Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes

Tumor necrosis factor-related apoptosis-inducing Ligand (TRAIL) has been sh own to exert potent cytotoxic activity against many tumor cell lines but no t-against normal cells, It has been hypothesized that this difference in TR AIL sensitivity between normal and transformed cells might be due to the ex pression:of the non-death-inducing TRAIL receptors (TRAIL-R) TRAIL-R3 and T RAIL-R4, presumably by competition for limited amounts of TRAIL, To assess the regulation of resistance versus sensitivity to TRAIL in primary as well as transformed keratinocytes, we examined TRAIL sensitivity, TRAIL recepto r expression, and intracellular signaling events induced by TRAIL. Although TRAIL induced apoptosis in primary as well as transformed keratinocytes, a marked difference in sensitivity could be observed with primary keratinocy tes (PK) being 5-fold less sensitive to TRAIL than transformed; keratinocyt es (TK). Yet both cell types exhibited similar TRAIL receptor surface expre ssion, suggesting that expression of TRAIL-R3 and TRAIL-R4 may not be the m ain regulator of sensitivity to TRAIL, Biochemical analysis of the signalin g events induced by TRAIL revealed that PK could be sensitized for TRAIL an d, similarly, for TRAIL-R1- and TRAIL-R2-specific apoptosis by pretreatment of the cells with cycloheximide (CHX). This sensitization concomitantly re sulted in processing of caspase-8, which did not occur in TRAIL-resistant P K, These data indicate that an early block of TRAIL-induced apoptosis was p resent in PK compared with TK or PK treated with CHX, Interestingly, cellul ar FLICE inhibitory protein (cFLIP) levels, high in PK and low in TK and se veral other squamous cell carcinoma cell lines,decreased rapidly after trea tment of PK with CHX, correlating viith the increase in TRAIL sensitivity a nd caspase-8 processing. Furthermore, ectopic expression of cFLIP long (cFL IP(L)) in TK by tansfection with a cFLIP(L) expression vector resulted in r esistance to TRAIL-mediated apoptosis of these cells, Thus, our results dem onstrate that TRAIL sensitivity in PK is primarily regulated at the intrace llular level rather than at the receptor level.