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. Author manuscript; available in PMC: 2010 Feb 17.
Published in final edited form as: Cell Cycle. 2009 Jun 30;8(11):1711–1719. doi: 10.4161/cc.8.11.8596

The transcription-independent mitochondrial p53 program is a major contributor to nutlin-induced apoptosis in tumor cells

Angelina V Vaseva 1, Natalia D Marchenko 1, Ute M Moll 1,*
PMCID: PMC2823264  NIHMSID: NIHMS172469  PMID: 19411846

Abstract

Strategies to induce p53 activation in tumors that retain wild-type p53 are promising for cancer therapy. Nutlin is a potent and selective pharmacological MDM2 inhibitor that competitively binds to its p53-binding pocket, thereby leading to non-genotoxic p53 stabilization and activation of growth arrest and apoptosis pathways. Nutlin-induced apoptosis is thought to occur via p53’s transcriptional program. Here we report that the transcription-independent mitochondrial p53 program plays an important role in Nutlin-induced p53-mediated tumor cell death. Aside from nuclear stabilization, Nutlin causes cytoplasmic p53 accumulation and translocation to mitochondria. Monoubiquitinated p53, originating from a distinct cytoplasmic pool, is the preferred p53 species that translocates to mitochondria in response to stress. Nutlin does not interfere with MDM2’s ability to monoubiquitinate p53, due to the fact that MDM2-p53 complexes are only partially disrupted and that Nutlin-stabilized MDM2 retains its E3 ubiquitin ligase activity. Nutlin-induced mitochondrial p53 translocation is rapid and associated with cytochrome C release that precedes induction of p53 target genes. Specific inhibition of mitochondrial p53 translocation by Pifithrin μ reduces the apoptotic Nutlin response by 2.5-fold, underlining the significance of p53’s mitochondrial program in Nutlin-induced apoptosis. Surprisingly, blocking the transcriptional arm of p53, either via α-Amanitin or the p53-specific transcriptional inhibitor Pifithrin α, not only fails to inhibit, but greatly potentiates Nutlin-induced apoptosis. In sum, the direct mitochondrial program is a major mechanism in Nutlin-induced p53-mediated apoptosis. Moreover, at least in some tumors the transcriptional p53 activities in net balance not only are dispensable for the apoptotic Nutlin response, but appear to actively block its therapeutic effect.

Keywords: nutlin, p53, MDM2, ubiquitination, mitochondria, apoptosis

Introduction

The tumor suppressor p53 is a master regulator of the cellular stress response and critically important in human cancer. p53 is a central protective node and its main function is to prevent the propagation of damaged cells that are potentially mutagenic by orchestrating defense pathways of apoptosis, cell cycle arrest, senescence and DNA repair.1–4 Upon activation by a diverse type of stress stimuli, p53 induces apoptosis via transcription-dependent and transcription-independent modes, two fundamentally different but synergistic mechanisms of action. In the nucleus, p53 governs a complex transcriptional program that includes apoptotic target genes such as Puma, Noxa and Bax, but also genes with non-apoptotic, cell cycle arrest or as yet poorly defined functions. In addition, stress-induced p53 translocates to mitochondria where it directly interacts with the multi-domain members of the Bcl2 family of mitochondrial permeability regulators to induce mitochondrial outer membrane permeabilization (MOMP).5–8

p53 itself is an attractive therapeutic target. Restoring p53 function in cancer cells with mutant p53 by small molecule or peptide modulators of p53 protein conformation was shown to induce tumor cell death in experimental systems. However, due to the high propensity of most tumor-associated p53 missense mutant proteins to denature and aggregate at body temperature,9 identifying pharmacologically relevant agents that properly refold mutant proteins in vivo has been difficult and so far none have been established. On the other hand, about 50% of human tumors retain wild-type p53, albeit with inadequate p53 function due to abnormalities in p53 regulation or defective signaling in the p53 pathway. One mechanism to suppress wild-type p53 function in tumors is based on overexpression of the E3 ligase MDM2, the key negative regulator of p53 stability and an inhibitor of p53 transcriptional activity. Strategies to activate p53 in wild-type tumors are highly promising and great efforts in pharmaceutical and academic research identified a number of lead compounds that have this effect (reviewed in refs. 10 and 11). The general concept of therapeutic activation of wild-type p53 is greatly strengthened by several sophisticated mouse models, where genetic restoration of wild-type p53 function after tumors are established in vivo leads to clinical tumor regression via induction of apoptosis and/or senescence.12–14 Several small molecule compounds with good cellular potency and selectivity were developed that activate wild-type p53 by interfering with the inhibitory p53-MDM2 complex. Nutlin 3a (here called Nutlin) is a highly specific, non-toxic MDM2 antagonist from the class of cis-imidazolines whose design was based on structural information of the complex. Nutlin liberates p53 by molecular mimickry, competitively binding to the relatively deep hydrophobic p53-binding pocket within the N-terminus of MDM2.15 This stabilizes and activates p53 in the absence of genotoxic stress to a degree comparable to genotoxic p53 activation. Nutlin has shown promising results in killing or arresting a broad array of wild-type p53 harboring human cancer cell lines in culture and in mouse xenografts.10,15,16

Mechanistically, Nutlin is currently thought to induce apoptosis via p53’s transcriptional program.10,15,16 Our study aimed at determining what if any role the mitochondrial p53 program might play during Nutlin-induced apoptosis. Our findings let us to conclude that the transcription-independent mitochondrial p53 program might be a therapeutically-relevant mechanism in Nutlin-induced apoptosis.

Results

Nutlin stabilizes p53 protein, induces p53 transcriptional activity and causes apoptosis in wild-type p53 harboring tumor cells

Nutlin was previously shown to induce apoptosis specifically in human cancer cell lines with wild-type but not mutant p53.10,15,16 We therefore first verified the effect of Nutlin in the wild-type p53 harboring human tumor cell lines used here, namely the acute myeloid leukemic line ML-1 and the colon carcinoma line RKO, representing hematopoietic and epithelial cell lineages. In ML-1 cells, p53 accumulation in crude extracts is detectable as early as 1 hour after 10 μM Nutlin treatment, a typical concentration used (Fig. 1A). Induction of the p53 transcriptional target proteins MDM2 and p21 starts at 2 and 3 hours respectively, and peaks at 12 hrs (Fig. 1A), in agreement with previous reports.15,16 Following p53 activation, Nutlin induces caspase activation and apoptosis after 8 hrs, as detected by PARP cleavage (Fig. 1A). After 24 hrs, 76% of ML-1 cells are TUNEL positive, indicating a strong response of these leukemic cells to Nutlin (Fig. 1B). Similar results were seen in RKO carcinoma cells, albeit with delayed kinetics of PARP cleavage (data not shown).

Figure 1.

Figure 1

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Nutlin stabilizes p53 protein, induces p53 transcriptional activity and causes apoptosis in wild-type p53 harboring tumor cells. (A) ML-1 cells were treated with 10 μM Nutlin for the indicated times. Cell lysates were analyzed by immunoblotting using the indicated antibodies. PCNA served as loading control. The two bands of p53 reflect the Arg/Pro codon 72 polymorphism of p53 in ML-1 cells. (B) ML-1 cells were treated with 10 μM Nutlin for 24 hours and processed for TUNEL staining. Left, representative example of TUNEL staining of ML-1 cells. Right, quantitation of apoptotic cells obtained from TUNEL assays; at least 500 cells each were counted. Error bars represent standard deviation of three random fields.

Nutlin causes nuclear and cytoplasmic p53 accumulation and does not interfere with monoubiquitination of cytoplasmic p53

Identical to treatment with DNA damage-inducing drugs, treatment of ML-1 and RKO cells with non-genotoxic Nutlin results in nuclear and cytoplasmic accumulation of p53 protein (Fig. 2A). Moreover, the p53-induced MDM2 protein becomes stabilized in nucleus and cytoplasm, consistent with its known dual localization (Fig. 2A).

Figure 2.

Figure 2

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Nutlin causes nuclear and cytoplasmic p53 accumulation and does not interfere with monoubiquitination of cytoplasmic p53. (A) Nutlin stabilizes wild-type p53 in both nucleus and cytoplasm. ML-1 and RKO cell were treated for 6 hours with 10 μM Nutlin followed by fractionation and immunoblotting. HDAC1 and Hsp90 serve as nuclear and cytoplasmic contamination markers, respectively. p53-induced MDM2 is present in both compartments. (B) Nutlin does not interfere with MDM2-mediated monoubiquitination of wild-type p53. From the experiment in (A), equal amounts of p53 were immunoblotted. The multi-band ‘ladder’ above non-ubiquitinated p53 represents multi-monoubiquitinated p53. The non-descript faint smear above is typical for polyubiquitinated p53. (C) Nutlin weakens but does not completely disrupt the endogenous p53-MDM2 complex. Lysates (500 μg each) of RKO cells treated with 10 μM Nutlin for 6 hours or left untreated were subject to reciprocal co-immunoprecipitations for MDM2 (left) and p53 (right), followed by immunoblotting as indicated.

We previously showed that in response to genotoxic stress, a cytoplasmic pool of MDM2-dependent (multi)-monoubiquitinated p53 becomes rapidly stabilized. This modification greatly promotes p53 translocation to mitochondria.17 After arrival at mitochondria, monoubiquitinated p53 is immediately deubiquitinated by mitochondrial HAUSP and thereby ready to induce outer mitochondrial membrane permeabilization by interacting with multi-domain Bcl-2 family members.17 Importantly, despite Nutlin’s known action of blocking p53 binding to MDM2, the pool of monoubiquitinated p53 in the cytoplasm of ML-1 and RKO cells was not affected by Nutlin treatment (Fig. 2B). Thus, in the presence of Nutlin MDM2 is still able to monoubiquitinate p53. The reasons for this surprising result are two-fold. First, in bi-directional co-immunoprecipitations with antibodies against MDM2 or p53, the endogenous MDM2-p53 complex was only partially but not completely disrupted by Nutlin. As shown in Figure 2C, endogenous p53-MDM2 complexes are still detectable in the presence of Nutlin, although the relative amount of p53 that co-immunoprecipitates with MDM2 in treated cells is reduced compared to that of untreated cells (Fig. 2C left, compare much higher MDM2 levels in treated versus untreated cells, but the same amount of co-precipitated p53). The same picture is seen in inverse co-precipitations for p53 (Fig. 2C right, despite the increased p53 level in treated cells, the rise in co-precipitated MDM2, although higher than in untreated cells, is disproportionately lower). Similar incomplete complex distribution by Nutlin was reported by Wade et al.18 Moreover, Nutlin-stabilized MDM2 retains its E3 ubiquitin ligase activity, as previously shown.16,19 MDM2 from Nutlin-treated cells unimpededly facilitates the degradation of MDMX, identical to MDM2 from untreated control cells.16 Thus, since the E3 ligase activity of MDM2 is not affected by Nutlin, the remaining low levels of complex formation between MDM2 and p53, although insufficient for mediating p53 polyubiquitination and destabilization, is sufficient for mediating p53 monoubiquitination which is known to promote p53 trafficking to mitochondria.

Nutlin induces mitochondrial translocation of p53

Thus, the fact that Nutlin treatment stabilizes cytoplasmic p53 without interfering with its monoubiquitination predicts that Nutlin also promotes translocation of p53 to mitochondria. Indeed, Nutlin induces significant mitochondrial p53 translocation in ML-1 and RKO cells (Fig. 3A). Of note, Nutlin-induced p53 translocation occurs as early as 1 hr after adding the drug and continues to increase over time (Fig. 3B). Coincident with rapid mitochondrial p53 translocation is the rapid mitochondrial release of Cytochrome C, reaching maximum even at the 1 hr time point. Cytochrome C release indicates mitochondrial outer membrane permeabilization and marks one of the earliest points of no return of the mitochondrial apoptotic pathway. Conversely, Nutlin induced upregulation of p53 transcriptional target proteins occurs at the earliest at 2–4 hours, as indicated by p21Cip1, considered to be the most sensitive p53 target gene, and corroborated by MDM2 (Fig. 1A).10,15,16 Together, this indicates that during Nutlin-induced apoptosis p53 translocation to mitochondria with its MOMP activity precedes the induction of the p53 transcriptional program.

Figure 3.

Figure 3

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Nutlin induces mitochondrial translocation of p53. (A) Nutlin induces p53 translocation to mitochondria. Mitochondrial fractions from RKO and ML-1 cells treated with 10 μM Nutlin or left untreated were prepared and analyzed by immunoblotting. PCNA and mthsp70 serve as loading control for crude extracts and mitochondria, respectively. (B) Time course of mitochondrial p53 translocation. ML-1 cells were treated with 10 μM Nutlin for the indicated times and mitochondrial fractions were analyzed as in (A). PCNA serves as loading control and nuclear contamination marker. The cytosol also contains PCNA due to nuclear rupture during fractionation. mthsp70 is the loading control for mitochondria.

Inhibition of mitochondrial p53 translocation by PFTμ inhibits nutlin-induced apoptosis

PFTμ is a small molecule inhibitor shown to specifically inhibit the direct pro-apoptotic actions of p53 at mitochondria by reducing the affinity of p53 towards its mitochondrial interaction partners Bcl-2, Bcl-xL and BAK.20,21 Pre-treatment with PFTμ was shown to rescue primary thymocytes in vitro and mice in vivo from lethal doses of γ-irradiation or DNA damaging agents.20,21 Thus, we used this inhibitor to determine the significance of the transcription-independent mitochondrial p53 death program during Nutlin-induced apoptosis. Indeed, cells pretreated with PFTμ prior to Nutlin exposure show significant inhibition of mitochondrial p53 translocation (Fig. 4A). Consequently, reduction of mitochondrial p53 translocation is associated with markedly depressed mitochondrial Cytochrome C release retaining now levels close to untreated cells, indicating reduced permeabilization of the outer mitochondrial membrane (Fig. 4A, last three lanes). Decreased TUNEL staining and decreased PARP cleavage further confirm that PFTμ pretreatment markedly inhibits Nutlin-induced apoptosis (Fig. 4B). Quantatification of the TUNEL scores reveals a 2.6 fold reduction in apoptosis (34% with Nutlin alone versus 13% with PFTμ pretreatment). In sum, these results confirm the importance of the direct mitochondrial p53 death program in Nutlin-induced apoptosis.

Figure 4.

Figure 4

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Inhibition of mitochondrial p53 translocation by PFTμ inhibits Nutlin-induced apoptosis. (A) Pre-treatment of ML-1 cells with PFTμ, a specific inhibitor of p53’s association with mitochondria, results in markedly reduced p53 translocation and reduced cytochrome C release in response to Nutlin treatment. ML-1 cells were pre-treated with 25 μM PFTμ for 2 hrs before adding 10 μM Nutlin for 12 hours, followed by mitochondrial fractionation and immunoblotting as indicated. PCNA serves as loading control and nuclear contamination marker. mthsp70 is the loading control for mitochondria. (B) Pretreatment of ML-1 cells with PFTμ inhibits the apoptotic response to Nutlin. ML-1 cells were pre-treated with 25 μM PFTμ for 2 hours followed by treatment with Nutlin for another 12 hours. Aliquots of cells were processed for TUNEL staining or lysed for immunoblotting. Top, representative example of TUNEL experiment. Bottom, quantitation of apoptotic cells obtained from TUNEL assays; at least 500 cells each were counted. Error bars represent standard deviation of 3 random fields. Immunoblot showing levels of PARP cleavage and p53. PCNA as loading control.

Surprisingly, transcriptional activities of p53 not only are dispensable for the apoptotic nutlin response, but actively block its mitochondrial p53 death program

The above findings suggest that the apoptotic p53 response to Nutlin might be largely transcription-independent, driven by the mitochondrial p53 pathway. To further test this notion, we used pharmacological inhibitors that block the transcriptional arm of p53 to determine whether the mitochondrial p53 program alone is sufficient to mediate the apoptotic Nutlin response. To this end, we first used α-Amanitin, a highly specific and potent inhibitor of the general RNA polymerase II transcription. Given the fact that under certain circumstances (cell type, time and dose-dependent) α-Amanitin itself can cause p53-dependent apoptosis by engaging the p53 mitochondrial pathway,5 we first performed careful titration experiments. The goal was to find a low dose and time of α-Amanitin treatment that by itself does not cause significant p53 stabilization and apoptosis, yet is sufficient to inhibit Nutlin-induced p53 transcription in ML-1 cells. As shown in Figure 5A top left, α-Amanitin pretreatment of ML-1 cells with 10 μM for 16 hrs blocked Nutlin-mediated p21 induction, indicating a profound blockade of p53 transcriptional activities (compare lanes 3 and 4), yet without stabilizing p53 levels much beyond those obtained with Nutlin alone. Surprisingly, however, compared to Nutlin alone significantly higher apoptosis occurs when cells are treated with the combination of α-Amanitin plus Nutlin, as determined by higher PARP cleavage (Fig. 5A top left). This indicates enhanced apoptosis under conditions when transcription is blocked. The result was confirmed by TUNEL staining (Fig. 5A right). α-Amanitin does not interfere with mitochondrial p53 translocation. To the contrary, in the presence of α-Amanitin, mitochondrial p53 translocation is enhanced, supporting the p53-dependence of the functional synergy (Fig. 5A bottom left).

Figure 5.

Figure 5

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Transcriptional activities of p53 are not only dispensable for the apoptotic Nutlin response, but can block its mitochondrial p53 death program. ML-1 cells undergo Nutlin-induced apoptosis despite inhibition of the p53 transcriptional program by α-Amanitin (A) or PFTα (B). (A) ML-1 cells were pretreated with 10 μM α-Amanitin for 16 hours, followed by 10 μM Nutlin for an additional 10 hours. Top left, Immunoblot of ML-1 lysates treated as indicated. p21 induction is the most sensitive indicator of p53 transcriptional activity. PARP cleavage indicates an apoptotic response. β-Actin and PCNA serve as loading controls. Representative example of TUNEL assay (Top right) and quantitation (Bottom right). Error bars represent standard deviation of 3 random fields. Bottom left, α-Amanitin does not interfere with mitochondrial translocation of p53 upon Nutlin treatment. Mitochondrial fractions were prepared from cells treated as in (A) and analyzed by immunoblotting. PCNA serves as loading control and nuclear contamination marker. mthsp70 as mitochondrial loading control. (B) ML-1 cells were pre-treated with 20 μM PFTα for 4 hours followed by 10 μM Nutlin for additional 10 hours. Top left, immunoblot indicating that pretreatment of ML-1 cells with PFTα represses p21 but induces PARP cleavage upon Nutlin treatment, while not altering p53 levels. Bottom left, PFTα does not interfere with Nutlin-induced p53 translocation to mitochondria. Top right, quantitation of TUNEL assay from ML-1 cells pre-treated with PFTα.

Finally, Pifithrin α (PFTα) is a specific small molecule inhibitor which selectively blocks p53-mediated transcription, as shown in diverse systems in cell culture and in vivo in mice.22–26 For example, PFTα was shown to rescue mice from lethal genotoxic stress caused by gamma irradiation.22 As expected, PFTα pretreatment does suppress Nutlin-induced p21 induction in ML-1 cells (Fig. 5B top left). Of note, pretreatment of ML-1 cells with PFTα does not affect Nutlin-induced cellular p53 stabilization (Fig. 5B top left) nor does it interfere with Nutlin-induced mitochondrial p53 translocation (Fig. 5B bottom). PFTα alone shows minimal apoptotic effect. Importantly, however, PFTα greatly potentiates Nutlin-induced apoptosis, as indicated by enhanced PARP cleavage (Fig. 5B top left) and about 3-fold increased TUNEL scores (Fig. 5B right, from 19% with Nutlin alone to 53% with PFTα plus Nutlin after 10 hrs). We conclude that at least in some human leukemia cells the p53 transcriptome is not only dispensable for the apoptotic Nutlin response, but in net balance can actively block the Nutlin-induced mitochondrial death program of p53. This might be of future clinical significance.

Discussion

MDM2 inhibitors of the Nutlin group are a promising new therapeutic strategy for human tumors retaining wild-type p53 status. Nutlin is a highly selective competitive binding inhibitor of the MDM2-p53 interaction that potently stabilizes and activates wild-type but not mutant p53 protein in tumor cells. In contrast to chemotherapeutics whose mechanism of action at least in part rests on genotoxic activation of p53, Nutlin’s ability to activate wild-type p53 in a non-genotoxic manner might render it a viable alternative to current cytotoxic chemotherapy with its high genotoxic burden and risk of developing secondary malignancies later in life. In fact, Nutlin’s potency and selectivity in single agent studies to induce growth inhibition on panels of cultured human tumor cells and shrinkage in nude mouse tumor xenografts has provided the proof-of-principle of this concept.10,15 As single agent Nutlin showed the most promising results in leukemias that rarely harbor alterations of the p53 gene.27–31 Nutlin also showed promise in neuroblastoma, a solid tumor characterized by essentially exclusive wild-type p53 status.32–34 Moreover, since Nutlin does not bind to p53 or induce post-translational modifications, Nutlin can also act in concert with conventional chemotherapeutics to activate p53, with the potential of improving efficacy or lowering the genotoxic burden by enabling dose reduction. For example, synergistic or additive effects with genotoxic drugs were found in in vitro studies of cultured and clinical samples of multiple myeloma,35 acute myelogenous leukemia31 and chronic lymphocytic leukemia.27,29 Clinical trials of Nutlin are ongoing.36,37

As discussed, activating p53 signaling by Nutlin is particularly promising in haematologic malignancies such as leukemias and lymphomas due to several favorable parameters that compound in this disease group: (i) p53 mutations are rare, (ii) inactivation of wild-type p53 by Mdm2 overexpression are frequent molecular events, e.g., in acute myeloid leukemia, (iii) downstream p53 signaling is intact and (iv) the cell-intrinsic response to Nutlin-induced p53 activation tends to be apoptosis rather than cell cycle arrest, favoring the preferred cytotoxic rather than a mere cytostatic outcome.

However, a big subset of wild-type p53 human tumor cells of non-hematopoietic lineages undergo cell cycle arrest in response to Nutlin, conferring only cytostatic rather than the desired cytotoxic antitumoral activity.10,15,28,29,31 Thus, elucidating the precise mechanism(s) of Nutlin-mediated apoptosis is critical for designing rational strategies on how to convert Nutlin from a cytostatic to a cytotoxic anticancer agent to improve its therapeutic utility. The apoptotic effects of Nutlin in wtp53 tumor cells are currently ascribed to the activation of transcriptional activities of p53 in the nucleus. Indeed, once freed from MDM2 by Nutlin, p53 accumulates in the nuclei of cancer cells, followed by activation of a broad wild-type p53-dependent transcriptional program that includes induction of the proapoptotic Puma, Noxa, Bax and DR5 genes, as well as repression of pro-survival genes.10,15,34 Here we show that the transcription-independent mitochondrial p53 program can be the major contributor to Nutlin-induced apoptosis, at least in some tumors cells including leukemia and colon carcinoma. Moreover, transcriptional activities of p53 are not only dispensable for the apoptotic Nutlin response, but actively block its mitochondrial p53 death program. In addition to nuclear stabilization, Nutlin treatment of ML-1 and RKO cells also caused significant cytoplasmic accumulation of p53 and rapid mitochondrial translocation within 1 hr associated with early MOMP activity. Induction of early response transcriptional p53 target genes started at approximately 2 hrs after Nutlin treatment, indicating that mitochondrial p53 translocation is the first event during Nutlin-induced apoptosis. Specific inhibition of mitochondrial p53 translocation by PFTμ, an inhibitor that blocks p53 interaction with Bcl-2 and Bcl-xL, markedly reduced the apoptotic Nutlin response by 2.5-fold, underlining the significance of p53’s mitochondrial program in Nutlin-induced apoptosis. We had shown earlier that MDM2-mediated monoubiquitination of p53 greatly promotes p53’s mitochondrial translocation and thus its direct mitochondrial apoptosis pathway.17 Thus, Salmena and Pandolfi raised the important concern whether therapeutic targeting of specific E3 ligases such as MDM2 might also disrupt MDM2’s role as p53 monoubiquitinase and thereby blunt the tumor suppression offered by mitochondrial p53.38 Fortunately our results show that Nutlin does not interfere with monoubiquitination of cytoplasmic p53 due to the fact that MDM2-p53 complexes are only partially disrupted and Nutlin-stabilized MDM2 retains its E3 ubiquitin ligase activity.16

Two earlier in vitro studies had also noted that in addition to the nuclear p53 death pathway, Nutlin can also induce mitochondrial p53 death functions.30,31,39 Using immunofluorescence-based mitochondrial colocalization and cycloheximide sensitivity, Kojima et al. concluded that a significant fraction of clinical samples of acute myeloid and chronic lymphocytic leukemias co-recruited the mitochondrial p53 death pathway to various degrees in response to Nutlin. Interestingly, samples in the primarily transcription-independent group responded significantly better to Nutlin-induced apoptosis.30,31 Going beyond this observation, we report here that at least in some human leukemia cells the p53 transcriptome not only is dispensable for the apoptotic Nutlin response, but in net balance can actively block the Nutlin-induced mitochondrial death program of p53. Specific blockade of p53-mediated transcription by PFTα potentiated Nutlin-induced apoptosis of ML-1 cells by several folds. This might be of therapeutic significance in future clinical trials of Nutlin or later generation Nutlin-type MDM2 inhibitors.

Very recently a large series of clinical samples of chronic lymphocytic leukemia was also reported to undergo drug-induced apoptosis via the transcription-independent mitochondrial p53 pathway in response to the clinically used chemotherapeutics chlorambucil and fludarabine and Nutlin, with PFTα potentiating the death response.39 Our results substantiate and expand these findings.

While other parameters can interfere with Nutlin-induced apoptosis sensitivity of tumor cells (such as overexpression of MDM2, Mdmx, delta Np73 and Notch-1, loss of p14ARF or abrogation of downstream apoptotic effectors11,40), our data indicate that p53’s transcriptional activity itself plays a counteracting role. Which p53 targets could be responsible for such a dampening effect? Likely a concert of several rather than a single target, but one of the strong candidates is the p53-induced upregulation of p21Cip1, cell cycle arrest and known to a powerful inducer of G1 and G2 block apoptosis.41 α-Amanitin or PFTα blocked p21 induction in our ML-1 cells. Other as of yet unknown transcriptional products might negatively affect the mitochondrial pro-apoptotic functions of p53.

In sum, our study further clarified the mechanism of action of Nutlin-induced cell death. We determined that transcription-independent mitochondrial p53 functions are important during Nutlin-induced apoptosis of some tumor cells. Moreover, blocking concomitant p53-mediated transcription might improve Nutlin’s therapeutic efficacy by enhancing an intrinsic apoptotic response or converting cytostatic responders into cytotoxic responders.

Materials and Methods

Cell culture and drugs

The human myeloid leukemia cell line ML-1 and the colorectal cancer cell line RKO were used. Both were cultured in RPMI medium (Gibco, Invitrogen), supplemented with 10% FBS (Gibco). Nutlin 3a was a gift from Dr. L. Vassilev, Roche Pharmaceuticals. PFTμ was a gift from Andrei Gudkov, Lerner Research Institute, OH. α-Amanitin and PFTα were purchased from Sigma (St. Louis, MO).

Immunoblotting and immunoprecipitation

Exponentially growing cells were treated as indicated, lysed with 0.5% Triton X-100 in phosphate-buffered saline supplemented with protease inhibitor cocktail (Roche) and 50 ng/ml ubiquitinaldehyde (Sigma) and sonicated. Standard ECL immunoblotting was performed. For immunoprecipitation, cells were lysed in the same buffer as above. One milligram of total protein was incubated overnight at 4°C with 1 μg of primary antibody and protein A/G agarose beads (Roche). In some instances, p53-specific FL393 antibody conjugated to agarose beads was used. Beads were washed 3 times in buffer (0.5% Triton X-100 in PBS) and proteins were solubilized by boiling in 50 μl of sample buffer prior to SDS-PAGE. The following antibodies were used: monoclonal DO1 and polyclonal FL393 against p53 (Santa Cruz), monoclonals Ab-1 (Calbiochem) and 2A10 (Santa Cruz) against MDM2, polyclonal anti-PARP (Cell Signaling), monoclonal anti-p21 Clone 187 (Santa Cruz). For loading controls antibodies against β-Actin (Ab-5, NeoMarkers), HDAC, mt hsp70 and hsp90 were used.

Nucler/cytoplasmic fractionations

Exponentially growing RKO or ML-1 cells were treated as indicated. Cells were washed with ice-cold TD buffer (135 mM NaCl, 5 mM KCl, 25 mM Tris-HCl, pH 7.6) and resuspended in 100 μl CaRSB buffer (10 mM NaCl, 1.5 mM CaCl2, 10 mM Tris-HCl, pH 7.5) supplemented with 2x protease inhibitor cocktail and ubiquitin-aldehyde. Then 100 μl 1% Triton X-100 in PBS was added, the suspension was gently mixed and nuclei were spun down at 700 rcf for 5 min at 4°C. The cytosolic fraction was transferred to a new tube and nuclei were lysed with 50 μl 0.5% Triton X-100 in PBS and sonicated. In immunoblots, monoclonal anti-HDAC1 (Affinity BioReagents) and monoclonal anti-Heat Shock Protein 90 (Calbiochem) were used as nuclear and cytoplasmic markers, respectively.

Mitochondrial purification

Cells were washed with TD buffer, resuspended in CaRSB buffer and incubated for 10 min to allow swelling. Cells were then homogenized with a loose-fit Dounce homogenizer and addition of 2.5x MS buffer (210 mM mannitol, 70 mM sucrose, 5 mM EDTA, 5 mM Tris, pH 7.6). The homogenate was cleared from nuclei by centrifugation at 1,000 g for 5 min at 4°C. The nucleus-free homogenate was subjected to discontinous sucrose gradient ultracentrifugation. Mitochondria fraction was carefully collected at the 1 M/1.5 M interface of the gradient.

TUNEL staining

Treated and untreated ML-1 and RKO cells were washed with PBS and 5 × 105 cells were cytospun onto glass slides and processed for TUNEL staining as recommended by the manufacturer (Roche). Staining was quantitated by measuring total pixels per field from 3 random fields (Zeiss Axiovision software).

Abbreviations

MDM2

murine double minute 2 gene

MOMP

mitochondrial outer membrane permeabilization

PARP

poly (ADP-ribose) polymerase

HAUSP

herpes-virus-associated ubiquitin-specific protease

TUNEL

terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling

Footnotes

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/8596

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