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Facilitation of Fas Mediated Apoptosis of Human Chondrocytes by the Proteasome Inhibitor and Actinomycin D
HYUN A. KIM, YONG H. KIM, and YEONG W. SONG
ABSTRACT. Methods. Chondrocytes were cultured from cartilages obtained at the time of joint replacement surgery for knee osteoarthritis (OA) or femur neck fracture. Fas receptor ligation was performed with agonistic anti-Fas antibody (clone CH-11) at concentrations ranging from 0.5 to 1.0 µg/ml. Mitogen activated protein kinase inhibitors SB203580 and PD98059, cycloheximide, bisindolylmaleimide, actinomycin D, or MG132 were added with anti-Fas to facilitate cell death. Chondrocyte surface expression of Fas was analyzed by FACS, and the expression of apoptosis related proteins analyzed by Western blot. Results. Cell death increased upon coculture with 0.5 µg/ml of anti-Fas and 0.2 µg/ml of actinomycin D or 20 µM MG132. Apoptosis potentiated by actinomycin D or MG132 was effectively inhibited by caspase inhibitors, implicating the involvement of the caspase cascade in chondrocyte apoptosis. Compared with untreated cells or actinomycin D treated cells, cells treated with MG132 showed distinct shifts in the distribution of surface Fas fluorescence. Although concentrations of Bcl-2, Bax, FLICE inhibitory protein (FLIP), and Fas ligand were unaffected by MG132 or actinomycin D, both treatments led to a significant increase of p53. The expression of the p53 response proteins, MDM2 and p21, was elevated in MG132 treated chondrocytes. Conclusion. Our results suggest that chondrocytes can be rendered sensitive to anti-Fas mediated apoptosis by the proteasome inhibitor MG132 and the transcription inhibitor actinomycin D. MG132 and actinomycin D show different characteristics in terms of apoptosis signaling. (J Rheumatol 2003;30:550-8) Key Indexing Terms:
FAS
From the Department of Internal Medicine, Hallym University College of Medicine; the Department of Orthopedic Surgery, Kangnam General Hospital Public Corporation; and the Department of Internal Medicine, Clinical Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea. Supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-C11-08-0007). H.A. Kim, MD, PhD, Assistant Professor of Internal Medicine, Department of Internal Medicine, Kangdong Sacred Heart Hospital, Hallym University; Y.H. Kim, MD, Department of Orthopedic Surgery, Kangnam General Hospital Public Corporation; Y.W. Song, MD, PhD, Professor of Internal Medicine, Department of Internal Medicine, Clinical Research Institute, Medical Research Center, Seoul National University, College of Medicine. Address reprint requests to Dr. Y.W. Song, Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Korea. E-mail: ysong@snu.ac.kr Submitted January 7, 2002; revision accepted September 6, 2002.
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