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Blood, 1 November 2008, Vol. 112, No. 9, pp. 3827-3834. Prepublished online as a Blood First Edition Paper on August 5, 2008; DOI 10.1182/blood-2008-05-156380.
NEOPLASIA p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism1 Department of Hematology, Royal Free and University College Medical School, London, United Kingdom; and 2 Department of Discovery Oncology, Hoffmann-La Roche, Nutley, NJ
The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin  , an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21CIP1. Surprisingly, pifithrin dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.
Chronic lymphocytic leukemia (CLL) was formerly thought to be an indolent disease but is now known to be characterized by a high rate of turnover, with 0.1% to 1% of the malignant clone being replaced each day.1,2 CLL comprises at least 2 subtypes, characterized by the extent of mutation within the variable region of the immunoglobulin heavy chain (IgVH) gene of the malignant cells. Patients with extensive mutation are associated with a good prognosis, whereas poor prognosis patients harbor IgVH genes with low levels of mutation and typically require early therapeutic intervention.1 Conventional drugs used in CLL therapy include the alkylating agent chlorambucil and the nucleoside analog fludarabine. The importance of the p53 pathway in the response to these drugs is underscored by the poor prognosis and drug resistance of CLL patients with mutations and/or deletions of the p53 genes or the gene encoding ATM protein kinase, the key upstream regulator of p53.3 p53 is up-regulated by a post-translational regulatory mechanism consequent to the induction of DNA damage.4,5 The p53 protein is a transcription factor that induces a G1 phase blockade, thus enabling DNA repair. The G1 blockade is mediated by up-regulation of the p53 target p21CIP1, an inhibitor of cyclin/cyclin-dependent kinase complexes.5 p53 also up-regulates transcription of proapoptotic BH3-only Bcl-2 family members Puma and Noxa.5 Puma and Noxa induce apoptosis by binding to and neutralizing the ability of antiapoptotic proteins, including Bcl-2 and Bcl-XL, to negatively regulate the proapoptotic Bax protein.6 Bax consequently undergoes a conformational change that allows its insertion into the mitochondrial outer membrane, resulting in the efflux of cytochrome c from the mitochondrial intermembrane space to the cytosol. Cytoplasmic cytochrome c binds to the Apaf-1 protein, enabling it to activate caspase 9, thus initiating a cascade of caspase activation events that results in apoptosis.6 The conventional view of p53-mediated apoptosis has emphasized its role as a transcription factor.5 In contrast, recent studies have identified a novel nontranscriptional mechanism that initiates apoptosis by direct binding of p53 to antiapoptotic Bcl-2 family proteins at the mitochondrial surface, resulting in Bax activation and apoptosis.7,8 This binding is dependent on the conformation of the DNA-binding domain of p53 and mutations that abolish p53's DNA binding and transcriptional activation function simultaneously impair its ability to interact with Bcl-2 family members.9,10
The accumulating evidence for transcription-independent apoptosis induction by p53 prompted us to determine its subcellular localization in CLL cells and whether selective pharmacological blockade of p53's transcriptional function blocked apoptotic killing. In the experiments described here, p53 was induced by treatment with chlorambucil, fludarabine, or nutlin 3a, a nongenotoxic agent that elevates p53 by direct inhibition of its interaction with the negative regulator Mdm211 and that induces p53-dependent apoptosis of CLL cells.12–14 Pifithrin
Materials
Tissue culture media and preformed polyacrylamide gels were from Invitrogen (Paisley, United Kingdom). PFT Patients and cell isolation CLL was diagnosed according to established clinical criteria.29 Age, sex, lymphocyte count, Rai staging, and IgVH gene mutation status on individual patients are summarized in Table 1. This study was approved by the Local Research Ethics Committee of the Royal Free Hospital. Written consent was obtained from patients prior to collection of heparinized peripheral blood samples in accordance with the Declaration of Helsinki. Procedures for the isolation of malignant cells and the determination of their purity have been described in detail.30 All isolates used contained more than 95% CD19/CD5-positive cells.
Normal T lymphocytes were isolated by negative selection using the MACS pan T-cell isolation kit (Miltenyi Biotec, Bisley, United Kingdom). Cell incubation CLL cells (107 mL–1) were routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U mL–1 penicillin, and 100 µg mL–1 streptomycin. Cultures were incubated for 24 hours at 37°C prior to addition of drugs. Incubations with chlorambucil or nutlin 3a were incubated for 18 hours. Incubations with fludarabine were for 48 hours, because induction of p53 by fludarabine was found in preliminary experiments to be delayed, compared with p53 induction by chlorambucil or nutlin 3a. In experiments designed to study subcellular distribution of p53 and other proteins, Bax conformation change or the suppression of p53 target induction by cytotoxic agents, 100 µM ZVAD, a pan-caspase inhibitor, was added to media to block protein degradation consequent to caspase activation. Protein extraction and cell fractionation Whole cell lysates were prepared by extraction of cell pellets with a buffer containing 2% nonidet P40, 0.5% sodium deoxycholate, and 0.2% sodium dodecyl sulfate as described.30 Subcellular fractionation was carried out by differential detergent fractionation (DDF) essentially as described.31 Fraction 1, consisting of cytosolic material, was obtained by extraction with buffer 1, which contained 0.02% digitonin. Fraction 2 (mitochondria plus membranous organelles) was extracted with buffer 2, which contained 0.5% triton X-100. Fraction 3 (nuclear material) was solubilized directly into gel loading buffer (300 mM sucrose, 250 mM Tris-HCl, pH 8.5, 0.5 mM EDTA, 80 mM dithiothreitol, 2% sodium dodecyl sulfate, and 0.2% Serva Blue G250) with heating at 70°C for 10 minutes. The DDF protocol was found by Waterhouse et al32 to give the most reproducible results in studies of apoptotic mechanisms. Protein concentrations were determined by a sensitive modification of the Lowry procedure.33 Coimmunoprecipitation of Bcl-2 and p53 Bcl-2 was immunoprecipitated by a modification of a published procedure.34 One hundred microliters of 5 µg mL–1 protein A/G (Calbiochem) in 100 mM sodium carbonate, pH 8.2, were added to the wells of a Nunc Immobilizer Amino Strip (VWR, Lutterworth, United Kingdom) and incubated at room temperature for 4 hours. The strips were then washed with 0.1% Tween 20 in TBS (20 mM Tris-HCl, pH 7.8, 137 mM NaCl). One microgram of hamster anti–Bcl-2 antibody (Clone 3F11; BD Biosciences, Oxford, United Kingdom) was added per well and allowed to bind to the immobilized protein A/G overnight at 4°C. The antibody-protein A/G complexes were crosslinked by addition of 50 µL of 20 mM dimethylpimelidate (Perbio, Cramlington, United Kingdom) in triethanolamine followed by incubation for 30 minutes at room temperature. The crosslinker was quenched by addition of 0.2 M ethanolamine (pH 8.2). The wells were then washed 3 times with 0.1 M sodium citrate (pH 3) and 3 times with 0.2% Tween 20 in TBS. Cells were lysed in a buffer35 containing 1% [(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS; Perbio, United Kingdom) and the lysates incubated in the prepared wells for 4 hour at 4°C. The supernatants were removed and stored for future analysis. Wells were washed 4 times with 0.2% Tween 20 in TBS. Forty microliters of gel loading buffer were added to each well and the plate heated at 70°C for 10 minutes prior to gel electrophoresis and western transfer. In control experiments we established that an isotype-matched control antibody failed to immunoprecipitate either Bcl-2 or p53. Quantitation of Bax conformation change Acquisition of the proapoptotic conformation of Bax was quantified by immunoprecipitation.35 Cells were lysed in 1% CHAPS buffer and immunoprecipitated essentially as described above ("Coimmunoprecipitation of Bcl-2 and p53") using the conformation-specific Bax 6A7 antibody (Calbiochem). Precipitates were analyzed by Western blotting using a pan-Bax antibody (Cell Signaling Technologies). Assays for apoptosis Apoptosis was quantified by Western blot assessment of the cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP).36,37 Late stage apoptosis was quantified morphologically, by counting of cells with or without evidence of nuclear condensation and fragmentation in Giemsa-stained cytospin preparations as described.38 A total of at least 800 cells were counted in 3 randomly selected fields. The percentage of apoptotic cells in each field was computed, and the data are presented as means plus or minus SEM. Gel electrophoresis and Western blotting Procedures for gel electrophoresis and western transfer have been described elsewhere.30 Antibodies against the following proteins were used: p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA); Poly (ADP ribose) polymerase, p21CIP1 (BD Biosciences); Puma and Mdm2 (Calbiochem) actin (Sigma-Aldrich); lactate dehydrogenase V (Abcam, Cambridge, United Kingdom); Hsp60 (Cambridge Biosciences, Cambridge, United Kingdom); cytochrome c, cytochrome c oxidase IV, histone 2A and lamin (Cell Signaling Technologies); Bcl-2, and horseradish peroxidase–conjugated secondary antibodies (DAKO, Ely, United Kingdom). When several blots were analyzed in a single experiment, they were simultaneously exposed to the same solution in a single glass cylinder using a rotisserie apparatus (Thermo Fisher Scientific, Loughborough, United Kingdom) in order to ensure uniformity of exposure to both primary and secondary antibodies. Immunoreactive bands were visualized by Enhanced Chemiluminescence (GE Healthcare, Little Chalfont, United Kingdom). As required by journal policy, backgrounds were not subtracted from the autoradiograms shown in the figures. However, backgrounds were subtracted prior to digital quantification of band intensities using Quantity 1 software (Bio Rad, Hemel Hempstead, United Kingdom).
Subcellular distribution of p53 Untreated CLL cells or cells treated with p53-inducing agents were partitioned by DDF ("Methods") into cytosolic, mitochondria plus organelle and nuclear fractions, designated as fractions 1, 2, and 3, respectively (Figure 1). Although nuclear p53 was clearly detectable, the major fraction of the p53 induced by chlorambucil or by fludarabine treatment (60% and 74%, respectively) was associated with fraction 2. The stability of this association is emphasized by the resistance to washing of the p53 localized to fraction 2 (Figure 1). The proapoptotic BH3-only Puma protein was constitutively expressed and was associated exclusively with fraction 2 in untreated or drug-treated cells. Western blot analysis established that the distribution of the nuclear markers lamin and histone 2A, the mitochondrial markers cytochrome oxidase subunit IV, heat-shock protein 60 (Hsp60) and Bcl-2 and the cytosolic marker lactate dehydrogenase V essentially validated the performance of the DDF procedure (Figure 1). Although we observed a minor fraction of Bcl-2 and Hsp60 in fraction 3, histone 2A and lamin were exclusively recovered in fraction 3, ruling out the possibility that the p53 detected in fractions 1 and 2 were the result of contamination by the nuclear fraction. In addition, the considerably higher ratio of p53 to lactate dehydrogenase band intensity in fraction 2 compared with fraction 1 rules out the possibility that p53 in fraction 2 resulted from contamination by the cytoplasmic fraction.
More than 85% of the p53 induced by nutlin 3a was also associated with fraction 2. Bcl-2 and Puma were again recovered exclusively in this fraction (Figure S1Figure S1; available on the Blood website; see the Supplemental Materials link at the top of the online article). Using confocal microscopy, Kojima et al13 have also shown the association of p53 with mitochondria in nutlin-treated CLL cells. Our data confirm these observations by an independent method and extend them to include cells treated with the clinically relevant cytotoxic agents chlorambucil and fludarabine. Preferential association of p53 with fraction 2 of cells treated with p53-elevating agents was demonstrated using cells from 14 additional patients (patients 1, 4-11, 13, 16, 17, 20 and 21; Table 1). The proportion of fraction 2-associated p53 in CLL cells treated with chlorambucil, fludarabine, or nutlin varied between 60% and 90% in different experiments. As a further control, untreated CLL cells were permeabilized with DDF buffer 1 and then incubated with recombinant p53 before proceeding with the DDF protocol, which included a washing step. Western blot analysis revealed that only a minor fraction of the p53 (< 10% of total) associated with fraction 2, the majority being recovered in fraction 1 (data not shown), the inverse of the distribution seen when intact CLL cells were treated with p53-elevating agents (Figure 1 and Figure S1Figure S1A). This experiment suggests that the association of p53 with fraction 2 is unlikely to be the result of adventitious association of soluble p53 with this fraction during the DDF procedure. Coimmunoprecipitation of p53 and Bcl-2 The Bcl-2 protein is largely localized to mitochondria.6 A physical association between induced p53 and Bcl-2 was shown by coimmunoprecipitation experiments, emphasizing the potential mechanistic significance of the predominantly mitochondrial localization of p53. Treatment of CLL cells with chlorambucil or nutlin 3a resulted in the induction of p53, whereas the inactive enantiomer nutlin 3b failed to do so (Figure 2A). Immunoprecipitation with an anti–Bcl-2 antibody resulted in co-precipitation of Bcl-2 and p53 from lysates of cells treated with chlorambucil or nutlin 3a, but not from untreated or nutlin 3b–treated lysates (Figure 2B). Omission of the anti–Bcl-2 antibody failed to precipitate either protein (Figure 2B). The selectivity of the Bcl-2-p53 coimmunoprecipitation is emphasized by the observation that the abundant mitochondrial protein Hsp60 was present in the supernatants remaining after immunoprecipitation (Figure 2C), but was entirely undetectable in the immunoprecipitates (Figure 2B). Similar results were obtained using cells from patients 10, 13, 15, 21, 22, 25, 28, and 29.
Some models of apoptosis regulation postulate that Bcl-2 can sequester Bax and that the release of the latter protein initiates pore formation and cytochrome c release.39 However, we were unable to detect Bax in immunoprecipitates of CLL cells (data not shown), thereby effectively ruling out the possibility that displacement of Bax consequent to binding of p53 to Bcl-2 may contribute to the induction of apoptosis.
PFT
We determined whether PFT
Puma was expressed in untreated cells and this expression was essentially unaffected by PFT (Figure 3A), suggesting that constitutive Puma expression was independent of p53.
Nutlin treatment resulted in an approximately 3-fold up-regulation of Puma. PFT
Dramatic up-regulation of p21CIP1 accompanied by a modest induction of Puma in CLL cells treated with fludarabine or chlorambucil and the blockade of these induction events by PFT
To confirm that constitutive expression of Puma by CLL cells was indeed p53 independent, we studied cells isolated from patient 23, a poor prognosis patient with unmutated IgVH and a deletion of the long arm of chromosome 17 (the site of the p53 gene), in more than 99% of the malignant cells. The patient's cells had completely lost inducibility of p53, p21CIP1 and Mdm2 in response to nutlin 3a in vitro (Figure S3Figure S3), but expressed Puma at a level similar to that seen in a cells from a patient (patient 25) with a functional p53 system (Figure 3B). Puma levels in patient 23's cells were unaffected by nutlin, PFT or a combination of these agents, whereas the cells from patient 25 showed the modest nutlin-induced increase in expression characteristic of CLL cells with functional p53. Identical results were observed using cells from 2 additional patients with 17p deletion and an undetectable p53 response (patients 24 and 28, not shown), confirming that substantial levels of p53-independent constitutive Puma expression could be maintained by CLL cells.
PFT
Western blot quantitation of cleavage of the caspase 3 substrate PARP provides a sensitive and accurate measure of an early event in apoptosis induction.36,37 Incubation of cells from patient 19 with PFT
Photomicrographs of cells treated with fludarabine in the presence or absence of PFT are shown in Figure S4Figure S4. Quantitative data, derived by evaluation of the percentage of apoptotic cells, are shown in Figure 5B, confirming the conclusion that PFT augmented apoptotic killing induced by fludarabine. Apoptosis induced by chlorambucil or nutlin were similarly enhanced by PFT (Figure 5B).
Clinically achievable concentrations of chlorambucil and of fludarabine are approximately 5 and 2 µM respectively.41,42 Additional experiments were therefore carried out to establish that PFT
PFT
A conformational change which results in exposure of the N-terminus of the proapoptotic Bax protein is a critical rate-determining event which results in cytochrome c release, caspase activation and apoptosis.6 We therefore carried out immunoprecipitation experiments using an antibody specific for the proapoptotic Bax conformation to determine whether the observed augmentation of apoptosis by PFT
PFT does not augment killing of CLL isolates lacking functional p53
The complete inability of the malignant cells from patient 23 to activate a p53 response is documented in Figure S3Figure S3. Nutlin 3a or fludarabine alone or in combination with PFT
PFT does not augment killing of normal T lymphocytes by nutlin 3a
Incubation of purified normal T lymphocytes with nutlin 3a did not result in the induction of apoptosis as quantified by PARP cleavage analysis (Figure 7C), in agreement with earlier observations.12 PFT
The data here show that the major fraction of p53 induced by treatment of CLL cells with genotoxic (chlorambucil, fluarabine) or nongenotoxic (nutlin 3a) agents associated stably with a cellular fraction enriched in mitochondria and other organelles. Immunoprecipitation studies showed that induced p53 bound to Bcl-2, which is itself largely associated with mitochondria.6 The proapoptotic Puma protein was constitutively expressed in a p53-independent manner and was modestly up-regulated following p53 induction. The up-regulation of Puma and also of p21Cip1 was substantially inhibited by PFT . Having confirmed the ability of PFT to block p53 target gene expression in CLL cells, we used this agent to assess the relative contributions of transcriptional and non-transcriptional mechanisms to p53-induced apoptosis of CLL cells. The observation that PFT markedly augmented apoptosis induction by all 3 p53-elevating agents was unexpected. Although PFT has been shown to block p53-dependent apoptosis in the majority of studies reported to date,15–28 the killing of acute myeloid leukemia cells by cytosine arabinoside43 and of mouse embryo fibroblasts and human glioblastoma cells by topotecan44 is augmented by this agent. It is therefore likely that the impact of PFT on apoptosis induction in different systems is highly dependent on the relative importance of pro- and antiapoptotic signaling pathways induced by p53 in a particular cell type.
Although the data here do not rule out a role for Puma in apoptosis induction, suppression of Puma elevation by PFT
The apparently paradoxical observation that PFT While p53 has conventionally been considered to regulate apoptosis in a positive manner,5,6 recent studies have emphasized the complex and dualistic nature of p53's transcriptional targets, which can either induce5,6 or block45 apoptosis, depending on the cellular context. The precise impact of p53-mediated transcription therefore depends on the selection of transcriptional targets, which is itself the result of the complex interactions between several variables which are highly dependent on the cellular context. These variables include the nature and extent of genotoxic damage, sites of post-translational modification of p53 (including phosphorylation and acetylation) and the spacing of p53 binding sites in individual promoters.46
Microarray studies have established that hundreds of genes are induced or repressed in a p53-dependent manner following irradiation of mice47 or of CLL cells.48 Furthermore, p53-dependent changes in gene expression were highly cell type-specific.47 Therefore, identification of the key p53 gene targets whose blockade by PFT
While the precise mechanisms involved in mediating p53's antiapoptotic actions require further clarification, the data here show clearly that its proapoptotic actions are augmented by pharmacological agents which block p53-dependent transactivation. The ability of PFT
Contribution: A.J.S., A.G.P., and R.G.W. designed studies, analyzed data, and wrote the paper; A.J.S., B.C.Y., S.M.H., E.P.N., J.D.H.-R., V.M.D., and R.G.W. carried out research; A.G.P., A.V.H., P.K., and K.C. recruited and obtained written consent from patients and collated clinical data; and L.T.V. analyzed data and synthesized the nutlins used in this study. Conflict-of-interest disclosure: L.T.V. is an employee of Hoffman-La Roche Inc. The other authors declare no competing financial interests. Correspondence: Dr R. Gitendra Wickremasinghe, Department of Hematology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, United Kingdom; e-mail: r.wickremasinghe{at}medsch.ucl.ac.uk.
This work was supported by the Leukaemia Research Fund, United Kingdom.
Submitted May 16, 2008; accepted June 24, 2008.
Prepublished online as Blood First Edition Paper, August 5, 2008
DOI: 10.1182/blood-2008-05-156380
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
B. Cell Death Differ. 2007;14:1529–1541.
