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membrane potential ( m) dissipation are early events in staurosporine-induced apoptosis of wild type and mutated p53 epithelial cells. J. F. Charlot, J. L. Prétet, ...
Apoptosis 2004; 9: 333–343  C 2004 Kluwer Academic Publishers

Mitochondrial translocation of p53 and mitochondrial membrane potential (m) dissipation are early events in staurosporine-induced apoptosis of wild type and mutated p53 epithelial cells J. F. Charlot, J. L. Pretet, ´ C. Haughey and C. Mougin EA 3181, Laboratoire de Biologie Cellulaire et Moleculaire ´ (IBCT, IFR 133), CHU Jean Minjoz, 2 Boulevard Fleming, 25030 Besan¸con Cedex, France

The mitochondrial localization of p53 is an important event in p53-dependent apoptosis. Some p53 mutants defective for transcription also facilitate apoptosis through changes of the mitochondria. Here, apoptosis of HeLa and CaSki cells (p53wt ), C33A and HaCat cells (p53mt ) and SaOs-2 cells (p53 deficient) was induced by 300 nM staurosporine. We showed that wild-type p53, as well as p53 mutants, were transiently located to the mitochondria with changes in the mitochondrial membrane potential (m). However, in C33A cells harboring a p53 mutated on its DNA binding domain, m collapse and SubG1 DNA content were reduced compared to p53wt cells, whereas no significant difference was observed in HaCat cells with a p53 mutated on UV hot spots. In addition, inhibition of the mitochondrial permeability transition pores by cyclosporine A significantly reduced the m loss and the sub-G1 DNA content in p53 positive cells. These results indicate that m collapse is an early and necessary event, which plays an important role in apoptosis of immortal mammalian cells. Keywords: apoptosis; depolarization; mitochondria; p53. Abbreviations: ST, staurosporine; CsA, cyclosporine A; MPT, mitochondrial permeability transition; MPTP, mitochondrial permeability transition pore; m, mitochondrial membrane potential; VDAC, voltage-dependent anion channel; ANT, adenine nucleotide translocase; AIF, apoptosis inducing factor; APAF-1, apoptosis protease activating factor; NAO, nonyl acridine orange; BSA, bovine serum albumin; PBS, phosphatebuffered saline; wt, wild-type; mt, mutated; HPV, human papillomavirus.

Introduction Mitochondria play a pivotal role in respiratory and oxidative functions of eukaryotic cells, as well as in apoptotic

Correspondence to: C. Mougin, Laboratoire de Biologie Cellulaire et Moleculaire, ´ CHU Jean Minjoz, 2 Boulevard Fleming, 25030 Besan¸con Cedex, France. Tel.: +333.81.66.91.11; Fax: +333 81 66 83 42; e-mail: [email protected]

death. To date, two main apoptotic cascades have been described. Firstly, extrinsic stimuli can induce trimerization of cell membrane-bound death receptors (such as Fas, TRAIL). Then, the death domain can attach adapter molecules, which in turn recruit initiator procaspase 8 inducing its activation. Effector caspases (like caspase 3) are subsequently cleaved which results in DNA fragmentation and death. Caspase 8 can also truncate Bid into tBid1 which mediates apoptotic mitochondrial dysfunctions after binding to cardiolipin.2 A second pathway also leads to mitochondrial dysfunction in response to intrinsic stimuli (such as DNA damage, reactive oxygen species). Indeed, mitochondria undergo permeabilization by opening of different mitochondrial membrane pores according to the death stimuli (intrinsic or extrinsic).3 This results in loss of mitochondrial membrane potential (m ) and release of apoptogenic factors, like cytochrome c , Smac/Diablo, AIF.4,5 Once in the cytosol, cytochrome c associates with procaspases, APAF-1 and ATP to form apoptosomes, which then cleave downstream caspases. However, it has been demonstrated that release of cytochrome c and subsequently caspase activation may occur before any detectable loss of m ,6–9 while release of AIF is strictly dependent on m (for review see10 ).11–14 The nature of pores implicated in the mitochondrial permeability transition (MPT) is not yet fully defined, and several models of pore are involved in regulated MPT. The mitochondrial permeability transition pores (MPTP) are constituted by an outer membrane voltagedependent anion channel (VDAC) and an inner membrane adenine nucleotide translocase (ANT). Mitochondrial benzodiazepine receptor in the outer membrane, cyclophilin D and cardiolipin in the mitochondrial matrix complete the MPTP. These channels can be inhibited with numerous compounds, like cyclosporine A (CsA) that targets cyclophilin D15,16 and rescues cells from apoptosis in some systems.5,17 On the other hand, members of the Apoptosis · Vol 9 · No 3 · 2004

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Bcl-2 superfamily control the mitochondrial membrane permeability by at least two mechanisms. First, proapoptotic proteins, like Bax, cause an increase in permeability by formation of conducting channels (for review see18 ).19 Thus, Bax channels, Bax/tBid pores and Bax/VDAC channels have been distinguished so far and cytochrome c can be shuttled through these channels.1,20–22 Second, anti-apoptotic proteins like Bcl2, Bcl-xL, which insert into the OMM, prevent an increase of permeability by directly closing the VDAC or by forming heterodimers with pro-apoptotic Bcl-2 family proteins.19,23 Other proteins such as p53 can also reside, at least transiently, in the mitochondria (for review see 24 ). Thus, Marchenko et al.25 have shown that a fraction of p53 localizes rapidly to mitochondria in response to death signals which precedes loss of m and cytochrome c release. More recently, Mihara et al.4 have shown that p53 can interact, at the mitochondrial level, with Bcl-2 and BclxL and p53 binds to Bcl-xL by its DNA binding domain. Tumor-derived p53 mutants severely impair in forming Bcl-xL complexes and reduce cell sensitivity to apoptotic stimulus.4 We have previously reported that staurosporine (ST) can induce apoptosis of human cervical carcinoma-derived cells, whether they are human papillomavirus (HPV) positive and p53 wild type (HeLa and CaSki cells) or whether they are HPV negative and p53 mutated (C33A).26 We next demonstrated in HPV positive cells that ST inhibited expression of MDM2 and E6 viral gene (that do not anymore favour p53 degradation), leading to increased levels of p53. Additionally, the expression of p53targeted genes, p21waf-1 and Bax, was increased while Bcl2 and Bcl-xL expression was lowered. Moreover, westernblotting performed on sub-cellular fractions showed a transient mitochondrial localization of p53, preceding cytochrome c release, caspase activation and DNA fragmentation. Finally, characteristic morphological signs confirmed the apoptosis execution.27 Given the evidence for a role of p53 in programmed cell death with sometimes dissipation of mitochondrial m , we explored by immunocytochemistry the temporal and spatial distribution of p53 in p53wt HeLa and CaSki cells and in p53mt C33A and HaCat cells upon ST exposure. Thereafter, we investigated the possible connection between p53 and changes in MPT.

Materials and methods Cell lines Five human cell lines, collected from ATCC (Rockville, MD), were used in this study. HeLa and CaSki cells are HPV positive cells derived from human cervical carci334 Apoptosis · Vol 9 · No 3 · 2004

nomas. Their p53 status is of wild type (p53wt ). C33A cells, also derived from human cervical carcinoma, are HPV negative and their p53 is not competent for transactivation because of mutation on DNA binding domain (amino acid 273). The epidermal immortalized HaCat cells present a mutated p53 on UV hot spots but not on the DNA binding domain. SaOs-2 cells, derived from osteosarcoma, are deficient in p53 and used as control. HeLa and CaSki cells were respectively cultured in RPMI (Bio Whittaker Europe, Verviers, France) and EMEM (Bio Whittaker) supplemented with 5% (v/v) fetal bovine serum (Bio Whittaker) and 2mM L-glutamine (Bio Whittaker). C33A cells were cultured in the same medium as Hela cells supplemented with 5% (v/v) sodium pyruvate (Sigma, St Louis, MO). HaCat and SaOs-2 cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum (Bio Whittaker) and 4mM L-glutamine (Bio Whittaker). All cells were incubated at 37◦ C under a humidified atmosphere of 95% air and 5% CO2 (v/v). They were routinely monitored and found to be free of mycoplasm infection.

Cell treatments Apoptosis induction. Staurosporine (ST) (Sigma) was dissolved in dimethyl sulfoxide. To induce apoptosis, cells were exposed to 300 nM ST for 15, 30 min, 1, 2, 3, 6 and 12 h to measure ψm variations and for 3, 6, 12 and 24 h to quantify the percentage of cells that present depolarized mitochondria and/or fragmented DNA. For immunocytochemistry studies, cells were treated during 15, 30 min, 1, 2, 4, 6, 8, 10 and 12 h. All experiments were performed at least three times. Inhibition of mitochondrial membrane depolarization. CsA (Novartis International AG, Basel, Switzerland) was diluted in DMSO. The five cell lines were cultured with ST (300 nM) plus CsA (10 µM) for 24 h and then analyzed for mitochondrial depolarization and sub G1 DNA content. The different cell lines treated for 24 h with only CsA were used as controls.

Immunocytochemistry studies Cells were plated onto glass coverslips and grown until 60% of confluence. As indicated in experiments, cells were rinsed twice with PBS before fixation and permeabilization with cold acetone/methanol (1:1) for 3 min. Cells were then washed 3 times with cold PBS and incubated for 20 min at room temperature (RT) in PBS-BSA 1%. Fixed cells were incubated for 1 h at RT with mouse primary anti-human p53 antibody (1:200; p53 DO-7, PharMingen, San Diego, CA) diluted in PBS-BSA 0.1%-Triton

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0.1% (v/v) referred to as PBT. Then, cells were washed 3 times with PBT before the addition of a secondary TRITC-conjugated goat anti-mouse antibody (1:100; DakoCytomation, Glostrup, Denmark) diluted in PBT. After 3 washes with PBT, cells were incubated for 30 min at RT with 200 nM Nonyl Acridine Orange (NAO) (Molecular Probes Europe, Leiden, The Netherlands) and 1 µg/ml Hoechst’s staining (Hoechst 33342; Molecular Probes Europe), both diluted in PBS, for mitochondria and DNA labelling respectively. Coverslips were then mounted using fluorescent mounting medium (DakoCytomation) for observation under a fluorescence microscope (Olympus BX51). Pictures were captured using the Olympus DP50 numeric camera and processed with the Analysis software (Media Cybernetics, Silver Spring, MD).

Flow cytometry At the indicated periods cells were washed twice with PBS, harvested by trypsinization (Bio Whittaker), washed again twice with PBS and centrifuged at 700g for 10 min. For measurement of mitochondrial ψm , a batch of cells was resuspended in 200 nM MitoTrackerRed CMXRos (Molecular Probes Europe) diluted in the appropriate culture medium according to the cell types and incubated at 37◦ C for 30 min. Then, cells were washed with PBS, centrifuged and resuspended in 400 µl PBS for flow cytometry analysis. Measurement of mean fluorescence intensity was used to appreciate the variations in ψm . The percentage of cells with depolarized mitochondria was furthermore calculated. For Sub-G1 DNA content analysis, another batch of cells was resuspended and fixed overnight in 70% (v/v) cold ethanol. Fixed cells were washed 3 times with cold PBS before the addition of 1 mg/ml RNaseA Dnase-free for 10 min and 10 µg/ml propidium iodide for 30 min. The percentage of cells with Sub-G1 DNA was calculated. Cells were analysed on a FACScan Epics Altra flow cytometer (Beckman Coulter). A minimum of 10 000 events was collected for each sample. The analyses performed on a gated cell population in order to discard cellular debris, were conducted using the expo-32 software.

Results Staurosporine-induced death of immortalized epithelial cells is associated with a transient mitochondrial accumulation of p53 followed by chromatin fragmentation To confirm the mitochondrial localization of p53 observed by western blot analysis in apoptosing p53 wild type HeLa and CaSki cells,27 we monitored the cellular

distribution of p53 by fluorescence microscopy. We also extended our study to p53 mutated C33A and HaCaT cell lines, likewise to p53-defective SaOs-2 cells used as controls. In parallel, apoptosis was confirmed by Hoechst staining. As shown in Figure 1A, p53 staining revealed an exclusively nuclear localization in p53wt malignant untreated cells, like HeLa cells. Mitochondrial localization occurred rapidly, within 30 min upon ST exposure and persisted at the mitochondrial level for at least 10 h. Beyond 12 h of ST exposure, p53 was again distributed throughout the nuclei, which appeared with condensed chromatin and visible blebbing. Similar results were observed in Caski cells (data not shown). This observation demonstrates that p53wt was translocated to mitochondria, suggesting that p53 may play a role in the apoptotic mitochondrial pathway of HeLa and CaSki cells. Importantly, immunocytochemical staining for mutated p53 did not exhibit strong differences in immortalized HaCaT cells and malignant C33A cells (Figure 1B and C). In both cell lines, p53mt was recruited to the mitochondria, as shown by merge of p53 and mitochondria labelings. However a slight delay (60 min) and a shorter duration (8 h) was observed in C33A cells harboring a p53 mutated on its DNA binding domain compared with p53wt HeLa and CaSki cells or HaCat cells with a p53 mutated on UV hot spots. Nuclear Hoechst staining confirmed death of these two immortal cell lines 8 to 12 h after ST treatment. These data suggest that, as wild-type p53, some mutated p53 are also directed to the mitochondria under ST exposure and might play a role at the mitochondrial level in the apoptotic outcome.25 As expected, p53 null SaOs-2 cells lacked detectable p53 expression (Figure 1D). Neither nuclear nor mitochondrial staining was observed. Nevertheless, many cells exhibited a condensed and fragmented chromatin after 12 h of death stimulus, suggesting that these cells underwent death by a p53-independent pathway.

An early mitochondrial membrane potential (ψm) loss is observed in cells treated with staurosporine It has been recently suggested that a reduction in ψm may be an important event in the apoptotic pathway and p53 can mediate the ψm changes. To investigate the requirement of ψm dissipation in ST-induced apoptosis, we used the MitoTrackerRed dye as an indicator of ψm and the intensity of fluorescence was measured by flow cytometry. As shown in Figure 2, variations in the mean fluorescence intensity of the mitochondrial potential sensitive dye confirmed dysfunctional changes in ψm . In each Apoptosis · Vol 9 · No 3 · 2004

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J. F. Charlot et al. Figure 1. Immunofluorescence microscopy showing transient mitochondrial relocalization of p53 in immortal mammalian cells upon staurosporine exposure. Cells were incubated with 300 nM ST for up to 12 h at 37◦ C. They were stained at the indicated times with anti-p53 DO-7 antibody, nonyl acridine orange (NAO) which binds to mitochondria and Hoechst 33342. They were examined under fluorescence microscopy at X400 magnification. Merge images were generated by superimposing coloured images of p53 (red), mitochondria (green) and DNA (blue). HeLa cells (plate A) and HaCat cells (plate B) exibit an overlay of green and red fluorescences (superposed image as yellow, arrowheads) from 30 min to 10 h of ST exposure, suggesting a mitochondrial localization of p53. C33A cells (plate C) demonstrate a delayed (60 min) and shorter (8 h) overlay. SaOs-2 cells (plate D) do not exibit p53 staining, but their morphology indicates the nuclear chromatin condensation (10 h). In each plate, arrows point the DNA cleavage. (Continued on next page.)

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p53 and mitochondrial depolarization Figure 1. (Continued).

cell line incubated in presence of 300 nM ST, an early transient hyperpolarization (15–30 min) was observed. This phase preceded the loss of ψm , which was slightly different between cell lines. After 12 h of ST-exposure, immortal HaCaT cells with p53 mutated on UV hot spots presented the highest (76%) ψm drop, whilst p53 defective SaOs-2 cells presented the lowest one (42%). The

reduction in ψm was also more important in malignant cells expressing wild-type p53 (70% for CaSki, and 64% for HeLa cells) than in those expressing p53 mutated on the DNA binding domain (50% for C33A cells). Interestingly, the early ψm dissipition in p53wt and p53mt cells treated with ST took place with the mitochondrial translocation of p53 (30–60 min).

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J. F. Charlot et al. Figure 2. Flow cytometry analysis of the mitochondrial membrane potential (ψm) variations, determined with the MitoTrackerRed dye, in HeLa, CaSki, C33A, HaCat and SaOs-2 cells exposed to 300 nM ST for 12 h. ST-induced apoptosis results in important changes corresponding to a loss of ψm, following a hyperpolarization phase, with differences according to the cell type. Results are representative of 3 independent experiments with error bars representing s.d. The arrow indicates the translocation of a p53 fraction to mitochondria within 30 to 60 min after ST exposure.

The loss of ψm precedes DNA fragmentation in epithelial cells We next monitored by kinetic studies the temporal relationship between mitochondrial depolarization and DNA fragmentation upon ST exposure. Cells undergoing mitochondrial depolarization and/or fragmented DNA revealed by the Sub-G1 DNA content were quantified by flow cytometry and results were expressed as percentage of the global cell population. As shown in Figure 3A, at any point of the time course study, the percentage of cells with depolarized mitochondria was always higher than the percentage of cells with fragmented DNA with the exception of SaOs-2 cells. There were however some differences among the cell lines (Figure 3B). At 24 h ST post-exposure, malignant C33A cells (of which p53 is mutated on its DNA binding site) with depolarized mitochondria were less numerous (60%) than malignant HeLa and CaSki cells (of which p53 is wild type) (80%). As for HaCaT cells harboring a p53 mutated on UV hot spots but not on its DNA binding site, the percentage of cells with mitochondrial ψm dissipation was of the same extent (80%) than HeLa and CaSki cells (Figure 3B left panel). This experiment also enabled us to demonstrate a delay in apoptosis progression. Again, the results differed among the cell lines: 82% of HaCaT cells, 70% of HeLa and CaSki cells and 40% of C33A cells presented a sub-G1 peak at 24 h of ST-treatment (Figure 3B right panel). When we focused especially on p53 deficient SaOs-2 cells, the effect of ST on permeability transition was reduced with only 34% of cells with depolarized mitochon338 Apoptosis · Vol 9 · No 3 · 2004

dria. Surprisingly, the percentage of SaOs-2 cells with DNA fragmentation was closed (60%) to that observed for CaSki and HeLa cells (Figure 3). In SaOs-2 cell line, the mitochondrial depolarization is likely a consequence rather than a cause of the apoptotic process which seems to be p53 independent. Cyclosporine A inhibits ψm dissipation and rescues cells from ST-induced apoptosis We then attempted to confirm that ψm dissipation had an essential role in ST-induced apoptosis of cells by incubating them with cyclosporine A (CsA) known to prevent permeability transition by targeting the cyclophilin D, a component of the MPTP. As shown in Figure 4 the addition of 10 µM CsA to 300 nM ST did reduce the ψm dissipation confirmed by the decreased percentage of cells with depolarized mitochondria, as compared with cells treated by ST alone (Figure 4A left panel). However this inhibition was not complete, indicating that the MPTP has a partial role in ST-induced ψm loss.5 Interestingly, in p53 positive cell lines, inhibition of mitochondrial depolarization was accompanied by a decrease of DNA fragmentation more or less important according to the cell type (Figure 4A right panel). To compare the behavior of the different cell lines, we expressed the above data as percentage of inhibition of depolarization and of DNA fragmentation (Figure 4B). Thus, the addition of CsA induced a similar decrease in the percentage of HeLa and CaSki cells with depolarized

p53 and mitochondrial depolarization Figure 3. Time course studies of mitochondrial membrane depolarization and DNA fragmentation during ST-induced apoptosis of CaSki, HeLa, C33A, HaCat and SaOs-2 cell lines. (A) The five cell lines were incubated with 300 nM ST for the indicated periods. A batch of cells was resuspended in MitoTrackerRed and another one in propidium iodide for analysis by flow cytometry (as described in experimental procedures). The percentage of cells with depolarized mitochondria and/or fragmented DNA increase upon ST exposure. The mitochondrial membrane depolarization (square) occurs before DNA fragmentation (circle) in p53 positive cell lines, but not in p53 deficient SaOs-2 cells. Graphs are representative of 3 independent experiments with error bars representing s.d. (B) Comparison of the behaviour of the different cell lines. Note that the DNA fragmentation in SaOs-2 cells is similar to that observed in HeLa or CaSki cells, while the mitochondrial membrane depolarization is much lower.

mitochondria (45–50%) and with a sub-G1 DNA content (40-45%). In C33A cells, CsA had the same effect on depolarization inhibition than that observed in malignant p53wt cells, but it led to a more intense cell death inhibition (65%) which might be partly explained by the lack of transcriptional activity of the mutated p53, unable to transactivate pro-apoptotic genes such as Bax (data not shown) and so, to counterbalance the CsA effect. HaCaT cells were less sensitive to CsA, since this compound did reduce by only 25% the number of cells with mitochondrial depolarization and by 20% those with DNA fragmentation. In contrast, the CsA did not affect the STinduced apoptosis of SaOs-2 cells (5% of inhibition on DNA fragmentation), whereas it caused a significant reduction of mitochondrial depolarization (45%). Thus, it

appeared again that SaOs-2 cells in presence of ST underwent nuclear fragmentation independently of mitochondrial changes.

Discussion In a previous study, we have shown, by western blotting on sub-cellular fractions, in dying HeLa and CaSki cells that p53wt was transiently localized to mitochondria and preceded cytochrome c release and caspase activation,27 which led us to investigate dysfunctional changes in ψm . The results presented here confirm that p53, either wild type or mutated, is rapidly translocated to mitochondria, Apoptosis · Vol 9 · No 3 · 2004

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J. F. Charlot et al. Figure 4. The MPTP inhibitor Cyclosporine A (CsA) reduces ST-induced mitochondrial depolarization and DNA fragmentation. (A) The five cell lines were incubated for 24 h in presence of 10 µM CsA (grey columns), 300 nM ST (black columns) or 300 nM ST plus 10 µM CsA (dashed columns). A batch of cells was resuspended in MitoTrackerRed and another one in propidium iodide for analysis by flow cytometry (as described in experimental procedures) to appreciate the percentage of cells with depolarized mitochondria (left panel) and with a sub-G1 DNA content (right panel). Cells treated with both ST and CsA exhibit an inhibition of the mitochondrial depolarization, compared to cells treated with ST alone. This process is accompanied by a DNA fragmentation inhibition, except for SaOs-2 cells. CsA by itself has no effect. Results are representative of 3 independent experiments with error bars representing s.d. (B) Results are expressed as the percentage of inhibition in cells treated with ST plus CsA compared to cells treated with ST alone in order to compare the behaviour of the different cell lines.

during staurosporine-induced death of immortalized epithelial cells. The nature of death investigated by Hoechst staining gives proof of apoptosis, since many nuclei appeared to contain condensed chromatin and visible blebbing under experimental conditions. Our study also provides evidence that ST-induced apoptosis involves changes in mitochondrial permeability, as shown by ψm dissipation following an early transient hyperpolarization phase, as already reported.23,28,29 Ly et al.10 suggested that ST might act on the MPTP channels and induce their transient closure resulting in failure of the mitochondria to 340 Apoptosis · Vol 9 · No 3 · 2004

maintain ATP/ADP exchange with the cytosol. Following their closure, pore opening is required for the release of apoptogenic factors and apoptosis progression.30–33 The opening of MPTP, as well as Bax/Bak oligomers and Bax-VDAC channels, is most of the time associated with ψm collapse, through a proton dissipation pathway.34,35 ψm drop was originally reported in many studies to be a crucial early event in the apoptotic cascade.36–38 Indeed, in PC6 cells treated with ST, ψm drop was coinciding with cytochrome c release.39 In addition translocation and oligomerization of Bax at the

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mitochondrial level, which can be induced by p53, were shown concomitantly with cytochrome c release in STtreated osteosarcoma cells.4 Moreover p53wt , that downregulates Bcl-2 and transactivates Bax,40 is able to form mitochondrial inhibitory complexes with Bcl-xL and Bcl-2. These two antiapoptotic proteins which act on VDAC and bind Bax or Bak cannot anymore inhibit the pore opening.5,41,42 Thus, Bcl-2 balance favors death. The results presented in this study suggest that mitochondrial p53 can induce mitochondrial transition permeability by forming an inhibitory complex with protective proteins of the mitochondrial membrane permeability, a conclusion based upon data derived from a variety of studies.4 Furthermore Marchenko et al.25 reported that the p53 mitochondrial localization is a marker of the p53-dependent apoptosis in many systems and does not occur during p53-independent apoptosis. The authors also demonstrated that targeting p53 to mitochondria is sufficient to induce death in p53 null cells. The present results highlight a significant difference between cells depending on their p53 status. Indeed, apoptosis was of greater extent in cells harboring a p53 with no mutation on its DNA-binding site (HeLa, CaSki and HaCat) than in C33A cells harboring a p53 mutated on its DNA-binding site. These differences might be first associated with the ability of p53 to bind or not Bcl-xL. The p53 domain implicated in such a binding belongs to the DNA-binding site and is especially mapped on the amino acid (aa) region 239–248. Deleting aa 239–248 from mitochondrial targeting p53 completely abrogated its Bcl-2/xL interaction and apoptotic activity in SaOs2 and HeLa cells.4 In C33A cells, the mutation in the DNA-binding site is mapped on aa 273.43 In these cells, p53 might be able to interact with Bcl2/xL. However, breast cancer-derived cell line, MDAMB 468, harboring also a mutant p53 R273H, showed no detectable endogenous p53-Bcl-2/xL complexes.4 Nevertheless, future experiments are required to determine if a p53-Bcl-2/xL interaction does exist in order to fully understand ST-induced death of p53mt cervical carcinoma derived cell line. The lower percentages of C33A cells with depolarized mitochondria and fragmented DNA upon ST exposure, compared with HeLa and CaSki cells, can also be due to the transcriptionally inactive mutant p53 as observed by the lack of Bax transactivation (data not shown). Another observation was DNA fragmentation of p53 null SaOs-2 cells of similar extent to that observed in CaSki and HeLa cells, which was not preceded by ψm disruption. The underlying mechanism of ST-induced death of SaOs-2 cells remains unknown. By inhibiting the VDAC-ANT channels with cyclosporine A,5,44,45 it appeared that ψm dissipation favours apoptotic cell death, with the exception of SaOs2 cells. However, the mitochondrial depolarization was

not completely abrogated. We hypothesize that Bax and Bak homo- and hetero-oligomers, as well as Bax-VDAC channels, which are CsA insensitive,46 can ensure the induction of mitochondrial depolarization. In HaCaT cells the involvement of these latter channels might predominate over MPTP channels whilst in cervical carcinomaderived cells, MPTP might have a predominant role. In SaOs-2 cells the DNA fragmentation was not significantly reduced in presence of CsA, leading us to conclude that mitochondrial pathway is not likely involved in the staurosporine effect. On the basis of our data (with the exception of SaOs2 cells), we suggest a model in which p53 exerts a role in the induction of mitochondrial membrane depolarization. The mutant p53 in C33A cells did not loose the possibility to localize to mitochondria, although its transcriptional activity was ineffective. This is in agreement with the data of Regula and Kirshenbaum who demonstrated on ventricular myocytes that p53 provoked a loss of ψm without de novo transcription and that m collapse was more important with a p53wt than with a p53mt defective for DNA binding.47 In the same way, Dumont et al. have showed that two polymorphic variants of p53 at the amino-acid 72 (Arg-Arg or Pro-Pro) were functionally distinct, the Arg 72 variant having a better ability to localize to mitochondria and to induce apoptosis than the Pro 72 variant.48 Very recently, Chipuk et al. revealed that the amino terminal proline-rich regulatory domain (amino acids 62 to 91) is required for the transcriptionindependent apoptotic activity of p53. Although, most p53 DNA binding domain mutants fail to induce apoptosis despite the presence of an intact proline-rich domain.49 Whether, the mitochondrial activity of p53 seems to be dependent on its status, mutations selected during tumor formation might abrogate both the transcriptional and the mitochondrial apoptotic activity of p53.4

Conclusion In conclusion, the results here strengthen the evidence for a mitochondrial relocalization of p53, whatever its status is, and for a dispensable p53 transcriptional activity in the apoptotic process. Nevertheless, the contribution of p53 in the mitochondrial apoptotic pathway remains to be clarified. From a therapeutic point of view, this knowledge would allow the use of ST in cancer therapies to modulate p53 transcription-independent apoptosis.

Acknowledgments

This work was supported by grants from Ligue Contre le Cancer (D´epartement du Doubs). J-F.C. received fellowships from Ligue Contre le Cancer (D´epartement du Apoptosis · Vol 9 · No 3 · 2004

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Doubs) and Association R´egionale pour l’Enseignement et la Recherche Scientifique (ARERS). We would like to thank Virginie Mougey for technical support. The authors thank students and all members of the laboratory for their help and encouragement during the course of this work. We are grateful to Dr. Sylvie Fauconnet and Dr. Isabelle Lascombe for helpful comments on this manuscript.

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