Science & Technology, Graduate School, Ajou University, Suwon 443-721, South ... Dept. of Biological science, Sookmyung women's University, Seoul 140-742.
Cancer Biology
Glutathione reverses delayed cell cycle progression by TTFA, a mitochondrial complex II inhibitor Hee Young Kim1, Hae-Ok Byun1, Jong-Jin Lim1 and Gyesoon Yoon1,2 1Dept. of Biochemistry and Molecular Biology, School of Medicine and 2Department of Molecular Science & Technology, Graduate School, Ajou University, Suwon 443-721, South Korea In aerobically growing cells, mitochondria play a pivotal role as ATP generator through oxidative phosphorylation (OXPHOS). Inhibition of the mitochondrial respiratory activity by specific inhibitors such as rotenone, antimycin A, and KCN has often been found to induce cell death in several cells. However, the effect of complex II inhibition on cellular fate has not been elucidated. In the present study, we investigated the effect of 2thenoyltrifluoroacetone (TTFA), a specific complex II inhibitor, on cell cycle progression and its underlying mechanism. Under asynchronous condition, TTFA delayed the cellular growth and eventually induced cell death, but did not change the pattern of cell cycle progression. However, when TTFA was applied to the cells synchronized at the late G1 phase by double-thymidine treatment, the time required for to return to nest G1 phase was lengthened from about 18 h to about 30 h. This was the result of its similar delayed effect (1.5 fold) on each phase (G1, S, and G2/M) of the cell cycle. Under exposure to TTFA decreased ATP level, increased production of reactive oxygen species (ROS), and related redox change of cellular glutathione were accompanied. The delayed effect was only slightly recovered by increasing ATP level with high glucose media, but was almost recovered (about 90%) by elevating intracellular glutathione using OTC. These results implied that oxidative stress generated by complex II inhibition might be mainly involved in delaying cell cycle progression.
G-80
G-82 Gene expression pattern of IL-18 gene over-expression in AML cells Yeon-Joo Yook, Min-Ji Seo, Jong-Hoon Park Dept. of Biological science, Sookmyung women's University, Seoul 140-742 In hematopoiesis, physiological cell death or apoptosis, can be induced by immunoregulatory cytokines. Interleukin (IL)-18, originally called IFN-r inducing factor (IGIF), is a novel proinflammatory cytokine, produced by activated macrophages and Kupper cells . IL-18 that is proinflammatory cytokines and concerned to cancer's immunity escaping is produced in KG-1(poor prognosis acute myeloid cell line) and not in NB4(good prognosis AML cell line). We exogeneously overexpress IL-18 in KG-1 and broadly estimate IL-18 effect on KG-1. Gene expression profiles between IL-18 treated KG-1 cells and IL-18 untreated KG-1 cells were analyzed by the use of an array consisting of 7488 human UniGene. Expression levels of 57 genes including stress related genes increased significantly at least 2 fold whereas expression of 48 genes decreased at least 2 fold relatively in IL-18 treated KG-1 cells. And we monitored significantly changed genes, CRYGC, NACA and NFKBIA, in several cancer cell lines.
G-83
Ginsenoside Rd enhances glutathione levels via NF-kappaB-dependent gamma-glutamylcysteine ligase induction
GAPDH acts as a negative regulator in the mTOR pathway through interacting with Rheb
Yuba Raj Pokharel and Keon Wook Kang BK21 Project Team, College of Pharmacy, Chosun University
Mi Nam Lee, Jae Yoon Kim, Sang Hoon Ha, Jung Hwan Kim, A-Ra Koh, Chang Sup Lee, Pann-Ghill Suh, Sung Ho Ryu Deptartment of Life Science and Division of Molecular and Life Sciences, POSTECH, Pohang, 790-784
Panax ginseng is widely used as herbal medicine in East Asia and the pharmacological effects of P. ginseng against certain chronic diseases might be explained by its antioxidative effects. Here, we show that ginsenoside Rd significantly increases both cellular glutathione (GSH) contents and the protein level of gamma-glutamylcysteine ligase (g-GCL) heavy chain in H4IIE cells (a rat hepatocyte cell line). Subcellular fractionation and Western blot analysis revealed that ginsenoside Rd increased the nuclear level of p65, but not of Nrf2. Moreover, ginsenoside Rd increased luciferase reporter gene activity in cells transfected with nuclear factor-kB (NF-kB) binding sitecontaining -1088 bp g-GCL promoter. However, ginsenoside Rd-inducible reporter activity was abolished when cells were transfected with NF-kB deletion mutant. These effects of ginsenoside Rd are suggested to underlie the putative anti-oxidative effect of Panax ginseng.
G-81
Rheb GTPase is a key player in the control of the cell growth and cell cycle progression. To understand the exact regulation mechanism of Rheb GTPases, we sought to identify the binding proteins of them. Here, we identified glyceraldehyde-3-phosphate dehydergenase (GAPDH) as a binding partner of Rheb. GAPDH binds to Rheb directly and its binding does not require Rheb nucleotide charging with either GTP or GDP. The substrate of GAPDH, glyceraldehyde-3-phosphate (G3P), reduced the interaction between GAPDH with Rheb. The inhibition of glycolytic flux using treatment of 2deoxyglucose or glucose depletion induced the interaction between Rheb and GAPDH, that this induced interaction was restored by co-incubation with G3P. We also found that overexpression of GAPDH inhibited the Rheb-induced activation of mTOR, that this negative effect of GAPDH resulted from binding with Rheb. In low glucose condition, increased Rheb-GAPDH binding reduced the Rheb-mTOR interaction and this effect was restored in high glucose condition. Taken together, we suggest that GAPDH acts as a negative regulator in the mTOR pathway through interaction with Rheb. This novel regulatory mechanism suggests that the mTOR pathway senses glucose sufficiency through direct interaction between glycolytic enzyme GAPDH and Rheb.
G-84
Gene expression profiling of human T-cells treated with mistletoe lectin in combination with anti-CD3/CD28 antibodies
Functional defects of mitochondria are involved in cellular transformation induced by oncogenic K-ras
Su-Yun Lyu1 and Won-Bong Park2 1International Research and Educational Institute for Integrated Medical Sciences (IREIIMS), Tokyo Women’s Medical University, Tokyo, Japan and 2Dept of Chemistry, Seoul Women’s University, Seoul, Korea
Jeong-Min Kim1, Hee Young Kim1, Yong-Hak Seo1, Su-Jae Lee3 and Gyesoon Yoon1,2 1Dept. of Biochemistry and Molecular Biology, School of Medicine and 2Department of Molecular Science & Technology, Ajou University, Suwon 443-721, 3Laboratory of Experimental Radiation Therapeutics, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, South Korea
Semiparasitic plants, mistletoes which are distributed in Europe and other East-Asian countries have long been recognized as therapeutic herbs. While the European mistletoe lectin has been studied intensively, less is known about Korean mistletoe lectin (Viscum album L. var coloratum agglutinin, VCA), especially as a suggested anticancer drug. This study aimed to identify the mechanisms of human T-cells when they were treated with VCA in combination with antiCD3/CD28 antibodies. Gene profiling was carried out via Affymetrix Human Genome U144 Plus 2.0 Array containing probes for 39,000 genes. Robust differences in gene expression were obtained by identifying genes that consistently changed by a minimum of 2-fold. Analysis of molecular function as designated by gene ontology (GO) identified differentially regulated mechanisms. When treated with VCA and anti-CD3/CD28 antibodies, human T-cells produced an increase (> 2 fold) in the expression of the following genes examined: PPP1R15A, DPF2, EP300, STK17A, TNFSF14, GADD45B, GLRX2, and GADD45B which are pro-apoptotic genes. In addition, the following genes which are anti-apoptotic were decreased: CD74, SPP1, CCL2, and API5. Also, VCA was found to induce inflammation cytokines (IL-1α, IL-1β, IL-6, IL8, INF-γand TNF) when used at low concentrations, but decreased the cytokines at high concentrations. The present study provides the first evidence that activated human T-cells are driven to undergo apoptosis after a treatment of high concentrations (10 µg/ml) of VCA. In addition, this concentration used to induce this apoptosis is not by itself proinflammatory.
Mitochondrial dysfunction has long been implicated in carcinogenesis since Warburg's hypothesis had been proposed. However, the detailed mechanisms on this phenomenon remain unclear, and it is still controversial whether the mitochondrial defect is a cause or a consequence whereas the role of oncogenes in cancer development has greatly been emphasized. Recently, we observed decreased expression levels of mitochondrial respiratory complexes in human HCC. In this study, therefore, we tried to explore whether and how functional defects of mitochondria were involved in oncogenic K-ras-induced cellular transformation. When Rat2 cells were infected by the retrovirus harboring oncogenic K-ras (V12), expression levels of mitochondrial respiratory proteins were decreased in a time- and dose-dependent manner. This result was well correlated with reduced respiratory activity and declined mitochondrial mass. Despite the defective mitochondria, total cellular ATP level was not changed. We also found that activity of lactate dehydrogenase (LDH) was dramatically augmented, implying that the cellular ATP level may be complemented through the activated LDH activity. Taken together, we conclude that functional defects of mitochondria and metabolic transition from mitochondrial oxidative phosphorylation to glycolytic energy production are clearly involved in oncogenic K-ras-induced cellular transformation of Rat2 cells.
Science and Technology for the Integration of Life｜
331
Poster Session
G-79
