INTRACELLULAR REGULATION OF TRAIL-INDUCED APOPTOSIS IN HUMAN-MELANOMACELLS

The observation that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family, induces apoptosis in a number of di fferent tumor cell types led us to compare the tumoricidal effects of TRAIL to those of other TNF family molecules on human melanoma cells. We found that a high proportion of the melanoma cell lines tested were killed by TRAIL, whereas all the melanoma lines were resistant to the other TNF family cytokines tested. TRAIL-induced death was characteri zed by caspase activation and cellular protein cleavage within minutes of TRAIL addition, and death could be completely inhibited by the cas pase inhibitors Ile-Glu-Thr-Asp (IETD) and Val-Ala-Asp (VAD), indicati ng the presence of a TRAIL receptor signaling pathway similar to that identified for Fas and TNF receptors. Specific TRAIL receptor expressi on was determined by RT-PCR, and the presence of mRNA encoding the ''p rotective'' TRAIL receptors did not correspond to resistance or sensit ivity to TRAIL-induced apoptosis. Addition of protein synthesis inhibi tors to TRAIL-resistant melanomas rendered them sensitive to TRAIL, in dicating that the presence or the absence of intracellular apoptosis i nhibitors may mediate resistance or sensitivity to TRAIL-mediated apop tosis. Expression of one such inhibitor, FLICE-inhibitory protein (FLI P), was highest in the TRAIL-resistant melanomas, while being low or u ndetectable in the TRAIL-sensitive melanomas. Furthermore, addition of actinomycin D to TRAIL-resistant melanomas resulted in decreased intr acellular concentrations of FLIP, which correlated with their acquisit ion of TRAIL sensitivity. Collectively; our results indicate that TRAI L-induced apoptosis occurs through a caspase signaling cascade and tha t resistance is controlled by intracellular regulators of apoptosis.