrosis. However, the consequences of apoptosis and necrosis are quite different for a whole organism. In the case of apoptosis, the cell content remains packed in the apoptotic bodies that are removed by marcrophages, and thereby inflammation does not occur; during necrosis, the cell membrane is ruptured, and the cytosolic constituents are released into the extracellular space provoking inflammation. Recently, inflammation and necrosis have been suggested to promote tumor growth. We investigated the molecular mechanism underlying cell death in response to glucose depletion (GD), a common characteristic of the tumor microenvironment. GD induced necrosis through production of reactive oxygen species (ROS) in A549 lung carcinoma cells. Inhibition of ROS production by N-acetyl-L-cysteine and catalase prevented necrosis and switched the cell death mode to apoptosis that depends on mitochondrial death pathway involving caspase-9 and caspase-3 activation, indicating a critical role of ROS in determination of GD-induced cell death mode. We demonstrate that protein kinase C-dependent extracellular regulated kinase 1/2 (ERK1/2) activation also switched GD-induced necrosis to apoptosis through inhibition of ROS production possibly by inducing manganese superoxide dismutase (SOD) expression and by preventing GD-induced degradation of cupper zinc SOD. Thus, these results suggest that GD-induced cell death mode is determined by the protein kinase C/ERK1/2 signal pathway that regulates MnSOD and CuZnSOD and that these antioxidants may exert their known tumor suppressive activities by inducing necrosis-to-apoptosis switch. J. Cell. Physiol. 211: 371–385, 2007. © 2007 Wiley-Liss, Inc.</P>

The suppressive effect of myeloid elf-1-like factor (MEF) in osteogenic differentiation

Kim, Youn-Jeong,Kim, Byung-Gyu,Lee, Seung-Jin,Lee, Ho-Kyung,Lee, Sang-Han,Ryoo, Hyun-Mo,Cho, Je-Yoel Liss 2007 Journal of Cellular Physiology Vol.211 No.1

<P>Myeloid Elf-1 like factor (MEF) is a member of the Ets transcription factor family. Ets family proteins control the expression of genes that are critical for biological processes such as proliferation, differentiation, and cell death. Some of Ets factors are also known to regulate bone development. In this study, we investigated the role of MEF in osteoblast differentiation. MEF expression was highest early in the differentiation of MC3T3-E1 osteoblasts and was reduced by treatment with BMP-2. The expression of MEF suppressed the alkaline phosphatase activity and expression induced by BMP-2 stimulation and mediated by Runx2. The expression of MEF also reduces osteocalcin mRNA levels, and mineralization in MC3T3-E1 cells. We found that the MEF-mediated suppression of osteogenic differentiation was critically related to Runx2 regulation. The MEF and Runx2 proteins physically interact to form a complex, and this interaction interferes with Runx2 binding to the cis-acting element OSE2 derived from the osteocalcin promoter. Co-transfection of MEF inhibited the 6xOSE2-luciferase reporter activity induced by Runx2. In addition, MEF stimulated the transcription of a negative mediator Msx2, and a transcriptional repressor, Mab21L1, and suppressed the transcription of a positive mediator, Dlx5 in osteoblast differentiation. MEF overexpression stimulated C2C12 cell proliferation. Together, our findings suggest that MEF promotes cell proliferation and functions as a negative regulator of osteogenic differentiation by directly interacting with Runx2 and suppressing its transcriptional activity. J. Cell. Physiol. 211: 253–260, 2007. © 2006 Wiley-Liss, Inc.</P>

Silibinin polarizes Th1/Th2 immune responses through the inhibition of immunostimulatory function of dendritic cells

Lee, Jun Sik,Kim, Sang Gap,Kim, Hyung Keun,Lee, Tae-Hyung,Jeong, Young-Il,Lee, Chang-Min,Yoon, Man-Soo,Na, Yong Jin,Suh, Dong-Soo,Park, Nam Cheol,Choi, In-hak,Kim, Gi-Young,Choi, Yung Hyun,Chung, Hae Liss 2007 Journal of Cellular Physiology Vol.210 No.2

<P>Silibinin is the primary active compound in silymarin. It has been demonstrated to exert anti-carcinogenic effects and hepato-protective effects. However, the effects of silibinin on the maturation and immunostimulatory activities exhibited by dendritic cells (DCs) remain, for the most part, unknown. In this study, we have attempted to determine whether silibinin can influence surface molecule expression, dextran uptake, cytokine production, capacity to induce T-cell differentiation, and the signaling pathways underlying these phenomena in murine bone marrow-derived DCs. Silibinin was shown to significantly suppress the expression of CD80, CD86, MHC class I, and MHC class II in the DCs, and was also associated with impairments of LPS-induced IL-12 expression in the DCs. Silibinin-treated DCs proved highly efficient with regard to Ag capture via mannose receptor-mediated endocytosis. Silibinin also inhibited the LPS-induced activation of MAPKs and the nuclear translocation of the NF-κB p65 subunit. Additionally, silibinin-treated DCs evidenced an impaired induction of Th1 response, and a normal cell-mediated immune response. These findings provide new insight into the immunopharmacological functions of silibinin, especially with regard to their impact on the DCs. These findings expand our current understanding of the immunopharmacological functions of silibinin, and may prove useful in the development of therapeutic adjuvants for acute and chronic DC-associated diseases. J. Cell. Physiol. 210: 385–397, 2007. © 2006 Wiley-Liss, Inc.</P>

Up-regulation of Bak and Bim via JNK downstream pathway in the response to nitric oxide in human glioblastoma cells

Jin, Hyeon-Ok,Park, In-Chul,An, Sungkwan,Lee, Hyung-Chahn,Woo, Sang-Hyeok,Hong, Young-Joon,Lee, Su-Jae,Park, Myung-Jin,Yoo, Doo-Hyun,Rhee, Chang-Hun,Hong, Seok-Il Liss 2006 Journal of Cellular Physiology Vol.206 No.2

<P>Nitric oxide (NO) is a chemical messenger implicated in neuronal damage associated with ischemia neurodegenerative disease and excitotoxicity. In the present study, we examined the biological effects of NO and its mechanisms in human malignant glioblastoma cells. Addition of a NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), induced apoptosis in U87MG human glioblastoma cells, accompanied by opening mitochondrial permeability transition pores, release of cytochrome c and AIF, and subsequently by caspase activation. NO-induced apoptosis occurred concurrently with significantly increased levels of the Bak and Bim. Treatment with SNAP resulted in sustained activation of JNK and its downstream pathway, c-Jun/AP-1. The expression of dominant-negative (DN)-JNK1 and DN-c-Jun suppressed the activation of AP-1, the induction of Bak and Bim, and the SNAP-induced apoptosis. In addition, de novo protein synthesis was required for the initiation of apoptosis in that the protein synthesis inhibitor, cycloheximide (CHX), inhibited NO-induced apoptotic cell death as well as up-regulation of Bak and Bim. These results suggest that NO activates an apoptotic cascade, involving sustained JNK activation, AP-1 DNA binding activity, and subsequent Bak and Bim induction, followed by cytochrome c and AIF releases and caspases cascade activation, resulting in human malignant brain tumor cell death. J. Cell. Physiol. 206: 477–486, 2006. © 2005 Wiley-Liss, Inc.</P>

Technique of semiautomatic surface reconstruction of the visible Korean human data using commercial software

Park, Jin Seo,Shin, Dong Sun,Chung, Min Suk,Hwang, Sung Bae,Chung, Jinoh WILEY-LISS, INC 2007 Clinical Anatomy Vol. No.

<P>This article describes the technique of semiautomatic surface reconstruction of anatomic structures using widely available commercial software. This technique would enable researchers to promptly and objectively perform surface reconstruction, creating three-dimensional anatomic images without any assistance from computer engineers. To develop the technique, we used data from the Visible Korean Human project, which produced digitalized photographic serial images of an entire cadaver. We selected 114 anatomic structures (skin [1], bones [32], knee joint structures [7], muscles [60], arteries [7], and nerves [7]) from the 976 anatomic images which were generated from the left lower limb of the cadaver. Using Adobe Photoshop, the selected anatomic structures in each serial image were outlined, creating a segmented image. The Photoshop files were then converted into Adobe Illustrator files to prepare isolated segmented images, so that the contours of the structure could be viewed independent of the surrounding anatomy. Using Alias Maya, these isolated segmented images were then stacked to construct a contour image. Gaps between the contour lines were filled with surfaces, and three-dimensional surface reconstruction could be visualized with Rhinoceros. Surface imperfections were then corrected to complete the three-dimensional images in Alias Maya. We believe that the three-dimensional anatomic images created by these methods will have widespread application in both medical education and research. Clin. Anat. 20:871–879, 2007. © 2007 Wiley-Liss, Inc.</P>

Electrophysiological correlates of behavioral response inhibition in patients with obsessive–compulsive disorder

Kim, Myung-Sun,Kim, Young Youn,Yoo, So Young,Kwon, Jun Soo WILEY-LISS, INC 2007 Depression and Anxiety Vol.24 No.1

<P>In this study, we have attempted to determine the electrophysiological correlates of behavioral response inhibition in patients with obsessive–compulsive disorder (OCD). To evaluate response inhibition ability, we have used the Go/NoGo task and measured N2 and P3 event-related potential (ERP) components. Both the OCD and control groups exhibited greater and more frontally distributed N2 and P3 amplitudes in the NoGo condition compared to what we observed in the Go condition. However, the patients with OCD also manifested reduced NoGo–N2 and Go–N2 amplitudes at the frontocentral electrode sites compared to the controls. In addition, the NoGo–N2 amplitudes were more posteriorly distributed in patients with OCD than in controls. The NoGo–N2 amplitudes and latencies measured at the central sites were also negatively correlated with the obsession score on the Yale–Brown Obsessive Compulsive Scale (Y-BOCS). The OCD and control groups were comparable with regard to Go–P3 and NoGo–P3 amplitude and latencies. Our findings suggest dysfunctions in frontal regions mediating response inhibition in OCD, consistent with the involvement of response inhibition in the pathophysiology of this disorder. In addition, NoGo–N2 seems to result in more accurate response inhibition measurements in patients with OCD than does NoGo–P3. Depression and Anxiety 24:22–31, 2007. © 2006 Wiley-Liss, Inc.</P>

Optimization of the concentration of autologous serum for generation of leukemic dendritic cells from acute myeloid leukemic cells for clinical immunotherapy

Choi, Bo-Hwa,Kang, Hyun-Kyu,Park, Jung-Sun,Kim, Sang-Ki,Pham, Than-Nhan Nguyen,Zhu, Xiao-Wei,Cho, Duck,Nam, Jong-Hee,Chung, Ik-Joo,Kim, Young-Jin,Rhee, Joon-Haeng,Kim, Hyeoung-Joon,Lee, Je-Jung Wiley-Liss 2006 JOURNAL OF CLINICAL APHERESIS Vol.21 No.4

<P>Clinical application of immunotherapy for acute myeloid leukemia (AML) requires the efficient induction of dendritic cells (DCs) from AML blast cells using in vitro culture. We examined the effect of autologous serum on the properties of leukemic DCs derived from leukemic cells of AML patients by culture in AIM-V medium with GM-CSF, IL-4, TNF-α, and 0, 2, 5, or 10% human autologous serum. The expressions of CD80, CD83, CD86, and HLA-DR were upregulated under all culture conditions; however, 10% autologous serum induced the highest expression levels of several molecules. The capacity of leukemic DCs to stimulate allogeneic T cells increased with increasing serum concentration. Stimulation of autologous CD3+ T cells with leukemic DCs grown in the presence of various concentrations of autologous serum resulted in induction of more IFN-γ-secreting cells than was the case for unprimed CD3+ T cells. Leukemic DCs cultured with 10% autologous serum induced the highest numbers of IFN-γ-secreting cells and CD8+CD56+ T cells from autologous T cells. These results suggest that culture of AML blast cells in the presence of autologous serum could be used to generate leukemic DCs for immunotherapy against AML. The highest serum concentration appeared optimal for generating the most potent leukemic DCs. J. Clin. Apheresis. © 2006 Wiley-Liss, Inc.</P>