Identification of p21 as a target of cycloheximide-mediated facilitation of CD95-mediated apoptosis in human malignant glioma cells

Human glioma cell lines differ in their requirement for the inhibition of p rotein synthesis to activate the CD95-dependent killing pathway. CD95 ligan d (CD95L) induced mitochondrial cytochrome c release and processing of casp ases 3, 7, 8 and 9 in LN-18 cells in the absence of an inhibitor of protein synthesis, cycloheximide (CHX). These biochemical changes were observed in LN-229 cells only in the presence of CHX. The viral caspase inhibitor, cyt okine response modifier (crm)-A, inhibited mitochondrial cytochrome c relea se, caspase processing and cell death under all conditions. Ectopic express ion of BCL-X-L prevented processing of caspase 8 in LN-18 cells but not in LN-229 cells. Thus, caspase 8 activation is amplified through the release o f cytochrome c in LN-18 cells but occurs mainly at the receptor in LN-229 c ells. In contrast to BCL-2, BCL-X-L, X-linked inhibitor-of-apoptosis protei n (XIAP) and FLICE-inhibitory protein (FLIP), the levels of the cyclin-depe ndent kinase (CDK) inhibitor, p21(Waf/Cip1), rapidly decreased in response to CHX. P21 antisense oligonucleotides promoted caspase activation and mito chondrial cytochrome c release and induced strong sensitization to CD95-med iated apoptosis. These data place potentiating effects of CHX (i) to the ac tivation of caspase 8 at the receptor in LN-229 cells as well as (ii) to a down-stream target at least in LN-18 cells, but probably both cell lines, t hat may be identical with p21(Waf/Cip1).