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Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death

Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement... Background: Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase- independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. Methods: We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 ΔTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. Results: Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. Conclusion: Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide. Background dependent apoptosis, the most well-studied form of PCD, The survival and homeostasis of multicellular organisms represents the principal suicide program in most physio- critically depends on programmed cell death (PCD) to logical settings [1], many cells have the ability to commit correctly remove cells that are potentially harmful or suicide by caspase-independent modes of PCD (ciPCD) which have fulfilled their function. Although caspase- [2]. ciPCD fulfills vital functions in processes such as the Page 1 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 negative selection of lymphocytes, the termination of cin, brefeldin A, ceramide, ionizing radiation, thapsi- immune responses, embryonic removal of interdigital gargin, Bax, Bad) but not all stimuli (reviewed in [24,25]). webs, regulation of bone growth, ovulation, and cellular turnover in the intestine [3]. Furthermore, ciPCD has In contrast to this relative wealth of available data on been implicated the pathology of hyperacute shock [4], apoptosis, the role of ER- vs. mitochondrially localized pancreatitis [5,6], cerebral and myocardial ischemia- Bcl-2 in ciPCD has not been investigated so far. Here, we reperfusion injury, epilepsy, Alzheimer's disease and utilize ciPCD elicited by ceramide as a model system, a other inflammatory injuries, as well as in the destruction lipid second messenger that has been recognized as of cells by pathogens such as HIV, vaccinia virus, Shigella important in radiation-induced elimination of tumor and Salmonella [3,7]. In contrast to apoptosis, the molec- cells [26,27]. We demonstrate for the first time that in ular mechanisms of ciPCD are just beginning to be contrast to wildtype Bcl-2, restricted expression of Bcl-2 unraveled, e. g. by the recent identification of RIP3, solely at mitochondria-, the ER-, or the cytosol/nucleus is enzymes of the energy metabolism, Nox1, CYLD, Bmf, insufficient to prevent ciPCD. Therefore, our data suggest and cathepsin and calpain proteases as candidate media- a combined requirement of mitochondria and the ER in tors of ciPCD [2,8-10]. However, and despite these ceramide-mediated ciPCD and implicate the existence of advances, a coherent picture of the molecular steps in a molecular crosstalk between both organelles. ciPCD is still lacking. Methods Reagents and constructs Mitochondria have been identified not only as essential elicitors of apoptosis, but also of ciPCD, e.g. by release of Highly purified human recombinant TNF (hTNF) was proteins such as AIF, EndoG and HtrA2/OMI, as well as by supplied by BASF Bioresearch. Benzyloxycarbonyl-Val- production of reactive oxygen species (ROS) [2,11-13]. It Ala-Asp-fluoromethylketone (zVAD-fmk) was obtained has been proposed that excess formation of ROS triggers from Bachem. Cycloheximide (CHX) was purchased from ciPCD by activation of the DNA repair enzyme PARP, fol- Sigma. Expression constructs cloned in the vector pRc/ lowed by intracellular depletion of NAD and ATP, CMV (Invitrogen) encoding wildtype Bcl-2 (Bcl-2 WT), nuclear translocation of AIF and finally, death [14]. Simi- cytosolic Bcl-2 lacking the transmembrane domain (Bcl-2 lar to mitochondria, the endoplasmic reticulum (ER) - as ΔTM) and Bcl-2 mutants restricted to the outer mitochon- the main site for critical cellular functions such as protein drial membrane (Bcl-2 ActA) or to the endoplasmic retic- folding, lipid biosynthesis, and calcium storage in the cell ulum (Bcl-2 cb5) were kindly provided by B. Leber - has also been implicated in the induction of both apop- (McMaster University, Hamilton, Canada). tosis [15] and ciPCD [2,12,13]. In fact, the ER may play a key role in certain types of ciPCD, as intracellular calcium Cell culture and transfections influx caused by ER stress induces activation of calpains, a Wildtype human leukemic Jurkat cells were originally family of calcium-dependent cytosolic proteases that can obtained from the American Type Culture Collection. Jur- elicit ciPCD [2]. kat cells stably transfected with the plasmid pSFFV-Bcl-2, overexpressing full length human Bcl-2 at levels 10-20- Members of the Bcl-2 family of proteins have long been fold over untransfected cells [28] were kindly provided by recognized as central regulators of mitochondrial apopto- S. Korsmeyer (Harvard Medical School, Boston, USA). sis, primarily by controlling the permeabilization of the Cells were maintained in a mixture of Click's/RPMI 1640 outer mitochondrial membrane [15]. They have also been (50/50% v/v) supplemented with 10% v/v FCS, 2 mM found localized at the ER, where they regulate apoptosis glutamine and 50 μg/ml each of streptomycin and penicil- in response to a range of cellular stresses, and also in the lin in a humidified incubator containing 5% w/v CO . nucleus [15], where they may fulfill yet unknown func- Transient transfections of wildtype Jurkat cells were per- tions. Similar to the established protective functions of formed by Amaxa nucleofection (Lonza Cologne), using mitochondrial Bcl-2, expression of Bcl-2 at the ER has solution V and program C-16. Jurkat cells stably express- been shown to protect cells from apoptosis, e. g. by regu- ing the various versions of Bcl-2 in pRc/CMV were lating the release of proapoptotic calcium, reducing cal- obtained by electroporation using a Gene pulser II (Bio- cium-uptake by mitochondria and subsequent calpain- Rad) followed by selection with Geneticin (Invitrogen) dependent apoptosis [15]. The role of Bcl-2 in mitochon- and were used as pool transfectants. drial vs. ER-mediated apoptosis has been further Caspase assays addressed in a number of studies by the use of Bcl-2 con- structs that specifically localize to mitochondria or to the Cells were lysed in a buffer containing 10 mM Hepes pH ER [16-23]. In these studies, Bcl-2 localizing to the ER was 7.4, 142 mM KCl, 5 mM MgCl , 1 mM EGTA, 0.2% v/v shown to interfere with apoptosis induction by some (c- NP40, 1 mM DTT and 2 mM Pefabloc. To generate posi- myc overexpression, etoposide, staurosporine, tunicamy- tive controls for activation, cytosolic extracts of untreated cells were equilibrated for 1 h at 30°C after the addition Page 2 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 of 1 mM dATP and 10 μM cytochrome c to permit activa- exclusively by the lipase acid sphingomyelinase, and clo- tion of caspases and subsequent cleavage of substrate pro- nogenicity of tumor cells is dramatically reduced by acti- teins. To measure caspase activity, 100 μl of caspase buffer vation of this pathway [29-32]. To evaluate the role of (20 mM Pipes, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, wildtype Bcl-2, we treated wildtype Jurkat cells with TNF 0.1% w/v CHAPS, 10% w/v sucrose, pH 7.2) containing in combination with the broad-spectrum caspase-inhibi- 100 μM zDEVD-afc or zIETD-afc (Calbiochem) were tor zVAD and the protein biosynthesis inhibitor CHX (to added to 5 μl of cytosolic extract (50 μg protein) and incu- sensitize the cells) and compared their survival to a Jurkat bated at 37°C. The release of afc was measured as emis- cell line that stably overexpresses wildtype Bcl-2 in the sion at 505 nm upon excitation at 405 nm using a vector pSFFV-neo [28]. We have previously confirmed that Labsystems Fluoroskan II fluorimeter equipped with a this treatment induces PCD exclusively by caspase-inde- thermostated plate reader. pendent mechanisms, but not by apoptosis [31,32]. In this study, we additionally verified this by measuring the Microscopy activities of the initiator-caspase 8 and the effector-caspase For documentation of cell morphology, images from 3. As shown in Fig. 1A, no caspase activity over back- unfixed cells were obtained using an Axiovert 100 micro- ground was detectable in TNF/CHX/zVAD-treated cells, scope (Zeiss) and a DS-5 M camera (Nikon). whereas the proapoptotic stimulus TNF/CHX as a control strongly activated both caspases. To monitor ceramide- Cytotoxicity assays mediated ciPCD, we decided to analyze distal events in For flow cytometric measurement of cell death, cells were the pathway (changes in cell morphology, loss of mem- seeded in six-well plates at 5 × 10 cells/well. Following brane integrity), since it was not clear whether Bcl-2 treatment, cells were collected by centrifugation and would affect proximal steps of the signaling chain (e. g. resuspended in PBS/5 mM EDTA containing 2 μg/ml pro- the generation of the lipid ceramide [31]). When analyzed pidium iodide (PI), and the red fluorescence was meas- microscopically, both untreated cell lines uniformly dis- ured on a FACSCalibur flow cytometer (BD Biosciences). played an intact cell morphology with round cells and Since we observed that measurement exclusively of PI- almost no irregular shapes or cell debris (Fig. 1B, left pan- positive cells did not account for a significant fraction of els). As expected, induction of ciPCD by TNF/CHX/zVAD highly disintegrated dead cells that - due to diffusion of strongly increased the amount of Jurkat wildtype cells the dye - had already become PI-negative again (shown e. with clear morphological irregularities and the "fried egg- g. in Fig. 1C, right panels, lower left quadrants), we alter- like" shape characteristic for necrosis-like ciPCD (Fig. 1B, natively measured the fraction of large, PI negative cells upper right panel; [32]). In contrast, the majority of (lower right quadrants) which represents viable, intact wildtype-Bcl-2-overexpressing Jurkat cells retained an cells. For each measurement, a total of 10.000 cells was intact cell morphology despite treatment (Fig. 1B, lower analyzed. right panel). As a more objective parameter for ciPCD, we measured uptake of PI as a marker for loss of plasma Immunoblots membrane integrity in dying cells. Again, wildtype-Bcl-2- Cells were collected and lysed in TNE buffer (50 mM Tris overexpressing Jurkat cells clearly displayed a higher pH 8.0, 150 mM NaCl, 1% v/v NP40, 2 mM EDTA) con- resistance to TNF/CHX/zVAD-induced ceramide-medi- taining 10 μg/ml pepstatin/aprotinin/leupeptin, 1 mM ated ciPCD than their parental counterparts (Fig. 1C, D), sodium orthovanadate and 5 mM NaF. After removal of demonstrating that in addition to its anti-apoptotic prop- insoluble material by centrifugation at 10.000 × g and erties, wildtype Bcl-2 also can protect against ciPCD. Our 4°C for 15 min, the protein concentration of the superna- results are supported by studies from other groups dem- tants was measured using a BCA assay (Pierce). 30 μg of onstrating that overexpression of wildtype Bcl-2 can delay cell protein per lane were resolved by electrophoresis on the onset of ceramide-induced ciPCD in yet other cell 12.5% w/v SDS polyacrylamide gels (SDS-PAGE). After types, such as L929 fibrosarcoma cells [33]. As a possible electrophoretic transfer to Protran nitrocellulose (What- mechanism, Denecker and coworkers have suggested a man), reactive proteins were detected using a monoclonal prolonged integrity of mitochondrial oxidative phospho- IgG1 antibody specific for human Bcl-2 (sc-7382, Santa rylation or complexation of the proapoptotic protein Cruz) and the ECL detection kit (GE Healthcare). BNIP3 by wildtype Bcl-2 [34]. Moreover, our results implicate that both mitochondria and the ER represent Results target organelles of wildtype Bcl-2 that may participate in Wildtype Bcl-2 protects from ceramide-mediated ciPCD ceramide-mediated ciPCD. In a first set of experiments, we examined the ability of full-length, wildtype Bcl-2 to protect human leukemic Jur- Transient expression of Bcl-2 constructs with restricted kat T cells from ceramide-mediated ciPCD elicited by subcellular localization TNF-R1, a system that we have intensively characterized in Since the above results did not yet provide information on previous studies. In this system, ceramide is generated a specific contribution of each organelle, we made use of Page 3 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 Wildtyp Figure 1 e Bcl-2 protects from ceramide-induced ciPCD Wildtype Bcl-2 protects from ceramide-induced ciPCD. (A) Activity of caspase-8 and -3 in wildtype Jurkat cells in response to TNF/CHX/zVAD inducing ciPCD or TNF/CHX as a proapoptotic stimulus. Cells were incubated with 100 ng/ml hTNF in combination with 2 μg/ml CHX and/or 50 μM zVAD-fmk for 4 h before activation of caspases -8 and -3 was deter- mined by measuring the cleavage of fluorogenic substrates (zIETD-afc and zDEVD-afc) over 120 minutes. Prior to stimulation, cells were preincubated with 50 μM zVAD-fmk for 30 min (for stimulations with TNF/CHX/zVAD) or medium (stimulations with TNF/CHX). For positive control, caspases were activated in vitro by adding cytochrome c and dATP (Cyt c/dATP) to the cell extracts. (B) Wildtype (untransfected) Jurkat cells and Jurkat cells overexpressing pSFFV-Bcl-2 (Bcl-2 WT) were left untreated or stimulated with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk for 20 h before micro- graphs of the cells were taken to document their morphology. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. As representative examples, one healthy cell and one cell undergoing ciPCD with necrosis-like mor- phology are marked by a black or a white arrow, respectively. (C) In parallel, uptake of PI was determined by flow cytometry as a marker for loss of plasma membrane integrity (see „Materials and Methods). The percentage of viable cells (PI-negative, large) is indicated in the lower right quadrants of the dot plots. One representative experiment out of three performed is shown. (D) Quantification of cell viability data. The bar graphs represent the means from all three independent experiments, error bars indicate the respective standard deviations. Page 4 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 a panel of Bcl-2 constructs that are specifically expressed tern in the cytosol, but also localized to the nucleus [21]. in distinct cellular compartments. The carboxyterminus of Having verified the integrity of the generated stable trans- wildtype Bcl-2 contains a stretch of hydrophobic amino fectants, we monitored their resistance against ceramide- acids (the "insertion sequence") that has been proposed induced ciPCD. As shown in Fig. 3B and Fig. 3C, all to anchor proteins in the cell membrane. Previously, Zhu untreated samples uniformly displayed a high percentage and coworkers have replaced this natural 21 amino acid of viable cells after 48 h (Fig. 3B, left panels). Induction of insertion sequence of wildtype Bcl-2 by the analogous 26 ciPCD by TNF/CHX/zVAD, however, resulted in an amino acid insertion sequence from Listeria monocytogenes almost complete loss of viability in untransfected cells. ActA to target Bcl-2 specifically to the outer mitochondrial Jurkat cells stably overexpressing pRc/CMV-encoded membrane (Bcl-2 ActA, Fig. 2A, B). Similarly, the con- wildtype Bcl-2 were clearly protected against ciPCD, iden- struct Bcl-2 cb5 (Fig. 2A, B) contains the 35 amino acid tical to Jurkat cells stably overexpressing wildtype Bcl-2 insertion sequence of the ER-specific isoform of rat from the distinct construct pSFFV- Bcl-2 (Fig. 1). Also, Jur- hepatic cytochrome b5, directing Bcl-2 to the cytosolic kat cells expressing only the vector control pRc/CMV did face of the ER. In addition, Zhu and coworkers con- not significantly differ in their response from untrans- structed a gene encoding a form of Bcl-2 that lacks the fected Jurkat cells, confirming that the observed resistance hydrophobic membrane-anchoring sequence (Bcl-2 ΔTM, of Bcl-2 WT-overexpressing cells was genuine and not due Fig. 2A, B) and which localizes to the cytosol [16]. We to artifacts caused by the transfection/stable selection pro- transiently nucleofected wildtype Jurkat cells with the vec- cedure. Remarkably, the transfectants overexpressing tor pRc/CMV encoding wildtype Bcl-2 or with empty pRc/ mitochondrially and ER-targeted Bcl-2 (ActA and cb5) CMV and determined their resistance against ceramide- were not substantially better protected than the vector induced ciPCD. In analyses for PI-uptake, cells transfected controls. Likewise, overexpression of cytosolic Bcl-2 ΔTM with wildtype Bcl-2 generally displayed a slightly higher resulted only in slightly better protection from ceramide- viability than vector-transfected cells (this was seen for elicited ciPCD, however not nearly at the level of wildtype untreated as well as TNF/CHX/zVAD-treated cells). How- Bcl-2 (Fig. 3B, C). Similar results were obtained at 24 and ever, this difference was only marginal, and no pro- 72 h of incubation, although with increased or reduced nounced protection from ciPCD was seen in comparison overall viability, and with Bcl-2 ΔTM showing a protection to vector-transfected control cells (Fig. 2C, D). These even more comparable to Bcl-2 ActA and cb5 (data not results were confirmed in independent experiments utiliz- shown), in summary arguing that efficient protection ing cotransfection of green fluorescent protein as a marker from ceramide-mediated ciPCD is conferred only by for transfection efficiency (data not shown). Since we wildtype Bcl-2, but not by organelle-restricted expression have previously found that transient transfection of Jurkat of Bcl-2. cells often occurs with low efficiency, we generated lysates from the transfectants and analyzed them by Western Discussion blot. As shown in Fig. 2E, Jurkat cells transfected with In this study, we show for the first time that Bcl-2-medi- wildtype Bcl-2 showed only a limited overexpression of ated protection from non-apoptotic, caspase-independent the construct relative to the endogenous Bcl-2 protein PCD requires the full-length, wildtype protein whereas a present in vector-transfected control cells, thereby limited expression of Bcl-2 at mitochondria, the ER, or the explaining their inadequate protection. cytosol/nucleus is not sufficient to prevent ciPCD elicited by ceramide. The failure of the organelle-specific con- Wildtype, but not organelle-specific Bcl-2 protects from structs to confer protection is not due to an inefficient ceramide-mediated ciPCD in stably transfected Jurkat cells level of expression (Fig. 3A) or an incorrect localization We therefore generated Jurkat cells that stably overexpress [21], and their functionality has been previously demon- the above panel of organelle-specific pRc/CMV-Bcl-2 con- strated for apoptotic PCD using the same stable transfect- structs [21]. For each transfectant cell line, we confirmed ants as analyzed here [21,22]. Moreover, the protection by overexpression of the corresponding Bcl-2 construct by wildtype Bcl-2 was independently seen in two distinct sta- Western blot analyses (Fig. 3A). We additionally verified bly transfected Jurkat lines, expressed from different vec- the assumed subcellular localization of Bcl-2 for each sta- tor backbones (pSFFV-neo, pRc/CMV). bly transfected cell line by confocal laser scanning micro- copy as described elsewhere [21]. In these experiments, The fact that expression of Bcl-2 restricted to mitochon- only the ER-specific mutant Bcl-2 cb5, but not mitochon- dria, the ER (or the cytosol/nucleus, if the results with Bcl- dria-targeted Bcl-2 ActA or cytosol-directed Bcl-2 ΔTM 2 ΔTM are additionally taken into account) is not effec- colocalized with the endoplasmic calcium ATPase SERCA. tively suppressing ceramide-induced ciPCD may suggest Likewise, only Bcl-2 ActA, but not Bcl-2 cb5 or Bcl-2 ΔTM that wildtype Bcl-2 interferes with this form of PCD at yet colocalized with mitochondrial cytochrome c, whereas another cellular site. However, this is unlikely, as wildtype Bcl-2 ΔTM displayed the expected diffuse expression pat- Bcl-2 has been detected only at mitochondria, the ER and Page 5 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 Tr Figure 2 ansient expression of Bcl-2 constructs with defined subcellular localization in Jurkat cells Transient expression of Bcl-2 constructs with defined subcellular localization in Jurkat cells. (A) Schematic repre- sentation of human wildtype Bcl-2 (Bcl-2 WT) localizing to both mitochondria and the ER, Bcl-2 ActA targeted to mitochon- dria, Bcl-2 cb5 expressed at the ER, and Bcl-2 ΔTM localized in the cytosol. The Bcl-2 homology domains BH1-BH4 are indicated together with the transmembrane domain (TM) for wildtype Bcl-2, which is replaced by amino acids from Listeria monocytogenes ActA, from rat cytochrome b5 (cb5) or deleted altogether in the other constructs. (B) amino acid sequence of the carboxyterminus of wildtype Bcl-2, Bcl-2 ActA, Bcl-2 cb5 and Bcl-2 ΔTM. The amino acids derived from ActA and cb5 are shown in bold. (C) Wildtype Jurkat cells were transiently nucleofected with empty vector pRc/CMV or with pRc/CMV encod- ing wildtype Bcl-2. 24 h after transfection, the cells were stimulated with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk or left untreated for another 24 h. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. PI-uptake was determined by flow cytometry and the percentage of viable cells is indicated in the lower right quadrants of the dot plots. One representative experiment out of three performed is shown. (D) Quantification of cell viability data. The bar graphs represent the means from all three independent experiments, error bars indicate the respective standard deviations. Due to the transfection procedure, the cells generally display a lower viability than untransfected cells (Fig. 1). (E) In parallel, expression of Bcl-2 in the transfectants was visualized by Western blot analysis. The band in vector transfectants rep- resents endogenous Bcl-2. Page 6 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 Figure 3 Wildtype, but not organelle-restricted Bcl-2 protects stably transfected Jurkat cells from ceramide-mediated ciPCD Wildtype, but not organelle-restricted Bcl-2 protects stably transfected Jurkat cells from ceramide-mediated ciPCD. (A) Expression of pRc/CMV-encoded Bcl-2 WT, Bcl-2 cb5, Bcl-2 ActA and Bcl-2 ΔTM-constructs in stably transfected Jurkat cells. Cell lysates were prepared from untransfected Jurkat cells or Jurkat cells stably transfected with empty vector or with Bcl-2 constructs targeted to the ER, mitochondria, both, or the cytosol as indicated. Expression of the constructs was verified by immunoblot with a Bcl-2-specific antibody (sc-7382, Santa Cruz). Multiple bands result from detection of the endog- enous Bcl-2 protein in addition to the construct (see untransfected Jurkat cells). (B) Flow cytometric analysis of PI-uptake in untransfected and stably transfected Jurkat cells. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. After that, ceramide-mediated ciPCD was induced by treatment with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk for 48 h, or the cells were left completely untreated. The percentage of viable cells is shown in the lower right quadrants of the dot plots. (C) Bar graphs showing the fraction of viable cells for each of the stably transfected lines depicted in (B). Similar results were obtained in two additional experiments with different incubation times (24 and 72 h), although with increased or reduced overall viability (data not shown). Page 7 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 the nucleus in numerous previous studies (reviewed in TNFR-1/Ceramide TNFR-1/Ceramide [15,35]). Alternatively, the exclusive protection by RIP1, RIP RIP1, RIP3 3, CYLD, L , CYLD, Liipox poxy yg gen enases ases, NA , NADPH Oxid DPH Oxidase ase//c-J c-Ju un n wildtype Bcl-2 may be explained by a combined require- Ki Kina nase ses, Bm s, Bmff,, E En ner erg gy y m me etta abol boliis sm m ment of Bcl-2 at both mitochondria and at the ER, suggest- ? ? ing that both organelles coordinately participate in ciPCD Bcl-2 Bcl-2 Bcl-2 Bcl-2 Bcl-2 Bcl-2 via a molecular crosstalk. This hypothesis appears espe- ER Mito Nucleus Crosstalk ? cially attractive because a similar crosstalk has been ? shown for apoptotic PCD, where the ER controls mito- chondrial apoptosis by releasing proapoptotic calcium + + ROS, PARP, NAD /ATP-depletion, AIF, ROS, PARP, NAD /ATP-depletion, AIF, Cathepsins, Calpains, EndoG, HtrA2/OMI Cathepsins, Calpains, EndoG, HtrA2/OMI [15]. Although calcium likewise appears as an attractive candidate molecule for ER-mitochondrial crosstalk in ciPCD ciPCD ciPCD (especially when considering that the calcium- dependent calpain proteases can elicit ciPCD [2]), the pre- Overview scheme depicting th the ER and mitocho ciPCD Figure 4 ndria in TNF-R1/ e proposed requirement ceramide-mediated of cise nature of this crosstalk will have to be clarified in Overview scheme depicting the proposed require- future studies. ment of the ER and mitochondria in TNF-R1/cera- mide-mediated ciPCD. Known proximal mediators of Notably, in autophagy, a distinct form of ciPCD, Bcl-2 has TNF-R1/ceramide-induced ciPCD are indicated, as are medi- ators of ciPCD that potentially act downstream of the ER been recognized to act as a negative regulator specifically and mitochondria. Only wildtype Bcl-2 simultaneously acting at the ER by binding to the autophagy-inducing protein at the ER, at mitochondria and at the nucleus efficiently Beclin-1 [15]. blocks the caspase-independent death signals of TNF-R1/ ceramide, whereas Bcl-2 constructs specifically localizing to Even though the role of Bcl-2 has been most intensively each organelle do not prevent ciPCD. This suggests that the studied in mitochondria and the ER, Bcl-2 also localizes to corresponding signaling pathways of TNF-R1/ceramide target the nucleus. However, apart from a recent study where it both the ER and mitochondria, and that both organelles par- was shown that nuclear Bcl-2 forms an integral part of ticipate in ciPCD via a molecular crosstalk. The nucleus may mitotic chromosomes [36], information on the functions represent a further organelle that participates in these signal- of nuclear Bcl-2 are scarce. Therefore, nuclear functions of ing pathways, yet its role remains to be confirmed. Bcl-2 may likewise be required for suppression of ciPCD in concert with its mitochondrial and ER-specific func- tions. Authors' contributions AD carried out immunoblots, transient transfections, flow In summary, the further exploration of the exact roles of cytometric analyses and analyzed data. JS carried out addi- mitochondrial, ER- and nucleus-specific Bcl-2 as well as tional flow cytometry, morphological analysis by micros- their potential crosstalk will provide further insight into copy and analyzed data. LT analyzed caspase activity. CB the molecular mechanisms by which ceramide-mediated provided critical reagents and participated in the design of ciPCD is executed. At the clinical level, this may directly the study. DA conceived and designed the experiments, prove beneficial also for radiation therapy by providing analyzed data and wrote the paper. All authors read and novel options to eliminate tumor cells that have become approved the final manuscript. resistant to apoptotic death signals. Acknowledgements We thank D. Kabelitz for his continuous support and encouragement. This Conclusion work was supported by grants from the from the Forschungskommission Based upon the data presented, we conclude that expres- der Medizinischen Fakultät and from the Deutsche Forschungsgemeinschaft sion of Bcl-2 at both the ER and mitochondria (and pos- to D. A. (SFB 415, A4). 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Nat Med 2005, peer reviewed and published immediately upon acceptance 11:477-478. cited in PubMed and archived on PubMed Central 27. Kolesnick R, Fuks Z: Radiation and ceramide-induced apopto- sis. Oncogene 2003, 22:5897-5906. yours — you keep the copyright 28. Armstrong RC, Aja T, Xiang J, Gaur S, Krebs JF, Hoang K, Bai X, Kors- BioMedcentral meyer SJ, Karanewsky DS, Fritz LC, Tomaselli KJ: Fas-induced acti- Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Oncology Springer Journals

Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death

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Springer Journals
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Copyright © 2009 by Deerberg et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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1748-717X
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10.1186/1748-717X-4-41
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19818125
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Abstract

Background: Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase- independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. Methods: We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 ΔTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. Results: Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. Conclusion: Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide. Background dependent apoptosis, the most well-studied form of PCD, The survival and homeostasis of multicellular organisms represents the principal suicide program in most physio- critically depends on programmed cell death (PCD) to logical settings [1], many cells have the ability to commit correctly remove cells that are potentially harmful or suicide by caspase-independent modes of PCD (ciPCD) which have fulfilled their function. Although caspase- [2]. ciPCD fulfills vital functions in processes such as the Page 1 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 negative selection of lymphocytes, the termination of cin, brefeldin A, ceramide, ionizing radiation, thapsi- immune responses, embryonic removal of interdigital gargin, Bax, Bad) but not all stimuli (reviewed in [24,25]). webs, regulation of bone growth, ovulation, and cellular turnover in the intestine [3]. Furthermore, ciPCD has In contrast to this relative wealth of available data on been implicated the pathology of hyperacute shock [4], apoptosis, the role of ER- vs. mitochondrially localized pancreatitis [5,6], cerebral and myocardial ischemia- Bcl-2 in ciPCD has not been investigated so far. Here, we reperfusion injury, epilepsy, Alzheimer's disease and utilize ciPCD elicited by ceramide as a model system, a other inflammatory injuries, as well as in the destruction lipid second messenger that has been recognized as of cells by pathogens such as HIV, vaccinia virus, Shigella important in radiation-induced elimination of tumor and Salmonella [3,7]. In contrast to apoptosis, the molec- cells [26,27]. We demonstrate for the first time that in ular mechanisms of ciPCD are just beginning to be contrast to wildtype Bcl-2, restricted expression of Bcl-2 unraveled, e. g. by the recent identification of RIP3, solely at mitochondria-, the ER-, or the cytosol/nucleus is enzymes of the energy metabolism, Nox1, CYLD, Bmf, insufficient to prevent ciPCD. Therefore, our data suggest and cathepsin and calpain proteases as candidate media- a combined requirement of mitochondria and the ER in tors of ciPCD [2,8-10]. However, and despite these ceramide-mediated ciPCD and implicate the existence of advances, a coherent picture of the molecular steps in a molecular crosstalk between both organelles. ciPCD is still lacking. Methods Reagents and constructs Mitochondria have been identified not only as essential elicitors of apoptosis, but also of ciPCD, e.g. by release of Highly purified human recombinant TNF (hTNF) was proteins such as AIF, EndoG and HtrA2/OMI, as well as by supplied by BASF Bioresearch. Benzyloxycarbonyl-Val- production of reactive oxygen species (ROS) [2,11-13]. It Ala-Asp-fluoromethylketone (zVAD-fmk) was obtained has been proposed that excess formation of ROS triggers from Bachem. Cycloheximide (CHX) was purchased from ciPCD by activation of the DNA repair enzyme PARP, fol- Sigma. Expression constructs cloned in the vector pRc/ lowed by intracellular depletion of NAD and ATP, CMV (Invitrogen) encoding wildtype Bcl-2 (Bcl-2 WT), nuclear translocation of AIF and finally, death [14]. Simi- cytosolic Bcl-2 lacking the transmembrane domain (Bcl-2 lar to mitochondria, the endoplasmic reticulum (ER) - as ΔTM) and Bcl-2 mutants restricted to the outer mitochon- the main site for critical cellular functions such as protein drial membrane (Bcl-2 ActA) or to the endoplasmic retic- folding, lipid biosynthesis, and calcium storage in the cell ulum (Bcl-2 cb5) were kindly provided by B. Leber - has also been implicated in the induction of both apop- (McMaster University, Hamilton, Canada). tosis [15] and ciPCD [2,12,13]. In fact, the ER may play a key role in certain types of ciPCD, as intracellular calcium Cell culture and transfections influx caused by ER stress induces activation of calpains, a Wildtype human leukemic Jurkat cells were originally family of calcium-dependent cytosolic proteases that can obtained from the American Type Culture Collection. Jur- elicit ciPCD [2]. kat cells stably transfected with the plasmid pSFFV-Bcl-2, overexpressing full length human Bcl-2 at levels 10-20- Members of the Bcl-2 family of proteins have long been fold over untransfected cells [28] were kindly provided by recognized as central regulators of mitochondrial apopto- S. Korsmeyer (Harvard Medical School, Boston, USA). sis, primarily by controlling the permeabilization of the Cells were maintained in a mixture of Click's/RPMI 1640 outer mitochondrial membrane [15]. They have also been (50/50% v/v) supplemented with 10% v/v FCS, 2 mM found localized at the ER, where they regulate apoptosis glutamine and 50 μg/ml each of streptomycin and penicil- in response to a range of cellular stresses, and also in the lin in a humidified incubator containing 5% w/v CO . nucleus [15], where they may fulfill yet unknown func- Transient transfections of wildtype Jurkat cells were per- tions. Similar to the established protective functions of formed by Amaxa nucleofection (Lonza Cologne), using mitochondrial Bcl-2, expression of Bcl-2 at the ER has solution V and program C-16. Jurkat cells stably express- been shown to protect cells from apoptosis, e. g. by regu- ing the various versions of Bcl-2 in pRc/CMV were lating the release of proapoptotic calcium, reducing cal- obtained by electroporation using a Gene pulser II (Bio- cium-uptake by mitochondria and subsequent calpain- Rad) followed by selection with Geneticin (Invitrogen) dependent apoptosis [15]. The role of Bcl-2 in mitochon- and were used as pool transfectants. drial vs. ER-mediated apoptosis has been further Caspase assays addressed in a number of studies by the use of Bcl-2 con- structs that specifically localize to mitochondria or to the Cells were lysed in a buffer containing 10 mM Hepes pH ER [16-23]. In these studies, Bcl-2 localizing to the ER was 7.4, 142 mM KCl, 5 mM MgCl , 1 mM EGTA, 0.2% v/v shown to interfere with apoptosis induction by some (c- NP40, 1 mM DTT and 2 mM Pefabloc. To generate posi- myc overexpression, etoposide, staurosporine, tunicamy- tive controls for activation, cytosolic extracts of untreated cells were equilibrated for 1 h at 30°C after the addition Page 2 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 of 1 mM dATP and 10 μM cytochrome c to permit activa- exclusively by the lipase acid sphingomyelinase, and clo- tion of caspases and subsequent cleavage of substrate pro- nogenicity of tumor cells is dramatically reduced by acti- teins. To measure caspase activity, 100 μl of caspase buffer vation of this pathway [29-32]. To evaluate the role of (20 mM Pipes, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, wildtype Bcl-2, we treated wildtype Jurkat cells with TNF 0.1% w/v CHAPS, 10% w/v sucrose, pH 7.2) containing in combination with the broad-spectrum caspase-inhibi- 100 μM zDEVD-afc or zIETD-afc (Calbiochem) were tor zVAD and the protein biosynthesis inhibitor CHX (to added to 5 μl of cytosolic extract (50 μg protein) and incu- sensitize the cells) and compared their survival to a Jurkat bated at 37°C. The release of afc was measured as emis- cell line that stably overexpresses wildtype Bcl-2 in the sion at 505 nm upon excitation at 405 nm using a vector pSFFV-neo [28]. We have previously confirmed that Labsystems Fluoroskan II fluorimeter equipped with a this treatment induces PCD exclusively by caspase-inde- thermostated plate reader. pendent mechanisms, but not by apoptosis [31,32]. In this study, we additionally verified this by measuring the Microscopy activities of the initiator-caspase 8 and the effector-caspase For documentation of cell morphology, images from 3. As shown in Fig. 1A, no caspase activity over back- unfixed cells were obtained using an Axiovert 100 micro- ground was detectable in TNF/CHX/zVAD-treated cells, scope (Zeiss) and a DS-5 M camera (Nikon). whereas the proapoptotic stimulus TNF/CHX as a control strongly activated both caspases. To monitor ceramide- Cytotoxicity assays mediated ciPCD, we decided to analyze distal events in For flow cytometric measurement of cell death, cells were the pathway (changes in cell morphology, loss of mem- seeded in six-well plates at 5 × 10 cells/well. Following brane integrity), since it was not clear whether Bcl-2 treatment, cells were collected by centrifugation and would affect proximal steps of the signaling chain (e. g. resuspended in PBS/5 mM EDTA containing 2 μg/ml pro- the generation of the lipid ceramide [31]). When analyzed pidium iodide (PI), and the red fluorescence was meas- microscopically, both untreated cell lines uniformly dis- ured on a FACSCalibur flow cytometer (BD Biosciences). played an intact cell morphology with round cells and Since we observed that measurement exclusively of PI- almost no irregular shapes or cell debris (Fig. 1B, left pan- positive cells did not account for a significant fraction of els). As expected, induction of ciPCD by TNF/CHX/zVAD highly disintegrated dead cells that - due to diffusion of strongly increased the amount of Jurkat wildtype cells the dye - had already become PI-negative again (shown e. with clear morphological irregularities and the "fried egg- g. in Fig. 1C, right panels, lower left quadrants), we alter- like" shape characteristic for necrosis-like ciPCD (Fig. 1B, natively measured the fraction of large, PI negative cells upper right panel; [32]). In contrast, the majority of (lower right quadrants) which represents viable, intact wildtype-Bcl-2-overexpressing Jurkat cells retained an cells. For each measurement, a total of 10.000 cells was intact cell morphology despite treatment (Fig. 1B, lower analyzed. right panel). As a more objective parameter for ciPCD, we measured uptake of PI as a marker for loss of plasma Immunoblots membrane integrity in dying cells. Again, wildtype-Bcl-2- Cells were collected and lysed in TNE buffer (50 mM Tris overexpressing Jurkat cells clearly displayed a higher pH 8.0, 150 mM NaCl, 1% v/v NP40, 2 mM EDTA) con- resistance to TNF/CHX/zVAD-induced ceramide-medi- taining 10 μg/ml pepstatin/aprotinin/leupeptin, 1 mM ated ciPCD than their parental counterparts (Fig. 1C, D), sodium orthovanadate and 5 mM NaF. After removal of demonstrating that in addition to its anti-apoptotic prop- insoluble material by centrifugation at 10.000 × g and erties, wildtype Bcl-2 also can protect against ciPCD. Our 4°C for 15 min, the protein concentration of the superna- results are supported by studies from other groups dem- tants was measured using a BCA assay (Pierce). 30 μg of onstrating that overexpression of wildtype Bcl-2 can delay cell protein per lane were resolved by electrophoresis on the onset of ceramide-induced ciPCD in yet other cell 12.5% w/v SDS polyacrylamide gels (SDS-PAGE). After types, such as L929 fibrosarcoma cells [33]. As a possible electrophoretic transfer to Protran nitrocellulose (What- mechanism, Denecker and coworkers have suggested a man), reactive proteins were detected using a monoclonal prolonged integrity of mitochondrial oxidative phospho- IgG1 antibody specific for human Bcl-2 (sc-7382, Santa rylation or complexation of the proapoptotic protein Cruz) and the ECL detection kit (GE Healthcare). BNIP3 by wildtype Bcl-2 [34]. Moreover, our results implicate that both mitochondria and the ER represent Results target organelles of wildtype Bcl-2 that may participate in Wildtype Bcl-2 protects from ceramide-mediated ciPCD ceramide-mediated ciPCD. In a first set of experiments, we examined the ability of full-length, wildtype Bcl-2 to protect human leukemic Jur- Transient expression of Bcl-2 constructs with restricted kat T cells from ceramide-mediated ciPCD elicited by subcellular localization TNF-R1, a system that we have intensively characterized in Since the above results did not yet provide information on previous studies. In this system, ceramide is generated a specific contribution of each organelle, we made use of Page 3 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 Wildtyp Figure 1 e Bcl-2 protects from ceramide-induced ciPCD Wildtype Bcl-2 protects from ceramide-induced ciPCD. (A) Activity of caspase-8 and -3 in wildtype Jurkat cells in response to TNF/CHX/zVAD inducing ciPCD or TNF/CHX as a proapoptotic stimulus. Cells were incubated with 100 ng/ml hTNF in combination with 2 μg/ml CHX and/or 50 μM zVAD-fmk for 4 h before activation of caspases -8 and -3 was deter- mined by measuring the cleavage of fluorogenic substrates (zIETD-afc and zDEVD-afc) over 120 minutes. Prior to stimulation, cells were preincubated with 50 μM zVAD-fmk for 30 min (for stimulations with TNF/CHX/zVAD) or medium (stimulations with TNF/CHX). For positive control, caspases were activated in vitro by adding cytochrome c and dATP (Cyt c/dATP) to the cell extracts. (B) Wildtype (untransfected) Jurkat cells and Jurkat cells overexpressing pSFFV-Bcl-2 (Bcl-2 WT) were left untreated or stimulated with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk for 20 h before micro- graphs of the cells were taken to document their morphology. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. As representative examples, one healthy cell and one cell undergoing ciPCD with necrosis-like mor- phology are marked by a black or a white arrow, respectively. (C) In parallel, uptake of PI was determined by flow cytometry as a marker for loss of plasma membrane integrity (see „Materials and Methods). The percentage of viable cells (PI-negative, large) is indicated in the lower right quadrants of the dot plots. One representative experiment out of three performed is shown. (D) Quantification of cell viability data. The bar graphs represent the means from all three independent experiments, error bars indicate the respective standard deviations. Page 4 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 a panel of Bcl-2 constructs that are specifically expressed tern in the cytosol, but also localized to the nucleus [21]. in distinct cellular compartments. The carboxyterminus of Having verified the integrity of the generated stable trans- wildtype Bcl-2 contains a stretch of hydrophobic amino fectants, we monitored their resistance against ceramide- acids (the "insertion sequence") that has been proposed induced ciPCD. As shown in Fig. 3B and Fig. 3C, all to anchor proteins in the cell membrane. Previously, Zhu untreated samples uniformly displayed a high percentage and coworkers have replaced this natural 21 amino acid of viable cells after 48 h (Fig. 3B, left panels). Induction of insertion sequence of wildtype Bcl-2 by the analogous 26 ciPCD by TNF/CHX/zVAD, however, resulted in an amino acid insertion sequence from Listeria monocytogenes almost complete loss of viability in untransfected cells. ActA to target Bcl-2 specifically to the outer mitochondrial Jurkat cells stably overexpressing pRc/CMV-encoded membrane (Bcl-2 ActA, Fig. 2A, B). Similarly, the con- wildtype Bcl-2 were clearly protected against ciPCD, iden- struct Bcl-2 cb5 (Fig. 2A, B) contains the 35 amino acid tical to Jurkat cells stably overexpressing wildtype Bcl-2 insertion sequence of the ER-specific isoform of rat from the distinct construct pSFFV- Bcl-2 (Fig. 1). Also, Jur- hepatic cytochrome b5, directing Bcl-2 to the cytosolic kat cells expressing only the vector control pRc/CMV did face of the ER. In addition, Zhu and coworkers con- not significantly differ in their response from untrans- structed a gene encoding a form of Bcl-2 that lacks the fected Jurkat cells, confirming that the observed resistance hydrophobic membrane-anchoring sequence (Bcl-2 ΔTM, of Bcl-2 WT-overexpressing cells was genuine and not due Fig. 2A, B) and which localizes to the cytosol [16]. We to artifacts caused by the transfection/stable selection pro- transiently nucleofected wildtype Jurkat cells with the vec- cedure. Remarkably, the transfectants overexpressing tor pRc/CMV encoding wildtype Bcl-2 or with empty pRc/ mitochondrially and ER-targeted Bcl-2 (ActA and cb5) CMV and determined their resistance against ceramide- were not substantially better protected than the vector induced ciPCD. In analyses for PI-uptake, cells transfected controls. Likewise, overexpression of cytosolic Bcl-2 ΔTM with wildtype Bcl-2 generally displayed a slightly higher resulted only in slightly better protection from ceramide- viability than vector-transfected cells (this was seen for elicited ciPCD, however not nearly at the level of wildtype untreated as well as TNF/CHX/zVAD-treated cells). How- Bcl-2 (Fig. 3B, C). Similar results were obtained at 24 and ever, this difference was only marginal, and no pro- 72 h of incubation, although with increased or reduced nounced protection from ciPCD was seen in comparison overall viability, and with Bcl-2 ΔTM showing a protection to vector-transfected control cells (Fig. 2C, D). These even more comparable to Bcl-2 ActA and cb5 (data not results were confirmed in independent experiments utiliz- shown), in summary arguing that efficient protection ing cotransfection of green fluorescent protein as a marker from ceramide-mediated ciPCD is conferred only by for transfection efficiency (data not shown). Since we wildtype Bcl-2, but not by organelle-restricted expression have previously found that transient transfection of Jurkat of Bcl-2. cells often occurs with low efficiency, we generated lysates from the transfectants and analyzed them by Western Discussion blot. As shown in Fig. 2E, Jurkat cells transfected with In this study, we show for the first time that Bcl-2-medi- wildtype Bcl-2 showed only a limited overexpression of ated protection from non-apoptotic, caspase-independent the construct relative to the endogenous Bcl-2 protein PCD requires the full-length, wildtype protein whereas a present in vector-transfected control cells, thereby limited expression of Bcl-2 at mitochondria, the ER, or the explaining their inadequate protection. cytosol/nucleus is not sufficient to prevent ciPCD elicited by ceramide. The failure of the organelle-specific con- Wildtype, but not organelle-specific Bcl-2 protects from structs to confer protection is not due to an inefficient ceramide-mediated ciPCD in stably transfected Jurkat cells level of expression (Fig. 3A) or an incorrect localization We therefore generated Jurkat cells that stably overexpress [21], and their functionality has been previously demon- the above panel of organelle-specific pRc/CMV-Bcl-2 con- strated for apoptotic PCD using the same stable transfect- structs [21]. For each transfectant cell line, we confirmed ants as analyzed here [21,22]. Moreover, the protection by overexpression of the corresponding Bcl-2 construct by wildtype Bcl-2 was independently seen in two distinct sta- Western blot analyses (Fig. 3A). We additionally verified bly transfected Jurkat lines, expressed from different vec- the assumed subcellular localization of Bcl-2 for each sta- tor backbones (pSFFV-neo, pRc/CMV). bly transfected cell line by confocal laser scanning micro- copy as described elsewhere [21]. In these experiments, The fact that expression of Bcl-2 restricted to mitochon- only the ER-specific mutant Bcl-2 cb5, but not mitochon- dria, the ER (or the cytosol/nucleus, if the results with Bcl- dria-targeted Bcl-2 ActA or cytosol-directed Bcl-2 ΔTM 2 ΔTM are additionally taken into account) is not effec- colocalized with the endoplasmic calcium ATPase SERCA. tively suppressing ceramide-induced ciPCD may suggest Likewise, only Bcl-2 ActA, but not Bcl-2 cb5 or Bcl-2 ΔTM that wildtype Bcl-2 interferes with this form of PCD at yet colocalized with mitochondrial cytochrome c, whereas another cellular site. However, this is unlikely, as wildtype Bcl-2 ΔTM displayed the expected diffuse expression pat- Bcl-2 has been detected only at mitochondria, the ER and Page 5 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 Tr Figure 2 ansient expression of Bcl-2 constructs with defined subcellular localization in Jurkat cells Transient expression of Bcl-2 constructs with defined subcellular localization in Jurkat cells. (A) Schematic repre- sentation of human wildtype Bcl-2 (Bcl-2 WT) localizing to both mitochondria and the ER, Bcl-2 ActA targeted to mitochon- dria, Bcl-2 cb5 expressed at the ER, and Bcl-2 ΔTM localized in the cytosol. The Bcl-2 homology domains BH1-BH4 are indicated together with the transmembrane domain (TM) for wildtype Bcl-2, which is replaced by amino acids from Listeria monocytogenes ActA, from rat cytochrome b5 (cb5) or deleted altogether in the other constructs. (B) amino acid sequence of the carboxyterminus of wildtype Bcl-2, Bcl-2 ActA, Bcl-2 cb5 and Bcl-2 ΔTM. The amino acids derived from ActA and cb5 are shown in bold. (C) Wildtype Jurkat cells were transiently nucleofected with empty vector pRc/CMV or with pRc/CMV encod- ing wildtype Bcl-2. 24 h after transfection, the cells were stimulated with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk or left untreated for another 24 h. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. PI-uptake was determined by flow cytometry and the percentage of viable cells is indicated in the lower right quadrants of the dot plots. One representative experiment out of three performed is shown. (D) Quantification of cell viability data. The bar graphs represent the means from all three independent experiments, error bars indicate the respective standard deviations. Due to the transfection procedure, the cells generally display a lower viability than untransfected cells (Fig. 1). (E) In parallel, expression of Bcl-2 in the transfectants was visualized by Western blot analysis. The band in vector transfectants rep- resents endogenous Bcl-2. Page 6 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 Figure 3 Wildtype, but not organelle-restricted Bcl-2 protects stably transfected Jurkat cells from ceramide-mediated ciPCD Wildtype, but not organelle-restricted Bcl-2 protects stably transfected Jurkat cells from ceramide-mediated ciPCD. (A) Expression of pRc/CMV-encoded Bcl-2 WT, Bcl-2 cb5, Bcl-2 ActA and Bcl-2 ΔTM-constructs in stably transfected Jurkat cells. Cell lysates were prepared from untransfected Jurkat cells or Jurkat cells stably transfected with empty vector or with Bcl-2 constructs targeted to the ER, mitochondria, both, or the cytosol as indicated. Expression of the constructs was verified by immunoblot with a Bcl-2-specific antibody (sc-7382, Santa Cruz). Multiple bands result from detection of the endog- enous Bcl-2 protein in addition to the construct (see untransfected Jurkat cells). (B) Flow cytometric analysis of PI-uptake in untransfected and stably transfected Jurkat cells. Prior to stimulation, the cells were preincubated for 60 min with 50 μM zVAD-fmk. After that, ceramide-mediated ciPCD was induced by treatment with 100 ng/ml hTNF in combination with 5 μg/ml CHX and 50 μM zVAD-fmk for 48 h, or the cells were left completely untreated. The percentage of viable cells is shown in the lower right quadrants of the dot plots. (C) Bar graphs showing the fraction of viable cells for each of the stably transfected lines depicted in (B). Similar results were obtained in two additional experiments with different incubation times (24 and 72 h), although with increased or reduced overall viability (data not shown). Page 7 of 9 (page number not for citation purposes) Radiation Oncology 2009, 4:41 http://www.ro-journal.com/content/4/1/41 the nucleus in numerous previous studies (reviewed in TNFR-1/Ceramide TNFR-1/Ceramide [15,35]). Alternatively, the exclusive protection by RIP1, RIP RIP1, RIP3 3, CYLD, L , CYLD, Liipox poxy yg gen enases ases, NA , NADPH Oxid DPH Oxidase ase//c-J c-Ju un n wildtype Bcl-2 may be explained by a combined require- Ki Kina nase ses, Bm s, Bmff,, E En ner erg gy y m me etta abol boliis sm m ment of Bcl-2 at both mitochondria and at the ER, suggest- ? ? ing that both organelles coordinately participate in ciPCD Bcl-2 Bcl-2 Bcl-2 Bcl-2 Bcl-2 Bcl-2 via a molecular crosstalk. This hypothesis appears espe- ER Mito Nucleus Crosstalk ? cially attractive because a similar crosstalk has been ? shown for apoptotic PCD, where the ER controls mito- chondrial apoptosis by releasing proapoptotic calcium + + ROS, PARP, NAD /ATP-depletion, AIF, ROS, PARP, NAD /ATP-depletion, AIF, Cathepsins, Calpains, EndoG, HtrA2/OMI Cathepsins, Calpains, EndoG, HtrA2/OMI [15]. Although calcium likewise appears as an attractive candidate molecule for ER-mitochondrial crosstalk in ciPCD ciPCD ciPCD (especially when considering that the calcium- dependent calpain proteases can elicit ciPCD [2]), the pre- Overview scheme depicting th the ER and mitocho ciPCD Figure 4 ndria in TNF-R1/ e proposed requirement ceramide-mediated of cise nature of this crosstalk will have to be clarified in Overview scheme depicting the proposed require- future studies. ment of the ER and mitochondria in TNF-R1/cera- mide-mediated ciPCD. Known proximal mediators of Notably, in autophagy, a distinct form of ciPCD, Bcl-2 has TNF-R1/ceramide-induced ciPCD are indicated, as are medi- ators of ciPCD that potentially act downstream of the ER been recognized to act as a negative regulator specifically and mitochondria. Only wildtype Bcl-2 simultaneously acting at the ER by binding to the autophagy-inducing protein at the ER, at mitochondria and at the nucleus efficiently Beclin-1 [15]. blocks the caspase-independent death signals of TNF-R1/ ceramide, whereas Bcl-2 constructs specifically localizing to Even though the role of Bcl-2 has been most intensively each organelle do not prevent ciPCD. This suggests that the studied in mitochondria and the ER, Bcl-2 also localizes to corresponding signaling pathways of TNF-R1/ceramide target the nucleus. However, apart from a recent study where it both the ER and mitochondria, and that both organelles par- was shown that nuclear Bcl-2 forms an integral part of ticipate in ciPCD via a molecular crosstalk. The nucleus may mitotic chromosomes [36], information on the functions represent a further organelle that participates in these signal- of nuclear Bcl-2 are scarce. Therefore, nuclear functions of ing pathways, yet its role remains to be confirmed. Bcl-2 may likewise be required for suppression of ciPCD in concert with its mitochondrial and ER-specific func- tions. Authors' contributions AD carried out immunoblots, transient transfections, flow In summary, the further exploration of the exact roles of cytometric analyses and analyzed data. JS carried out addi- mitochondrial, ER- and nucleus-specific Bcl-2 as well as tional flow cytometry, morphological analysis by micros- their potential crosstalk will provide further insight into copy and analyzed data. LT analyzed caspase activity. CB the molecular mechanisms by which ceramide-mediated provided critical reagents and participated in the design of ciPCD is executed. At the clinical level, this may directly the study. DA conceived and designed the experiments, prove beneficial also for radiation therapy by providing analyzed data and wrote the paper. All authors read and novel options to eliminate tumor cells that have become approved the final manuscript. resistant to apoptotic death signals. Acknowledgements We thank D. Kabelitz for his continuous support and encouragement. This Conclusion work was supported by grants from the from the Forschungskommission Based upon the data presented, we conclude that expres- der Medizinischen Fakultät and from the Deutsche Forschungsgemeinschaft sion of Bcl-2 at both the ER and mitochondria (and pos- to D. A. (SFB 415, A4). 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Radiation OncologySpringer Journals

Published: Oct 9, 2009

References