Abstract
The chemotherapeutic cisplatin causes renal dysfunction and renal proximal tubular cell (RPTC) apoptosis. The goal of these studies was to examine the role of p53, caspase 3, 8, and 9, and mitochondria in the signaling of cisplatin-induced apoptosis. Cisplatin (50 μM) produced time-dependent apoptosis in RPTCs, causing cell shrinkage, a 50-fold increase in caspase 3 activity, a 4-fold increase in phosphatidylserine externalization, and 5- and 15-fold increases in chromatin condensation and DNA hypoploidy, respectively. Mitochondrial membrane potential and ATP levels did not change at any time during cisplatin exposure. Caspase 8 and 9 activities also did not increase during treatment. Cisplatin increased nuclear p53 expression 4 h after treatment, preceding both caspase 3 activation and chromatin condensation. Treatment with the p53 inhibitor α-2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone (PFT) before cisplatin exposure inhibited p53 nuclear expression at 4, 8, and 12 h and inhibited phosphatidylserine externalization and caspase 3 activation at 12 h. Neither DEVD-fmk nor ZVAD-fmk inhibited cisplatin-induced p53 nuclear expression. Both DEVD-fmk and ZVAD-fmk completely inhibited caspase 3 activity but, like PFT, partially inhibited cisplatin-induced chromatin condensation, annexin V labeling, and DNA hypoploidy after 24 h. These data demonstrate that at least 50% of cisplatin-induced apoptosis in RPTC is mediated by p53 and that p53 activates caspase 3 independently of either caspase 9 or 8 or mitochondrial dysfunction. Furthermore, 50% of cisplatin-induced RPTC apoptosis is independent of p53 and caspases 3, 8, and 9.
Footnotes
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This work was supported by a National Research Service Award DK-10079 (to B.S.C.) and by a National Institute of Environmental Health Sciences Award ES-04410 (to R.G.S.).
- Abbreviations:
- RPTC
- renal proximal tubular cell
- PFT
- p53 inhibitor α-(2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone
- FITC
- fluorescein isothiocyanate
- DMSO
- dimethyl sulfoxide
- PBS
- phosphate-buffered saline
- PI
- propidium iodide
- JC-1
- 5,5′6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazocarbocyanine iodide
- Received January 15, 2002.
- Accepted March 1, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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