|14 September 2000, Volume 19, Number 39, Pages 4461-4468|
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|Matrix detachment induces caspase-dependent cytochrome c release from mitochondria: inhibition by PKB/Akt but not Raf signalling|
|Marjatta Rytömaa, Kerstin Lehmann and Julian Downward|
Signal Transduction Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
Correspondence to: J Downward, Signal Transduction Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
Detachment of epithelial cells from extracellular matrix results in induction of apoptosis ('anoikis') which can be blocked by expression of activated Ras or PKB/Akt. Here we show that detachment causes release of cytochrome c from mitochondria in MDCK cells. This is blocked by caspase inhibitors, suggesting a role for caspases upstream of mitochondria in the initiation of anoikis, in accord with the ability of dominant negative FADD to inhibit this form of cell death. Bulk activation of caspase-8 following detachment lags behind cytochrome c release, and is likely the result of a mitochondrial positive feed back loop. Matrix detachment also induces Bax translocation to mitochondria in a caspase-dependent manner. Expression of activated Ras or PKB/Akt blocks all the detectable events on the detachment-induced apoptosis signalling pathway, suggesting that PKB/Akt acts at an early point in the pathway, providing the signal normally generated by matrix attachment. Strong activation of Raf can also protect MDCK cells from detachment induced apoptosis, but this occurs at a point downstream of cytochrome c release from mitochondria, and is clearly distinct from the effect of PKB/Akt. Oncogene (2000) 19, 4461-4468
anoikis; apoptosis; Ras; Akt; PKB; Raf
Untransformed adherent cells commonly engage a cell death programme when detached from the extracellular matrix (Boudreau et al., 1995; Frisch and Francis, 1994; Meredith et al., 1993; Meredith and Schwartz, 1997). This type of apoptosis has been named anoikis (Frisch and Francis, 1994). Matrix binding to cell surface integrins normally triggers a survival signal for cells through the activation of phosphoinositide 3-OH kinase (PI 3-kinase) (Khwaja et al., 1997), possibly mediated in part by p125FAK, the focal adhesion kinase (Frisch et al., 1996). Downstream of PI 3-kinase, the protein kinase PKB/Akt is responsible for transmitting the matrix-induced survival signal, as has also been found to be the case in several other anti-apoptotic systems (Downward, 1998). A number of substrate proteins for PKB/Akt have been reported which may be involved in its anti-apoptotic effects (reviewed in Datta et al., 1999).
Detachment induced apoptosis is suppressed in epithelial cells transformed by ras or src oncogenes (Frisch and Francis, 1994). Mutationally activated Ras protein protects cells from anoikis by stimulating PI 3-kinase through direct interaction with the catalytic p110 subunit, leading to activation of PKB/Akt (Khwaja et al., 1997) and thereby mimicking the protective effects of matrix even in detached cells. Ras proteins are known to directly engage several different effector pathways including both the well characterized Raf/MAP kinase system and PI 3-kinase/PKB/Akt (Marshall, 1996); Ras transformation appears to involve the synergistic effects of activating these multiple signalling systems (Rodriguez-Viciana et al., 1997; White et al., 1995). Many tumour cells may also acquire resistance to detachment-induced and other forms of apoptosis through deletion of PTEN, a phosphatidylinositol trisphosphate 3-phosphatase which reverses the action of PI 3-kinase and hence is required to keep PKB/Akt activity in check (Maehama and Dixon, 1999).
It is possible that removal of the PKB/Akt mediated survival signal originating from activated integrins leads to the ability of a constitutive pre-existing death signal to be transmitted as far as execution events. Alternatively, there could also be direct induction of a death signal by detachment: cell death would result from the combined effect of inducing a death signal at the same time as removing the matrix-derived survival signal. Expression of a dominant negative form of the Fas adaptor protein FADD or over-expression of SODD, a silencer of death domains, have been recently reported to block detachment induced apoptosis in MDCK cells (Frisch, 1999; Rytömaa et al., 1999). This suggests the involvement of a death domain containing protein, possibly a death receptor, in the induction of apoptosis following loss of the adhesion generated PKB/Akt survival signal.
In this study we further investigate the mechanism by which apoptosis is induced following epithelial cell detachment from extracellular matrix, and how Ras signalling pathways impact on this to block cell death. We show that detachment induces both translocation of the pro-apoptotic Bcl-2 family protein Bax to, and release of cytochrome c from, mitochondria. Both events are dependent on caspase activity, suggesting that caspase activation is an initiating event in this process, acting upstream of effects on the mitochondria. Ras and PKB/Akt block the death signal at a very early stage, upstream of Bax and cytochrome c movements. By contrast, strong activation of Raf can protect cells from apoptosis, but acts downstream of cytochrome c release. It is proposed that detachment triggers apoptosis through a process analogous to death receptor killing in type II cells (Schulze-Osthoff et al., 1998), where a mitochondrial amplification loop is required, and that PKB/Akt and Raf impact on different stages of this pathway.
We have previously shown that detachment of epithelial cells from extracellular matrix leads to the activation of a caspase cascade, and that this can be blocked by dominant negative FADD (Rytömaa et al., 1999), perhaps suggesting the involvement of a death receptor pathway. To further investigate the mechanism of anoikis we wanted to study the possible involvement of cytochrome c release from the inter-membrane space of mitochondria in detachment-induced apoptosis. It has been suggested that in some cell types the oligomerization of death receptors leading to formation of death inducing signalling complex (DISC) initially activates only a very small amount of caspase-8 (Scaffidi et al., 1998). In these so called type II cells, cytochrome c would then be released from the mitochondria to the cytoplasm, probably due to the cleavage of Bid by caspase-8 (Li et al., 1998; Luo et al., 1998) and translocation of Bid and Bax to mitochondria (Desagher et al., 1999; Eskes et al., 1998; Goping et al., 1998; Wolter et al., 1997). Subsequently, binding of cytochrome c to Apaf-1 in the presence of ATP would then lead to activation of caspase-9 (Li et al., 1997) and thus downstream effector caspases. The formation of this apoptosome might then lead to stronger activation of caspase-8 either because of cleavage by caspase-9 or other downstream caspases, or possibly due to direct interaction of the Apaf-1/cytochrome c/ATP complex with caspase-8. By contrast, in type I cells, the initial activation of caspase-8 at the DISC is much more robust and is able to activate downstream caspases directly without requirement for an amplification step involving mitochondria.
As shown in Figure 1, the release of cytochrome c can be detected after just 2 h in suspension, with the amount of cytochrome c in the cytoplasm increasing with longer times in suspension. Cytochrome c release is detected prior to caspase-8 activation and DNA fragmentation (Figure 1); this could indicate that detachment-induced apoptosis in MDCK cells is similar to that reported for Fas activation in type II cells (Scaffidi et al., 1998). The initial activation of caspase-8 would thus be under the detection limit and the activated form only detected when cytochrome c is released from mitochondria to cytoplasm, resulting in activation of downstream caspases and further activation of caspase-8. Alternatively, the caspase-8 activation could occur entirely downstream of cytochrome c release from mitochondria, with no initiating function.
Detachment induced apoptosis has been shown to be blocked by activated Ras via PI-3-kinase and protein kinase B (Khwaja et al., 1997) as well as by dominant negative FADD (Rytömaa et al., 1999). Thus we were interested to see how the expression of these proteins would affect cytochrome c release and caspase-8 activation. As shown in Figure 2, dominant negative FADD blocked the release of cytochrome c and prevented also the detectable activation of caspase-8. Interestingly, the expression of V12 Ras or activated PKB/Akt was also able to block cytochrome c release and activation of caspase-8. For comparison we also show the effect of these proteins in detachment induced apoptosis measured by DNA fragmentation ELISA assay. To address whether an initiating caspase activity was involved upstream of cytochrome c release in detachment-induced apoptosis, we studied the effect of a broad specificity caspase inhibitor z-VAD on cytochrome c release. As shown previously (Rytömaa et al., 1999) z-VAD is able to block the bulk activation of caspase-8 (Figure 2d) and detachment induced apoptosis (Figure 2e). More importantly, z-VAD was also able to substantially block the release of cytochrome c from mitochondria when cells were pre-treated with the inhibitor for 1 h before detaching the cells. To make sure that the cytoplasmic fractions did not contain mitochondrial proteins the blots were probed with an antibody against mitochondrial marker protein, cytochrome c oxidase subunit IV. As shown in Figure 2f only fractions containing unbroken cells, and thus also mitochondria, showed the presence of cytochrome c oxidase subunit IV. To further investigate the importance of caspase-8 in the release of cytochrome c from mitochondria we compared the effect of a broad spectrum caspase inhibitor (z-VAD), caspase-8 inhibitor (z-IETD), and caspase-9 inhibitor (z-LEHD), z-VAD and z-IETD were able to block caspase-8 activation while z-LEHD did not (Figure 2g). Caspase-8 inhibitor, as well as caspase-9 inhibitor, was able to decrease the amount of cytochrome c released to cytoplasm following detachment from extracellular matrix (Figure 2g). However, at the concentration used (20 M) it was not as effective as z-VAD at 100 M concentration. This indicates that the release of cytochrome c from mitochondria following detachment of epithelial cells from extracellular matrix is dependent on the activation of upstream caspases, with caspase-8 one likely candidate, although other caspases are also likely to be involved (see Discussion).
Since the protective effect of V12 Ras is somewhat stronger than the effect of activated PKB/Akt on DNA fragmentation, caspase-8 activation and cytochrome c release from mitochondria, we were interested in whether Raf, another downstream effector of Ras, might also have some protective effects on anoikis signalling. To investigate this, MDCK cells stably expressing a conditionally activatible form of Raf (Raf-ER) were generated. In these cells the kinase domain of Raf is fused to the hormone binding domain of a mutant oestrogen receptor; this allows the kinase to be activated when cells are treated with 4-hydroxytamoxifen (4-HT), while remaining inactive in the absence 4-HT (Woods et al., 1997). As shown in Figure 3, these cells undergo anoikis normally in the absence of 4-HT, showing cytochrome c release, caspase-8 activation and DNA fragmentation. However, when cells are pre-treated with 4-HT for 1 or 24 h before detachment they show significant protection from anoikis. One hour pre-treatment is already enough to block the strong detectable activation of caspase-8. However, the detachment induced release of cytochrome c can still be detected in cells where Raf has been activated, indicating that the protective effect of Raf is downstream of cytochrome c release. As a control for Raf activity, phosphorylation and activation of the mitogen activated protein kinases ERK1 and ERK2 was followed: the level of phosphorylation of ERKs 1 and 2 decreases in detached cells in the absence of 4-HT, but can be restored by the activation of Raf-ER by 4-HT in detached cells. To control the cell fractionation the absence of mitochondrial marker protein, cytochrome c oxidase subunit IV, in the cytoplasmic fractions was verified by Western blotting (Figure 3f). As a control the presence of the marker protein is shown in the fractions containing unbroken cells.
Since translocation of Bax from cytoplasm to mitochondria has been suggested to be important in the release of cytochrome c from the inter-membrane space of mitochondria (Hsu et al., 1997), we studied the subcellular localization of Bax during detachment-induced apoptosis. As shown in Figure 4, increase in the amount of Bax in the mitochondrial fraction can be seen in wild type MDCK cells when they were kept in suspension for 7 h. The caspase inhibitor z-VAD was able to block this translocation (Figure 4), further supporting the importance of activation of initiating upstream caspases as part of the triggering of apoptosis following detachment from matrix. In addition, the expression of activated forms of PKB/Akt and Ras prevents the detachment induced translocation of Bax to mitochondria, suggesting that these proteins provide a survival signal that impacts at the earliest possible stage of the death signalling pathway triggered by detachment. To confirm that the increase of Bax in the mitochondrial fraction of detached MDCK cells was not due to contaminating cytoplasmic proteins we reprobed the blot with an antibody against cytoplasmic protein p120GAP. We could not see any p120GAP in mitochondrial fractions even though it was easily detected in cytoplasmic fractions (Figure 4).
Detachment of epithelial cells from extracellular matrix has been reported to lead to activation of a caspase cascade which can be blocked by expression of dominant negative FADD (Rytömaa et al., 1999) or over-expression of SODD (silencer of death domains) (Frisch, 1999). Both these molecules contain death domains which can interact with death domains present in the cytoplasmic portion of death receptors, thereby blocking transmission of the apoptosis inducing signal. It is therefore possible that detachment-induced apoptosis is triggered through a death receptor pathway, with the caveat that over-expression of dominant negative FADD or SODD could result in them interacting with other critical partners in addition to death receptors.
Triggering of death receptors leads to rapid activation of initiator caspases such as caspase-8 or - 10 as part of the death inducing signalling complex (DISC) at the membrane (Schulze-Osthoff et al., 1998). In type II cells, this initial activation of caspases leads to cleavage and activation of the pro-apoptotic Bcl-2 family member Bid, resulting in release of cytochrome c from mitochondria, formation of the apoptosome containing cytochrome c, caspase-9 and Apaf1, and then subsequent activation of downstream caspases such as caspase-3 and possible further activation of initiator caspases as part of a feed back amplification loop. In detached MDCK cells, the time course of release of cytochrome c from mitochondria and activation of caspase-8 suggests that much of the caspase-8 activation is occurring downstream of cytochrome c release (Figure 1). This caspase-8 activation, and also DNA fragmentation, is blocked by treatment of the cells with the broad specificity caspase inhibitor z-VAD-fmk, as would be expected (Figure 2). However, the release of cytochrome c from mitochondria is also clearly strongly reduced by z-VAD treatment, suggesting that a caspase activity is required upstream of cytochrome c release, and that activation of a caspase is required as an initiating event in detachment induced apoptosis. This provides further support for a model in which detachment of epithelial cells from extracellular matrix results in activation of death receptors and hence initiator caspases such as caspase-8 in the DISC, which then trigger cytochrome c release from mitochondria and apoptosome activation leading to further activation of both upstream and downstream caspases.
Attempts to further define the exact role of caspase-8 in the triggering of apoptosis by detachment have been made using more specific peptide-based inhibitors of caspases (Figure 2g). z-IETD, which is designed to preferentially inhibit caspase-8, blocks activation of caspase-8 as expected and also partially inhibits the release of cytochrome c from mitochondria, although not as effectively as z-VAD. A caspase-9 inhibitor, z-LEHD, also partially inhibits cytochrome c release and significantly reduces caspase-8 activation. A difficulty in interpreting these data is that the specificity of each inhibitor is poorly defined and there is clearly cross inhibition. The ability of z-LEHD to reduce caspase-8 activation could be due to inhibition of the apoptosome mediated amplification loop, or simply the fact that z-LEHD has some activity towards caspase-8 itself. Similarly, z-IETD reduction in cytochrome c release may be due to effects of caspase-8 upstream of mitochondria, or may be due to direct inhibition of other caspases. However, the data do suggest that since appreciable cytochrome c release is still seen under conditions where there is little caspase-8 activation, that caspase-8 is unlikely to be the only caspase acting upstream of cytochrome c release. The identity of other caspases can only be guessed at, but other initiating caspases such as caspase-10 could clearly be involved.
A very early event detected in several types of cells in response to diverse apoptotic stimuli is the translocation of the pro-apoptotic Bcl-2 family member Bax from the cytosol to the mitochondrial outer membrane (Hsu et al., 1997; Wolter et al., 1997). Forcing Bax translocation to the mitochondria will induce apoptosis (Goping et al., 1998), suggesting that this can be a critical death signalling event. An activating conformational change in Bax can be triggered by interaction with Bid (Desagher et al., 1999). We show here that Bax translocation from cytosol to particulate fraction is also induced by detachment of MDCK cells from matrix (Figure 4). In keeping with the model outlined above, the translocation of Bax is also blocked by z-VAD-fmk, suggesting that this event is also dependent on initiating caspase activity. The simplest explanation would be that Bax translocation is caused by interaction with Bid which has been activated following cleavage by caspase-8; evidence for Bid cleavage following detachment from matrix has been reported recently (Frisch, 1999).
Expression of activated Ras or PKB/Akt both block induction of all the events on the death signalling pathway studied here: Bax translocation, cytochrome c release, bulk caspase-8 activation and DNA fragmentation. Activated PKB/Akt has recently been reported to inhibit cytochrome c release from mitochondria in Rat1a fibroblasts treated with ultraviolet light, etoposide or on induction of Myc expression in the absence of serum (Kennedy et al., 1999). The effect of PKB/Akt is clearly well upstream of caspase-9, one putative substrate for the kinase (Cardone et al., 1998), suggesting that caspase-9 is not likely to be an important target for PKB/Akt in this system: this ties in with the lack of conservation of the PKB/Akt substrate motif in caspase-9 in mammalian species other than human (Fujita et al., 1999; LM Martins and J Downward, unpublished observations). Since MDCK cells do not express detectable BAD, phosphorylation of this protein (Downward, 1999) is also unlikely to account for the protective effects of PKB/Akt, although the phenotype of the activated PKB/Akt expressing cells is very similar to those in which anti-apoptotic Bcl-2 family members such as Bcl-2 or Bcl-XL have been over-expressed. Many other PKB/Akt substrates have been identified that may be able to explain its protective effect (reviewed in Datta et al., 1999) but the identity of the critical targets in this system remains unclear.
With regard to apoptosis signalling, MDCK cells expressing activated Ras or PKB/Akt behave as though they are still attached even when they are in suspension. This is consistent with the observation that matrix attachment normally induces PKB/Akt activity (Khwaja et al., 1997), so cells expressing activated PKB/Akt or Ras continue to receive a signal normally generated by attachment to matrix despite being in suspension. The PKB/Akt activity, whether stimulated by attachment to matrix, expression of activated PKB/Akt or activated Ras (through its ability to activate PI 3-kinase (Rodriguez-Viciana et al., 1994)) provides a survival signal that is presumably sufficient to overcome any death signals that the cells are receiving. In normal cells which have been detached from matrix, the PKB/Akt survival signal is lost and the cells appear to receive an apoptotic signal from death receptors, resulting in their commitment to apoptosis. The death signal experienced by detached cells could normally be present also in attached cells, but be suppressed downstream by the PKB/Akt pathway. Alternatively, the death signal could itself be induced following detachment, perhaps as a result of changes in the association state of death receptors due to alterations in membrane or cytoskeletal organization in suspended cells.
It is noticeable that the protection from apoptosis caused by Ras is greater than that by PKB/Akt, suggesting that other effectors of Ras are contributing to the survival signal. Previously we had failed to see any protection in this system by an activated Raf construct, Raf-CAAX, which was capable of causing ERK activation in suspension (Khwaja et al., 1997). Using a more potent inducible Raf construct, in which the regulatory domain has been removed and two regulatory tyrosine residues changed to aspartic acids, we found that short term activation of Raf could protect from detachment induced apoptosis. Interestingly, Raf activation did not prevent cytochrome c release from mitochondria following detachment, so the mechanism of protection would appear to be fundamentally different from that provided by PKB/Akt. B-Raf has recently been reported to protect Rat-1 fibroblasts from serum deprivation induced apoptosis at a point downstream of cytochrome c release from mitochondria (Erhardt et al., 1999). The point at which Raf impacts on apoptosis regulation may, however, differ in different cell types: in another epithelial cell line, MCF-10A, Raf is able to block detachment-induced cytochrome c release from mitochondria (A Schulze and J Downward, manuscript in preparation). The nature of the target of this Raf signalling remains unclear, although the rapidity of the effect makes it unlikely that changes in gene expression are involved; it is also uncertain whether this effect of strong Ras activation is relevant to normal physiological signal strengths through this pathway.
Materials and methods
Gag-PKB cDNA was provided by B Burgering and P Coffer (University of Utrecht). The V12 Ras expression vector was generated as described previously (Rodriguez-Viciana et al., 1994). Dominant negative FADD was provided by V Dixit (Genentech, South San Francisco, CA, USA). GFP tagged [DD] form of Raf-ER (Woods et al., 1997) in pBabe-puro was provided by Martin McMahon (DNAX Research Institute, Palo Alto, CA, USA).
Early passage Madin-Darby canine kidney (MDCK) cells were provided by J Taylor-Papadimitriou (ICRF) and were maintained in DMEM supplemented with 10% foetal bovine serum (FBS). Cell lines stably expressing V12 H-Ras, gag-PKB, and dnFADD were generated as previously described (Khwaja et al., 1997; Rytömaa et al., 1999). To generate cell stably expressing Raf-ER, MDCK cells were first infected with an amphotropic vector (pWZL-Neo-EcoR, a gift of Manuel Serrano) allowing subsequent infection with ecotropic viruses. Retrovirus stock was obtained by transfecting Raf-ER in pBabe-puro into GP+E cells. Target MDCK cells were then infected with the supernatant containing Raf-ER and incubated at 37°C for 7 h. Sixteen hours later, infected cell populations were selected with 2.5 g/ml of puromycin. After selection with antibiotic GFP expressing cells were selected by two rounds of fluorescence activated cell sorting (FACS). The expression of Raf-ER was detected by Western blotting using an antibody against Ostrogen receptor (Santa Cruz). Raf-ER expressing cells were cultured in phenol red-free DMEM supplemented with 10% foetal bovine serum.
DNA fragmentation assay
Subconfluent MDCK cells were detached using trypsin/EDTA, washed and resuspended with DMEM supplemented with 10% FBS as described previously (Khwaja et al., 1997). Cells were maintained in suspension in poly(2-hydroxyethyl methacrylate) (polyHEMA) coated plates. Coating was carried out with two rounds of 10 mg/ml polyHEMA in ethanol and subsequent washing with DMEM. MDCK cells were kept in suspension for 8 h. One hundred M z-VAD-fluoromethylketone (z-VAD, Bachem), 20 M z-IETD-fmk (Calbiochem), or LEHD-fmk (Calbiochem) were added 1 h prior to replating the cells, when indicated. Raf-ER expressing cells were treated with 100 nM 4-hydroxytamoxifen (4-HT) for 1 or 24 h when indicated. DNA fragmentation ELISA was performed using the Cell Death Detection ELISA kit (Boehringer Mannheim) according to the manufacturer's instructions. Lysates from 2´103 cells/point were assayed. The results represent at least three independent experiments.
After indicated treatment of cells they were lysed with sample buffer (50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), boiled for 5 min, and proteins were separated on 15% SDS-PAGE with subsequent transfer to PVDF membrane. Membrane was blocked by drying, incubated with anti-caspase-8 antibody (C-20, Santa Cruz Biotechnology) overnight at +4°C followed by incubation with peroxidase-coupled secondary antibody (Pierce). The activated form of caspase-8 was detected by Amersham ECL enhanced chemiluminescent detection as described by the supplier.
Cytochrome c release
Rapid preparation of cytosolic fraction was done according to Samali et al., 1999. Cells were washed and resuspended in 100 l of mitochondrial buffer (70 mM Tris base, 0.25 M sucrose and 1 mM EDTA, pH 7.4). An equal volume of digitonin (0.2 mg/ml dissolved in MES.buffer: 19.8 mM EGTA, 19.8 mM EDTA, 0.25 M D-mannitol and 19.8 mM MES, pH 7.4) was added to the samples for 5 min. After centrifugation at 900 g for 2 min, the supernatant was centrifuged further at 20 000 g for 5 min to obtain the cytosolic fraction. Samples were then prepared for Western blotting.
Subcellular fractions were prepared as described previously (Samali et al., 1999). Cells were washed in buffer A (100 mM sucrose, 1 mM EGTA, 20 mM MOPS pH 7.4) and resuspended in buffer B (buffer A plus 5% Percoll, 0.01% digitonin and a cocktail of protease inhibitors: 10 M aprotinin, 10 M pepstatin A, 10 M leupeptin, 25 M calpain inhibitor I, and 1 mM phenylmethylsulfonyl fluoride). After 15 min incubation on ice, unbroken cells and nuclei were pelleted by centrifugation at 2500 g for 10 min. The supernatant was centrifuged further at 15 000 g for 15 min to pellet mitochondria. Samples were then prepared for Western blotting.
Protein concentration was determined by Bio-Rad Protein Assay and equal amounts of protein/sample were used. SDS-PAGE sample buffer (50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) was added to the samples and they were boiled in a water bath for 5 min. Lysates were stored at -20°C until further analysis. Proteins were separated on 10% Bis-Tris Gel (NuPAGE Electrophoresis System, Novex) with MES Running buffer according to manufacturers instructions with subsequent transfer to PVDF membrane. Membrane was blocked by drying, incubated with anti-cytochrome c antibody (PharMingen, clone 7H8.2C12), anti-Bax antibody (Transduction Laboratories), anti-Active MAPK (Promega), anti-cytochrome c oxidase subunit IV (Molecular Probes), or anti-p120GAP (PW6) overnight at +4°C followed by incubation with peroxidase-coupled secondary antibody (Pierce) and detected by Amersham ECL enhanced chemiluminescent detection as described by the supplier.
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Figure 1 Time course of detachment induced cytochrome c release, caspase-8 activation, and apoptosis. Cytosolic fractions of wild type MDCK cells were prepared as indicated in Materials and methods. After cells were kept in suspension for 0-8 h the release of cytochrome c (a) or activation of caspase-8 by detecting the cleaved p20 subunit (arrow) (b) were detected by immunoblotting. (c) Detachment induced apoptosis of MDCK cells was followed by DNA fragmentation ELISA assay
Figure 2 Effect of caspase inhibitor, dominant negative FADD and activated Ras and PKB/Akt on detachment induced cytochrome c release, caspase-8 activation and apoptosis. (a and b) Release of cytochrome c from mitochondria to cytoplasm was detected in wild type MDCK cells and MDCK cells stably expressing dnFADD, gag-PKB, or V12 Ras after 7 h in suspension. Also shown is the effect of 100 M z-VAD: cells were pre-treated with z-VAD for 1 h before detaching from the matrix. (c and d) Activation of caspase-8 after MDCK cells were kept in suspension for 7 h was measured by detecting the cleaved p20 subunit (arrow) of activated caspase-8 by immunoblotting. (e) Detachment induced apoptosis of MDCK cells was followed by DNA fragmentation ELISA assay. (f) To show that the cytoplasmic fractions were free of mitochondrial proteins, blots were reprobed with anti-cytochrome c oxidase subunit IV (ox). As a control the presence of cytochrome c oxidase subunit IV in the fraction containing unbroken cells is shown. (g) Effect of caspase inhibitors in caspase-8 activation (upper panel) and cytochrome c release (lower panel). Cells were pre-treated with inhibitors for 1 h before detaching from the matrix. z-IETD and z-LEHD were used at 20 M and z-VAD at 100 M concentration
Figure 3 Effect of Raf on detachment induced cytochrome c release, caspase-8 activation, and apoptosis. Release of cytochrome c (a) and activation of caspase-8 (b) from mitochondria to cytoplasm was detected in Raf-ER expressing MDCK cells after 7 h in suspension. In detached cells Raf-ER was not activated or the activation was induced by treatment with 100 nM 4-hydroxytamoxifen (4-HT) for 1 or 24 h prior to detachment. (c) Activation of ERK1/2 in detached Raf-ER expressing MDCK cells with or without 4-hydroxytamoxifen treatment. Cells were kept in suspension for 7 h. Activated ERK was detected with an antibody that recognizes only the activated, phosphorylated, forms of ERK1/2. (d) Detachment induced apoptosis of MDCK cells was followed by DNA fragmentation ELISA assay. (f) To show that the cytoplasmic fractions were free of mitochondrial proteins, blots were reprobed with anti-cytochrome c oxidase subunit IV (ox). As a control the presence of cytochrome c oxidase subunit IV in the fraction containing unbroken cells is shown
Figure 4 Detachment induced translocation of Bax to mitochondria. Mitochondrial fractions of MDCK cells were prepared, the proteins were separated on SDS-PAGE and the amount of Bax present was detected by immunoblotting (top panel). To show that the mitochondrial fractions were free of cytoplasmic proteins, blots were reprobed with anti-p120GAP (middle panel). As a control the presence of p120GAP in the cytoplasmic fraction is shown (bottom panel)
|Received 11 January 2000; revised 17 July 2000; accepted 17 July 2000|
|14 September 2000, Volume 19, Number 39, Pages 4461-4468|
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