ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert potent cytotoxic activity against many tumor cell lines but not against normal cells. It has been hypothesized that this difference in TRAIL sensitivity between normal and transformed cells might be due to the expression of the non-death-inducing TRAIL receptors (TRAIL-R) TRAIL-R3 and TRAIL-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 receptor 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 keratinocytes (PK) being 5-fold less sensitive to TRAIL than transformed keratinocytes (TK). Yet both cell types exhibited similar TRAIL receptor surface expression, suggesting that expression of TRAIL-R3 and TRAIL-R4 may not be the main regulator of sensitivity to TRAIL. Biochemical analysis of the signaling events induced by TRAIL revealed that PK could be sensitized for TRAIL and, similarly, for TRAIL-R1- and TRAIL-R2-specific apoptosis by pretreatment of the cells with cycloheximide (CHX). This sensitization concomitantly resulted in processing of caspase-8, which did not occur in TRAIL-resistant PK. These data indicate that an early block of TRAIL-induced apoptosis was present in PK compared with TK or PK treated with CHX. Interestingly, cellular FLICE inhibitory protein (cFLIP) levels, high in PK and low in TK and several other squamous cell carcinoma cell lines, decreased rapidly after treatment of PK with CHX, correlating with the increase in TRAIL sensitivity and caspase-8 processing. Furthermore, ectopic expression of cFLIP long (cFLIP(L)) in TK by transfection with a cFLIP(L) expression vector resulted in resistance to TRAIL-mediated apoptosis of these cells. Thus, our results demonstrate that TRAIL sensitivity in PK is primarily regulated at the intracellular level rather than at the receptor level.
Subject(s)
Intracellular Signaling Peptides and Proteins , Keratinocytes/drug effects , Membrane Glycoproteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/analysis , Caspase 8 , Caspase 9 , Caspase Inhibitors , Cells, Cultured , Cycloheximide/pharmacology , GPI-Linked Proteins , Humans , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/analysis , Receptors, Tumor Necrosis Factor, Member 10c , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor Decoy ReceptorsABSTRACT
Disappearance of antigen presenting cells (APC) from the lymph node occurs following antigen specific interactions with T cells. We have investigated the regulation of CD95 (Apo-1/Fas) induced apoptosis during murine dendritic cell (DC) development. Consistent with the moderate levels of CD95 surface expression and low, or absent, MHC class II expression, immature DC in bone marrow cultures were highly sensitive to CD95 induced apoptosis, but insensitive to class II mediated apoptosis. In contrast, mature splenic, epidermal and bone marrow derived DC were fully resistant to CD95 induced cell death, but sensitive to class II induced apoptosis. Although caspase 3 and 8 activation was detected in immature DC undergoing CD95L-induced apoptosis, the pan-caspase inhibitor zVAD-fmk did not inhibit the early events of CD95-induced mitochondrial depolarisation or phosphatidyl serine exposure and only partially inhibited the killing of immature DC. In contrast, zVAD-fmk was completely effective in preventing CD95L mediated death of murine thymocytes. Collectively, these data do not support a major role of CD95: CD95L ligation in apoptosis of mature DC, but rather emphasise the existence of distinct pathways for the elimination of DC at different stages of maturation.
Subject(s)
Apoptosis/immunology , Dendritic Cells/cytology , Dendritic Cells/immunology , Histocompatibility Antigens Class II/metabolism , fas Receptor/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Apoptosis/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Caspases/metabolism , Cell Differentiation/immunology , Cross-Linking Reagents/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Dendritic Cells/metabolism , Histocompatibility Antigens Class II/analysis , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mitochondria/metabolism , fas Receptor/analysisABSTRACT
Evidence for the operation of expanded trinucleotide repeats in the pathogenesis of bipolar affected disorder has recently been found at the molecular genetic level. For the screening of these repeat motifs in genomes of patients with bipolar affective disorder, we established a modified PCR-based fingerprinting technique, called triplet repeat enhanced arbitrarily primed PCR (TREAP-PCR). Using this approach, 40 patients suffering from bipolar affective disorder (ICD10: F31) and 15 healthy controls were investigated. Interindividual polymorphisms generated by TREAP-PCR seemed to depend on the type of triplet. Using CCG triplet primers, polymorphisms could be observed more often in the genomes of patients compared with controls, whereas no significant differences could be found using primers of the CAG or AAT type. These data might indicate the existence of subgroups of manic-depressive patients based on molecular genetic differences.
