Differential effect of oxidative stress on the apoptosis of early and late passage human diploid fibroblasts: implication of heat shock protein 60 Young-Hee Lee 1,2 , Jung-Chang Lee 1,2 , Hyun-Ju Moon 1,2 , Ji-Eun Jung 1,2 , Manju Sharma 3 , Byung-Hyun Park 4 , Ho-Keun Yi 1,2 and Eun-Chung Jhee 1,2 * 1 Department of Oral Biochemistry, School of Dentistry, Chonbuk National University, Korea 2 Institute of Oral-bio Science, School of Medicine, Chonbuk National University, Korea 3 Department of Pediatric, School of Medicine, Chonbuk National University, Korea 4 Department of Biochemistry, School of Medicine, Chonbuk National University, Korea Since an attenuated response to stress is a characteristic of senescence, a cellular senescence model was used to examine the mechanism of resistance against oxidative stress using human diploid fibroblasts (HDF). With increasing passage, the HDF showed increased production of reactive oxygen species (ROS). Late passage HDF were resistant to the lethal effects of oxidative stress, showing less cleavage of pro-caspase-3 and PARP than those of early ones. Since heat shock proteins (Hsps) are not only cytoprotective but also interfere with the apoptotic cascade, the expression patterns of Hsps during cellular senescence were next examined. Oxidative stress induced a decrease in the mitochondrial Hsp60 levels with a concomitant increase in the cytosolic Hsp60 levels in the early passage HDF, but not in late ones. To show that the resistance to oxidative stress is a specific effect of Hsp60, the levels of Hsp60 were knocked down by siRNA. As expected the Hsp60 knock-down cells were more resistant to oxidative stress. These findings show that Hsp60 is a key player in the resistance mechanism against oxidative stress and aging. Copyright # 2008 John Wiley & Sons, Ltd. key words — apoptosis; cellular senescence; heat shock protein 60; human diploid fibroblasts; oxidative stress INTRODUCTION A decrease in the capacity to respond to stress is characteristic of senescence. 1 Therefore, identification of the stress response at both the cellular and molecular levels is important for understanding the process of senescence. Heat shock proteins (Hsps) are produced in response to a variety of stimuli such as heat, heavy metals, and oxidants. 2–4 They serve as scaffolding or chaperone proteins to preserve the integrity of the essential intracellular proteins from various forms of stress. The Hsps are broadly classified into distinct families according to their apparent molecular weights, amino acid sequences, and functions. 5 Some Hsps are constitutively expressed but others are induced only after stress. It has been suggested that this protective function of Hsps reflects their ability to regulate several forms of cell death, particularly apoptosis. In general, Hsp27 and Hsp70 have been shown to prevent apoptosis, 6–9 whereas Hsp60 has both anti- and pro-apoptotic roles. 10,11 Hsp60 is primarily a mitochondrial protein that is important for folding key proteins after import into the mitochondria. 12,13 It is clear that a significant amount of Hsp60 is also present in the cytosol. 14 Cytosolic Hsp60 inhibits apoptosis by sequestering Bax and preventing its translocation to the mitochondrial membrane. 15 On the other hand, mitochondrial Hsp60 promotes the maturation of pro-caspase-3, cell biochemistry and function Cell Biochem Funct 2008; 26: 502–508. Published online 9 April 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/cbf.1473 * Correspondence to: E.-C. Jhee, Ph.D., Department of Oral Bio- chemistry, School of Dentistry, Chonbuk National University, 634-18 Geumam-Dong, Deokjin-Gu, Jeonju, Jeonbuk 561-712, Korea. Tel: þ82-63-270-4032, Fax: þ82-63-270-4033. E-mail: agjhee@chonbuk.ac.kr Copyright # 2008 John Wiley & Sons, Ltd. Received: 12 November 2007 Revised 2 January 2008 Accepted 31 January 2008