Abstract
Recent studies suggest that the aryl hydrocarbon receptor (AhR) modulates susceptibilities to some pro-apoptotic agents. AhR-containing murine hepatoma 1c1c7 cultures underwent apoptosis following exposure to tumor necrosis factor-alpha (TNFalpha) + cycloheximide (CHX). In contrast, Tao cells, an AhR-deficient variant of the 1c1c7 line, were refractory to this treatment. AhR sense/antisense transfection studies demonstrated that AhR contents influenced susceptibility to the pro-apoptotic effects of TNFalpha + CHX. 1c1c7 cells and all variants expressed comparable amounts of TNF receptor-1 and TRADD. However, no cell line expressed FADD, and consequently pro-caspase-8 was not activated. AhR content did not influence JNK and NF-kappaB activation. However, Bid and pro-caspase-9, -3, and -12 processing occurred only in AhR-containing cells. Analyses of cathepsin B and D activities in digitonin-permeabilized cultures and the monitoring of cathepsin B/D co-localization with Lamp-1 indicated that TNFalpha + CHX disrupted late endosomes/lysosomes in only AhR-containing cells. Stabilization of acidic organelles with 3-O-methylsphingomyelin inhibited TNFalpha + CHX-induced apoptosis. The cathepsin D inhibitor pepstatin A suppressed in vitro cleavage of Bid by 1c1c7 lysosomal extracts. It also delayed the induction of apoptosis and partially prevented Bid cleavage and the activation of pro-caspases-3/7 in cultures treated with TNFalpha + CHX. Similar suppressive effects occurred in cultures transfected with murine Bid antisense oligonucleotides. These studies showed that in cells where pro-caspase-8 is not activated, TNFalpha + CHX can initiate apoptosis through lysosomal disruption. Released proteases such as cathepsin D trigger the apoptotic program by activating Bid. Furthermore, in the absence of exogenous ligand, the AhR modulates lysosomal disruption/permeability.
Highlights
Somal membrane permeabilization, causing release of resident proteases into the cytosol and induction of apoptosis
Analyses of aryl hydrocarbon receptor (AhR)-deficient cell lines suggest that AhR content, in the absence of exogenous ligands, regulates cell morphology [35], progression through G1 [35, 36], and susceptibility to the pro-apoptotic effects of ceramide [37], Fas ligand, CD95 cross-linking antibody [38], and the lysosomal photosensitizer N-aspartyl chlorine 6 (NPe6) [25]
We have demonstrated that the AhR-containing murine hepatoma cell line 1c1c7 undergoes apoptosis in photodynamic therapy (PDT) protocols employing the lysosomal sensitizer NPe6 [9, 25]
Summary
TNF␣, tumor necrosis factor-␣; ␣MEM, ␣-minimal essential medium; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; AhR, aryl hydrocarbon receptor; AMC, 7-amino-4-methylcoumarin; CA-074, L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-L-isoleucyl-L-proline; CA-074-Me, methyl ester of CA-074; CHX, cycloheximide; DAME, diazoacetyl-DL-2-aminohexanoic acid methyl ester; E-64, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane; E-64d, ethyl ester of E-64; HA14-1, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4Hchromene-3-carboxylate; EMSA, electrophoretic mobility shift assay; LDH, lactate dehydrogenase; NPe6, N-aspartyl chlorin e6; PDT, photodynamic therapy; 3-O-MeSM, 3-O-methylsphingomyelin; SMase, sphingomyelinase; TNFr, tumor necrosis factor receptor; Z-FA-fmk, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; JNK, c-Jun NH2terminal kinase. Because restoration of AhR expression restored susceptibility to PDT-induced acidic organelle disruption and apoptosis, it appeared that AhR content regulated lysosome fragility [25]. The 1c1c7 model proved to be a unique system for investigating the contributions of non-caspase-8 pathways to TNF␣-mediated Bid cleavage and apoptosis, and for identifying the AhR as a putative modifier of lysosomal permeability
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