Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells.

The lack of cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) in mice increases renal proximal tubular cell death and enhances sensitivity to acute renal failure produced by the chemotherapeutic agent cisplatin. We used primary cultures of mouse renal proximal tubular cells (MPTC) grown in optimized culture conditions to investigate the cellular basis for increased apoptosis in p21 knockout mice. Cisplatin (15 microM) activated caspase-3 but not caspase-8 or caspase-9 and produced phosphatidylserine externalization, chromatin condensation, and nuclear fragmentation in wild-type [p21(+/+)] MPTC. Caspase-3 activation and apoptosis were accelerated in cisplatin-treated MPTC lacking p21 [p21(-/-) MPTC]. In contrast to p21(+/+) MPTC, cisplatin activated caspase-9 but not caspase-8 in p21(-/-) MPTC before caspase-3 activation. The caspase-3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone (DEVD-fmk) inhibited caspase-3 activity but did not abolish apoptosis in p21(+/+) and p21(-/-) MPTC. General caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (ZVAD-fmk) inhibited caspase activity and decreased chromatin condensation by 51% in p21(-/-) but not in p21(+/+) MPTC. However, cisplatin-induced phosphatidylserine externalization was not inhibited by ZVAD-fmk in p21(-/-) MPTC. We conclude that 1) in the presence of p21, cisplatin activates caspase-3 through a mechanism independent of caspase-8 or caspase-9; 2) in the absence of p21, caspase-9 activation precedes caspase-3 activation; 3) the lack of p21 accelerates caspase-3 activation and cisplatin-induced MPTC apoptosis; and 4) MPTC apoptosis is caspase independent in the presence of p21 but partially dependent on caspases in the absence of p21.