Abstract
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family. It induces apoptosis primarily of transformed but not of normal cells and may therefore be a promising anti-cancer drug. Studying the role of TRAIL in apoptosis of keratinocytes, we detected TRAIL transcripts and protein in both normal human keratinocytes and transformed keratinocyte cell lines HaCaT and KB. Although normal keratinocytes were resistant to TRAIL, HaCaT and KB cells underwent apoptosis following TRAIL exposure. When HaCaT and KB cells were pretreated with the pro-inflammatory cytokine interleukin-1 (IL-1), cells became resistant to TRAIL-induced apoptosis. IL-1 significantly induced activation of the transcription factor NFkappaB in transformed keratinocytes. Moreover, the proteasome inhibitor MG132, which inhibits IL-1-induced NFkappaB activation, completely prevented the protective effect of IL-1. Thus, IL-1 appears to protect transformed keratinocytes from the cytotoxic effect of TRAIL via activation of NFkappaB. These data suggest that NFkappaB activation may protect cells from TRAIL-induced apoptosis and indicate a TRAIL receptor-independent pathway, which allows cells to escape the cytotoxic effect of TRAIL. Because IL-1 is secreted by a variety of tumor cells and is also released by inflammatory cells participating in the tumor-host immune response, tumors under these conditions could become resistant to TRAIL.
Highlights
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),1 called APO-2 ligand, is a recently identified member of the tumor necrosis factor (TNF) family that was originally characterized by its ability to induce apoptosis [1, 2]
To evaluate the responsiveness of keratinocytes to TRAIL, human keratinocytes (HNK), HaCaT and KB cells were exposed to recombinant TRAIL, and 16 h later apoptosis was determined using an apoptosis detection ELISA kit
Interleukin-1 Protects from TRAIL-mediated Killing—Because activation of the transcription factor nuclear factor-B (NFB) recently was found to prevent TNF-induced apoptosis (28 –30), we addressed whether transformed keratinocytes, which are susceptible to TRAIL, can escape TRAIL-induced apoptosis through the same pathway
Summary
Cells—Long term cultures of foreskin HNKs were prepared according to the method described by Boyce et al [20]. Reagents—Recombinant human TRAIL protein was obtained from Immunex Corp., Seattle WA. Flow Cytometry—Aliquots of 2 ϫ 105 cells were incubated with antibodies directed against DR4 and DR5, respectively, for 45 min on ice. Purified mouse IgG was used as an isotype control. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and incubated with antibodies of interest. RNA was extracted from HNK, HaCaT or KB cells, and Northern blot analysis was performed using cDNA probes encoding for TRAIL and -actin, respectively (a). Western blot analysis was performed using antibodies directed against TRAIL and ␣-tubulin, respectively (b). Binding reactions were carried out by addition of 2 g of poly(dI-dC) (Boehringer Mannheim) and 104 cpm of 32P-labeled, double-stranded oligonucleotide to the nuclear protein extracts for 20 min at 22 °C. Competition analysis was performed by adding 40 molar excess amounts of unlabeled oligonucleotides
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