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Anti‐apoptotic defense of bcl‐2 gene against hydroperoxide‐induced cytotoxicity together with suppressed lipid peroxidation, enhanced ascorbate uptake, and upregulated Bcl‐2 protein
Author(s) -
Saitoh Yasukazu,
Ouchida Rika,
Kayasuga Atsushi,
Miwa Nobuhiko
Publication year - 2003
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.10506
Subject(s) - apoptosis , transfection , intracellular , downregulation and upregulation , lipid peroxidation , programmed cell death , microbiology and biotechnology , cytotoxicity , ascorbic acid , reactive oxygen species , biology , cell culture , chemistry , biochemistry , in vitro , oxidative stress , gene , genetics , food science
Although it is well known that Bcl‐2 can prevent apoptosis, the Bcl‐2's anti‐apoptotic mechanism is not fully understood. Here, we investigate the mechanism of oxidant‐induced cell death and to investigate the role of Bcl‐2 in the tert ‐butyl hydroperoxide ( t ‐BuOOH)‐induced oxidant injury in Rat‐1 fibroblasts and their bcl‐2 transfected counterparts, b5 cells. Treatment with t ‐BuOOH causes mitochondrial disfunction and induced morphological features consistent with apoptosis more markedly in Rat‐1 cells than in b5 cells. The hydroperoxide t ‐BuOOH at concentrations less than 100 nM for as long as 48 h or with higher concentrations (up to 100 μM) for only 3 h induces death in Rat‐1 cells, whereas their bcl‐2 transfectants were significantly resistant to cytotoxicity by both time and all concentration other than 100 μM. The similar results were obtained also for DNA strand cleavages as detected by TUNEL stain. The bcl‐2 transfectants significantly suppressed t ‐BuOOH‐induced increases in both lipid peroxidation and caspase‐3 activation 3 and 1 h after t ‐BuOOH exposure, respectively, but failed to suppress either caspase‐1 activation or an enhanced production of the intracellular reactive oxygen species (ROS). Intracellular uptake of [1‐ 14 C] ascorbic acid (Asc) into the bcl‐2 transfectants was superior to that into the non‐transfectants always under examined conditions regardless of serum addition to culture medium and cell density. Upregulation of Bcl‐2 proteins was rapidly induced after t ‐BuOOH exposure in the transfectants, but not in non‐transfectants, and restored till 24 h to the normal Bcl‐2 level. Thus suppressions of both lipid peroxidation and the subsequent cell death events such as caspase‐3 activation and DNA cleavage were concerned with the inhibitory effects of Bcl‐2 on the t ‐BuOOH‐induced cytotoxicity. And some of these events may correlate with Bcl‐2 expression‐induced partial enhanced anti‐oxidant cellular ability including enrichment of intracellular Asc and oxidative stress‐induced upregulation of Bcl‐2 protein. On the other hand, ROS production and caspase‐1 activation were not related to cytoprotection by Bcl‐2. J. Cell. Biochem. 89: 321–334, 2003. © 2003 Wiley‐Liss, Inc.