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Originally published In Press as doi:10.1074/jbc.M503305200 on June 27, 2005

J. Biol. Chem., Vol. 280, Issue 35, 31230-31239, September 2, 2005
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p53-dependent Caspase-2 Activation in Mitochondrial Release of Apoptosis-inducing Factor and Its Role in Renal Tubular Epithelial Cell Injury*

Rohit Seth{ddagger}§, Cheng Yang{ddagger}§, Varsha Kaushal{ddagger}§, Sudhir V. Shah{ddagger}§, and Gur P. Kaushal{ddagger}§||

From the Departments of {ddagger}Medicine and Biochemistry, University of Arkansas for Medical Sciences and §Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205

Received for publication, March 25, 2005 , and in revised form, June 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-{alpha} markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-{alpha} or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis-inducing factor (AIF)1 is a highly conserved mitochondrial inter-membrane flavoprotein encoded by nuclear DNA (1, 2). In response to a specific apoptotic stimulus, AIF is translocated to the cytosol and then to the nucleus. When in the nucleus, AIF associates with chromatin (3) and the nuclear protein cyclophilin A (4). This interaction enables AIF to induce nuclear chromatin condensation and large scale DNA fragmentation (2, 5). AIF without these interactions loses its ability to degrade DNA (4). In addition to its endonuclease function in apoptosis, AIF has NADH oxidase activity confined to the mitochondria in normal cells (1). Recently, AIF has also been shown to participate in the respiratory chain complex I activity (6).

Overexpression of recombinant AIF by transient transfection, microinjection, or adenoviral infection results in chromatinolysis and cell death without caspase activation (1, 7, 8). However, the question of whether the release of mitochondrial AIF into the cytosol and the nucleus is dependent on caspase activation is not fully resolved. The release of AIF has been shown to be caspase-independent in some cell types (9). These studies were supported by the evidence that caspase inhibitors (10, 11) and Apaf-1- or caspase-3-deficient neuronal and mouse embryonic fibroblasts were unable to prevent mitochondrial release of AIF in response to death signals (7, 12). These studies suggest that AIF translocation is independent of caspase-9 and caspase-3 activation in these cells. However, recent evidence has also suggested the role of caspases in the mitochondrial release of AIF. Mitochondrial release of AIF was blocked by pancaspase inhibitor in HeLa and Jurkat cells treated with staurosporine or actinomycin D (13, 14). In response to an apoptotic stimulus, many mitochondrial intermembrane proteins including cytochrome c, Smac/Diablo, Omi/HtrA2, endonuclease G, and AIF are released upon permeabilization of the mitochondrial outer membrane. Although proapoptotic molecules of the Bcl-2 family can regulate the mitochondrial permeabilization (15, 16), little is known on the mechanism and the temporal sequence of the release of the mitochondrial intermembrane proteins. Recent evidence showed that the mitochondrial membrane permeabilization induced by Bax/Bak or tBid resulted in selective release of cytochrome c, Smac/Diablo, and Omi/HtrA2 (14, 17) but not AIF, and subsequent caspase activation was required for the mitochondrial release of AIF (14). The identity of caspase involved in the release of AIF was not determined in these studies.

Nephrotoxicity is one of the major side effects when the chemotherapeutic drug cisplatin is used for the treatment of solid tumors of a wide range of tissues (18, 19). The cellular and molecular mechanisms responsible for drug-induced nephrotoxicity are not well understood. Mitochondrial dysfunction is an important component of cisplatin nephrotoxicity to proximal tubules in vivo (20-23) and in vitro (24-26). Cisplatin has been shown to induce the mitochondrial release of proapoptotic molecules, including cytochrome c (27-31), Omi/HtrA2 (32), Smac/Diablo (33), and AIF (11). The cisplatin-induced mitochondria-dependent intrinsic pathway of caspase activation and cell death was blocked significantly by caspase inhibitors in many cell types (34-38). However, the mechanism of cisplatin-induced mitochondrial release of AIF and the role of caspases in its release is not known. We have recently demonstrated a marked increase in cisplatin-induced caspase-3 and -2 activation in renal tubular epithelial cells (RTEC) (39). This study in RTEC provided a unique model to examine the role of caspase-2 and -3 in the mitochondrial release of AIF. Thus, in the present study, we have explored cisplatin-induced release of mitochondrial AIF to the cytosol and the nucleus and the role of caspases in this process. We provide evidence that cisplatin-induced caspase-2 activation but not caspase-3 activation is involved in the mitochondrial release of AIF. We have further explored the mechanism of caspase-2 activation and demonstrated the role of p53 and its responsive gene p53-induced death domain protein (PIDD) on caspase-2 activation that participates in the subsequent release of mitochondrial AIF in RTEC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents
LLC-PK1 cells obtained from ATCC were cultured as described in our previous studies (39). The cells were grown in Gibco medium 199 supplemented with 10% heat-inactivated fetal calf serum. Cultures were maintained in a humidified incubator gassed with 5% CO2 and 95% air at 37 °C and fed with fresh medium at intervals of 48-72 h. Experiments were performed with cells grown to 80% confluence. Caspase substrates were purchased from Peptide International (Louisville, KY), and antibodies to caspase-3, AIF, histone H1A, {alpha}-tubulin, {beta}-actin, and proteins of the Bcl-2 family were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) Antibodies to p53, Ser-15-phosphorylated p53, and active caspase-3 were obtained from Cell Signaling Technology (Beverly, MA). Antibody to cytochrome c oxidase subunit IV was from Molecular Probes, Inc. (Eugene, OR). Caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone (Z-VDVAD-fmk), and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone were obtained from Enzyme System Products (Livermore, CA).

Induction of Cisplatin-induced Injury
The cell culture medium was replaced with fresh medium containing serum, and cells were incubated either without or with cisplatin at various concentrations (25-200 µM) for the period of time indicated (1-24 h). In initial studies, we determined the optimum exposure time and the suitable concentration of cisplatin. To determine the effect of inhibitors, cells were treated with the inhibitors for 10 min prior to the addition of cisplatin (50 µM).

Preparation of Primary Cultures of Caspase-3 (-/-) and Wild-type (+/+) RTEC
Primary cultures of RTEC were prepared from caspase-3 (-/-) and wild-type (+/+) mice. Caspase-3 (-/-) mice were generously provided by Richard Flavell, Ph.D. All procedures for the preparation of primary cultures were performed under sterile conditions as described (40). Briefly, renal cortices were minced and incubated with 0.5 mg/ml collagenase and 0.5 mg/ml soybean trypsin inhibitor for 30 min. After the removal of large, undigested fragments by gravity, the cell suspension was washed first with Hanks' solution containing 10% horse serum and then with Dulbecco's modified Eagle's medium by centrifugation, and the cell pellets were suspended in growth medium containing a serum-free mixture of Dulbecco's modified Eagle's medium, Ham's F-12 (1:1), 2 mM glutamine, 15 mM HEPES, 5 µg/ml transferrin, 5 µg/ml insulin, 50 mM hydrocortisone, 500 units/ml penicillin, and 50 mg/ml streptomycin. The cells previously isolated by this procedure were identified as being predominantly of proximal tubular origin (41). The cell suspension was then plated into tissue culture flasks. After 24 h, the mono-layers were washed several times to remove unattached cells, and new growth medium was added. The cultures became confluent in 5-7 days.

Subcellular Fractionation
Isolation of Nuclear Fraction—The nuclei were prepared by a minor modification of the previously described procedure (42). LLC-PK1 cells were gently scraped using the rubber policeman, harvested by centrifugation, and washed twice with PBS. The cells were resuspended in homogenization buffer containing 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM HEPES, pH 7.5; supplemented with 1x protease inhibitor mixture (Sigma); and homogenized with 30-35 strokes of a Dounce homogenizer (Wheaton). To establish the number of stokes for cell permeabilization, the trypan blue exclusion method (0.4% trypan blue solution in PBS diluted 1:10 with cell suspension), which discriminates between permeabilized (stained) cells and intact cells (unstained), was used. The suspension was then transferred to Eppendorf centrifuge tubes and centrifuged at 500 x g for 10 min to pellet nuclei and unbroken cells. The nuclear fraction was adjusted to the final concentration of 0.25 M sucrose and 0.35% Triton X-100 and layered on top of a discontinuous sucrose density gradient prepared with 0.32, 0.8, and 1.2 M sucrose in a Beckman centrifuge. The tubes were centrifuged at 40,000 x g for 2 h. The nuclei were recovered at the interface of 0.8 M and 1.2 M sucrose. Samples were stored at -70 °C before being used for Western blot analysis.

Isolation of Mitochondria—Mitochondria were isolated by sucrose density gradient centrifugation essentially as described (43). Briefly, cells were washed with PBS containing 1 mM EDTA and resuspended in isotonic homogenization buffer supplemented with 1x protease inhibitor mixture (Sigma). Cells were broken with 30-35 strokes of a Dounce homogenizer (Wheaton), and the suspension was centrifuged at 2,000 x g in an Eppendorf centrifuge at 4 °C. The supernatant was then centrifuged at 13,000 x g at 4 °C for 10 min. The pellet was resuspended in 1 ml of homogenization buffer and layered on top of a discontinuous sucrose gradient consisting of 20 ml of 1.2 M sucrose, 10 mM HEPES, pH 7.5, 1 mM EDTA, and 0.1% bovine serum albumin on top of 17 ml of 1.6 M sucrose, 10 mM HEPES, pH 7.5, 1 mM EDTA, and 0.1% bovine serum albumin. The samples were centrifuged at 27,000 rpm for 2 h at 4 °C using a Beckman SW28 rotor. Mitochondria recovered at the 1.6-1.2 M sucrose interface were washed and resuspended in homogenization buffer. This procedure results in the mitochondrial preparation with very little contamination of other organelles. Contamination of mitochondria in the nuclear fraction was determined by immunoblotting for cytochrome oxidase subunit IV, an integral membrane protein of the mitochondria.

Immunofluorescence Localization of AIF and Caspase-3
Cells were grown on sterile glass coverslips and treated with cisplatin for various time points in the presence and absence of inhibitors. Following treatments, the cells were washed in PBS and fixed in 2% paraformaldehyde in PBS for 15 min. After washing twice in PBS, the cells were permeabilized for 1 h in blocking buffer containing 1% bovine serum albumin, 1% goat serum, 0.1% saponin, 1 mM CaCl2, 1 mM MgCl2, and 2 mM NaV2O5 in PBS. The cells were then incubated with mouse monoclonal anti-AIF antibody (1:200) and rabbit anti-caspase-3 (active) antibody (1:200) for 1 h in a 37 °C humidified incubator. After three washes in washing buffer containing 1% bovine serum albumin and 0.1% saponin in PBS, the cells were incubated at 37 °C in a humidified incubator for 1 h with a 1:500 dilution of Alexa Fluor-conjugated secondary antibody (goat anti-mouse Alexa Fluor 488 for AIF and goat anti-rabbit Alexa Fluor 594 for caspase-3) in blocking solution and again washed with washing buffer. The nuclei were stained with 0.5 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) for 5 min, and the cells were washed twice in washing buffer. Coverslips were then mounted on slides using antifade mounting medium (Molecular Probes). Localization of active caspase-3 and morphological changes of the nuclei were analyzed using a Zeiss deconvoluted microscope.

Determination of Caspase-2 and Caspase-3 Activity
Cells were harvested by centrifugation, and the pellets were washed in cold PBS twice. The washed cell pellets were lysed with 20 mM HEPES, pH 7.5, containing 10% sucrose, 0.1% CHAPS, 2 mM dithiothreitol, 0.1% Nonidet P-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A at 4 °C. The supernatants obtained were used to determine the activities of caspase-2 and -3 by fluorometric assay using the following amino-4-methylcoumarin (AMC)-tagged substrates: VDVAD-AMC for caspase-2 and DEVD-AMC for caspase-3 (45). The enzyme extracts containing 50 µg of protein were incubated with 100 mM HEPES, pH 7.4, containing 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol, and 50 µM caspase substrate in a total reaction volume of 0.25 ml. The reaction mixture was incubated for 60 min at 30 °C. At the end of the incubation, the amount of liberated fluorescent group, AMC, was determined using a fluorescent spectrofluorometer (PerkinElmer Life Sciences) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. AMC was used as a standard. Based on the standard curve made with fluorescence reading with free AMC, the data for caspase activity are expressed as nmol of AMC liberated when 50 µg of protein extract was incubated with 50 µM of substrate for 60 min at 30 °C.

Western Blot Analysis
The cell lysates were prepared as described above for the caspase assay, and equal amounts of protein samples were resolved by SDS-polyacrylamide gel electrophoresis using 8% polyacrylamide gels as previously described (44). The proteins were electrophoretically transferred to a Transblot membrane (Bio-Rad), processed for immunoblotting with specific antibodies, and detected using the ECL system as previously described (39).



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  		FIG. 1.
AIF expression in response to cisplatin. Cells were treated with 50 µM cisplatin (CP) (A) for various time periods and with various cisplatin concentrations ranging from 25 to 100 µM for 12 h as indicated (B). Cell lysates (50 µg of protein) were analyzed for AIF expression by Western blot analysis using antibody to AIF. Blots for {alpha}-tubulin confirm equal loading of the samples.

 
Adenoviral Infection of Cells
Recombinant adenoviral vectors carrying p53, green fluorescent protein, lacZ, and AIF were generous gifts from Dr Ruth S. Slack. The amplification of packaged adenovirus was achieved by infection of HEK 293 cells. Adenoviruses were released from the cells by repeated freezing and thawing of the cells, and a titer of 1.1 x 1012 plaque-forming units/ml was produced. The adenoviral infectivity titers for LLC-PK1 cells were determined using procedures as described (46). LLC-PK1 cells growing at 70-80% confluence were infected with adenoviral vectors at a multiplicity of infection of 50 plaque-forming units/cell. The presence of the recombinant adenovirus was confirmed by Western blot. When the adenovirus was used for the experiment, the efficiency of the infection was confirmed by contransfection of Ad-GFP or Ad-LacZ followed by 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal) staining for {beta}-galactosidase expression. The cells were infected with adenovirus p53 or transfected with p53 small interfering RNA (siRNA) 36 h prior to the treatment of cisplatin.

Reverse Transcription-PCR
Total RNA from the cultured cells was obtained by using the RNeasy Mini Kit (Qiagen, Inc., Chatsworth, CA). Approximately 1 µg of total RNA was used for reverse transcription. The expression levels of various genes were determined by reverse transcription-PCR. The following primer pairs were used for amplifying gene-specific amplicons: GGAAGCTCAGGATGTTCGAG-3' and the complementary downstream primer 5'-GTTTCTGCATCACCCAGGTT-3' for PIDD, 5'-CAGCTCCAAGAGGTTTTTCG-3' and 5'-CCAGCATCACTCTCCTCACA-3' for caspase-2, and 5'-AGCCATGTACGTAGCCATCC-3' and downstream primer 5'-TCTCAGCTGTGGTGGTGAAG-3' for {beta}-actin used as an internal control. To ensure that the PCR had not reached a saturation point that would skew the quantitation, aliquots were removed after certain cycle numbers and analyzed in series on an agarose gel. Then the minimum cycle that initially gives optimal product was used in the experiment.

RNA Interference
LLCPK1 cells were plated in a 6-well plate with complete medium. When the cells were 60% confluent, old medium was replaced with fresh medium without serum and antibiotics. siRNAs were designed or obtained from commercial sources to interfere with the expression of AIF, p53, and caspase-2. AIF siRNA sequence 5'-AUGCAGAACUCCAAGCACGTT-3' (sense strand) was designed using online software (BLOCK-iTTM RNAi Designer from Invitrogen). Commercially available siRNAs for AIF, caspase 2, p53, and AIF from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) were also used in this study according to the manufacturer's instructions. Briefly, Lipofectamine (10 µl) and siRNA (10 pmol in 10 µl) in 0.5 ml of serum-free medium were mixed and incubated at room temperature for 20 min. The cells in each well were then transfected with this mixture. After 12 h, fetal bovine serum was added to a final concentration of 10%, and on day 2, the medium in the cells was replaced with fresh medium before cisplatin treatment.

Cell Viability and Cell Death Assays
Cells (5,000/well) were plated in 96-well dishes and treated with 25 µM caspase inhibitors (Z-VAD-fmk or Z-VDVAD-fmk) or p53 inhibitor pifithrin-{alpha} prior to the treatment with 50 µM cisplatin. Inhibition of cell proliferation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Roche Molecular Biochemicals, Laval, Canada) according to the manufacturer's protocol. For the detection of apoptosis, cells were grown on glass coverslips in 6-well plates. The cells were treated with caspase inhibitors or pifithrin-{alpha} for 1 h prior to treatment with 50 µM cisplatin for 12 h. Apoptosis was detected on the basis of nuclear morphology. Cells were fixed with 4% paraformaldehyde and stained with DAPI to reveal fragmented and condensed nuclei.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Translocation of Mitochondrial AIF to Nuclei—We have examined the role of AIF and its translocation during cisplatin injury to RTEC. AIF protein is constitutively expressed in RTEC, and its basal expression was markedly increased by cisplatin injury in a time- and dose-dependent manner (Fig. 1). Down-regulation of AIF with its siRNA provided marked protection against cisplatin-induced cell death (Fig. 2, A-C) when tested at multiple time points at 8, 12, and 16 h following cisplatin treatment (Fig. 2, C and D). The effect of AIF siRNA was specific, since scrambled siRNA did not change the expression of AIF. Also, AIF siRNA did not affect the expression of caspase-3 (Fig. 2A). Immunofluorescence analysis of control and cisplatin-treated cells revealed translocation of AIF from the mitochondria into the nuclei of many cells (Fig. 3). As shown, control cells reveal exclusively mitochondrial punctate staining for AIF. No nuclear staining for AIF or active caspase-3 was seen (Fig. 3, A and B, Control). When cells were treated with cisplatin and co-immunostained with anti-AIF and anti-active caspase-3, a significant number of cells displayed nuclear staining for AIF as well as for active caspase-3 (Fig. 3, A and B, Cisplatin), indicating translocation of AIF to the nuclei. Staining with DAPI revealed many condensed and fragmented nuclei. Most of these apoptotic nuclei displayed colocalization for AIF and active caspase-3 (Fig. 3, A and B, Cisplatin).

Z-VAD-fmk and Pifithrin-{alpha} Block AIF Translocation—To examine the role of caspases and p53 in AIF translocation, we first determined the effect of their inhibition in the mitochondrial release of AIF. Prior treatment of pancaspase inhibitor Z-VAD-fmk in cisplatin-treated cells did not show nuclear staining for AIF and active caspase-3. This suggests that Z-VAD-fmk was able to prevent AIF translocation to the nuclei as well as block caspase-3 activation and nuclear fragmentation (Fig. 3A, Cisplatin + ZVAD). The p53 inhibitor pifithrin-{alpha}, which blocks p53 transcriptional activity, showed the same effect as observed with Z-VAD-fmk, preventing the nuclear features of apoptosis, caspase activation, and translocation of AIF to the nucleus (Fig. 3B, Cisplatin + Pifithrin). Western blot analysis of the subcellular fractions also revealed translocation of AIF from the mitochondria to the nuclei (Fig. 3C). In control cells, AIF was primarily present in the mitochondria, and a very small amount was detected in the cytosol and the nuclei. In cisplatin-treated cells, AIF was predominantly detected in the cytosol and the nuclei. Pancaspase inhibitor Z-VAD-fmk and p53 inhibitor pifithrin-{alpha} markedly prevented the release of AIF from the mitochondria to the cytosol and the nuclei. Blots for cytochrome c oxidase subunit IV, {alpha}-tubulin, and histone H1A were included to confirm equivalent loading for the mitochondrial, cytosolic, and nuclear fractions, respectively. There was some cross-contamination that became evident when mitochondrial marker cytochrome c oxidase subunit IV was assessed in the nuclear fractions, and nuclear marker histone H1A was assessed in the mitochondrial fractions by immunoblotting (not shown). These studies further suggest that p53 and caspases may be involved in the translocation of AIF to the nucleus.

p53 Triggers Caspase-2 and Caspase-3 Activation and AIF Translocation—In response to a variety of death stimuli, p53 is stabilized and rapidly activated in many cell types (47, 48). One important mechanism through which p53 promotes caspase activation is by transactivation of proapoptotic proteins that primarily involve the mitochondria-dependent apoptotic pathway (48, 49). Cisplatin treatment markedly induced p53 and its phosphorylation in RTEC (Fig. 4A, a). Also, transfection of normal cells with a recombinant adenoviral vector carrying an expression cassette for p53 (Ad-p53) increased expression of p53 as well as its phosphorylation (Fig. 4A, b). Control Ad-LacZ transfection did not induce p53 expression (data not shown). To determine the role of p53 in cisplatin-induced caspase-2 and -3 activation, we utilized p53 siRNA to down-regulate p53 expression by RNA interference. The p53 siRNA efficiently and specifically silenced p53 expression (Fig. 4B, a), since p53 siRNA silenced p53-responsive p21 expression without affecting p73 expression (Fig. 4B, a). Cisplatin-induced marked activation of caspase-3 and caspase-2 that we have previously shown in these cells (39) was significantly blocked by p53 siRNA as well as by the p53 inhibitor pifithrin-{alpha} (Fig. 4B, b). To further ascertain whether p53 promotes caspase-2 and -3 activation, we transfected RTEC with Ad-p53 or the control Ad-LacZ. Overexpression of p53 with Ad-53 resulted in caspase-2 and -3 activation (Fig. 4B, b). In addition, overexpression of p53 resulted in marked AIF translocation and cell death that was effectively prevented by pifithrin-{alpha} and Z-VAD-fmk (Fig. 4C). These studies indicate that p53 activation is involved upstream of the cisplatin-induced caspase-2 and -3 activation and is associated with AIF translocation and cell death.



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  		FIG. 2.
AIF siRNA provides marked protection from cisplatin-induced cell death. A, LLC-PK1 cells grown in 6-well plates were transfected with AIF siRNA and treated with and without 50 µM cisplatin for 12 h as described under "Materials and Methods." Scrambled siRNA was used as a control. Cell lysates (50 µg of protein) were analyzed for AIF, caspase-3, and {beta}-actin by Western blot analysis using antibodies to AIF and {beta}-actin, respectively. Caspase-3 was used to confirm specificity of AIF ablation. Anti-{beta}-actin was used to confirm equal loading of the samples. B, MTT assays were used to determine the relative amounts of viable cells after down-regulation of AIF by its siRNA. Cells were treated with cisplatin (CP) at 8, 12, and 16 h following transfection with AIF siRNA as described under "Materials and Methods." Scrambled siRNA was used as a control. Results are mean ± S.E. (n = 4, independent samples). *, p < 0.01 is for AIF siRNA plus cisplatin versus 12-h cisplatin-treated cells. C, the appearance of apoptotic nuclei (fragmented and condensed nuclei) was scored by staining with DAPI after down-regulation of AIF by transfection with its siRNA. The percentage of apoptotic nuclei averaged from 20 fields visualized are shown. Scrambled siRNA was used as a control. Results are mean ± S.E. (n = 4, independent samples). *, p < 0.005 for cisplatin-treated cells versus cisplatin plus AIF siRNA.

 
Thus, it was of interest to examine whether caspase-3 and/or caspase-2 are involved in the translocation of AIF from the mitochondria to the nuclei.



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  		FIG. 3.
A, cisplatin induces AIF translocation to nucleus and Z-VAD-fmk prevents cisplatin-induced AIF translocation and nuclear fragmentation. Cells were untreated or treated with 50 µM cisplatin in the presence and absence of Z-VAD-fmk for 12 h. Cells were stained with anti-AIF (green) and anti-active caspase-3 (red) followed by DAPI (blue) for nuclei, and images were overlaid as shown. Control cells show clear staining with DAPI for intact nuclei (blue) and clear punctate staining for AIF (green) exclusively in the mitochondria without nuclear staining and no staining for active caspase-3. Cisplatin-treated cells show many fragmented and condensed nuclear staining with DAPI (blue) and prominent staining for nuclear AIF (green) that co-localizes with nuclei (blue), active caspase-3 (red) staining that localizes with nuclei (blue), and active caspase-3 (red) staining that co-localizes with nuclear AIF (green), which together appears as yellow. For the cisplatin plus Z-VAD group, prior treatment of pancaspase inhibitor did not show nuclear fragmentation and nuclear staining for AIF and caspase-3 in cisplatin-treated cells. *, view of the cisplatin-induced AIF and caspase-3-stained fragmented nuclei at higher magnification. B, p53 inhibitor pifithrin-{alpha} prevents cisplatin-induced AIF translocation and nuclear fragmentation. Cells were treated with 50 µM cisplatin in the presence and absence of pifithrin-{alpha} for 12 h. Cells were stained with anti-AIF (green) and anti-active caspase-3 (red) and DAPI (blue) for nuclei, and images were overlaid as shown. As indicated, p53 inhibitor pifithrin-{alpha} prevents nuclear localization of AIF and inhibits caspase-3 activation and nuclear fragmentation. Control cells show clear punctate staining for AIF exclusively in the mitochondria with no staining for AIF in the nuclei and no staining for active caspase-3. Cisplatin-treated cells show prominent staining for nuclear AIF (green) and active caspase-3 (red). The yellow color indicates colocalization of AIF and active caspase-3 in the nuclei. For the cisplatin plus pifithrin-{alpha} group, pifithrin-{alpha}-treated cells do not show nuclear fragmentation and nuclear staining for AIF and caspase-3. *, magnified view of the AIF and caspase-3-stained fragmented nuclei of cisplatin-treated cells shown in the cisplatin group. C, analysis of the AIF translocation by subcellular fractionation. Cells were treated with 25 µM Z-VAD-fmk or pifithrin-{alpha} prior to the treatment with 50 µM cisplatin. Mitochondrial, cytosolic, and nuclear fractions were isolated as described under "Materials and Methods" and 10 µg of mitochondrial proteins, 10 µg of nuclear proteins, and 50 µg of cytosolic proteins were electrophoresed and immunoblotted for AIF. Blots for cytochrome c oxidase subunit IV, {alpha}-tubulin, and histone H1A confirm equivalent loading for the mitochondrial, cytosolic, and nuclear fractions, respectively.

 



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  		FIG. 4.
A, a, cisplatin induces p53 and its activation. Cells were treated with 50 µM cisplatin for the times indicated. Cell lysates (50 µg) were subjected to Western blot using antibodies specific to p53 and phospho-p53. b, overexpression of p53 induces p53 activation. Cells were transfected with Ad-p53 in the presence and absence of 50 µM cisplatin, and cell lysates (50 µg protein) were subjected to Western blot using antibodies specific to p53 and phospho-p53. B, cisplatin or p53 overexpression activates caspase-2 and -3, and p53 inhibition prevents cisplatin-{alpha}-induced caspase-2 and -3 activation. a, cells were transfected with p53 siRNA as described in "Materials and Methods," and cell lysates (50 µg of protein) were electrophoresed and blotted for p53, p21, and p73. Transfection of p53 siRNA down-regulates p53 expression as well as its responsive p21 expression but not p73 expression. Blot for {alpha}-tubulin confirms equal loading of the samples. b, cells were treated with cisplatin or cisplatin plus pifithrin-{alpha} or infected with Ad-53 or infected with Ad-LacZ for control, or transfected with p53 siRNA as indicated and described under "Materials and Methods." The inhibition of p53 was accomplished by treatment with pifithrin-{alpha} or by transfection with p53 siRNA. Caspase-2 and -3 activities in cell lysates (50 µg of protein) were determined using caspase-2 substrate VDVAD-AMC and caspase-3 substrate DEVD-AMC. Caspase activities are shown as relative units. The results are mean ± S.E. (n = 4, independent samples). *, p < 0.005 compared with cisplatin-treated cells. C, pifithrin-{alpha} or caspase-2 inhibitor prevent AIF translocation in p53-overexpressing cells. Cells were infected with Ad-p53 in the presence or absence of pifithrin-{alpha} or Z-VDVAD-fmk. Control cells were infected with Ad-LacZ as described under "Materials and Methods." The cells were immunostained with anti-AIF and anti-caspase-3 antibodies together with DAPI for nuclear staining. Cells infected with Ad-LacZ show clear punctate staining for AIF exclusively in the mitochondria with no staining for AIF in the nuclei and no staining for active caspase-3. Cells infected with Ad-p53 show prominent staining for nuclear AIF (green) and active caspase-3 (red). For the Ad-p53 plus pifithrin-{alpha} or Ad-p53 plus Z-VAD-fmk group, cells infected with Ad-p53 in the presence of pifithrin-{alpha} or Z-VAD show no nuclear staining for AIF or active caspase-3.

 
Caspase-3-deficient Cells Do Not Block AIF Translocation—To examine the role of caspase-3 in the translocation of AIF, we prepared primary cultures of kidney proximal tubular epithelial cells from caspase-3 (-/-) and caspase-3 (+/+) mice. Immunocytochemistry revealed cisplatin-induced translocation of AIF to the nuclei in both caspase-3 (+/+) (Fig. 5A) and caspase-3 (-/-) cells (Fig. 5B), suggesting that the knock-out of the caspase-3 gene does not affect AIF translocation. Cisplatin caused cell death in both caspase-3 (+/+) and caspase-3 (-/-) cells. In addition, as revealed by DAPI staining, only caspase-3 (+/+) cells showed typical morphological changes associated with apoptosis, including nuclear chromatin condensation and nuclear fragmentation. Unlike caspase-3 (+/+) cells, caspase-3 (-/-) cells showed nuclear condensation but no nuclear fragmentation, implying that caspase-3 may be involved in the fragmentation of the nuclei. AIF staining was observed in all condensed nuclei in caspase-3 (-/-) cells. In fact, activation of caspase-3 was previously shown to cause an apoptotic nuclear morphology, which could be blocked by a peptide inhibitor of caspase-3 (50). The pancaspase inhibitor Z-VAD-fmk or pifithrin-{alpha} prevented transport of AIF to the nucleus in both caspase-3 (+/+) and caspase-3 (-/-) cells (Fig. 5, A and B). These studies indicated that p53-controlled activation of caspase(s) other than caspase-3 is involved in the translocation of AIF to the nucleus.



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  		FIG. 5.
Z-VAD-fmk or pifithrin-{alpha} prevents cisplatin-induced AIF translocation in caspase-3 (+/+) cells and in caspase-3 (-/-) cells. Primary cultures of kidney proximal tubular epithelial cells prepared from caspase-3 (+/+) mice (A) and caspase-3 (-/-) mice (B) were treated with 50 µM cisplatin for overnight in the presence and absence of 25 µM Z-VAD-fmk or pifithrin-{alpha}. The cells were immunostained with anti-AIF (green) and anti-caspase-3 (red) antibodies together with DAPI (blue) for nuclear staining. Nuclear staining of AIF (green) is demonstrated by the co-localization of AIF (green) and nuclei (blue, stained by DAPI). Nuclear staining of AIF (green) overlaps the nuclear staining of active caspase-3 (red), and when overlaid, the appearance of the yellow color indicates co-localization of AIF and active caspase-3.

 
p53-responsive PIDD-mediated Caspase-2 Activation Involved in AIF Translocation—A recent study demonstrated that the p53-responsive gene PIDD is involved in caspase-2 activation (51). Caspase-2 activation requires the formation of a complex that contains the adapter protein RAIDD and a p53-responsive death domain-containing protein PIDD. The overexpression of PIDD has been shown to result in spontaneous activation of caspase-2 (51). Thus, we examined PIDD expression in response to cisplatin injury to RTEC. In cisplatin-treated cells, the p53 phosphorylation was followed by increased mRNA expression of PIDD. Pifithrin-{alpha} or p53 siRNA markedly reduced PIDD expression whereas overexpression of p53 with Ad-p53 elevated the levels of PIDD mRNA (Fig. 6). These manipulations for p53 expression did not affect caspase-2 mRNA levels. These studies are in agreement with the previous observation that p53 through its responsive gene PIDD can control activation of caspase-2. To examine the role of caspase-2 activation in AIF translocation, we utilized its siRNA to down-regulate caspase-2 expression (Fig. 6a) and used VDVAD-fmk to inhibit caspase-2 activity. Caspase-2 siRNA efficiently and specifically silenced caspase-2 expression but did not affect caspase-3 expression (Fig. 6a). Also, scrambled siRNA had no effect on caspase-2 and -3 expression. The inhibition of caspase-2 activity by VDVAD-fmk and the specific silencing of caspase-2 expression (Fig. 7) prevented the translocation of AIF to the nucleus in RTEC (Fig. 7) and markedly prevented cisplatin-induced cell death. Thus, p53, whose expression is up-regulated in cisplatin injury to RTEC, can mediate the release of AIF through the activation of caspase-2. Down-regulation of caspase-2 with its siRNA provided marked protection against cisplatin-induced cell death (Fig. 7, C and D) when tested at multiple time points at 8, 12, and 16 h following cisplatin treatment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrate that cisplatin injury to RTEC increased basal levels of AIF expression and induced translocation of AIF from the mitochondria to the nuclei. The inhibition of AIF by gene silencing with its siRNA significantly prevented cisplatin-induced cell death, suggesting that AIF plays an important role in renal tubular epithelial cell injury. Although cytochrome c is also released in response to cisplatin, inhibition of cytochrome c-mediated caspase-9 and caspase-3 activation provided only partial protection from cisplatin-induced apoptosis (35, 39, 52-54), suggesting the role of AIF in cell death.



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  		FIG. 6.
Inhibition of p53 or down-regulation of p53 inhibits PIDD mRNA expression. Cells were infected with Ad-p53 or transfected with p53 siRNA 36 h prior to the treatment of cisplatin or cisplatin together with pifithrin-{alpha}. PIDD, caspase-2, and {beta}-actin gene expression were determined by reverse transcription-PCR as described under "Materials and Methods."

 
Our results reveal that p53 is involved in the cisplatin-induced regulation of AIF translocation. In normal RTEC cells, p53 is present at low levels, and, following cisplatin treatment, wild-type p53 accumulates and becomes activated. The treatment of cells with the p53 inhibitor pifithrin-{alpha} markedly blocked AIF translocation. In addition, overexpression of p53 in RTEC induced AIF translocation, and down-regulation of p53 using its siRNA suppressed AIF translocation. These studies further extend the previous observation that DNA damage-induced AIF translocation depends on the presence of p53 (10).

We have previously shown that caspase-3 and caspase-2 are predominantly activated in cisplatin injury to RTEC (44). We now demonstrate that the p53 inhibitor pifithrin-{alpha} or down-regulation of p53 by its siRNA not only significantly inhibited cisplatin induced caspase-2 and -3 activation but also blocked AIF translocation. Overexpression of p53 also induced activation of caspase-2 and -3 in RTEC. These studies suggest that p53 functions upstream of caspase-2 and -3 and is involved in regulation of cisplatin-induced caspase-2 and -3 activation.

It is well documented that p53 regulates transactivation of a multitude of proapoptotic genes that encode proteins for BH3-only proteins of the Bcl-2 family, death receptors, and other factors involved in different steps of the apoptotic pathway (55). p53 can also regulate apoptosis by transcriptional repression of Bcl-2 and IAPs (inhibitor of apoptosis protein) (56). In addition, p53 has an extranuclear role to regulate transcription-independent apoptosis. The extranuclear p53 can directly activate Bax (57) or directly binds to BclxL and Bcl-2 proteins to induce mitochondrial permeabilization and release cytochrome c (58). The p53-responsive genes that control the expression of Bax, Bok, Noxa, Puma, Apaf1, and p53AIP1 (47, 59, 61) can promote caspase-3 activation by induction of the mitochondria-dependent intrinsic apoptotic pathway. Thus, cisplatin-induced caspase-3 activation observed in RTEC can be attributed to p53 activation, since p53 inhibitor or gene silencing of p53 in RTEC markedly prevents caspase-3 activation.

A recent study has demonstrated that p53 controls the expression of death domain-containing adaptor protein PIDD that, in association with another adaptor protein, RAIDD, recruit procaspase-2 by interaction with its prodomain (51). The resulting ternary complex activates caspase-2 without interdomain cleavage of caspase-2 (51). Our studies show that the expression of the p53-responsive gene PIDD is induced by cisplatin as well as by overexpression of p53 in RTEC. Together, these studies suggest that p53 is involved in cisplatin-induced activation of caspase-2 and -3. Since p53 inhibition prevents translocation of AIF, we hypothesized that p53-mediated caspase activation may play a role in AIF translocation. Indeed, the pancaspase inhibitor Z-VAD-fmk significantly blocked cisplatin-induced translocation of AIF and provided marked protection in cisplatin-induced cell death. We further explored whether caspase-2 or -3 or both are involved in p53-dependent translocation of AIF.

The executioner caspase, caspase-3, is activated in cisplatin injury, and p53 contributes significantly to this activation. However, caspase-3 activation is not involved in the translocation of AIF to the nuclei. This became evident from studies that demonstrated that AIF was translocated to the nucleus in primary caspase-3 (-/-) cells treated with cisplatin. Also, the caspase-3 inhibitor Z-DEVD-fmk did not prevent the translocation of AIF to the nucleus. These studies are in agreement with previous findings that show that Apaf-1 (-/-) and caspase-3 (-/-) cells treated with apoptosis inducers staurosporine or etoposide did not prevent AIF release from the mitochondria (12). Both p53 inhibitor and pancaspase inhibitor Z-VAD-fmk prevented translocation of AIF to the nucleus. These studies further support that p53-induced caspase-3 activation is not involved in translocation of AIF.

Our findings demonstrate that p53-mediated caspase-2 but not caspase-3 activation engage in the release of AIF. The specific function of caspase-2 to release AIF was confirmed by using a specific inhibitor of caspase-2 as well as by down-regulating caspase-2 expression using caspase-2 siRNA. Several studies have provided compelling evidence that caspase-2 acts upstream of mitochondrial release of cytochrome c (62-67). Caspase-2, like caspase-8, is capable of cleaving the proapototic Bcl-2 family member Bid (65-67) that triggers mitochondrial membrane permeabilization. However, incubation of purified caspase-2 with isolated mitochondria can trigger the release of cytochrome c, AIF, and Smac/Diablo independent of Bid and other cytosolic factors (67), suggesting that caspase-2 can directly cause mitochondrial membrane permeabilization. Indeed, a recent study has demonstrated that caspase-2 directly permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids (62).

In C. elegans, AIF homolog WAH-1 is released into the cytosol and nucleus by the BH3 domain protein EGL-1 in a caspase (CED-3)-dependent manner (68). However, in mammalian cells, the dependence of caspases for AIF translocation is not well established and needs further exploration. Many studies are of the view that AIF mediates caspase-independent cell death (8, 9), whereas other recent studies show that AIF translocation to the nucleus is caspase-dependent (9, 13, 14). The pancaspase inhibitor Z-VAD-fmk has been shown to prevent actinomycin- and staurosporine-induced AIF release and cell death in HeLa cells. A recent study showed that the pancaspase inhibitor prevented the actinomycin- and staurosprine-induced mitochondrial release of AIF and endonuclease G but not cytochrome c, Smac/Diablo, and HtrA2/Omi in HeLa cells (7, 8). AIF release induced by introduction of cytochrome c into living Jurkat cells by square wave pulse electropermeabilization were prevented by Z-VAD-fmk (60), suggesting the involvement of caspases. Thus, more studies are required to resolve whether there are different mechanisms to release AIF from the mitochondria.



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  		FIG. 7.
A, inhibition of caspase-2 by Z-VDVAD-fmk or down-regulation of caspase-2 by its siRNA prevent AIF translocation. Cells were treated with caspase-2 inhibitor (25 µM) or transfected with caspase-2 siRNA prior to the treatment of 50 µM cisplatin. The cells were double immunostained with anti-AIF (green) and anti-caspase-3 (red) followed by staining with DAPI (blue). Nuclear staining of AIF (green) is demonstrated by the co-localization of AIF (green) and nuclei (blue, stained by DAPI). Nuclear staining of AIF (green) colocalizes with the nuclear staining of active caspase-3 (red), and the appearance of yellow color indicates co-localization of AIF and active caspase-3. B, pancaspase inhibitor Z-VAD-fmk, caspase-2 inhibitor Z-VDVAD-fmk, or p53 inhibitor pifithrin-{alpha} markedly prevent cisplatin-induced cell death. Cells were untreated or treated with 50 µM cisplatin in the presence and absence of 25 µM of Z-VAD-fmk or Z-VDVAD-fmk or pifithrin-{alpha} for 12 h. MTT assays were used to determine the relative amounts of viable cells as described under "Materials and Methods." Results are mean ± S.E. (n = 4, independent samples). *, p < 0.005 compared with cisplatin-treated cells. C, MTT assays were used to determine the relative amounts of viable cells after down-regulation of caspase-2 by its siRNA. Cells were treated with cisplatin (CP) at 8, 12, and 16 h following transfection with caspase-2 siRNA as described under "Materials and Methods." Scrambled siRNA was used as a control. Results are mean ± S.E. (n = 4, independent samples). *, p < 0.02 is for caspase-2 siRNA plus cisplatin-versus cisplatin-treated cells. D, the appearance of apoptotic nuclei (fragmented and condensed nuclei) was scored by staining with DAPI after down-regulation of caspase-2 by transfection with its siRNA. Percentage of apoptotic nuclei averaged from 20 fields visualized are shown. Scrambled siRNA was used as a control. Results are mean ± S.E. (n = 4, independent samples). *, p < 0.01 for cisplatin treated cells versus cisplatin plus caspase-2 siRNA.

 
In summary, the data presented in the present study demonstrate that AIF translocation from the mitochondria to the nuclei plays an important role in cisplatin injury to RTEC. p53 is an essential mediator of AIF translocation to the nucleus in response to cisplatin in RTEC. Although cisplatin-induced p53 plays a role in the activation of caspase-2 and -3, only caspase-2 is capable of mitochondrial release of AIF. The p53-responsive gene PIDD is involved in the mechanism of cisplatin-induced caspase-2 activation.


   FOOTNOTES
 
* This work was supported by NIDDK, National Institutes of Health, Grant DK-58324 and an American Heart Association (Affiliate) grant (to G. P. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Medicine, 4301 W. Markham St., Slot 501, University of Arkansas for Medical Sciences, Little Rock, AR 72205. Tel.: 501-257-5834; Fax: 501-257-5827; E-mail: kaushalgurp{at}uams.edu.

1 The abbreviations used are: AIF, apoptosis-inducing factor; RTEC, renal tubular epithelial cell(s); PIDD, p53-induced protein with a death domain; Smac, second mitochondria-derived activator of caspase; Z, benzyloxycarbonyl; fmk, fluoromethylketone; DAPI, 4', 6'-diamidino-2-phenylindole; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Diablo, direct IAP-binding protein with low pl; HtrA2, high temperature requirement A2; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,3-diphenyltetrazolium bromide; siRNA, small interfering RNA; AMC, amino-4-methylcoumarin; IAP, hihibitor of apoptosis protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Ruth Slack for the gift of recombinant adenoviral vectors. The authors thank Randy Haun, Ph.D., for critical review of the manuscript and Cindy Reid for secretarial assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D. R., Aebersold, R., Siderovski, D. P., Penninger, J. M., and Kroemer, G. (1999) Nature 397, 441-446[CrossRef][Medline] [Order article via Infotrieve]
  2. Penninger, J. M., and Kroemer, G. (2003) Nat. Cell Biol. 5, 97-99[CrossRef][Medline] [Order article via Infotrieve]
  3. Ye, H., Cande, C., Stephanou, N. C., Jiang, S., Gurbuxani, S., Larochette, N., Daugas, E., Garrido, C., Kroemer, G., and Wu, H. (2002) Nat. Struct. Biol. 9, 680-684[CrossRef][Medline] [Order article via Infotrieve]
  4. Cande, C., Vahsen, N., Kouranti, I., Schmitt, E., Daugas, E., Spahr, C., Luban, J., Kroemer, R. T., Giordanetto, F., Garrido, C., Penninger, J. M., and Kroemer, G. (2004) Oncogene 23, 1514-1521[CrossRef][Medline] [Order article via Infotrieve]
  5. Daugas, E., Nochy, D., Ravagnan, L., Loeffler, M., Susin, S. A., Zamzami, N., and Kroemer, G. (2000) FEBS Lett. 476, 118-123[CrossRef][Medline] [Order article via Infotrieve]
  6. Vahsen, N., Cande, C., Briere, J. J., Benit, P., Joza, N., Larochette, N., Mastroberardino, P. G., Pequignot, M. O., Casares, N., Lazar, V., Feraud, O., Debili, N., Wissing, S., Engelhardt, S., Madeo, F., Piacentini, M., Penninger, J. M., Schagger, H., Rustin, P., and Kroemer, G. (2004) EMBO J. 23, 4679-4689[CrossRef][Medline] [Order article via Infotrieve]
  7. Joza, N., Susin, S. A., Daugas, E., Stanford, W. L., Cho, S. K., Li, C. Y., Sasaki, T., Elia, A. J., Cheng, H.Y., Ravagnan, L., Ferri, K. F., Zamzami, N., Wakeham, A., Hakem, R., Yoshida, H., Kong, Y. Y., Mak, T. W., Zuniga-Pflucker, J. C., Kroemer, G., and Penninger, J. M. (2001) Nature 410, 549-554[CrossRef][Medline] [Order article via Infotrieve]
  8. Cregan, S. P., Dawson, V. L., and Slack, R. S. (2004) Oncogene 23, 2785-2796[CrossRef][Medline] [Order article via Infotrieve]
  9. Cande, C., Vahsen, N., Garrido, C., and Kroemer, G. (2004) Cell Death Differ. 11, 591-595[Medline] [Order article via Infotrieve]
  10. Cregan, S. P., Fortin, A., MacLaurin, J. G., Callaghan, S. M., Cecconi, F., Yu, S. W., Dawson, T. M., Dawson, V. L., Park, D. S., Kroemer, G., and Slack, R. S. (2002) J. Cell Biol. 158, 507-517[Abstract/Free Full Text]
  11. Yu, S. W., Wang, H., Poitras, M. F., Coombs, C., Bowers, W. J., Federoff, H. J., Poirier, G. G., Dawson, T. M., and Dawson, V. L. (2002) Science 297, 200-201[Free Full Text]
  12. Susin, S. A., Daugas, E., Ravagnan, L., Samejima, K., Zamzami, N., Loeffler, M., Costantini. P., Ferri, K. F., Irinopoulou, T., Prevost, M. C., Brothers, G., Mak, T.W., Penninger, J., Earnshaw, W. C., and Kroemer, G. (2000) J. Exp. Med. 192, 571-580[Abstract/Free Full Text]
  13. Arnoult, D., Tatischeff, I., Estaquier, J., Girard, M., Sureau, F., Tissier, J. P., Grodet, A., Dellinger, M., Traincard, F., Kahn, A., Ameisen, J. C., and Petit, P. X. (2001) Mol. Biol. Cell 12, 3016-3030[Abstract/Free Full Text]
  14. Arnoult, D., Parone, P., Martinou, J. C., Antonsson, B., Estaquier, J., and Ameisen, J. C. (2002) J. Cell Biol. 159, 923-929[Abstract/Free Full Text]
  15. Kuwana, T., and Newmeyer, D. D. (2003) Curr. Opin. Cell Biol. 15, 691-699[CrossRef][Medline] [Order article via Infotrieve]
  16. Green, D. R., and Kroemer, G. (2004) Science 305, 626-629[Abstract/Free Full Text]
  17. Uren, R. T., Dewson, G., Bonzon, C., Lithgow, T., Newmeyer, D. D., and Kluck, R. M. (2004) J. Biol. Chem. 280, 2266-2274[Medline] [Order article via Infotrieve]
  18. Rozencweig, M., von Hoff, D. D., Slavik, M., and Muggia, F. M. (1997) Ann. Intern. Med. 86, 803-812
  19. Safirstein, R., Winston, J., Moel, D., Dikman, S., and Guttenplan, J. (1987) Int. J. Androl. 10, 325-346[Medline] [Order article via Infotrieve]
  20. Gordon, J. A., Gattone, V. H 2nd. (1986) Am. J. Physiol. 250, F991-F998[Medline] [Order article via Infotrieve]
  21. Satoh, M., Kashihara, N., Fujimoto, S., Horike, H., Tokura, T., Namikoshi, T., Sasaki, T., and Makino, H. (2003) J. Pharmacol. Exp. Ther. 305, 1183-1190[Abstract/Free Full Text]
  22. Davis, C. A., Nick, H. S., and Agarwal, A. (2001) J. Am. Soc. Nephrol. 12, 2683-2690[Abstract/Free Full Text]
  23. Sugiyama, S., Hayakawa, M., Kato, T., Hanaki, Y., Shimizu, K., and Ozawa, T. (1989) Biochem. Biophys. Res. Commun. 159, 1121-1127[CrossRef][Medline] [Order article via Infotrieve]
  24. Brady, H. R., Kone, B. C., Stromski, M. E., Zeidel, M. L., Giebisch, G., and Gullans, S. R. (1990) Am. J. Physiol. 258, F1181-F1187[Medline] [Order article via Infotrieve]
  25. Kruidering, M., Van de Water, B., de Heer, E., Mulder, G. J., and Nagelkerke, J. F. (1997) J. Pharmacol. Exp. Ther. 280, 638-649[Abstract/Free Full Text]
  26. Zhang, J. G., and Lindup, W. E. (1993) Biochem. Pharmacol. 45, 2215-2222[CrossRef][Medline] [Order article via Infotrieve]
  27. Schwerdt, G., Freudinger, R., Schuster, C., Silbernagl, S., and Gekle, M. (2003) Kidney Int. 63, 1725-1735[CrossRef][Medline] [Order article via Infotrieve]
  28. Nishikawa, M., Nagatomi, H., Chang, B. J., Sato, E., and Inoue, M. (2001) Arch. Biochem. Biophys. 387, 78-84[CrossRef][Medline] [Order article via Infotrieve]
  29. Park, M. S., De Leon, M., and Devarajan, P. (2002) J. Am. Soc. Nephrol. 13, 858-865[Abstract/Free Full Text]
  30. Baek, S. M., Kwon, C. H., Kim, J. H., Woo, J. S., Jung, J. S., and Kim, Y. K. (2003) J. Lab. Clin. Med. 142, 178-186[CrossRef][Medline] [Order article via Infotrieve]
  31. Nowak, G. (2002) J. Biol. Chem. 277, 43377-43388[Abstract/Free Full Text]
  32. Cilenti, L., Kyriazis, G. A., Soundarapandian, M. M., Stratico, V., Yerkes, A., Park, K. M., Sheridan, A. M., Alnemri, E. S., Bonventre, J. V., and Zervos, A. S. (2005) Am. J. Physiol. Renal Physiol. 288, F371-F379[Abstract/Free Full Text]
  33. Justo, P., Sanz, A., Lorz, C., Gomez-Garre, D., Mezzano, S., Egido, J., and Ortiz, A. (2003) Kidney Int. Suppl. 86, S52-S56[Medline] [Order article via Infotrieve]
  34. Fukuoka, K., Takeda, M., Kobayashi, M., Osaki, T., Shirato, I., Soejima, A., Nagasawa, T., and Endou, H. (1998) Life Sci. 62, 1125-1138[CrossRef][Medline] [Order article via Infotrieve]
  35. Nowak, G., Price, P. M., and Schnellmann, R. G. (2003) Am. J. Physiol. 285, F440-F450
  36. Zhan, Y., van de Water, B., Wang, Y., and Stevens, J. L. (1999) Oncogene 18, 6505-6512[CrossRef][Medline] [Order article via Infotrieve]
  37. Kim, J. S., Lee, J. M., Chwae, Y. J., Kim, Y. H., Lee, J. H., Kim, K., Lee, T. H., Kim, S. J., and Park, J. H. (2004) Biochem. Pharmacol. 67, 1459-1468[CrossRef][Medline] [Order article via Infotrieve]
  38. Jagjit, S., Gill, J. S., and Windebank, A. J. (1998) J. Clin. Invest. 101, 2842-2850[Medline] [Order article via Infotrieve]
  39. Kaushal, G. P., Kaushal, V., Hong, X., and Shah, S. V. (2001) Kidney Int. 60, 1726-1736[CrossRef][Medline] [Order article via Infotrieve]
  40. Bergin, E., Levine, J. S., Koh, J. S., and Lieberthal, W. (2000) Am. J. Physiol. 278, F758-F768
  41. Sheridan, A. M., Schwartz, J. H., Kroshian, V. M., Tercyak, A. M., Masino, S., and Lieberthal, W. (1993) Am. J. Physiol. 265, F342-F350[Medline] [Order article via Infotrieve]
  42. Wood, E. R., and Earnshaw, W. C. (1990) J. Cell Biol. 111, 2839-2850[Abstract/Free Full Text]
  43. Desagher, S. Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., and Martinou, J.-C. (1999) J. Cell Biol. 144, 891-901[Abstract/Free Full Text]
  44. Kaushal, G. P., Singh, A. B., and Shah, S. V. (1998) Am. J. Physiol. 274, F587-F595[Medline] [Order article via Infotrieve]
  45. Stennicke, H. R., and Salvesen, G. S. (2000) Methods Enzymol. 322, 91-100[Medline] [Order article via Infotrieve]
  46. Cregan, S. P., MacLaurin, J., Gendron, T. F., Callaghan, S. M., Park, D. S., Parks, R. J., Graham, F. L., Morley, P., and Slack, R. S. (2000) Gene Ther. 7, 1200-1209[CrossRef][Medline] [Order article via Infotrieve]
  47. Slee, E. A., O'Connor, D. J., and Lu, X. (2004) Oncogene 23, 2809-2818[CrossRef][Medline] [Order article via Infotrieve]
  48. El-Deiry, W. S. (2003) Oncogene 22, 7486-7495[CrossRef][Medline] [Order article via Infotrieve]
  49. Schuler, M., Green, D. R. (2001) Biochem. Soc. Trans. 29, 684-688[CrossRef][Medline] [Order article via Infotrieve]
  50. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T.-T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37-43[CrossRef][Medline] [Order article via Infotrieve]
  51. Tinel, A., and Tschopp, J. (2004) Science 304, 843-846[Abstract/Free Full Text]
  52. Sauerwald, T. M., Betenbaugh, M. J., and Oyler, G. A. (2002) Biotechnol. Bioeng. 77, 704-716[CrossRef][Medline] [Order article via Infotrieve]
  53. Takeda, M., Kobayashi, M., Shirato, I., Osaki, T., and Endou, H. (1997) Arch. Toxicol. 71, 612-621[CrossRef][Medline] [Order article via Infotrieve]
  54. Cummings, B. S., and Schnellmann, R. G. (2002) J. Pharmacol. Exp. Ther. 302, 8-17[Abstract/Free Full Text]
  55. Sax, J. K., and El-Deiry, W. S. (2003) Cell Death Differ. 10, 413-417[CrossRef][Medline] [Order article via Infotrieve]
  56. Johnstone, R. W., Ruefli, A. A., and Lowe, S. W. (2002) Cell 108, 153-164[CrossRef][Medline] [Order article via Infotrieve]
  57. Chipuk, J. E., Kuwana, T., Bouchier-Hayes, L., Droin, N. M., Newmeyer, D. D., Schuler, M., and Green, D. R. (2004) Science 303, 1010-1014[Abstract/Free Full Text]
  58. Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P., and Moll, U. M. (2003) Mol. Cell 11, 577-590[CrossRef][Medline] [Order article via Infotrieve]
  59. Yakovlev, A. G., Di Giovanni, S., Wang, G., Liu, W., Stoica, B., and Faden, A. I. (2004) J. Biol. Chem. 279, 28367-28374[Abstract/Free Full Text]
  60. Gabriel, B., Sureau, F., Casselyn, M., Teissie, J., and Petit, P. X. (2003) Exp. Cell Res. 289, 195-210[CrossRef][Medline] [Order article via Infotrieve]
  61. Vousden, K. H., and Lu, X. (2002) Nat. Rev. Cancer 2, 594-604[CrossRef][Medline] [Order article via Infotrieve]
  62. Enoksson, M., Robertson, J. D., Gogvadze, V., Bu, P., Kropotov, A., Zhivotovsky, B., and Orrenius, S. (2004) J. Biol. Chem. 279, 49575-49578[Abstract/Free Full Text]
  63. Robertson, J. D., Enoksson, M., Suomela, M., Zhivotovsky, B., and Orrenius, S. (2002) J. Biol. Chem. 277, 29803-29809[Abstract/Free Full Text]
  64. Lassus, P., Opitz-Araya, X., and Lazebnik, Y. (2002) Science 297, 1352-1354[Abstract/Free Full Text]
  65. Lin, C. F., Chen, C. L., Chang, W. T., Jan, M. S., Hsu, L. J., Wu, R. H., Tang, M. J., Chang, W. C., and Lin, Y. S. (2004) J. Biol. Chem. 279, 40755-40761[Abstract/Free Full Text]
  66. Wagner, K. W., Engels, I. H., and Deveraux, Q. L. (2004) J. Biol. Chem. 279, 35047-35052[Abstract/Free Full Text]
  67. Guo, Y., Srinivasula, S. M., Druilhe, A., Fernandes-Alnemri, T., and Alnemri, E. S. (2002) J. Biol. Chem. 277, 13430-13437[Abstract/Free Full Text]
  68. Wang, X., Yang, C., Chai, J., Shi, Y., and Xue, D. (2002) Science 298, 1587-1592[Abstract/Free Full Text]

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