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. 2005 Feb 14;168(4):545-51.
doi: 10.1083/jcb.200407162. Epub 2005 Feb 7.

Protein synthesis persists during necrotic cell death

Xavier Saelens  1 , Nele Festjens, Eef Parthoens, Isabel Vanoverberghe, Michael Kalai, Frank van Kuppeveld, Peter Vandenabeele
Affiliations

Protein synthesis persists during necrotic cell death

Xavier Saelens et al. J Cell Biol. .

Abstract

Cell death is an intrinsic part of metazoan development and mammalian immune regulation. Whereas the molecular events orchestrating apoptosis have been characterized extensively, little is known about the biochemistry of necrotic cell death. Here, we show that, in contrast to apoptosis, the induction of necrosis does not lead to the shut down of protein synthesis. The rapid drop in protein synthesis observed in apoptosis correlates with caspase-dependent breakdown of eukaryotic translation initiation factor (eIF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate eIF2-alpha. In necrosis induced by tumor necrosis factor, double-stranded RNA, or viral infection, de novo protein synthesis persists and 28S ribosomal RNA fragmentation, eIF2-alpha phosphorylation, and proteolytic activation of PKR are absent. Collectively, these results show that, in contrast to apoptotic cells, necrotic dying cells retain the opportunity to synthesize proteins.

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Figures

Figure 1.
Figure 1.
dsRNA and TNF induce necrosis in JB6 and FADD-deficient Jurkat cells. (A) Microscopic analysis. JE cells were treated with anti-Fas antibody to induce apoptosis (top), JB6 cells with dsRNA (middle), and FADD-deficient cells with TNF (bottom). Photomicrographs were taken after 4 (JE) and 6 h (JB6 and FADD-deficient cells). Apoptotic cells showing blebbing of the cell membrane and necrotic cells displaying swelling are indicated (closed arrows). In the middle and right columns, cells with nuclear PI staining are indicated with open arrowheads. (B) Cells were treated with anti-Fas (JE), dsRNA (JB6), or TNF (FADD−) or left untreated (CTR) for the indicated time periods. ROS production was determined by measuring the fluorescence intensity of rhodamine 123 in PI-negative cells. Bars represent mean values from three experiments, relative to time 0 (set to 100%). (C) Cells, pretreated for 30 min with 25 μM zVAD-fmk, quinoline-Val-Asp-O-phenoxy (q-VD-OPH), or DMSO, were stimulated with anti-Fas (JE), dsRNA (JB6), or TNF (FADD−) for 8 h. Control (CTR) cells were not stimulated. Error bars equal SD of the mean (n = 3).
Figure 2.
Figure 2.
Translation is rapidly shut down in apoptosis but not in necrosis. (A) Protein synthesis in Jurkat cells was analyzed by pulse-labeling cells for 30 min with 10 μCi 35S-methionine in normal medium at various time points after stimulation. Pulse-labeling was also performed on unstimulated cells treated for 1 h with 10 μg/ml CHX and on cells stimulated for 6 h with anti-Fas, dsRNA, or TNF together with CHX added 1 h before addition of 35S-methionine. After labeling, cells were lysed and 30-μg samples were resolved by SDS-PAGE followed by Coomassie staining and autoradiography. Coomassie and radioactivity signals from gels were quantified by densitometry and phosphorimaging, respectively, and the ratio of 35S-counts over total protein in each lane was calculated (top). Cell death from cells used for pulse-labeling experiments was determined by trypan blue exclusion (bottom). (B) The rate of protein synthesis remains constant during TNF-induced necrosis in mouse L929 fibrosarcoma cells. L929sAFas cells were seeded at 2.5 × 105 cells/ml in suspension plates on the day before labeling with 35S-methionine. 30-min pulse-labeling was performed at different time points after stimulation with 100 ng/ml anti-Fas (left) or 5,000 IU/ml TNF (right) to induce apoptosis and necrosis, respectively (top). CHX (10 μg/ml; 1 h)-treated cells were also included. Cell lysates were prepared and analyzed by measuring scintillation counts of TCA-precipitated cell extracts (anti-Fas–treated cells) or by SDS-PAGE followed by Coomassie staining and autoradiography (TNF-treated cells). 35S scintillation counts of anti-Fas–treated cells are depicted (left). For TNF-treated cells, 35S counts in each lane of the gels were quantitated using a phosphorimager and represented as mean values from three independent experiments (right). Cell death in the same experiment was monitored by trypan blue exclusion (bottom). Data in A and B represent mean values and error bars indicate the SD of the mean (n = 3).
Figure 3.
Figure 3.
Translation initiation factors eIF4G, eIF2-α, PKR, and PARP remain intact in necrosis but not in apoptosis. (A) Western blot analysis of total cell lysates reveals the cleavage of eIF4G into a 45-kD fragment (eIF4G C-FAG) in time after anti-Fas treatment of JE cells (left). Asterisks denote an aspecific band. The phosphorylation status of eIF2-α was monitored using an antibody specific for eIF2-α phosphorylated at Ser51 (P-eIF2-α). The total amount of eIF2-α was also assessed on the same blot after inactivation of the P-eIF2-α signal by overnight treatment of the blot with 0.1% NaN3. The ratio of phosphorylated over total eIF2-α signal per lane was determined by densitometric analysis, normalized to 1 for time 0, and is indicated below each lane. Immunoblots for PKR and PARP show the appearance of a 40-kD PKR fragment (PKR-Nterm) and of an 85-kD fragment of PARP in apoptosis. (B) Ribosomal 28S RNA is cleaved in apoptosis but not in necrosis. 10 μg of total RNA isolated from cells treated as indicated was analyzed by agarose gel electrophoresis followed by ethidium bromide staining. Arrows indicate the position of 28S and 18S rRNA. Arrowheads indicate cleavage products in the anti-Fas–treated sample. CTR, untreated.
Figure 4.
Figure 4.
Coxsackievirus induces necrosis-like death. Cells were infected with CVB (multiplicity of infection of 20) for 1 h, washed, and incubated for the time points indicated. (A) CVB replicates in JE and JB6 cells. Y-axis shows virus titer (in 50% tissue culture infective dose/ml). (B) Microscopic analysis of JE (top) and JB6 (bottom) cells infected with CVB for 16 h. Closed arrows indicate swollen cells; open arrowheads indicate cells with nuclear PI staining. (C) Jurkat cells do not display caspase activity following CVB infection. As a control, a sample from anti-Fas–treated JE cells was also analyzed (A-Fas). (D) Kinetics of cell death following CVB infection of JE and JB6 cells, as measured by permeability to trypan blue. (E) Translation rate of JE (left) and JB6 (right) cells infected in D. Coomassie and radioactivity signals from SDS-PAGE gels were quantified by densitometry and phosphorimaging, respectively, and the ratio of 35S-counts over total protein in each lane were calculated. Data in the graph are from one experiment that was performed three times, giving similar results. (F) Fate of eIF4G (top) and PARP (bottom) in CVB-infected JE (left) and JB6 (right) cells. Note that eIF4G is not cleaved into the apoptotic 45-kD fragment, but, instead, into a 100-kD fragment (eIF4GCVB). A-Fas, apoptotic lysates.
Figure 5.
Figure 5.
Induction of apoptosis in CVB-infected cells leads to a drop in virus titer, whereas enhancing necrosis by dsRNA-treatment of infected JB6 cells does not affect virus propagation. CVB-infected JE (A and C) or JB6 (B and D) cells were left untreated (−) or stimulated with anti-Fas or dsRNA, respectively, for 8 h, starting 2 h after infection. (A and B) Cells were pulse-labeled with 35S-methionine, and the amount of incorporated 35S in the protein fraction of cell lysates and cell death was determined (top). Effects on eIF4G and PARP were analyzed by immunoblotting (bottom). (C and D) CVB titers from JE (C) and JB6 (D) infected cells stimulated with anti-Fas or dsRNA or left untreated (ctr). Error bars equal SD of the mean (n = 3).
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