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Cells differ in the time required to execute cell death after receipt of a death signal. One reason may be the requirement for de novo synthesis of components of the death pathway. TSU-Pr1 prostate cancer cells treated with okadaic acid demonstrated activation of caspase-3, PARP cleavage, and nuclear fragmentation by 24 h and apoptosis by 72 h. Levels of procaspase-3 and procaspase-7, the precursor molecules of two effector caspases, were not depleted during apoptosis. Levels of procaspase-3 and -7 mRNA increased steadily in TSU-Pr1 cells up to 72 h after exposure to okadaic acid. Nuclear run-off experiments showed that the increase in mRNA was not due to transcriptional activation of caspase-3 and -7 mRNA. Antisense caspase-3 and caspase-7 oligodeoxynucleotides caused a depletion of procaspases-3 and -7 and a delay in apoptosis of TSU-Pr1 cells. Caspase antisense oligodeoxynucleotides inhibited apoptosis to a similar extent as peptide inhibitors of cysteine proteases. Synthesis of procaspases-3 and -7 was necessary to sustain programmed cell death in TSU-Pr1 prostate cancer cells.

Tumour necrosis factor (TNF) elicits multiple biological effects through two distinct cell surface receptors, TNF-R1 (p55) and TNF-R2 (p75). Most TNF-mediated biological responses, such as cell death, gene induction, antiviral activity and cytokine production, have been attributed to TNF-R1 (refs 1-5). Gene targeting of this receptor confirms its role in the lethality attributable to low doses of lipopolysaccharide after sensitization with D-galactosamine; surprisingly, the toxicity of high doses of lipopolysaccharide was unaffected. The function of TNF-R2 is less well understood, although there are data supporting a role in T-cell development and the proliferation of cytotoxic T lymphocytes. To clarify the physiological role of TNF-R2, we have generated mice deficient in this receptor by gene targeting. The TNF-R2-/- mice show normal T-cell development and activity, but we find that they have increased resistance to TNF-induced death. Additionally, such mice injected subcutaneously with TNF show a dramatic decrease in tissue necrosis, indicating that this receptor plays a role in the necrotic effects of TNF.