Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:43 |只看该作者 |倒序浏览 |打印

作者：Gra yna Nowak, Peter M. Price,  Rick G. Schnellmann作者单位：Departments of Pharmaceutical Sciences and Internal Medicine, University of Arkansas for MedicalSciences, Little Rock, Arkansas 72205; and Departmentof Pharmaceutical Sciences, Medical University of South Carolina, Charleston,South Carolina 29425 + q$ p' w: s+ J" Y1 Q. w

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      【摘要】
   The lack of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 (p21) in mice increases renal proximal tubular cell death and enhances sensitivityto acute renal failure produced by the chemotherapeutic agent cisplatin. Weused primary cultures of mouse renal proximal tubular cells (MPTC) grown inoptimized culture conditions to investigate the cellular basis for increasedapoptosis in p21 knockout mice. Cisplatin (15 µM) activated caspase-3 butnot caspase-8 or caspase-9 and produced phosphatidylserine externalization,chromatin condensation, and nuclear fragmentation in wild-type [p21( / )]MPTC. Caspase-3 activation and apoptosis were accelerated in cisplatin-treatedMPTC lacking p21 [p21(-/-) MPTC]. In contrast to p21( / ) MPTC, cisplatinactivated caspase-9 but not caspase-8 in p21(-/-) MPTC before caspase-3activation. The caspase-3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone (DEVD-fmk) inhibited caspase-3 activity but did not abolish apoptosis inp21( / ) and p21(-/-) MPTC. General caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (ZVAD-fmk) inhibited caspase activityand decreased chromatin condensation by 51% in p21(-/-) but not in p21( / )MPTC. However, cisplatin-induced phosphatidylserine externalization was notinhibited by ZVAD-fmk in p21(-/-) MPTC. We conclude that 1 ) in thepresence of p21, cisplatin activates caspase-3 through a mechanism independent of caspase-8 or caspase-9; 2 ) in the absence of p21, caspase-9 activation precedes caspase-3 activation; 3 ) the lack of p21 accelerates caspase-3 activation and cisplatin-induced MPTC apoptosis; and 4 ) MPTC apoptosis is caspase independent in the presence of p21 butpartially dependent on caspases in the absence of p21.
      【关键词】 renal proximal tubular cells caspase activation DNA damage knockout mice8 ?7 M7 s' q2 q
   CISPLATIN IS A WIDELY USED anticancer agent in the treatment ofmany solid tumors and metastatic cancers( 6, 19 ). Cisplatin administrationcauses cell-cycle arrest at different phases of the cell cycle and inducesapoptosis in a variety of cancer cells( 6 ). The major disadvantage ofthis antineoplastic agent is a dose-dependent and cumulative nephrotoxicity,which manifests primarily as proximal tubule morphological damage (oncosis and apoptosis) and physiological dysfunction( 1, 3, 8, 10, 20, 21, 30, 38, 45 ). Multiple mechanisms havebeen implicated in cisplatin-induced nephrotoxicity, including inhibition of protein synthesis, DNA damage, oxidative stress, mitochondrial dysfunction,and alterations in signal transduction pathways involved in apoptosis( 12, 17, 36, 39, 41, 45, 48 ). Lower concentrations ofcisplatin induce apoptosis, whereas concentrations 50 µM cause oncosisin renal proximal tubular cells ( 7, 25 ). Cisplatin-inducedapoptosis in this cell type is preceded by activation of caspase-3, but notcaspase-1 ( 3, 4, 8, 32, 44, 48 ), and is prevented by bcl-2( 48 ). Activation of caspase-8and caspase-9 by cisplatin has been reported in the LLC-PK 1 cellline ( 14 ).
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The cyclin-dependent kinase inhibitor p21 WAF1/CIP1 plays a pivotal role in cell differentiation, DNA repair, and apoptosis throughregulation of the cell cycle. The p21 WAF1/CIP1 protein isconstitutively present at low levels in the nucleus of most cells as a complexwith cyclin, cyclin-dependent kinase, and proliferating cell nuclear antigen( 23, 49 ). p21 WAF1/CIP1 is known to be a mediator of p53 tumor suppressor function and has beenimplicated in apoptosis caused by numerous agents. DNA damage caused byradiation and various chemotherapeutic drugs activates p53, which upregulatesp21 WAF1/CIP1 mRNA and protein levels. Induction ofp21 WAF1/CIP1 is required for the p53-dependent arrest in theG 1 phase of the cell cycle after DNA damage( 2, 46 ). However, transcriptionaland posttranscriptional changes in p21 WAF1/CIP1 expression afterDNA damage can also be induced in a p53-independent manner( 11, 29 ).
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p21 WAF1/CIP1 has differential effects on the sensitivity of cancer cells to ionizing radiation, cisplatin, and other antineoplastic agents, such as adriamycin, taxol, and vincristine, protecting against celldeath in some models and not in others( 7, 8, 37 ). For example, increasedlevels of p21 WAF1/CIP1 enhance sensitivity of hepatoma andosteosarcoma cells to cisplatin ( 22 ). In contrast, in coloncancer and embryonic fibroblast cells, increased sensitivity to cisplatin andother chemotherapeutic agents is associated with the lack ofp21 WAF1/CIP1 ( 7, 37 ).3 N  w$ O; B7 o* E) L
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p21 WAF1/CIP1 is rapidly induced in the murine kidney in response to acute renal failure (ARF) produced by ischemia-reperfusion, ureteralobstruction, and cisplatin( 29 ). Thep21 WAF1/CIP1 induction decreases renal damage after cisplatin- andischemia-induced ARF in murine models( 26, 28 ). In contrast, the lack ofthe p21 WAF1/CIP1 gene accelerates progression to ARF and chronic renal failure, increases morphological and functional damage to proximaltubular cells and the kidney, and results in a higher mortality rate( 26 - 28 ).The mechanisms of the protective effects of p21 WAF1/CIP1 are notclear. It was shown that cells having DNA damaged by cisplatin administrationenter the cell cycle, but cell-cycle progression is inhibited in the presence of p21 WAF1/CIP1. In contrast, kidney cells lackingp21 WAF1/CIP1 progress from the G 1 to S phase of the cellcycle after cisplatin-induced damage( 28 ). Therefore, it has beenproposed that p21 WAF1/CIP1 decreases cisplatin nephrotoxicity bydecreasing the number of injured cells that enter the cell cycle and undergomitosis with damaged DNA, which results in cell death by either apoptosis oroncosis ( 28 ).

The aim of this study was to examine the cellular basis for increased celldeath in p21 WAF1/CIP1 knockout mice by investigating the temporalactivation and the role of caspase-3, caspase-8, and caspase-9 duringcisplatin-induced apoptosis in mouse renal proximal tubular cells (MPTC)obtained from p21 WAF1/CIP1 knockout and wild-type mice.) g# k' Q0 p' c8 n# L8 ?- ?

MATERIALS AND METHODS0 k* M' i* N3 Y

Materials. Cell isolation media, cell culture media, horse serum, and soybean trypsin inhibitor were supplied by Life Technologies (GrandIsland, NY). Collagenase type 4 and L -ascorbic acid-2-phosphate magnesium salt were provided by Worthington Biochemical (Freehold, NJ) andWako Bio-Products (Richmond, VA), respectively. Cisplatin [ cis -diamminedichloroplatinum(II)], Percoll, methyl - D -glucopyranoside, propidium iodide, and cell culturehormones were purchased from Sigma (St. Louis, MO). Ethidium homodimer and4',6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained fromMolecular Probes (Eugene, OR). Caspase-3 [Asp-Glu-Val-Asp(DEVD)-7-amino-4-trifluoromethylcoumarin (AFC)] and caspase-8 [Ile-Glu-Thr-Asp(IETD)-AFC] fluorometric substrates, AFC, and buffers for caspase assays werepurchased from BioVision (Palo Alto, CA). Caspase-9 substrate [Leu-Glu-His-Asp (LEHD)-AFC] was obtained from Calbiochem (La Jolla, CA). Caspase-3 [DEVD-fluoromethylketone (fmk)] and pan-caspase [Z-Val-Ala-Asp(OCH3)-fmk (ZVAD-fmk)] inhibitors were purchased from R&D Systems (Minneapolis, MN).Antibodies against cleaved forms of caspase-3, caspase-8, and caspase-9 weresupplied by Cell Signaling Technology (Beverly, MA), and an anti-p21 antibodywas supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Methyl - D -[U- 14 C]glucopyranoside (MGP; specific activity230 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ).The sources of the other reagents have been described previously( 34 ).6 l% W4 A4 v: }8 Y# N5 w2 H6 y/ F5 _
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Animals. Adult mice carrying a deletion of a large portion of thep21 WAF1/CIP1 gene, in which neither p21 WAF1/CIP1 mRNA nor p21 WAF1/CIP1 protein is expressed, were obtained from Dr. Philip Leder (Harvard Medical School, Boston, MA)( 5 ). Mice homozygous for thep21 WAF1/CIP1 deletion were selected from the offspring ofheterozygous matings using Southern blotting of tail DNA as describedpreviously ( 5 ). The animalsused in these studies were housed at the Veterinary Medical Unit at the JohnL. McClellan Memorial Veterans Hospital (Little Rock, AR). Wild-typehomozygous mice 129Sv (obtained from The Jackson Laboratory, Bar Harbor, ME)were used as controls. Female mice (12-18 wk old) were used in this study.
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Isolation of proximal tubules. Mouse renal proximal tubules wereisolated by a modification of the method described by Sheridan et al.( 40 ). The basal isolationmedium was Hanks' solution and a 50:50 mixture of DMEM and Ham's F-12 nutrient mix (DMEM/F-12) supplemented with 29 mM NaHCO 3, 15 mM HEPES, andpenicillin G (150 U/ml). The medium was adjusted to pH 7.4 while being gassedwith 95% O 2 -5% CO 2 and was diluted to 295mosmol/kgH 2 O before filter sterilization.
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The animals were anesthetized with halothane, and both kidneys wereremoved, dissected under sterile conditions, and placed in ice-cold Hanks'solution gassed with 95% O 2 -5% CO 2. Cortical tissue wasremoved, placed in fresh ice-cold Hanks' solution, and finely minced with ascalpel blade. Minced tissue was incubated for 30 min at 37°C (withshaking) in digestion medium consisting of Hanks' solution, collagenase type 4(140 U/ml), and soybean trypsin inhibitor (0.75 mg/ml). Large undigestedfragments of cortical tissue were separated by gravity after the mixing ofequal volumes of the tissue suspension and ice-cold 10% horse serum in Hanks'solution. Following sedimentation of undigested tissue, the supernatantcontaining cortical tubules was collected and centrifuged for 2 min x 50 g at 4°C. The pellet was washed with ice-cold Hanks' solution,centrifuged for 2 min x 50 g at 4°C, washed again withDMEM/F-12, centrifuged, and resuspended in DMEM/F-12 medium. Cortical tubuleswere purified by gradient centrifugation in a 40% Percoll/60% DMEM/F-12 at36,000 g x 20 min at 4°C. The band containing renal proximal tubules was collected and washed twice with DMEM/F-12, and the finalpellet was resuspended in DMEM/F-12.& M. J$ }* N2 ?9 o9 r2 Y
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Culture conditions. The culture medium was a 50:50 mixture of DMEM/F-12 nutrient mix without phenol red and pyruvate, supplemented with 15mM NaHCO 3, 15 mM HEPES, 1 mM glucose, and 5 mM lactate (pH 7.4, 295mosmol/kgH 2 O). Renal proximal tubule segments were plated in 35-mmculture dishes (0.3 mg protein/dish) and grown in optimized conditions asdescribed previously for rabbit renal proximal tubular cells( 34 ). Culture dishes wereconstantly swirled on an orbital shaker to improve media oxygenation. Human transferrin (5 µg/ml), selenium (5 ng/ml), hydrocortisone (50 nM), bovineinsulin (5 µg/ml), and L -ascorbic acid-2-phosphate (50 µM)were added to the medium immediately before daily media change (2ml/dish).1 U* [8 q: N2 f0 @( Y  J

O 2 consumption. Confluent MPTCmonolayers were gently detached from the dishes with a rubber policeman,suspended in 37°C culture medium, and transferred to the O 2 consumption (Q O 2 ) measurement chamber.Q O 2 was measured polarographically using a Clark-typeelectrode as described previously( 33, 34 ). UncoupledQ O 2 was used as a marker of the activity of electron transfer through the respiratory chain and was measured after addition ofcarbonyl cyanide p -(trifluoromethoxy)phenylhydrazone (2 µM).+ t; J) ~+ Q5 n* _  w) Z

Active Na transport. Ouabain-sensitiveQ O 2 was used as a marker of active Na transport as described previously( 33 ). Ouabain-sensitive Q O 2 was measured in the presence of 1 mM ouabain andcalculated as a difference between basal and ouabain-insensitiveQ O 2.

Na -dependent glucose uptake. Glucose uptakewas assessed using a nonmetabolizable glucose analog, MGP, as describedpreviously ( 34 ). In brief,confluent monolayers of MPTC were washed with 37°C glucose-free culturemedium and incubated (with shaking) in glucose-free medium containing 1 mM MGPand 0.2 µCi/ml of [ 14 C]MGP for 30 min at 37°C in a 5%CO 2 -95% air atmosphere. Following washing, the amount of[ 14 C]MGP associated with the renal proximal tubular cell monolayerwas determined by liquid scintillation spectrometry.$ X9 q9 g( O4 C! [" l! V+ E; }

Cisplatin treatment of MPTC. MPTC monolayers reached confluence within 6 days and were treated with cisplatin on day 7 of culture. Samples of MPTC were taken at various time points (between 4 and 18 h) ofcisplatin exposure. Caspase inhibitors DEVD-fmk and Z-VAD-fmk were added 0.5 hbefore cisplatin treatment. Control MPTC were treated with diluent (DMSO; 0.1%final concentration).

Measurement of caspase activities. Caspase-3-, caspase-8-, and caspase-9-like activities were quantified by fluorometric detection of AFCafter cleavage from DEVD-AFC, IETDAFC, and LEHD-AFC, respectively. In brief,media were aspirated from culture dishes, MPTC monolayers were washed twicewith ice-cold PBS, scraped from the culture dishes, resuspended and lysed incell lysis buffer (10 min on ice, BioVision, Palo Alto, CA), and centrifuged at 15,000 g for 10 min at 4°C. The pellet was discarded, and thesupernatant was used for caspase assays. The supernatant samples wereincubated for 1 h at 37°C in the presence of the reaction buffer optimizedfor caspase activity assays (BioVision), 10 mM dithiothreitol, and 50 µMDEVD-AFC (for measurements of caspase-3-like activity), 50 µM IETD-AFC (formeasurement of caspase-8-like activity), or 50 µM LEHD-AFC (for measurementof caspase-9-like activity). The samples were read in a fluorometer at 380/500nm (excitation/emission), and the amount of product cleaved under linearconditions was determined from the AFC standard curve.- m! g9 \6 S( R# I/ Y  k+ _( m; p
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Immunoblotting. Immunoblot analysis was used for the measurement of protein levels of p21 and cleaved (active) caspase-3, caspase-8, andcaspase-9 in renal proximal tubular cell homogenates. Renal proximal tubularcell homogenates were lysed and boiled for 10 min in Laemmli sample buffer (60mM Tris·HCl, pH 6.8, containing 2% SDS, 10% glycerol, 100 mM -mercaptoethanol, and 0.01% bromophenol blue)( 18 ), and proteins wereseparated using SDS-PAGE. Following electroblotting of the proteins to anitrocellulose membrane, blots were blocked for 1 h in 50 mM Tris-bufferedsaline (pH 7.5) containing 0.5% casein and 0.1% Tween 20 (blocking buffer) andincubated overnight at 4°C in the presence of primary antibodies dilutedin the blocking buffer. Following washing with 50 mM Tris-buffered salinecontaining 0.05% Tween 20, the membranes were incubated for 1 h withanti-rabbit or anti-mouse IgG coupled to horseradish peroxidase and washedagain. The supersignal chemiluminescent system (Pierce, Rockford, IL) was usedfor protein detection, and scanning densitometry was utilized for thequantification of results.9 ~( _. ~0 O  F6 [. a2 {: k, ~
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Assessment of oncosis. The release of LDH into the culture medium was measured as a marker of oncosis, as described previously ( 31 ). Oncosis was alsoassessed by measuring the permeability of the MPTC plasma membrane to ethidiumhomodimer. As plasma membrane permeability increases, ethidium homodimerenters the cell and binds to DNA, producing a bright red fluorescence. After24 h of cisplatin exposure, MPTC monolayers were incubated with 25 µMethidium homodimer for 15 min on ice, and cells were processed forfluorescence analysis. In brief, media were aspirated and MPTC were scrapedfrom the culture dishes, washed twice with ice-cold PBS, and resuspended inPBS. Cell-associated fluorescence was analyzed by flow cytometry (BDFACSCalibur) using excitation at 488 nm and emission at 590 nm. For each sample, 10,000 events were counted.

Assessment of apoptosis. Apoptosis was assessed using twodissimilar markers: plasma membrane phosphatidylserine externalization measured by annexin V/propidium iodide binding assay and chromatin condensation and nuclear fragmentation measured by immunocytochemistry. MPTCwere washed twice with a binding buffer consisting of (in mM) 10 HEPES (pH7.4), 140 NaCl, 5 KCl, 1 MgCl 2, and 1.8 CaCl 2. Followingresuspension in the binding buffer, MPTC were incubated in the presence ofpropidium iodide (2 µg/ml) for 15 min on ice, washed three times with thebinding buffer, and incubated in the presence of annexin V-FITC (125 ng/ml) for 10 min at room temperature. MPTC were washed three times with the bindingbuffer and processed for flow cytometry. Propidium iodide and annexin V-FITCfluorescence was quantified by flow cytometry using excitation at 488 nm andemission at 590 and 530 nm for propidium iodide and annexin V-FITC,respectively. For each sample, 10,000 events were counted. Cells positive forannexin V and negative for propidium iodide were considered apoptotic.8 M& B+ a1 f& r; a6 v  `

MPTC nuclei were visualized by DAPI staining. The monolayers were fixed in3.7% formaldehyde for 15 min, rinsed with PBS, and incubated with 8 µM DAPIfor 2 h at room temperature. Following staining, MPTC monolayers were washedwith PBS, coverslips were applied, and nuclei were evaluated using a Zeissfluorescent microscope (Axioscope). Total and condensed or fragmented nuclei were counted in six to eight different areas of every monolayer using twoplates per each experimental group from three independent MPTC isolations.

Biochemical assays. Protein concentration was determined using bicinchoninic acid assay and bovine serum albumin as the standard. CellularDNA content was determined using a PicoGreen dsDNA Quantitation Kit (MolecularProbes). Monolayers were solubilized in 0.1 M Tris·HCl (pH 7.4)containing 0.15 M NaCl and 0.05% Triton X-100, and DNA was determined in celllysates according to the manufacturer's protocol.
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Statistical analysis. Data are presented as means ± SE andwere analyzed for significance using ANOVA. Multiple means were compared usingthe Student-Newman-Keuls test. Statements of significance were based on P tubules isolated on a given dayrepresent a separate experiment ( n = 1) consisting of data obtainedfrom two plates.

RESULTS4 g: [  Z/ {- x

Metabolic characteristics of primary cultures of MPTC grown inoptimized culture conditions. Previously, we showed that primary culturesof rabbit renal proximal tubular cells maintain many in vivo differentiatedfunctions of renal proximal tubules when grown under optimized cultureconditions ( 33, 34 ). Improving culture mediaoxygenation (by shaking the dishes) in conjunction with decreasing glucoseconcentration and supplementing with lactate and ascorbic acid results in areduction in glycolysis, an in vivo-like respiration, and an increase intransport functions. Therefore, we modified the original culture conditions( 33 ) used to grow MPTC inprimary culture to improve the growth, oxidative metabolism, and transportfunctions in these cells. The modifications included 1 ) decreasingglucose concentration in the culture media from 5 to 1 mM, 2 )supplementing media with 5 mM lactate (a major substrate for the kidney invivo) and 50 µM ascorbic acid phosphate, and 3 ) shaking the dishesto improve media oxygenation.8 ]/ Z" ~" Q( N) d- e, K" O

Protein and DNA contents of confluent MPTC monolayers grown in the improvedculture conditions are illustrated in Table1. Basal and uncoupled Q O 2 were used asmarkers of oxidative metabolism and electron transport through the respiratorychain, respectively ( Table 1 ).Both basal and uncoupled Q O 2 were similar toQ O 2 in freshly isolated mouse renal proximal tubules(basal Q O 2 : 32 ± 2 nmolO 2 ·min - 1 ·mgprotein - 1; uncoupled Q O 2 :48± 6 nmol O 2 ·min - 1 ·mg protein - 1 ) and rabbit renal proximal tubular cells grownunder optimized conditions( 34 ). Ouabain-sensitiveQ O 2 was used as a marker of active Na transport in MPTC. Ouabain-sensitive Q O 2 in confluentcultures of MPTC grown in the improved culture conditions( Table 1 ) was equivalent toouabain-sensitive Q O 2 in freshly isolated mouse renalproximal tubules (14 ± 1 nmolO 2 ·min - 1 ·mgprotein - 1 ) and rabbit renal proximal tubular cells grownunder optimized conditions( 30 ). Na -dependentglucose uptake was measured using a nonmetabolizable glucose analog MGP. MGPuptake in confluent quiescent MPTC ( Table1 ) was similar to MGP uptake in primary cultures of rabbit renalproximal tubular cells ( 34 ).Phlorizin (1 mM) inhibited MGP uptake in MPTC by 97 ± 1%. Cellulardensity, respiration, active Na transport, andNa -dependent glucose uptake in MPTC isolated from mice lackingp21 WAF1/CIP1 were equivalent to those in MPTC from p21( / ) mice( Table 1 ). These data show thatprimary cultures of MPTC grown in optimized conditions maintain in vivo-likerespiration and active Na transport of renal proximal tubules inthe presence and absence of p21 WAF1/CIP1.! e- n6 E7 S& w+ H6 {7 w/ h
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Table 1. Metabolic characteristics of primary cultures of MPTC isolated fromwild-type and p21 WAF1/CIP1 knockout mice and grown for 7 days inoptimized culture conditions7 @+ H7 }! D1 e6 x# {/ {

Cisplatin exposure and p21 WAF1/CIP1 protein levels. Protein levels of p21 WAF1/CIP1 in MPTC from p21( / ) mice increased twofold at 4 h of exposure to 15 µM cisplatin and remained increased untilthe end of the treatment (18 h) ( Fig.1 A ). Caspase inhibitors had no effect oncisplatin-induced increases in p21 levels( Fig. 1 A ).p21 WAF1/CIP1 protein was absent in control and cisplatin-treatedMPTC from p21(-/-) mice ( Fig. 1 B ).& [# w) d5 n: H4 x. r
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Fig. 1. Effect of cisplatin on protein levels of p21 WAF1/CIP1 in primarycultures of mouse renal proximal tubular cells (MPTC) expressing ( A )and lacking ( B ) p21 WAF1/CIP1. MPTC were treated with 15µM cisplatin, and samples were taken at 4, 8, 12, and 18 h of exposure forimmunoblot analysis. The experiment was repeated 3 times with similar results.DEVD, Asp-Glu-Val-Asp; fmk, fluoromethylketone; ZVAD, Z-Val-Ala-Asp(OCH3).

Cisplatin-induced caspase activation in p21( / )MPTC. Caspase activation in MPTC was assessed by measuring caspaseactivities and evaluating the formation of caspase cleavage products during cisplatin exposure.( ^& U- T9 A  H- P; O  d

Caspase-3, caspase-8, and caspase-9 activities were assessed in MPTC aftercisplatin (5, 10, and 15 µM) exposure and in MPTC treated with the vehicle(0.1% DMSO, controls). No significant changes in caspase-3 activity wereobserved in MPTC treated with DMSO. Caspase-3 activity was not altered during the first 8 h of cisplatin treatment in MPTC( Fig. 2 A ). At 12 h,caspase-3 activity was unaffected in MPTC treated with 5 and 10 µMcisplatin but increased fivefold in MPTC incubated with 15 µM cisplatin( Fig. 2 A ). At 18 h,caspase-3 activity increased 11-fold in MPTC incubated with 10 µM cisplatinand was 22-fold higher in MPTC treated with 15 µM cisplatin( Fig. 2 A ). Immunoblotanalysis showed the presence of significant amounts of the cleaved (active)caspase-3 (17-20 kDa) in MPTC treated with 15 µM cisplatin for 18 h( Fig. 2 C ). The caspaseinhibitors DEVD-fmk and ZVAD-fmk decreased caspase-3 processing during 15µM cisplatin exposure ( Fig.2 C ).

Fig. 2. Effect of cisplatin on caspase-3-like activity in primary cultures of MPTCexpressing ( A ) and lacking ( B ) p21 WAF1/CIP1. MPTCwere treated with 0, 5, 10, and 15 µM cisplatin, and samples were taken at4, 8, 12, and 18 h of cisplatin exposure for measurements of caspase-3-likeactivity. Control MPTC were treated with vehicle (DMSO) alone. Values aremeans ± SE of 4-5 experiments. Values with different letters aresignificantly different ( P C and D : protein levels of cleaved (active) caspase-3 (17-20 kDa) duringcisplatin (15 µM) exposure in primary cultures of MPTC expressing( C ) and lacking ( D ) p21 WAF1/CIP1. Presented blotsare representative of results obtained from 3 independent experiments.8 \  B* e: D  p/ P
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To elucidate whether caspase-8 and caspase-9 play a role in cisplatin-induced caspase-3 activation in MPTC, we examined the time course ofthe activation of caspase-8 and caspase-9 during cisplatin exposure comparedwith the activation of caspase-3. There was no significant change in caspase-8activity during 12 h of exposure at any concentration of cisplatin, whereas caspase-3 activity was markedly increased (Figs. 2 A and 3 A ). Cisplatin at 10and 15 µM increased caspase-8 activity two- and threefold, respectively,after 18 h of treatment ( Fig. 3 A ). Nevertheless, immunoblot analysis showed no cleavageproducts of caspase-8 (10 kDa) in MPTC exposed to 15 µM cisplatin for 18 h( Fig. 3 C ). Theseresults show that caspase-8 activation either does not occur incisplatin-treated MPTC or caspase-8 activation by cisplatin is minor andfollows caspase-3 activation.
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Fig. 3. Effect of cisplatin on caspase-8-like activity in primary cultures of MPTCexpressing ( A ) and lacking ( B ) p21 WAF1/CIP1. MPTCwere treated with 0, 5, 10, and 15 µM cisplatin, and samples were taken at4, 8, 12, and 18 h of cisplatin exposure for measurements of caspase-8-likeactivity. Control MPTC were treated with vehicle (DMSO) alone. Values aremeans ± SE of 3-4 experiments. Values with different letters aresignificantly different ( P C and D : absence of cleaved (active) caspase-8 (10 kDa) during cisplatin(15 µM) exposure in primary cultures of MPTC expressing ( C ) andlacking ( D ) p21 WAF1/CIP1. Presented blots arerepresentative of results obtained from 3 independent experiments. C,untreated Jurkat cells; PC, positive control (Jurkat cells treated withetoposide).
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No activation of caspase-9 occurred during an 18-h exposure of MPTC to 5and 10 µM cisplatin ( Fig.4 A ). An 18-h treatment with 15 µM cisplatin increasedMPTC caspase-9 activity 2.3-fold ( Fig.4 A ). However, immunoblot analysis demonstrated only thepresence of procaspase-9 (47 kDa) and no cleaved caspase-9 (35 and 37 kDa) inMPTC exposed to 15 µM cisplatin for 18 h( Fig. 4 C ). Based onthe caspase activity and immunblot assays, we conclude that cisplatinactivates caspase-3 without activation of caspase-9 and caspase-8 in p21( / )MPTC.

Fig. 4. Effect of cisplatin on caspase-9-like activity in primary cultures of MPTCexpressing ( A ) and lacking ( B ) p21 WAF1/CIP1. MPTCwere treated with 0, 5, 10, and 15 µM cisplatin, and samples were taken at4, 8, 12, and 18 h of cisplatin exposure for measurements of caspase-9-likeactivity. Control MPTC were treated with vehicle (DMSO) alone. Values aremeans ± SE of 6 experiments. Values with different letters aresignificantly different ( P C and D : protein levels of procaspase-9 ( top band) and cleaved(active) caspase-9 ( bottom bands; 35-37 kDa) during cisplatin (15µM) exposure in primary cultures of MPTC expressing ( C ) andlacking ( D ) p21 WAF1/CIP1. Presented blots arerepresentative of results obtained from 3 independent experiments. C,untreated Jurkat cells; PC, positive control (Jurkat cells treated withcytochrome c ).; O! H' G2 O* |5 x& t* t- _2 U
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Cisplatin-induced caspase activation in p21( - / - )MPTC. Exposure to 15 µM cisplatin for 8 h resulted in a threefoldincrease in caspase-3 activity in p21(-/-) MPTC, whereas no activation ofcaspase-3 occurred at this time point in p21( / ) MPTC ( Fig. 2, A and B ). At 12 h of treatment, caspase-3 activity increasedfourfold and sixfold in the presence of 10 and 15 µM cisplatin,respectively ( Fig.2 B ). Caspase-3 activity continued to increase and, at 18h of exposure, was 14-fold higher in cells treated with 15 µM cisplatinthan in controls and was threefold higher than in p21( / ) MPTC( Fig. 2, A and B ). Immunoblot analysis demonstrated the presence of thecleaved form of caspase-3 at 12 h of 15 µM cisplatin exposure in p21(-/-)MPTC and a further increase in protein levels of the active caspase-3 at 18 hof cisplatin treatment ( Fig.2 D ). Similar to p21( / ) MPTC, the caspase inhibitors DEVD-fmk and ZVAD-fmk decreased caspase-3 cleavage during cisplatin exposurein p21(-/-) MPTC ( Fig.2 D ).
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No increase in caspase-8 activity was observed in p21(-/-) MPTC during thefirst 8 h of cisplatin treatment ( Fig.3 B ). After 12 h of exposure to 15 µM cisplatin,caspase-8 activity increased threefold in p21(-/-) MPTC, whereas no activation of caspase-8 occurred in p21( / ) MPTC at this time point( Fig. 3, A and B ). At 18 h of treatment, caspase-8 activity in p21(-/-) MPTC treated with 15 µM cisplatin was fourfold higher than in respectivecontrols and threefold higher than in p21( / ) MPTC undergoing the sametreatment ( Fig. 3, A and B ). However, immunoblot analysis showed no cleaved(active) form of caspase-8 in p21(-/-) MPTC exposed to 15 µM cisplatin for18 h ( Fig. 3 D ).

No increase in caspase-9 activity occurred during the first 8 h ofcisplatin treatment in p21(-/-) MPTC ( Fig.4 B ). After 12 h of cisplatin (15 µM) exposure,caspase-9 activity increased sevenfold in p21(-/-) MPTC, whereas no increasein caspase-9 activity occurred in p21( / ) MPTC( Fig. 4, A and B ). At 18 h of treatment, caspase-9 activity in p21(-/-)MPTC treated with 15 µM cisplatin was eightfold higher than in controls andtwofold higher than in p21( / ) MPTC undergoing the same treatment( Fig. 4, A and B ). In contrast to p21( / ) MPTC, immunoblot analysisdemonstrated the presence of cleaved forms of caspase-9 (35 and 37 kDA) in p21(-/-) MPTC starting at 4 h of the cisplatin exposure( Fig. 4 D ). The caspaseinhibitors DEVD-fmk and ZVAD-fmk did not block cisplatin-induced caspase-9 processing in p21(-/-) MPTC ( Fig.4 D ). These results demonstrate that the lack ofp21 WAF1/CIP1 results in the acceleration and greater extent ofactivation of caspase-3 and the activation caspase-9 by cisplatin.

Assessment of cisplatin-induced MPTC apoptosis by annexin V/propidium iodide binding. Cisplatin-induced MPTC apoptosis was assessed usingannexin V binding as a marker of phosphatidylserine externalization. Cisplatin(15 µM) induced 18 ± 6 and 25 ± 9% apoptosis in p21( / ) MPTCat 12 and 18 h of exposure, respectively ( Fig. 5 A ). The caspaseinhibitors DEVD-fmk and ZVAD-fmk had no effect on the number of annexinV-positive cells at 12 or 18 h of cisplatin exposure( Fig. 5 A ). Theseresults suggest that caspase inhibition does not block cisplatin-inducedapoptosis in MPTC.  l% l0 g' a& [7 D6 F

Fig. 5. Effects of cisplatin (15 µM), DEVD-fmk (50 µM), and ZVAD-fmk (50µM) on annexin V-FITC binding in primary cultures of MPTC expressing( A ) and lacking ( B ) p21 WAF1/CIP1. The graphs showthe percentage of annexin V-FITC-positive and propidium iodide (PI)-negativeMPTC as determined by flow cytometry. Values are means ± SE of 3experiments. Values with different letters are significantly different( P

Annexin V binding in p21(-/-) MPTC was 26 ± 9 and 30 ± 9% at12 and 18 h of cisplatin treatment, respectively, and was caspase inhibitorindependent ( Fig. 5 B ).Furthermore, the apoptosis in control p21(-/-) MPTC (i.e., spontaneousapoptosis) was increased 1.4-fold at both 12- and 18-h time points compared with control p21( / ) MPTC ( Fig.5 ). These results show that the lack of p21 acceleratescisplatin-induced caspase-independent apoptosis in MPTC.0 w$ @" O% X$ [* j; }( \. N

LDH release was 4 ± 2% in p21( / ) MPTC and 4 ± 1% inp21(-/-) MPTC after 24 h of cisplatin (15 µM) exposure and was notdifferent from LDH release in controls (1 ± 1%). Uptake of ethidiumhomodimer in MPTC treated with 15 µM cisplatin for 24 h (4 ± 1%) wasnot different from that in controls (3 ± 1%). These results show thatthe plasma membrane was not compromised by cisplatin exposure and support theconclusion that cisplatin induced apoptosis in MPTC in the absence ofoncosis.
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Assessment of cisplatin-induced MPTC chromatin condensation and nuclearfragmentation. In p21( / ) MPTC, nuclear changes occurred in 32 ±1% of the cells treated with 15 µM cisplatin for 18 h (Figs. 6 B and 7 A ), an amountsignificantly greater (45 ± 2%) than in p21(-/-) MPTC (Figs. 6 F and 7 B ). Monolayerspretreated with DEVD-fmk or 50 µM ZVAD-fmk before cisplatin (15 µM)treatment and caspase activities and chromatin condensation were assessedafter 18 h of exposure. DEVD-fmk and ZVAD-fmk prevented the increase incaspase-3 activity and procaspase-3 cleavage in cisplatin-treated MPTCregardless of the presence or absence of p21 WAF1/CIP1 ( Fig. 2, A and B ). Similarly, ZVAD-fmk and DEVD-fmk abolishedcisplatin-induced increases in LEHD-ase( Fig. 4, A and B ) and IETD-ase ( Fig. 3, A and B ) activities in both p21( / ) andp21(-/-) MPTC. However, neither DEVD-fmk nor ZVAD-fmk blocked procaspase-9 processing in p21(-/-) MPTC treated with cisplatin( Fig. 4 D ). These datasuggest that blocking caspase-3 activation in cisplatin-treated MPTC preventsincreases in caspase-8 and caspase-9 activities and support our conclusionthat increases in caspase-8-like and caspase-9-like activities are secondaryto caspase-3 activation.# W3 R, R' o1 X
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Fig. 6. Effect of caspase inhibitors on cisplatin-induced changes in nuclearmorphology after 18 h of cisplatin exposure in primary cultures of MPTC. MPTCwere pretreated with caspase inhibitors (50 µM DEVD-fmk and 50 µMZVAD-fmk) for 30 min before cisplatin (15 µM) treatment. Control MPTC weretreated with vehicle (DMSO) alone. MPTC were fixed and stained with4',6-diamidino-2-phenylindole dihydrochloride, and nuclei were evaluatedusing a fluorescent microscope. A : control [p21( / )]. B :cisplatin [p21( / )]. C : DEVD-fmk cisplatin [p21( / )]. D :ZVAD-fmk cisplatin [p21( / )]. E : control [p21(-/-)]. F :cisplatin [p21(-/-)]. G : DEVD-fmk cisplatin [p21(-/-)]. H :ZVAD-fmk cisplatin [p21(-/-)]. Microphotographs are representative of resultsobtained from 3 independent experiments. Magnification: x 400.* K  ]# L' b/ ?4 z: P$ w6 l
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Fig. 7. Effect of caspase inhibitors on the percentage of cells displayingchromatin condensation and nuclear fragmentation after 18 h of cisplatinexposure in primary cultures of MPTC expressing ( A ) and lacking( B ) p21 WAF1/CIP1. MPTC were pretreated with caspaseinhibitors (50 µM DEVD-fmk and 50 µM ZVAD-fmk) for 30 min beforecisplatin (15 µM) treatment. Control MPTC were treated with vehicle (DMSO)alone. See Fig. 6 for sampleprocessing. Values are means ± SE of 3 experiments. Values withdifferent letters are significantly different ( P/ A/ S# e9 f. I: q1 P4 Y* r- M3 @3 Z
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Treatment of MPTC with DEVD-fmk and ZVAD-fmk for 18 h had no effect onnuclear morphology in control MPTC (Figs. 5, A and B,and 7, A and B ). Pretreatment of p21( / ) MPTC with DEVD-fmk orZVAD-fmk before cisplatin exposure had no effect on the number of cells withchromatin condensation and nuclear fragmentation (Figs. 6, C and D,and 7 A ). Similarly,pretreatment with both inhibitors did not prevent cisplatin-induced MPTCshrinkage and detachment from the monolayer (data not shown). These data suggest that cisplatin-induced apoptosis in p21( / ) MPTC is caspaseindependent.. c% N5 D7 {; T4 Q4 A8 v  O2 Q
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Pretreatment of p21(-/-) MPTC with DEVD-fmk had no significant effect oncisplatin-induced chromatin condensation and nuclear fragmentation despite thedecrease in caspase-3 processing and complete inhibition of caspase-3 activity(Figs. 2, B and D, 6 G, and 7 B ). However,pretreatment with ZVAD-fmk before cisplatin exposure decreased nuclearcondensation in p21(-/-) MPTC 51% at 18 h (Figs. 6 H and 7 B ). These resultssuggest that, in addition to caspase-independent apoptosis, another mechanism(s) is responsible for the increased cisplatin sensitivity ofp21(-/-) MPTC. The other mechanism is caspase dependent, but the pathway isindependent of caspase-3, -8, and -9 activation.

DISCUSSION, e& g7 |" j; \: \- k

ARF after ischemia or cisplatin administration is associated with a rapidinduction of the cell cycle inhibitory protein p21 WAF1/CIP1 geneand overexpression of p21 WAF1/CIP1 protein ( 26, 29 ). p21 WAF1/CIP1 induction is associated with cell-cycle interruption and arrest in theG 1 phase of the cell cycle ( 46 ). Induction ofp21 WAF1/CIP1 after cisplatin administration has a protective effecton the murine kidney as mice lacking p21 WAF1/CIP1 display a morerapid onset of ARF and have a higher mortality rate than wild-type animals( 27 ). The proximal tubule segment of the nephron is the most sensitive to cisplatin and undergoes severedamage in response to cisplatin-based chemotherapy ( 10 ). In vivo studiesdemonstrated that proximal tubular cells in mice lackingp21 WAF1/CIP1 exhibited greater cell death than proximal tubularcells in wild-type mice ( 28 ).Our in vitro studies show that low concentrations of cisplatin cause cell death by apoptosis, but not oncosis, and that the lack ofp21 WAF1/CIP1 increases the extent of apoptosis by 40% in primarycultures of MPTC. Thus our results suggest that p21 WAF1/CIP1 playsa protective role by decreasing cisplatin-induced cell death. Furthermore, ourdata demonstrate that the increase in apoptosis in mice lackingp21 WAF1/CIP1 is the result of a direct effect of cisplatin on therenal proximal tubular cells.5 ~# c4 h0 S5 }% n1 V6 k5 k) O9 p
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The lack of p21 WAF1/CIP1 is associated with the acceleration ofcaspase activation and apoptosis. Caspase proteases play an important role inapoptosis by degrading specific structural, regulatory, and DNA repairproteins within the cell. Caspase-3 is one of the key executioners ofapoptosis and is responsible for the proteolytic cleavage of proteinsessential for cell survival. The best known pathways of caspase-3 activationare those through proteolytic cleavage of the caspase-3-inactive zymogen byinitiator caspases, caspase-8 and caspase-9. Caspase-8 is recruited afteractivation of receptors belonging to a family of "deathreceptors." Caspase-9 is activated as a result of mitochondrialdysfunction and release of cytochrome c from the mitochondrialintermembrane space. Activated caspase-8 and caspase-9 initiate theproteolytic activity of other downstream caspases, including caspase-3 andcaspase-6. Therefore, the activation of caspase-8 or caspase-9 precedesactivation of caspase-3 during caspase-8- or caspase-9-initiated apoptosis. While cisplatin-induced apoptosis in MPTC is associated with activation ofcaspase-3, caspase-8 is not cleaved to the enzymatically active form despiteincreases in IETD-ase activity that accompany apoptosis in MPTC in thepresence or absence of p21 WAF1/CIP1. Therefore, we conclude that 1 ) the increases in IETD-ase activity were not due to caspase-8, 2 ) caspase-8 is not activated by cisplatin in MPTC, and 3 )caspase-3 is not activated by caspase-8.

Similarly, our data show that caspase-9 was not processed to the activeform in p21( / ) MPTC treated with cisplatin. Therefore, the increases inLEHD-ase activity at 18 h of cisplatin exposure were not due to caspase-9activation and caspase-3 activation occurred without caspase-9. In contrast,caspase-9 was cleaved in cisplatin-treated p21(-/-) MPTC and caspase-9processing preceded caspase-3 activation. These results suggest the sequential activation of caspase-9 and caspase-3 in p21(-/-) MPTC but not in p21( / )MPTC after cisplatin exposure. These data are somewhat in contrast to theresults of Kaushal and colleagues( 14 ), who demonstrated theactivation of caspase-8, caspase-9, and caspase-3 by cisplatin inLLC-PK 1 cells. This discrepancy may be due to differences betweenimmortalized cell lines and primary cultures, including differences incellular metabolism. It is also possible that caspase-8 and caspase-9 inwild-type 18 hof exposure). However, similar to our results, Kaushal et al. reported thatthe activation of caspase-3 preceded that of caspase-8 and caspase-9 andconcluded that caspase-3 activation may be initiated by a caspase-independentpathway or caspases other than caspase-8 and caspase-9.# p) x" s! z4 y3 C- C
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Our results suggest that caspase-3 activation by cisplatin in MPTC may bepartially regulated through a p21 WAF1/CIP1 -dependent mechanismbecause the lack of functional p21 WAF1/CIP1 protein acceleratescaspase-3 activation. This is in agreement with reports thatp21 WAF1/CIP1 overexpression suppresses caspase activation andapoptosis ( 47, 48 ). However, despitecaspase-3 activation during cisplatin-induced apoptosis, caspase-3 does notappear to be involved in the execution of apoptosis in either p21( / ) MPTC orp21(-/-) MPTC. This conclusion is based on the observation that a caspase-3inhibitor, DEVD-fmk, blocked caspase-3 activation but did not prevent MPTCphosphatidylserine externalization, chromatin condensation, and cell shrinkage (Figs. 5 and 7; data not shown). Therefore,we conclude that caspase-3 is activated by cisplatin but is not required for cisplatin-induced apoptosis in MPTC. Caspase-3-independent apoptosis has beenreported in other models, including X-irradiation-induced apoptosis in ratembryonic fibroblasts ( 16 ) andcisplatin-induced apoptosis in rabbit primary cultures of renal proximaltubular cells ( 4, 32 ). Furthermore, geneticdeletion of caspase-3 and caspase-9 does not alter apoptosis duringembryogenesis and fetal development in mice, further supporting the existence of caspase-3- and caspase-9-independent mechanisms of apoptosis ( 35 ). Apoptosis induced by avariety of stimuli in human breast cancer MCF-7 cells bypasses the need forcaspase-3 activation and proceeds through the activation of caspase-7 andcaspase-6 ( 24 ).

We determined whether other caspases mediate cisplatin-induced apoptosis inMPTC and whether p21 WAF1/CIP1 plays a role in these events. Ourresults demonstrate that the broad spectrum caspase inhibitor ZVAD-fmk had noprotective effects against cisplatin-induced phosphatidylserineexternalization, chromatin condensation, and cell shrinkage in MPTC expressingfunctional p21 WAF1/CIP1 despite completely inhibiting caspase-3activation. These observations suggest that caspases do not play a role inapoptosis in MPTC expressing p21 WAF1/CIP1. In contrast, incisplatin-treated MPTC lacking p21 WAF1/CIP1, ZVAD-fmk decreased cell shrinkage (data not shown) and nuclear fragmentation by 51% but did notoffer any protection against phosphatidylserine externalization. These resultssuggest that the lack of p21 WAF1/CIP1 results in the activation ofa ZVAD-fmk-sensitive caspase that accelerates and potentiatescisplatin-induced chromatin and nuclear condensation but notphosphatidylserine externalization. Because caspase-9 is activated bycisplatin in MPTC lacking p21 WAF1/CIP1 and inhibited by ZVAD-fmk,mitochondria-dependent activation of caspase-9 may be involved in mediatingnuclear changes. Furthermore, these results suggest that phosphatidylserine externalization and nuclear condensation are mediated by different mechanisms.However, ZVAD-fmk provided only 50% protection against cell shrinkage andnuclear fragmentation, which suggests that 50% of cisplatin-induced apoptosisin MPTC lacking p21 WAF1/CIP1 is caspase independent. In contrast,cisplatin-induced apoptosis in MPTC expressing functionalp21 WAF1/CIP1 was entirely caspase independent.
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An alternate mechanism for the antiapoptotic effect ofp21 WAF1/CIP1 may be through the inhibition of the activation of thecyclin A-cyclin-dependent kinase 2 (Cdk2) complex( 13 ). DNA damage by cisplatinand cell cycle progression in injured cells is blocked by the binding offunctional p21 WAF1/CIP1 to the cyclin A-Cdk2 complex and inhibitionof Cdk2 activity. Caspase-3-mediated cleavage of p21 WAF1/CIP1 prevents inhibition of the cyclin A-Cdk2 complex and leads to apoptosis( 13 ). In kidney cells, thelack of p21 WAF1/CIP1 leads to progression from the G 1 to S phase of the cell cycle after cisplatin-induced DNA damage and cell injuryin vivo ( 28 ). Similarly, inprimary culture of MPTC, the lack of p21 WAF1/CIP1 increased thenumber of cells in S phase [17% in p21(-/-) MPTC vs. 3% in p21( / ) MPTC] and G 2 /M phase [15% in p21(-/-) MPTC vs. 2% in p21( / ) MPTC] of thecell cycle at 18 h of cisplatin exposure (data not shown). In contrast,overexpression of p21 WAF1/CIP1 or transfection of cells with ap21 WAF1/CIP1 mutant resistant to caspase-3 cleavage suppressescyclin A-Cdk2 activity and subsequent apoptosis ( 13 ). Caspase inhibitorsDEVD-fmk and ZVAD-fmk did not block cisplatin-induced cell cycle progressionto G 2 /M phase in MPTC lacking p21 WAF1/CIP1 (data notshown).
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In conclusion, this study shows that cisplatin treatment in wild-type MPTCactivates caspase-3 through a caspase-8- and caspase-9-independent pathway andinduces apoptosis. However, caspase activation is not the mechanism by whichcisplatin induces apoptosis in MPTC expressing functionalp21 WAF1/CIP1. Lack of p21 WAF1/CIP1 stimulates caspase-9activation and accelerates caspase-3 activation in cisplatin-induced MPTCapoptosis, which suggests that p21 WAF1/CIP1 functions, in part,through decreasing activation of caspase(s). Cisplatin-induced apoptosis inthe absence of p21 WAF1/CIP1 is caspase-3 independent but dependent, in part, on other caspase(s). Our study also suggests that some pharmacological agents activate caspases and induce apoptosis but producenephrotoxicity through mechanisms other than caspase-mediated proteolysis.
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ACKNOWLEDGMENTS+ O! f/ Z! }! a" L* |; ?2 g9 s
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We thank Dr. Philip Leder (Harvard Medical School, Boston, MA) forproviding several heterozygous mice carrying thep21 WAF1/CIP1 gene deletion and for providing a probe forscreening and Dr. Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD) for cloned mouse p21 WAF1/CIP1 cDNA. We also thank Ashley B.Whitlow (UAMS, Little Rock, AR) for assistance with flow cytometry.# O# p: t" K' D8 @/ b  @) S
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