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Originally published In Press as doi:10.1074/jbc.M200401200 on March 27, 2002

J. Biol. Chem., Vol. 277, Issue 22, 19346-19352, May 31, 2002
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Activation of Caspase-9 Is Required for UV-induced Apoptosis of Human Keratinocytes*

Leonid A. Sitailo, Shalini S. Tibudan, and Mitchell F. DenningDagger

From the Department of Pathology and the Cardinal Bernardin Cancer Center, Skin Cancer Research Program, Loyola University Medical Center, Maywood, Illinois 60153

UV radiation from the sun activates both the membrane death receptor and the intrinsic or mitochondrial apoptotic signaling pathways in epidermal keratinocytes, triggering apoptosis and affording protection against skin cancer formation. We have investigated the involvement of caspase-9 in the UV death effector pathway in human keratinocytes, since this is the initiating caspase in the mitochondrial pathway required for UV-induced apoptosis in some, but not all, cell types. UV radiation triggered activation of caspase-3, caspase-9, and caspase-8 with similar kinetics, although the rank order of activation was caspase-3 > caspase-9 > caspase-8. Inhibition of caspase-9 with either the peptide inhibitor benzyloxycarbonyl-Leu-Glu(OCH3)-His-Asp(OCH3)-fluoromethyl ketone, or expression of a catalytically inactive caspase-9 by retroviral transduction, protected normal keratinocytes from UV-induced apoptosis. HaCaT keratinocytes harboring mutant p53 alleles were also protected from UV-induced apoptosis by the dominant negative caspase-9. The dominant negative caspase-9 blocked UV-induced activation of caspase-3, caspase-9, and caspase-8, and also protected cells from the loss of mitochondrial membrane potential. In contrast, the dominant negative caspase-9 did not protect from anti-Fas-induced apoptosis or caspase activation. These results identify caspase-9 as the critical upstream caspase initiating apoptosis by UV radiation in human keratinocytes, the relevant cell type for this important environmental carcinogen.

* This study was supported by National Institutes of Health Grant CA83784 (to M. F. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: 2160 S. First Ave., Cardinal Bernardin Cancer Center, Rm. 304, Loyola University Medical Center, Maywood, IL 60153. Tel.: 708-327-3358; Fax: 708-327-3158; E-mail: mdennin@lumc.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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