Original Article

Oncogene (2008) 27, 6571–6580; doi:10.1038/onc.2008.249; published online 28 July 2008

There is a Corrigendum (4 July 2016) associated with this article.

There is a Retraction (13 February 2017) associated with this article.

PRIMA-1MET induces mitochondrial apoptosis through activation of caspase-2

J Shen1, H Vakifahmetoglu2, H Stridh1,3, B Zhivotovsky2 and K G Wiman1

  1. 1Department of Oncology-Pathology, Cancer Center Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden
  2. 2Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden

Correspondence: Professor KG Wiman, Department of Oncology-Pathology, Karolinska Institute, Cancer Center Karolinska (CCK), R8:04 Karolinska University Hospital, Stockholm SE-171 76, Sweden. E-mail: klas.wiman@ki.se

3Current address: Amgen, Stockholm 169 27, Sweden.

Received 7 April 2008; Revised 16 June 2008; Accepted 26 June 2008; Published online 28 July 2008.

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Abstract

p53 mutations occur frequently in human tumors. The low-molecular-weight compound PRIMA-1MET reactivates mutant p53, induces apoptosis in human tumor cells and inhibits tumor xenograft growth in vivo. Here, we show that PRIMA-1MET induces mutant p53-dependent mitochondria-mediated apoptosis through activation of caspase-2 with subsequent cytochrome c release and further activation of downstream caspase-9 and caspase-3. Inhibition of caspase-2 by a selective inhibitor and/or siRNA prevents cytochrome c release on PRIMA-1MET treatment and causes a significant reduction in PRIMA-1MET-induced cell death. Our findings highlight a chain of cellular events triggered by PRIMA-1MET that lead to apoptotic cell death. This should facilitate further development and optimization of efficient PRIMA-1MET-based anticancer drugs.

Keywords:

apoptosis, cancer therapy, caspase-2, mutant p53, PRIMA-1MET

Abbreviations:

Δψm, mitochondrial membrane potential; FACS, fluorescence-activated cell sorting; G3PDH, glyceraldehyde-3phosphate dehydrogenase; PI, propidium iodide; fmk, fluoromethyl ketone

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Introduction

The p53 tumor suppressor gene is frequently mutated in human tumors (see www-p53.iarc.fr and http://p53.free.fr). p53 mutation allows evasion from apoptosis in response to cellular stress and may confer increased resistance to cancer therapy (al-Shabanah et al., 1995; Bergh et al., 1995; Guimaraes and Hainaut, 2002; Olivier et al., 2006). Pharmacological reactivation of mutant p53 should induce tumor cell apoptosis and thus eliminate tumors (Bykov et al., 2003). This notion is supported by recent studies in mice showing that restoration of wild-type p53 expression in tumors in vivo has a significant antitumor effect, despite the presence of other genetic alterations (Ventura et al., 2007; Xue et al., 2007). PRIMA-1 is a low-molecular-weight compound that reactivates mutant p53 and inhibits growth of human tumor xenografts in mice (Bykov et al., 2002). Mutant p53 reactivation by PRIMA-1 can trigger both transcription-dependent (Bykov et al., 2002) and transcription-independent (Chipuk et al., 2003) apoptosis. A methylated form of PRIMA-1, PRIMA-1MET, is more active than the original compound (Bykov et al., 2005b). Yet the precise mechanism of PRIMA-1MET-induced apoptosis is unknown.

p53 can induce apoptosis both through activation of the receptor-mediated (extrinsic) and the mitochondrial (intrinsic) pathway (Fulda and Debatin, 2006). The p53-mediated extrinsic pathway involves upregulation of the Fas/CD95 death receptor and formation of the death-inducing signaling complex, followed by sequential activation of caspase-8 and caspase-3. In the intrinsic pathway, induction of proapoptotic Bcl-2 family proteins, such as PUMA and Bax, and downregulation of Bcl-2 trigger permeabilization of the outer mitochondrial membrane. This is followed by release of cytochrome c from mitochondria to the cytoplasm where it binds to Apaf-1, leading to the activation of initiator caspase-9, which in turn activates executioner caspases (Read et al., 2002). The intrinsic and extrinsic pathways are linked, supporting the idea of converging rather than distinct pathways (Gross et al., 1999).

Caspase-2 seems to have a crucial role in triggering apoptosis through the intrinsic pathway (Lassus et al., 2002). This enzyme acts upstream of mitochondria by inducing Bid cleavage, mitochondrial translocation of Bax and subsequent cytochrome c release (Guo et al., 2002; Lassus et al., 2002; Robertson et al., 2002). Caspase-2-deficient mice are viable and the only observed defects were resistance of germ cells and oocytes to death after treatment with chemotherapeutic drugs (Bergeron et al., 1998). Activation of caspase-2 was suggested to occur in the PIDDosome, a protein complex consisting of the death-domain-containing protein PIDD, a p53 target that promotes apoptosis, and the adaptor protein RAIDD (Tinel and Tschopp, 2004).

Here, we investigated the mechanism of PRIMA-1MET-induced apoptosis with focus on the mitochondrial and death receptor pathways. We show that PRIMA-1MET induces mutant p53-dependent activation of caspase-2, followed by loss of mitochondrial membrane potential (Δψm), release of cytochrome c and activation of caspase-3. Mutant p53-dependent induction of PUMA and Bax by PRIMA-1MET forms a parallel signaling pathway converging on mitochondria.

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Results

PRIMA-1MET induces mutant p53-dependent loss of Δψm

First, we examined the kinetics of PRIMA-1MET-induced cell death in H1299 human lung adenocarcinoma cells carrying exogenous His175 mutant p53 and in the parental p53 null cells using propidium iodide (PI) staining and fluorescence-activated cell storing (FACS) analysis. Treatment of H1299-His175 cells with PRIMA-1MET for 24h resulted in the accumulation of cells with a sub-G1 DNA content (well-known biochemical marker of apoptosis) in a dose-dependent manner (Figure 1a, upper panel). At this time point, ~39% of H1299-His175 cells treated with 25μM PRIMA-1MET accumulated in the sub-G1 fraction, whereas only ~9% of PRIMA-1MET-treated p53 null H1299 cells had a sub-G1 DNA content (Figure 1a, upper panel). Treatment with 50μM PRIMA-1MET increased apoptosis in the H1299-His175 cells up to ~62% compared with ~16% for the parental H1299 cells (Figure 1a, upper panel). PRIMA-1MET also caused an increase of cells with sub-G1 DNA content in a time-dependent manner (Figure 1a, lower panel).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

PRIMA-1MET induces mutant p53-dependent loss of Δψm and cytochrome c release. (a) PRIMA-1MET-induced cell death in H1299-His175 () and H1299 () cells. FACS analysis of sub-G1 apoptotic cells using PI staining 24h posttreatment with indicated doses of PRIMA-1MET (upper panel), and with 25μM PRIMA-1MET at indicated time points (lower panel). Results are mean±s.e. (n=3) from three independent experiments. (b) FACS analysis of loss of Δψm (%) using TMRE staining at 12h with indicated doses of PRIMA-1MET (upper panel) and time course of loss of Δψm (%) with 25μM PRIMA-1MET (lower panel). Results are mean±s.e. (n=3) from three independent experiments. (c) Western blot analysis of Bax and PUMA expression following treatment with indicated concentrations of PRIMA-1MET for 16h (Bax) or 8h (PUMA). β-actin was used as control for equal loading. (d) Cells were treated with 25μM PRIMA-1MET for 16h and stained for cytochrome c (green). DAPI (blue) was used for staining of nuclei. Representative fluorescence microscope images of stained cells are shown. Bar: 10μm. Statistically significant difference at Pless than or equal to0.05 according to the independent t-test is indicated by **.

Full figure and legend (259K)

Next, we investigated whether PRIMA-1MET induced changes in the Δψm by FACS analysis using tetramethylrhodamine ethyl (TMRE) staining. As shown in Figure 1b (upper panel), PRIMA-1MET triggered mutant p53-dependent loss of Δψm in a dose-dependent manner. At 12h, loss of Δψm was detected in ~19 and 12% of H1299-His175 and parental H1299 cells treated with 25μM PRIMA-1MET, respectively (Figure 1b, upper panel). However, at the same time point, 37.5μM PRIMA-1MET induced loss of Δψm in ~26% of the H1299-His175 cells and only in ~14% of the H1299 cells (Figure 1b, upper panel). An even greater difference in loss of Δψm between H1299-His175 and H1299 cells was observed after treatment with 50 or 75μM PRIMA-1MET for 12h (Figure 1b, upper panel). PRIMA-1MET-induced Δψm loss was time-dependent. At 24h, 25μM PRIMA-1MET-induced loss of Δψm in ~35% of the H1299-His175 cells and in ~15% of the p53 null H1299 cells (Figure 1b, lower panel). At 30h loss of Δψm was detected in ~45% of the H1299-His175 cells as compared with ~12% of the H1299 cells (Figure 1b, lower panel).

Thus, these results demonstrate that PRIMA-1MET induces accumulation of sub-G1 cells and loss of Δψm in a dose- and time-dependent manner in the mutant p53-expressing cells, both changes characteristic for apoptosis. Similar mutant p53-dependent loss of Δψm was observed in two other human tumor cell lines of different origin and carrying exogenous mutant p53, namely human Saos-2-His 273 osteosarcoma and SKOV-His 175 ovarian carcinoma cells (Supplementary Figures S1a and b, left panel). No significant effect of PRIMA-1MET was detected in the corresponding parental p53 null Saos-2 and SKOV-TA cells.

As PRIMA-1MET-induced death of H1299-His175 cells engages the mitochondrial pathway, it was of interest to investigate the possible involvement of Bcl-2 proteins in this experimental system. Hence, the status of several Bcl-2 family proteins was analyzed. The appearance of a distinct band corresponding to the 15kDa cleavage fragment of Bid was observed in H1299-His175 cells at 24h of treatment with 50μM PRIMA-1MET, but not in the parental cells (Supplementary Figure S2). Furthermore, treatment with PRIMA-1MET induced expression of both Bax and PUMA in H1299-His 175 cells and not in the parental H1299 cells (Figure 1c).

PRIMA-1MET induces mutant p53-dependent redistribution of cytochrome c

To further study the involvement of mitochondria in PRIMA-1MET-induced cell death, we assessed the release of cytochrome c in PRIMA-1MET-treated H1299 and H1299-His175 cells using immunofluorescence staining. Exposure of H1299-His175 cells to 25μM PRIMA-1MET for 16h triggered release of cytochrome c from mitochondria to the cytoplasm as shown by the diffuse staining pattern (Figure 1d). At later time points, these cells displayed formation of pyknotic nuclei indicative of apoptosis, and the punctate staining of cytochrome c was significantly reduced (data not shown). In contrast, no cytochrome c release was detected in the parental p53 null H1299 cells after treatment with the same concentration of PRIMA-1MET for up to 24h. In addition, no nuclear changes were observed in the parental H1299 cells (data not shown).

PRIMA-1MET-induced apoptosis involves activation of caspases

To determine whether the observed mitochondria-mediated apoptosis induced by PRIMA-1MET involves caspase activation, we analyzed PRIMA-1MET-treated H1299-His175 and parental H1299 cells by FACS using FLICA, which binds to active forms of all caspases. Treatment with 25 or 50μM PRIMA-1MET for 24h resulted in a significant (P<0.01) caspase activation in the H1299-His175 cells. PRIMA-1MET (25μM) induced appearance of ~12% caspase-positive H1299-His175 cells, as compared with ~5% for the H1299 cells (data not shown). Treatment with 50μM PRIMA-1MET resulted in ~80% caspase-positive H1299-His175 cells and ~15% caspase-positive p53 null H1299 cells (data not shown). To examine activation of individual caspases in this experimental system, we treated H1299 and H1299-His175 cells with 37.5μM PRIMA-1MET and assessed activation of caspase-2, -3, -9 and -8 using the fluorogenic caspase substrates zVDVAD-AMC, zDEVD-AMC, zLEHD-AMC and zIETD-AMC, respectively. Activation of caspase-2 was detected in H1299-His175 cells already at 8h and reached a more pronounced activation plateau after 12h, whereas only a weak increase was observed in the p53 null H1299 cells (Figure 2a). We obtained similar results using two other cell lines, that is, Saos-2-His 273 and SKOV-His 175 (Supplementary Figures S1a and b, right panel). No significant effect of PRIMA-1MET was detected in the corresponding parental p53 null Saos-2 and SKOV-TA cells. Caspase-9 was activated at 8h in H1299-His175 cells but not in the H1299 cells (Figure 2b). Significant caspase-3 activation was observed at 12h in the H1299-His175 cells as compared with H1299 cells (Figure 2c). No caspase-8 activity was detected in either of the cell lines at 24h of PRIMA-1MET treatment (Figure 2d). The latter result suggests that activation of the death receptor pathway is not required for PRIMA-1MET-induced cell death.

Figure 2.
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Activation of caspases on PRIMA-1MET treatment. H1299-His175 () and H1299 () cells (106/ml) were treated with 37.5μM PRIMA-1MET and harvested at the indicated time points. Fold increase in caspase activity was assessed by the release of AMC from (a) VDVAD-AMC for caspase-2 activity, (b) LEVD-AMC for caspase-9 activity, (c) DEVD-AMC for caspase-3-like activity, and (d) IEHD-AMC for caspase-8 activity. Data are mean±s.e. (n=2) from two independent experiments. Statistically significant differences at Pless than or equal to0.1 and Pless than or equal to0.05 according to the independent t-test are indicated by * and **, respectively.

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Next, we analyzed whether PRIMA-1MET treatment had any effect on PIDD expression. We observed a slight increase in the levels of the 100kDa full-length PIDD protein in PRIMA-1MET-treated H1299-His175 cells. Furthermore, a time-dependent accumulation of a band corresponding to the 37kDa cleavage fragment PIDD-CC of PIDD was observed in these cells on PRIMA-1MET treatment. This was not detected in the parental cell line, which, however, is characterized by the appearance of the 51kDa fragment PIDD-C. We detected a small amount of the 37kDa PIDD-CC fragment in untreated H1299 cells; however, PRIMA-1MET treatment did not increase its levels (Figure 3). In combination, these observations demonstrate that PRIMA-1MET treatment induces the activation of caspases. Moreover, the results indicate that caspase-2 is the most apical caspase activated by PRIMA-1MET.

Figure 3.
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PRIMA-1MET triggers PIDD processing in a mutant p53-dependent manner. Western blot analysis of PIDD expression in p53 null parental H1299 cells and mutant p53-expressing H1299-His175 cells on treatment with 25 or 50μM PRIMA-1MET at the indicated time points. G3PDH was used as control for equal loading. Cleavage products of PIDD (PIDD-C, 51kDa; PIDD-CC, 37kDa) are indicated by arrows.

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Inhibition of caspase-2 blocks PRIMA-1MET-induced activation of caspase-3 and caspase-9

To further analyze the relationship between the initiator and the effector caspases in PRIMA-1MET-induced apoptosis, we examined the effect of the most selective caspase-2 inhibitor (zVDVAD-fmk) and of the caspase-3 inhibitor (zDEVD-fmk) on the activation of caspase-2, -3 and -9 in PRIMA-1MET-treated H1299-His175 cells. Pre-incubation with the caspase-2 inhibitor significantly suppressed activation of caspase-2 and caspase-3 on PRIMA-1MET treatment (Figures 4a and b). In contrast, pre-incubation with caspase-3 inhibitor blocked the activation of caspase-3, and had no significant effect on the activation of caspase-2 (Figures 4d and e). Both inhibitors at least partially blocked the activation of caspase-9 (Figures 4c and f).

Figure 4.
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Inhibition of caspase-2 blocks activation of caspase-3 and 9. H1299-His175 cells were treated with 37.5μM PRIMA-1MET in the presence of caspase-2 inhibitor (zVDVAD-fmk) (ac), or caspase-3 inhibitor (zDEVD-fmk) (df) and harvested after 24h. Caspase activity was assessed by the release of AMC from VDVAD-AMC (a and d) for caspase-2 activity, DEVD-AMC (b and e) for caspase-3-like activity and LEVD-AMC (c and f) for caspase-9 activity. Results are mean±s.e. (n=2) from two independent experiments. Statistically significant differences at Pless than or equal to0.1 and Pless than or equal to0.05 according to the independent t-test are indicated by * and **, respectively.

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Inhibition or silencing of caspase-2 attenuates PRIMA-1MET-induced apoptosis

To investigate the role of caspase-2 in our experimental system, several apoptosis-related features in PRIMA-1MET-treated H1299-His 175 cells were investigated in the presence of zVDVAD-fmk. No effect of the caspase-2 inhibitor on PRIMA-1MET-induced loss of Δψm in H1299-His175 cells was observed (Figure 5a). However, compared with PRIMA-1MET treatment alone, pre-incubation of cells with caspase-2 inhibitor resulted in a significant reduction in cytochrome c release (Figure 5c). In contrast to caspase-2 inhibitor, pre-incubation of cells with the caspase-3 inhibitor zDEVD-fmk had only a minor effect on cytochrome c release, indicating that caspase-2 activation is required to initiate cytochrome c release in the mutant p53-expressing H1299 cells. However, FACS analysis revealed that the sub-G1 apoptotic cell population was equally reduced in the presence of either caspase-2 or caspase-3 inhibitor following PRIMA-1MET treatment (Figure 5b).

Figure 5.
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Inhibition of caspase-2 prevents PRIMA-1MET-induced cytochorome c release and cell death. H1299-His175 cells were treated with 37.5μM PRIMA-1MET in the presence or absence of caspase-2 (zVDVAD-fmk), or caspase-3 (zDEVD-fmk) inhibitor. (a) FACS analysis of loss of Δψm using TMRE staining after 16h of PRIMA-1MET treatment. (b) FACS analysis of sub-G1 apoptotic cells using PI staining after 24h of PRIMA-1MET treatment. Data are mean±s.e. (n=3) from three independent experiments. (c) Western blot analysis showing cytochrome c distribution in supernatants and mitochondrial pellets (upper panel) after 16h of PRIMA-1MET treatment. Quantification of protein levels is shown in the lower panel. Statistically significant difference at Pless than or equal to0.05 according to the independent t-test is indicated by **.

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To further confirm the requirement for caspase-2 activation in PRIMA-1MET-induced mitochondrial changes and apoptosis, we silenced caspase-2 using siRNA. Efficient silencing by two different siRNAs was confirmed by western blotting (Figure 6a). This silencing was specific and did not affect expression levels of other caspases, such as caspase-3 and caspase-8 (Supplementary Figure S3). In line with our results obtained with zVDVAD-fmk, knockdown of caspase-2 by either of the two siRNAs resulted in a significant reduction of cytochrome c release in PRIMA-1MET-treated H1299-His175 cells (Figure 6b). In addition, silencing of caspase-2 significantly reduced the fraction of sub-G1 apoptotic cells on PRIMA-1MET treatment (Figure 6c). Taken together, these findings suggest a role for caspase-2 as an initiator caspase of critical importance for the apoptotic outcome of PRIMA-1MET treatment in H1299-His175 cells.

Figure 6.
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siRNA silencing of caspase-2 attenuates PRIMA-1MET-induced apoptosis. H1299-His175 cells were treated with two different siRNAs for caspase-2 (C-2a and C-2b) for 24h earlier to 24h treatment with 37.5μM PRIMA-1MET. A scrambled siRNA (si ctrl) was used as a control. (a) Western blot analysis of caspase-2 expression. G3PDH was used as control for equal loading. (b) Western blot analysis of cytochrome c distribution in supernatants and pellets (upper panel). Quantification of protein levels (lower panel). (c) FACS analysis of sub-G1 apoptotic cells using PI staining. Data are mean±s.e. (n=3) from three independent experiments. Statistically significant difference at Pless than or equal to0.1 according to the independent t-test is indicated by *.

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To determine the significance of the previously observed upregulation of PUMA in PRIMA-1MET-treated H1299-His175 cells (Figure 1c), we examined the response to PRIMA-1MET after siRNA knockdown of PUMA. Efficient silencing of PUMA using siRNA was confirmed by western blotting (Figures 7a). We observed a markedly attenuated apoptotic response to PRIMA-1MET on PUMA knockdown, as shown by a reduction in the sub-G1 fraction of cells (Figure 7b). However, the combination of both PUMA siRNA and caspase-2 inhibitor did not result in any additional protection from cell death. This indicates that although both PUMA and caspase-2 are involved in the apoptotic process, other mutant p53-dependent pathways may be activated on treatment with PRIMA-1MET.

Figure 7.
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Effect of PUMA and caspase-2 inactivation on PRIMA-1MET-induced apoptosis. (a) H1299-His175 cells were treated with PUMA siRNA for 24h earlier to 24h treatment with either 37.5μM PRIMA-1MET in the absence or presence of caspase-2 inhibitor (zVDVAD-fmk). A scrambled siRNA (si ctrl) was used as a control. (a) Western blot analysis of PUMA expression. β-actin was used as control for equal loading. (b) FACS analysis of sub-G1 apoptotic cells using PI staining. Data are mean±s.e. (n=3) from three independent experiments. Statistically significant difference at Pless than or equal to0.1 according to the independent t-test is indicated by *.

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Discussion

Here, we demonstrate that PRIMA-1MET triggers mutant p53-dependent apoptosis in human tumor cells through the activation of the mitochondrial pathway, that is, through the loss of Δψm and release of cytochrome c (Figure 8). These effects are dependent on mutant p53, as they were observed in the mutant p53-expressing H1299-His175, Saos-2-His273 and SKOV-His175 cells but not in their corresponding parental p53 null cells (Supplementary Figures S1a and b, left panel). Moreover, PRIMA-1MET failed to induce apoptosis in KRC/Y renal carcinoma cells expressing Phe176 mutant p53 (Supplementary Figure S1c, left panel), in agreement with our earlier findings that PRIMA-1 is unable to rescue this mutant (Bykov et al., 2002). Thus, these data demonstrate that PRIMA-1MET induces mitochondrial apoptosis in tumor cell lines of different origin harboring mutant p53.

Figure 8.
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Model for PRIMA-1MET-induced apoptotic signaling pathways. PRIMA-1MET targets mutant p53 and triggers several parallel apoptotic pathways. (i) Activation of caspase-2 leads to cytochrome c release and caspase-3 activation. PIDD, a transcription target of p53, may play a key role in activation of caspase-2 on PRIMA-1MET treatment. (ii) Upregulation of PUMA and Bax induces mitochondrial translocation of Bax, which also leads to release of mitochondrial cytochrome c, and further activation of downstream caspases. (iii) PRIMA-1MET can presumably trigger other apoptotic pathways as well in mutant p53-expressing cells. The concerted activation of these pathways result in a robust apoptotic response.

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The significance of p53 function for the activation of caspases in general, and caspase-2 and caspase-8 in particular, has previously been demonstrated (Ding et al., 2000; Schuler et al., 2000; Tyagi et al., 2006). In this study, we found that PRIMA-1MET treatment induced activation of caspase-2, -9 and -3 in H1299-His175 cells. Although a cleaved product of Bid was detected on treatment, no significant caspase-8 activation was observed. It has previously been shown that Bid can be cleaved by several proteases, including caspase-2, caspase-3, cathepsins and calpains (Yin, 2006), thus suggesting that the effect of PRIMA-1MET does not involve activation of the death receptor pathway. Our finding that PRIMA-1MET induced caspase-2 activity in several cell lines carrying mutant p53, that is, H1299-His175, Saos-2-His273 and SKOV-His175 (Figure 2; Supplementary Figure S1), suggests that mutant p53 reactivation is fundamental for caspase-2 activation on PRIMA-1MET treatment.

The identification of caspase-2 as an apical caspase activated in PRIMA-1MET-induced apoptosis is in line with an earlier study reporting that p53 is essential for caspase-2 activation (Vakifahmetoglu et al., 2006). Moreover, the early activation of caspase-2 on PRIMA-1MET treatment supports the previously suggested pre-mitochondrial function of this enzyme. This assumption was further strengthened by our data demonstrating that both caspase-2 inhibition and siRNA knockdown blocked PRIMA-1MET-induced cytochrome c release and downstream caspase activation. In addition, knockdown of caspase-2 attenuated PRIMA-1MET-induced cell death.

How does PRIMA-1MET-mediated mutant p53 reactivation lead to activation of caspase-2? The recent identification of the PIDDosome complex suggested a link between p53 and activation of caspase-2 (Tinel and Tschopp, 2004). PIDD is a p53 target gene (Lin et al., 2000). We observed a minor increase in full-length PIDD protein following PRIMA-1MET treatment in H1299-His 175 cells (Figure 3). However, it was recently shown that autoprocessing of PIDD rather than its upregulation is essential for stimulation of cell death (Tinel and Tschopp, 2004). Notably, PRIMA-1MET treatment caused increased levels of the small PIDD-CC fragment in treated H1299-His175 cells but not in the parental p53 null cells. The formation of this fragment is strongly correlated with the apoptotic response of cells, for which caspase-2 activation is a prerequisite (Tinel and Tschopp, 2004). Furthermore, silencing of PIDD partially inhibited the accumulation of H1299-His175 cells with sub-G1 DNA content on PRIMA-1MET treatment (data not shown). These results suggest that PIDD is involved in the observed activation of caspase-2 on PRIMA-1MET treatment. Further studies are required to examine the effect of PRIMA-1MET on the PIDDosome in mutant p53-expressing cells.

Although the caspase-2 and caspase-3 inhibitors suppressed PRIMA-1MET-induced cell death, they did not have any effect on loss of Δψm. Similarly, siRNA-mediated silencing of caspase-2 also failed to affect Δψm on PRIMA-1MET treatment (data not shown). These observations, together with the finding that inhibition of caspase-2 only partially inhibited cytochrome c release and cell death, suggest that PRIMA-1MET also triggers activation of a caspase-2-independent signaling pathway for execution of apoptosis.

A number of possible scenarios could account for this observation. First, the proapoptotic Bcl-2 family members Bax and PUMA are transcriptional targets of p53 (Nakano and Vousden, 2001; Yu et al., 2001). More recent study has indicated that PUMA can dislodge p53 from Bcl-xL (Chipuk et al., 2005), resulting in p53-dependent transcription. It is also possible that on its release from Bcl-xL, p53 could directly target mitochondria and activate other transcription-independent apoptotic pathways. In agreement with our earlier results (Bykov et al., 2005a), PRIMA-1MET induced expression of both Bax and PUMA in a mutant p53-dependent manner in the H1299 cells. Thus, Bax and PUMA are p53 targets that may trigger mitochondrial apoptosis independently of caspase-2 activation. As we observed a significant decrease in cell death when either PUMA or caspase-2 was inactivated, both activation of caspase-2 and upregulation of proapoptotic p53 targets are presumably required for a full apoptotic response to PRIMA-1MET. However, no further reduction in cell death was detected on inactivation of both caspase-2 and PUMA, suggesting that additional pathway(s) are activated on PRIMA-1MET treatment. This possibility needs further investigation.

In conclusion, PRIMA-1MET induces signaling cascades that target mitochondria and engages the intrinsic apoptotic pathway (Figure 8). Our results shed new light on how PRIMA-1MET triggers mutant p53-dependent apoptosis at the molecular level. This information should facilitate further development and optimization of PRIMA-based anticancer drugs for treatment of mutant p53-carrying tumors.

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Materials and methods

Cell culture

The following cell lines were used: p53 null H1299 human lung adenocarcinoma cells and the subline H1299-His175 carrying exogenous His175 mutant p53, p53 null Saos-2 human osteosarcoma cells and the subline Saos-2-His273 carrying exogenous His273 mutant p53, p53 null SKOV-TA human ovarian carcinoma cells and the subline SKOV-His175 carrying exogenous His175 mutant p53 and KRC/Y renal carcinoma cells carrying endogenous Phe176 mutant p53. The cells were grown in Iscove's modified Dulbecco's medium supplemented with 10% heat-inactivated fetal bovine serum, 2mM L-glutamine and 2.5μg/ml plasmocin in a humidified 5% CO2 atmosphere at 37°C. The cells were seeded in 6-well plates at 10000cells/cm2 and cultured for 16h before addition of PRIMA-1MET. The caspase-2 inhibitor zVDVAD-fmk and the caspase-3 inhibitor zDEVD-fmk (Enzyme Systems Products, CA, USA) were added at 20μM 1–2h before experiments.

Reagents

The following antibodies were used: polyclonal anti-G3PDH Ab (Nordic Biosite, Sweden); anti-cytochrome c mAb, clone 7H8.2C12 (BD Biosciences, CA, USA); anti-PUMA mAb clone ab9643 (Abcam, UK); anti-Bax rabbit polyclonal AB clone N-20 (Santa Cruz, CA, USA); anti-PIDD mAb clone Anto-1 (Alexis Biochemicals, Switzerland); anti-β-Actin mAb clone AC-15 (Sigma, Sweden); FITC-conjugated rabbit polyclonal anti-mouse Ig (DAKO, Denmark), and horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, UK). PRIMA-1MET (Bykov et al., 2005b) was used as a mutant p53-targeting compound in all experiments.

Western blotting

Cells were harvested, washed in phosphate-buffered saline (PBS) and lysed for 30min on ice in lysis buffer (20mM Tris–HCl, pH 8.0, 2mM EDTA, 138mM NaCl, 10% glycerol, 1% Triton X-100 and 1 × complete protease inhibitors (Roche Diagnostics, IN, USA). Cell extracts were centrifuged at 14000 r.p.m. for 8min at 4°C followed by determination of protein concentration using the BSA assay (Pierce, CA, USA). Equal amounts of protein from each sample were mixed with Laemmli's loading buffer, boiled for 5min, and subjected to SDS–polyacrylamide gel electrophoresis at 130V followed by electroblotting to nitrocellulose membranes for 2h at 100V. Membranes were blocked for 1h with 5% non-fat milk in PBS at room temperature and probed with primary antibodies. All antibodies were diluted in PBS containing 5% non-fat milk, and 0.05% Tween-20. Blots were visualized by ECL (Amersham Biosciences, Sweden).

Mitochondrial cytochrome c release

Cells (1 × 106) treated as indicated were washed in ice-cold PBS, resuspended in 100μl of buffer (5mM Tris–HCl, pH 7.4, 50mM KCl, 5mM MgCl2, 1mM EGTA, 5mM succinate, 1mM KH2PO4 and 140mM manitol), and permeabilized with digitonin (0.01%) for 5min at room temperature. Mitochondria and nuclei were removed from the soluble cytosolic fraction by centrifugation at 16000 × g for 5min. Mitochondrial pellets and supernatants were analyzed by western blotting.

DAPI staining and immunofluorescence

Cells were treated for 16h on coverslips, fixed for 15min in 4% paraformaldehyde on ice, washed in PBS, and permeabilized with 0.1% Triton X-100. Blocking of nonspecific binding of antibodies was performed by incubation of cells in PBS containing 10mM HEPES, 3% BSA and 0.3% Triton X-100 at room temperature for 60min. Incubations with primary (1:100) and secondary antibodies (1:200) were performed at 4°C overnight and at room temperature for 60min, respectively. Among all steps, cells were washed 3 × 10min with PBS at room temperature. Stained sections were mounted using Vectashield H-1000 containing DAPI stain (Vector Laboratories, CA, USA), and examined under a Zeiss Axioplan 2 fluorescence microscope equipped with an AxioCam HRm Camera (Carl Zeiss, Germany). The software used for analysis was Axio Vision 4.2.

Flow cytometry

The Δψm was measured by tetramethylrhodamine ethyl staining (TMRE; Molecular Probes, Netherlands). Cells were harvested and loaded with 20nM TMRE at the indicated time points for 45min at 37°C, and analyzed on a FACScanflow cytometer (BD Biosciences, CA, USA). For analysis of PI staining, cells were harvested, washed in PBS, fixed in ice-cold 50% ethanol overnight at 4°C, and incubated in PI solution (50μg/ml PI and 0.1% (w/w) Na-citrate in PBS) in presence of RNase A (0.25mg/ml) for 30min at 37°C. Flow cytometric analysis was carried out using 536nm excitation and 617nm emission filters, and the Cell Quest software (BD Biosciences, CA, USA).

Measurement of caspase activities

Cells were treated with PRIMA-1MET, trypsinized, washed and incubated with the CaspaTag TM pan-caspase assay kit (Ichemicon, UK). Caspase-positive cells were assessed by FLICA staining and FACS. Data were analyzed using Cell Quest. Measurements of caspase-2, -9, -3 and -8-like activities using cleaved fluorogenic caspase substrates (Peptide Institute, Japan) were carried out as follows: 5 × 105 cells were washed with ice-cold PBS, resuspended in 25μl of PBS and loaded on a microtiter plate. Next, fluorogenic substrates VDVAD-AMC (50μM), DEVD-AMC (50μM), LEHD-AMC (50μM) or IETD-AMC (50μM) dissolved in 100mM HEPES, pH 7.25, 10% sucrose, 10mM DTT, 0.1% CHAPS or 100mM MES, pH 6.5, 10% polyethylene glycol, 10mM DTT and 0.1% CHAPS were added. Substrate cleavage was monitored by AMC liberation in a Fluoroscan II plate reader (Labsystems, Sweden) using 355nm excitation and 460nm emission wavelengths. Fluorescence units were converted to picomole of AMC using a standard curve generated with free AMC. Data from duplicate samples were analyzed by linear regression.

Gene silencing by siRNA

Silencing of caspase-2 or PUMA was achieved by transfection of 21-nucleotide siRNA duplexes using the Hiperfect transfection reagent (Qiagene, Ireland). A scrambled siRNA was used as control. Downregulation of caspase-2 or PUMA expression was confirmed by western blotting.

Statistical analysis

All experiments were performed at least twice. Data were analyzed by Origin (Originlab, USA). Statistically significant differences according to the independent t-test at Pless than or equal to0.1 and Pless than or equal to0.05 are indicated by * and **, respectively.

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Acknowledgements

This study was supported by grants from the Swedish and Stockholm Cancer Societies, the Swedish Research Council, the EU 6th and 7th Framework Programs, and Karolinska Institutet. This publication reflects the author's views and not necessarily those of the EC partners. The information in this document is provided as is and no guarantee or warranty is given that the information is fit for any particular purpose. The user thereof uses the information at its sole risk and liability. The Community is not liable for any use that may be made of the information contained herein.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)