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First published online 18 January 2005
doi: 10.1242/jcs.01640

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Induction of apoptosis by TNF receptor 2 in a T-cell hybridoma is FADD dependent and blocked by caspase-8 inhibitors

Bart Depuydt, Geert Van Loo^*, Peter Vandenabeele and Wim Declercq

Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB) and Gent University, 9052 Ghent-Zwijnaarde, Belgium

View larger version (13K): [in a new window]   Fig. 1. Both TNF rceptors induce apoptosis in PC60 R1R2 cells. PC60 R1R2 cells were treated for 20 hours with 100 ng/ml hTNF or its receptor-specific muteins (500 ng/ml). The percentage of apoptotic cells was determined using propidium iodide exclusion.

View larger version (41K): [in a new window]   Fig. 2. Both TNFR1- and TNFR2-mediated apoptosis in PC60 R1R2 cells are blocked by the caspase inhibitor zVAD-fmk. (A) PC60 R1R2luc cells were seeded in microtiter plates and left untreated or were stimulated with saturating amounts of hTNF (100 ng/ml) in the presence (100 µM) or absence of the synthetic peptide caspase inhibitors zVAD-fmk, zDEVD-fmk, zYVAD-cmk, zIETD-fmk or the granzyme B inhibitor zAAD-fmk. Cells were cultured for 20 hours before cell death was measured by cytofluorometric analysis of PI uptake. (B) The experimental procedure was the same as in A. Cells were incubated with hTNF or its receptor-specific muteins R32WS86T or D143NA145R (500 ng/ml) in the absence or presence of zVAD-fmk. (C) Cells were treated for 7 hours in the presence of 100 ng/ml hTNF, in the presence or absence of peptide inhibitors zDEVD-fmk or zVAD-fmk. Cell lysates were analyzed by means of western blotting with a caspase-3 antibody. Pro is the unprocessed procaspase-3, and p20 is the processed form with the black arrowhead showing the normal p20 subunit and the white arrowhead showing a shifted band indicative of a covalent bond between the p20 and zDEVD-fmk. (D) The experimental procedure was the same as in A. Cells were lysed 6 hours after stimulation and luciferase activity determined.

View larger version (22K): [in a new window]   Fig. 3. TNFR1- and TNFR2-mediated apoptosis in PC60 R1R2 cells are both inhibited by the cowpox caspase inhibitor CrmA. (A) PC60 R1R2 cells were transfected with a combination of the plasmids pCAGGSCrmA and pNFconluc. After selection and subcloning, cell lines were isolated that expressed the CrmA protein and produced elevated levels of luciferase activity after TNF stimulation (named PC60 R1R2 CrmALuc). Control cells were obtained by only transfecting pNFconluc and subsequent screening for luciferase inducibility (PC60 R1R2 luc). Untreated cells were lysed and 100 µg of protein was subjected to CrmA immunoblot. The arrowhead shows the position of the CrmA protein. (B) PC60 R1R2luc and PC60 R1R2CrmAluc cells were cultured in the absence or presence of hTNF (100 ng/ml) or receptor specific muteins (500 ng/ml) for 20 hours and the percentage of propidium iodide-positive cells was determined.

View larger version (26K): [in a new window]   Fig. 4. Both TNFR1- and TNFR2-mediated apoptosis in PC60 R1R2 cells is FADD dependent. (A) PC60 R1R2 were electroporated with: pUT651, a ß-gal expression plasmid, plus pCDNAI (control) or plus pCDNAFADD-DN, encoding dominant negative FADD (80-205). 48 hours after transfection cells were lysed and 150 µg of protein was submitted to immunoblotting using a monoclonal FADD-specific antibody. (B) 24 hours after electroporation, cells were stimulated overnight either with 500 ng/ml R32WS86T, 500 ng/ml D143NA145R or 100 ng/ml hTNF. Subsequently, cells were stained with X-gal and ß-gal-positive blue cells were examined microscopically for apoptotic features, such as membrane blebbing and nuclear changes, after which the percentage of induced apoptotic cells was determined (500 blue cells were counted in each measurement).

View larger version (12K): [in a new window]   Fig. 5. TNFR2-mediated apoptosis is not dependent on endogenous production of TNF or other death-inducing ligands. PC60 R1R2 cells were treated with hTNF or its muteins as in Fig. 1 and the resulting amount of cell death determined. (A) Incubation in the absence or presence of 5 µg/ml hTNFR1 antagonizing monoclonal antibody htr-5. (B) Incubation in the absence or presence of 10 µg/ml cycloheximide (CHX).

View larger version (19K): [in a new window]   Fig. 6. TNFR2 stimulation does not sensitize PC60 R1R2 cells for a subsequent TNFR1-mediated apoptotic signal. (A) PC60 R1R2 cells, preincubated for 14 hours in medium or medium containing 500 ng/ml of the TNFR2-specific mutein D143NA145R were stimulated with 100 ng/ml hTNF, the TNFR1 specific mutein R32WS86T (500 ng/ml) or a combination of both receptor specific muteins. (B) Cells preincubated as in A were exposed to a dilution range of hTNF as indicated. (C) Western blot, revealed with anti-TRAF2, of lysates of PC60 R1R2 cells that were treated for 14 hours with hTNF (100 ng/ml) or its receptor specific muteins (500 ng/ml).

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