Dependence of Cisplatin-Induced Cell Death In Vitro and In Vivo on Cyclin-Dependent Kinase 2
Peter M. Price*,,
Fang Yu,
Philipp Kaldis,
Eiman Aleem,
Grayna Nowak,
Robert L. Safirstein*, and
Judit Megyesi*,
Departments of * Internal Medicine, Physiology, and Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas; and Mouse Cancer Genetics Program, National Cancer Institute, Frederick, Maryland
Address correspondence to: Dr. Peter M. Price, Department of Medicine, University of Arkansas for Medical Sciences, VA Medical Center-Research Section, 4300 West 7th Street, Room GC 147, Little Rock, AR 72205. Phone: 501-257-4800; Fax: 501-257-4822; E-mail: PricePeterM{at}uams.edu
Received for publication February 21, 2006.
Accepted for publication June 29, 2006.
Cisplatin is one of the most effective chemotherapeutics, butits usefulness is limited by its toxicity to normal tissues,including cells of the kidney proximal tubule. The purpose ofthese studies was to determine the mechanism of cisplatin cytotoxicity.It was shown in vivo that cisplatin administration induces upregulationof the gene for the p21 cyclin-dependent kinase (cdk) inhibitorin kidney cells. This protein is a positive effector on thefate of cisplatin-exposed renal tubule cells in vivo and invitro; adenoviral transduction of p21 completely protected proximaltubule cells from cisplatin toxicity. Herein is reported thatcdk2 inhibitory drugs protect kidney cells in vivo and in vitro,that transduction of kidney cells in vitro with dominant-negativecdk2 also protected, and that cdk2 knockout cells were resistantto cisplatin. The cdk2 knockout cells regained cisplatin sensitivityafter transduction with wild-type cdk2. It is concluded thatcisplatin cytotoxicity depends on cdk2 activation and that themechanism of p21 protection is by direct inhibition of cdk2.This demonstrated the involvement of a protein that previouslywas associated with cell-cycle progression with pathways ofapoptosis. It also was demonstrated that this pathway of cisplatin-inducedcell death can be interceded in vivo to prevent nephrotoxicity.
Cisplatin is one of the most effective chemotherapeutic agentsagainst testicular and bladder tumors, head and neck, ovarian,breast, and lung cancers, and refractory non-Hodgkin’slymphomas (1,2). The major adverse effect of cisplatin use isnephrotoxicity, in which kidney proximal tubule cells are especiallysensitive (3). It is likely that its anticancer activity dependson formation of DNA intrastrand cross-links (4). Several distinctmechanisms have been proposed for cisplatin cytotoxicity inrenal tubule cells, including direct DNA damage (5), caspaseactivation (6), mitochondrial dysfunction (7), formation ofreactive oxygen species (8), effects on the endoplasmic reticulum(9), and activation of TNF- apoptotic pathways (10). However,it is unclear whether cisplatin nephrotoxicity depends on anyof these pathways or these apoptotic cascades merely amplifymore proximal initiated cell death signals.
We have shown in vivo that kidney cells entered the cell cycleafter cisplatin administration and that the gene for the p21Cip1/WAF1cell-cycle inhibitor was induced simultaneously (11). The p21protein interacts with several members of the cell cycle toregulate cell-cycle progression (12–14), and its inductionis a positive effector on the fate of renal tubule cells bothin vivo and in vitro (15,16). In addition, we recently reportedthat cells cultured from mouse proximal tubules were completelyprotected from cisplatin cytotoxicity by adenoviral transductionof human p21 (17). The activity of cyclin-dependent kinase 2(cdk2), a serine/threonine kinase whose main function is thephosphorylation of substrates necessary for cell-cycle progression(reviewed in reference [18]), is inhibited by binding with p21.We now report that cisplatin cytotoxicity in vitro depends oncdk2 activity and that both functional and morphologic nephrotoxicityin vivo can be prevented using cdk2-inhibitory drugs. The dependenceof the apoptotic pathway on cdk2 was demonstrated by (1) usingseparate interactive moieties of p21, in which only the cdk2binding region effectively protected (19); (2) using differentcdk2 inhibitory drugs (17); (3) using a transducing cdk2 dominantnegative adenovirus; and (4) using cdk2 knockout cells. Theseresults demonstrated that induction of cisplatin cytotoxicityin kidney cells depends on cdk2 activity and can be preventedboth in vitro and in vivo by cdk2 inhibition.
Animals and Administration of Drugs
Experiments were performed on 10- to 12-wk-old wild-type 129Svmice that weighed 22 to 28 g. The mice were maintained on astandard diet, and water was available freely. Cisplatin wasadministered by a single intraperitoneal injection of 20 mg/kg,a dosage that produces severe acute renal failure in mice (11).Some mice also received daily intravenous injections of purvalanol(Tocris Cookson, Inc., Ellisville, MO) at 30 mg/kg dissolvedat 4 mg/ml in a 5:7:13 (vol/vol/vol) mixture of DMSO:PEG400:10%Kollidon 17PF. Animals were killed painlessly with methods ofeuthanasia approved by the Panel on Euthanasia of the AmericanVeterinary Medical Association. The induction of acute renalfailure was monitored by following creatinine concentrationin serum that was obtained by retro-orbital bleeding using acommercial kit (Sigma Diagnostics No. 555, St. Louis, MO).
Morphologic Assessment
At various times after cisplatin treatment, kidneys were removed,immersed in 4% neutral-buffered formaldehyde, and fixed for48 to 72 h. The tissues were paraffin embedded and processedfor light microscopy. Sections of 5 µm were stained withhematoxylin-eosin and periodic acid-Schiff, and histologic criteriawere determined (20). The following parameters were chosen asindicative of morphologic damage to the kidney after cisplatininjection: Brush border loss, red blood cell extravasation,tubule dilation, tubule degeneration, tubule necrosis, and tubularcast formation. These parameters were evaluated as describedpreviously (20) on a scale from 0 to 4: Not present (0), mild(1), moderate (2), severe (3), and very severe (4). Each parameterwas determined on at least five different animals by a histologistwho was blinded to the source of the sections. Statistical significancewas assessed by the two-sided t test for independent samples.
Cell Culture
Cells were grown at 37°C in 5% CO2. Mouse kidney proximaltubule cells (TKPTS) (21) were cultured in DMEM + Ham’sF-12 medium supplemented with 50 µU/ml insulin and 7%FBS. Mouse embryonic fibroblasts (MEF) that were derived fromcdk2 knockout mice (22) were grown in DMEM with 10% FBS. Thesecells were obtained from cdk2 (–/–) mouse embryos(13.5 d postcoitus) and were spontaneously immortalized in a3T3 protocol after approximately 10 passages (22). Cisplatinwas added to cultures, when indicated, to a final concentrationof 25 µM (TKPTS) or 35 µM (MEF) when cells wereapproximately 75% confluent, and the cells were grown for anadditional 24 h. TKPTS and MEF that expressed wild-type cdk2had similar sensitivity to cisplatin; apoptotic cell death occurredto the same extent 24 h after exposure to 25 µM cisplatin.Adenoviruses were added to a final multiplicity of infectionof 100 18 h before cisplatin. Purvalanol and roscovitine weredissolved in DMSO and added 4 h before cisplatin to a finalconcentration of 9 or 45 µM, respectively. As reportedpreviously (17), roscovitine extended the cell cycle, ratherthan causing cell-cycle arrest. Purvalanol had a similar effecton the TKPTS. Except as noted, culture conditions followed thesame schedule: Cultured cells were maintained for 30 h aftersplitting before adenovirus was added, cell-cycle inhibitorswere added at 44 h, cisplatin was added at 48 h, and treatmentswere as indicated. The cells were harvested 72 h after splitting.
Cell Death Determination FACS Analysis.
Cells were harvested by trypsinization, pooled with the culturemedium that contained floating cells, and collected by centrifugation(10 min, 500 x g). The cell pellets were resuspended in 0.3ml of PBS that contained 5 mM EDTA, and 0.7 ml of ethanol wasadded. Cells were incubated at 4°C for 16 h, collected bycentrifugation (10 min, 2000 rpm), and resuspended in 0.5 mlof PBS-EDTA. RNase A was added (50 µl, 10 mg/ml) and thesuspension was incubated at 25°C for 30 min. Propidium iodidewas added (450 µl, 100 µg/ml), and the samples wereanalyzed using FACSCalibur (Becton Dickinson, Rockville, MD).At least 10,000 cells were analyzed for each culture condition(Figures 2 and 3), and values (in Figure 2) were based on threeseparate cultures. The percentage of cells in sub-G1/G0 (apoptoticfraction) (17,23), G1/G0, S, and G2/M phases was determinedusing a cell-cycle analysis program (WinMDI 2.8; Scripps Institute,San Diego, CA). The cells in the subdiploid (sub-G1/G0) regionof the histogram are classified as apoptotic, probably as aresult of the generation and loss of low molecular weight DNAfragments during apoptosis. These analyses were performed onat least four separate cultures.
Figure 2. Bar graph of FACS analyses for apoptosis (percentage of cells in sub-G1/G0 fraction). Values represent means (± SE) of cells that were treated with and without 24 h of cisplatin (as indicated), using either TKPTS (lanes 1 through 6) or mouse embryonic fibroblasts (MEF) that were derived from cdk2 knockout mice (lanes 7 through 9). In addition to possible cisplatin exposure, cells were untreated (lanes 1 and 7); treated with 100 multiplicity of infection adenovirus that expressed p21 (lane 2), wild-type cdk2 (lanes 5, 8, and 9), or DN-cdk2 (lane 6); treated with 45 µM roscovitine (Lane 3); or treated with 9 µM purvalanol (lanes 4 and 9), as described in Materials and Methods.
Figure 3. Representative analyses for apoptosis. Cells (TKPTS) were untreated (A, C, and E) or treated with 25 µg/ml cisplatin for 24 h (B, D, and F). (A and B) Light microscopy of cells before harvesting (scale indicated). (C and D) Hoechst 33258 staining of formaldehyde-fixed cells that were grown on coverslips (scale indicated; typical apoptotic nuclei indicated by arrows); (E and F) FACS analysis of propidium iodide–stained ethanol-fixed cells. Cell-cycle analysis (cells in G0 + G1, S, and G2 + M phases) shown in first diagram.
Hoechst Staining.
Cells were grown on coverslips as described above. After timesas indicated, cells were rinsed with PBS, fixed for 5 min withneutral-buffered formaldehyde, and stained with Hoechst 33258(10 µg/ml in PBS) for 10 min. Under these conditions,Hoechst 33258 stains the nuclei of all cells blue when examinedby fluorescence microscopy using a DAPI filter. The nuclei ofapoptotic cells display chromatin condensation and/or nuclearfragmentation, whereas normal cell nuclei are stained homogeneously.Apoptosis was assessed in at least 300 cells using a fluorescencemicroscope with a DAPI filter and a x40 objective.
Light Microscopy.
Cells were photographed with an inverted microscope (Nikon EclipseTE200, Melville, NY) using Hoffman optics before harvesting.
Adenoviruses
The p21-expression adenovirus was obtained from Dr. Wafik El-Deiry(University of Pennsylvania, Philadelphia, PA [13]). Green fluorescenceprotein (GFP)-expression adenoviruses that expressed p21, p21fragments, and cdk2 were constructed in our laboratory accordingto protocols and materials supplied by Dr. Bert Vogelstein (JohnsHopkins, Baltimore, MD [24]). Human p21 cDNA was obtained fromDr. Bert Vogelstein (13); human cdk2 wild-type and cdk2 dominantnegative (DN-cdk2) cDNA were obtained from Dr. Sander van denHeuvel (Massachusetts General Hospital, Boston, MA [25]). Thewild-type and DN-cdk2 adenoviruses were constructed by insertionof a BamHI fragment that contained the cDNA for the proteininto the Bgl II site of the pAdTrack-CMV plasmid. After selectionfor clones with the proper orientation of cDNA insertion byrestriction analysis, adenoviruses were constructed as describedabove.
Western Blot Analysis
Proteins were extracted from TKPTS and MEF using a lysis bufferthat contained 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5% NP-40with phosphatase inhibitor I and II, and proteinase inhibitor(Sigma, St. Louis, MO). Western blot analyses were as describedpreviously (17,19). In brief, protein concentration was determinedusing a Bio-Rad protein assay (Hercules, CA). Protein (100 µg/lane)was electrophoresed using 12% SDS-polyacrylamide gel and transferredto polyvinylidene difluoride membrane. After blocking with 5%nonfat dry milk in TBST, the membrane was incubated at 4°Covernight with primary antibody. After washing, horseradishperoxidase–conjugated secondary antibody was applied.Proteins that bound to the secondary antibody were visualizedusing ECL (Amersham Biosciences, Piscataway, NJ).
Kinase Assay for cdk2
For assay of in vivo activity, kidneys were homogenized witha Teflon glass homogenizer in buffer that contained 10 mM HEPES(pH 7.6), 25 mM KCl, 1 mM EDTA, 10% glycerol, 1.8 M sucrose,0.15 mM spermine, 0.5 mM spermidine, 0.5 mM dithiothreitol,and phosphatase and proteinase inhibitors as described above.The homogenate was layered over 2.2 ml of this buffer, and nucleiwere pelleted by centrifugation at 24,000 rpm in an SW60 rotor.Nuclei from two mouse kidneys were resuspended in 1 ml of coldlysis buffer (as discussed in Western Blot Analysis), sonicated,and centrifuged for 10 min in an Eppendorf centrifuge. The supernatantwas used for histone H1 kinase activity (see next). For assayof in vitro activity, TKPTS were washed twice with PBS and lysedin cold lysis buffer (as discussed in Western Blot Analysis).Samples were kept on ice for 30 min and centrifuged for 30 minat 10,000 x g. The supernatant was used for histone H1 kinaseactivity (see next).
Kinase activity of cdk2 was assayed by a modified histone H1kinase assay (19,26,27). Briefly, protein extracts (200 µg)were immunoprecipitated by agarose-immobilized anti-cdk2 antibodyfor 4 h at 4°C with constant rocking and washed three timeswith lysis buffer and once with kinase buffer that contained20 mM Tris-Cl (pH 7.4), 10 mM MgCl2, and 1 mM dithiothreitol.Agarose beads were resuspended in 20 µl of kinase bufferthat contained 2 µg of histone H1, 20 µM ATP, and10 µCi -32P-ATP and incubated for 30 min at 30°C.Samples were boiled and electrophoresed by PAGE as describedabove and autoradiographed.
Correlation of cdk2 Activity with Cell Death after Cisplatin Exposure In Vitro and In Vivo
Cdk2 activity was determined in TKPTS and in mouse kidney cellnuclei before and after cisplatin treatment as a function ofhistone H1 kinase activity associated with an anti-cdk2 immunoprecipitate(Figure 1). The background cdk2 activity in TKPTS (Figure 1A,lane 1) was unaffected by transduction with a GFP-expressionadenovirus (lane 2) but was increased by treatment with cisplatin(lane 3). Transduction of cells with adenoviruses that expressedfull-length p21 (lane 4), the cdk2-inhibitory region of p21(lane 5), or DN-cdk2 (lane 7) before cisplatin exposure loweredcdk2 activity to below background levels. Similarly, treatmentwith purvalanol and cisplatin virtually eliminated cdk2 activity(lane 9). However, transduction with adenovirus that expresseda p21 protein that was missing the cdk2-inhibitory region (lane6) or wild-type cdk2 (lane 8) had no effect on the rise of cisplatin-inducedcdk2 activity. A similar effect on cdk2 activity by cisplatinand purvalanol also was found in vivo (Figure 1B). Without cisplatintreatment, a background level of cdk2 activity was measured(Figure 1B, lanes 1, 2, and 7). Treatment with cisplatin for24 or 48 h increased the cdk2 activity (lanes 3 and 5, respectively).Treatment with purvalanol without cisplatin (lane 2), with 24h of cisplatin (lane 4), or with 48 h of cisplatin (lane 6)lowered cdk2 activity to control levels, although not to theextreme extent that was found in vitro. A control in which immunoprecipitationwas performed without anti-cdk2 antibody did not contain anymeasurable kinase activity (lane 8).
Figure 1. (A) Cyclin-dependent kinase (Cdk2) activity in mouse kidney proximal tubule cells (TKPTS) correlates with cell death after cisplatin treatment. Background activity in untreated cells (lane 1), cells after transduction with a green fluorescence protein (GFP)-expression adenovirus (lane 2), or cells that were treated with 9 µM purvalanol (lane 9). Cells were treated for 24 h with cisplatin (lanes 3 through 9). In addition to cisplatin, cells were transduced with adenoviruses that expressed full-length p21 (lane 4), the cdk2-inhibitory region of p21 (lane 5), the C-terminal region of p21 missing the cdk2-inhibitory region (lane 6), dominant negative cdk2 (DN-cdk2; lane 7), or wild-type cdk2 (lane 8). (B) Cdk2 activity in mouse kidney nuclei after cisplatin treatment. Mice either were not treated with cisplatin (lanes 1, 2, and 7) or were treated with 20 mg/kg cisplatin (lanes 3 through 6) for 24 h (lanes 3 and 4) or 48 h (lanes 5 and 6). In addition, some mice received daily intravenous injections of purvalanol at 30 mg/kg (lanes 2, 4, and 6) as described in Materials and Methods. A control was included in which agarose beads that did not contain anti-cdk2 antibody was used for immunoprecipitation of extract (lane 8).
Protection of Cultured TKPTS by cdk2 Inhibition
Apoptosis was determined by FACS analysis (Figures 2 and 3)of propidium iodide–stained TKPTS, as described in Materialsand Methods. Morphologic changes were confirmed by light microscopy,and apoptotic changes in nuclear structure were confirmed byfluorescence microscopy of Hoechst 33258–stained cells(Figure 3).
TKPTS that were cultured for 72 h in the absence of cisplatinhad a low level of apoptosis (3.2 ± 0.85%) as determinedby FACS analysis (Figure 2, lane 1). Transduction with p21-adenoviruslowered the percentage of apoptotic cells to 0.76 ± 0.20%(Figure 2, lane 2). The addition of cdk inhibitors had littleeffect on background apoptosis; roscovitine slightly loweredthe level of apoptosis to 1.82 ± 0.74% (Figure 2, lane3), and purvalanol decreased the percentage of apoptotic cellsto 0.36 ± 0.10% (Figure 2, lane 4).
Administration of cisplatin in the absence of cdk inhibitionincreased apoptosis to 27.6 ± 4.6% of the cells (Figure 2,lane 1). However, addition of p21-adenovirus 18 h before cisplatintreatment or treatment with roscovitine or purvalanol 4 h beforecisplatin reduced the level of apoptosis to background levels:3.8 ± 1.0% (Figure 2, lane 2), 1.89 ± 0.74 (Figure 2,lane 3), and 0.8 ± 0.15% (Figure 2, lane 4), respectively.Addition of adenovirus-expressing GFP had no effect on the percentageof cells in apoptosis (data not shown).
Because the p21 protein had been reported to bind and repressseveral proteins with proapoptotic functions and chemical cdk2inhibitors may inhibit other kinases with potential proapoptoticfunctions, we transduced TKPTS with wild-type and DN-cdk2 todetermine whether specifically inhibiting cellular cdk2 activitywould protect from cisplatin cytotoxicity. Transduction usingadenovirus that expressed these cdk2 had no effect on the lowlevels of background apoptosis that was detected without cisplatintreatment (Figure 2, lanes 5 and 6). Similarly, wild-type cdk2had little effect on cytotoxicity in the presence of cisplatin,in which 22.6 ± 3.8% of the cells were apoptotic (Figure 2,lane 5). However, transduction with DN-cdk2 reduced the cisplatin-inducedapoptosis to 1.3 ± 0.5% (Figure 2, lane 6).
The recent demonstration of the viability of mice that are homozygoticfor a knockout of the cdk2 gene showed that this kinase is notrequired for cell-cycle progression (22,28). Embryonic fibroblaststhat were derived from cdk2 knockout mice were resistant tocisplatin, in which apoptosis occurred in only 2.0 ±0.4% of cells (Figure 2, lane 7). To determine whether thisrelative insensitivity to cisplatin cytotoxicity depended onthe lack of cdk2, the cells were transduced with an adenovirusthat expressed wild-type cdk2. This converted the cells to cisplatinsensitive, in which cisplatin induced apoptosis in 18.3 ±3.8% of the cells (Figure 2, lane 8). Because exposure of cellsto adenovirus per se also can induce apoptosis, we confirmedthat the increased sensitivity to cisplatin was induced by cdk2and not by adenovirus by exposing these cells to cdk2 inhibitor(purvalanol), which lowered the cisplatin-induced apoptosisto 1.3 ± 0.3% in the presence of transduced wild-typecdk2 (Figure 2, lane 9). We also confirmed that the MEF knockoutcells reacted to cisplatin by inducing p21 (Figure 4) to thesame extent that TKPTS are stimulated, even though these knockoutcells largely are protected from cisplatin cytotoxicity.
Figure 4. Induction of p21 in cultured cells. Cdk2 knockout MEF were untreated (lane 1), or treated with 25 µM cisplatin for 24 h (lane 2). TKPTS were treated with 25 µM cisplatin for 24 h (lane 3) as a positive control for p21 induction. Proteins were electrophoresed using 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane. After blocking with 5% nonfat dry milk in TBST, membranes were incubated at 4°C overnight with primary antibodies to p21 and tubulin. After washing, horseradish peroxidase–conjugated second antibody was applied. Proteins that bound to the second antibody were visualized using ECL. Tubulin (55 kD) was used as a loading control. The 21-kD p21 and 55-kD tubulin are indicated.
Cdk2 Inhibition Ameliorates Cisplatin Nephrotoxicity
We next determined whether a similar mechanism of cdk2 dependencewas responsible for cisplatin-induced nephrotoxicity in vivo.Purvalanol (0.8 mg) was injected into the tail vein of mice(129/Sv, male, 22 to 28 g) followed by cisplatin injection (intraperitoneally,20 mg/kg) 24 h later. Mice were given purvalanol daily and killed1, 2, or 3 d after cisplatin injection. Creatinine (Figure 5)and blood urea nitrogen (BUN) levels were determined using serumthat was collected from the retro-orbital vein. Mice that receivedcisplatin but were not treated with purvalanol had elevatedcreatinine levels at day 2 (1.33 ± 0.11) and day 3 (2.17± 0.19). Mice that were treated with purvalanol cdk2inhibitor had significantly lower creatinine levels at day 2(0.55 ± 0.10; P = 0.0005) and day 3 (0.66 ± 0.22;P = 0.0008). In fact, creatinine levels in the mice that weretreated with cisplatin plus cdk2 inhibitor were not significantlydifferent from levels in untreated control mice (P = 0.12 andP = 0.19 at days 2 and 3, respectively). The rise in BUN alsowas significantly reduced by purvalanol (control 28.3 ±1.7, day 2 88.6 ± 4.1 versus 67.5 ± 6.1 [P = 0.01];day 3 149.8 ± 3.5 versus 75.9 ± 13.6 [P = 0.0007]comparing mice without and with purvalanol, respectively). After5 d of cisplatin, creatinine levels in purvalanol-treated micestill were within the normal range, whereas creatinine levelsin mice without purvalanol treatment were starting to decline.Similarly, BUN levels in both populations were declining (datanot shown).
Figure 5. Serum creatinine levels after cisplatin administration. Values, in milligrams per deciliter, at each time point represent means (± SE) of at least six mice. Comparing statistical differences between non–purvalanol-treated mice and untreated control, P = 0.00005 at day 2 and P = 0.000015 at day 3; between non–purvalanol- and purvalanol-treated mice, P = 0.0005 at day 2 and 0.0008 at day 3; between purvalanol-treated mice and untreated control, P = 0.12 at day 2 and 0.19 at day 3.
Necrosis also was markedly reduced in purvalanol-treated mice,showing that both kidney function and morphology (Figure 6)were protected. An assessment of the morphologic differences(Figure 7) demonstrated that all measured parameters of nephrotoxicitywere lowered by purvalanol, most significantly necrosis, redblood cell extravasation, and tubular dilation. The quantitativemorphology shown in Figure 7 seems to depict more severe injurythan illustrated by the morphology displayed in Figure 6 orby the functional injury (creatinine) in Figure 5. In the cisplatin+ purvalanol group, one mouse had areas of the kidney that wereinjured much greater than we observed in other mice from thisgroup. Although the morphologic injury to the kidney was notsevere enough to affect physiologic function (BUN and creatininelevels), inclusion of these data affected quantitative morphologyvalues, although there still were statistical differences betweentreated and untreated groups. Because this damage was not typicalin the cisplatin + purvalanol group, it was not illustratedin Figure 6.
Figure 6. Morphology of kidney 72 h after cisplatin injection. Representative sections of cisplatin treated (top) or purvalanol + cisplatin treated (bottom). Scale is indicated.
Figure 7. Morphologic evaluation of mouse kidneys 72 h after cisplatin injection expressed on a scale from 0 to 4. Values represent means (± SE) of kidney sections from at least five mice from each treatment. Statistically significant differences are indicated: *P = 0.00001; **P = 0.02; ***P = 0.00009.
We have reported that p21 is highly induced in kidney aftercisplatin administration and that p21 induction by itself isprotective (20). Therefore, we investigated whether purvalanolinduced p21 and whether purvalanol protection depended on p21by using p21 knockout mice. Purvalanol did not induce p21 higherthan background in wild-type mice that were not treated withcisplatin (data not shown), and the cdk2 inhibitor protectedp21 knockout mice from cisplatin nephrotoxicity (BUN after 3d of cisplatin: 154.5 ± 8.1 without purvalanol; 106.6± 16.5 with purvalanol; P = 0.010), showing that thisinhibitor is not dependent on p21 induction to protect kidneycells in vivo. The BUN values after cisplatin treatment werehigher in p21 knockout than those that we found in p21-competentmice, probably because (1) p21 knockout mice are more sensitiveto cisplatin-induced nephrotoxicity (20) and (2) purvalanolis not as effective in eliminating cdk2 activity in vivo asit is in vitro (compare Figure 1B, lanes 1 and 2, with Figure 1A,lanes 1 and 9), although it lowers cdk2 activity (compare Figure 1B,lanes 3 and 4 or lanes 5 and 6) so that p21 induction, combinedwith purvalanol treatment, more efficiently lowers cdk2 activity.
We previously reported that induction of the p21 cdk inhibitorin kidney after acute renal failure ameliorates the nephrotoxiceffects of both cisplatin administration and ischemia-reperfusion(15,20). Using cultured cells that were derived from mouse kidneyproximal tubules, we also demonstrated that p21 transductionand cdk2 inhibitors before cisplatin exposure could protectfrom cytotoxicity (17). We now demonstrate that cisplatin exposurecaused an increase in cdk2 activity, both in vitro and in vivo(Figure 1), that the mechanism of cisplatin cytotoxicity dependson cdk2 activity, and that protection from cisplatin toxicityby p21 is directly dependent on cdk2 inhibition (Figure 2).This mechanism was confirmed by the following evidence: (1)Several different cdk2-inhibitory drugs and proteins are protective,(2) transduction using DN-cdk2 is cytoprotective, and (3) cdk2knockout cells are not sensitive to doses of cisplatin thatcause apoptosis in wild-type cells.
The expression of p21 has been associated with both protectionfrom and induction of apoptosis (17,29, and references within).Similarly, cell-cycle inhibitory drugs such as roscovitine,olomoucine, and purvalanol, which are potent and selective inhibitorsof cell-cycle kinases cdk2 and cdc2, prevent apoptosis in avariety of cell types but induce apoptosis in others (17,30and references within). The spectrum of kinases inhibited bythese agents include not only cdk2 but also cdk1 (cdc2) andcdk5 (27,31–35). Because of the similarity of the proteins,they also are likely to inhibit cdk3, although this was notinvestigated. In addition, the p21 protein interacts with severalother proteins through other domains (36). The cdk2 interactivedomain was localized between amino acids 49 and 71 (37) by deletionanalysis and 49 and 79 (38) by crystallography. We have reported(19) that a 54–amino acid fragment of p21 (amino acids38 to 91) binds and inhibits cdk2 and also protects from cisplatincytotoxicity. It therefore was likely that cdk2 inhibition wasa primary mechanism for protection by p21 and cdk2 inhibitorydrugs, but solely on the basis of these results, other kinaseinhibition could not be ruled out.
The cDNA and the gene for the cdk2 protein have been isolated(39), and have led to the development of a DN-cdk2 (40) thatconsists of a single amino acid change that inactivates thephospho-transfer reaction. These DN-cdk2 mutants have providedprotection from growth factor deprivation–induced apoptosisin human endothelial cells (41), staurosporine- or TNF-–inducedapoptosis in HeLa cells (42,43), and from hypoxia-induced apoptosisin cardiomyocytes (44). At the same time, these results do notprovide definite proof of the involvement of cdk2 in the apoptoticpathway. First, DN-cdk1 and -cdk3 have provided similar protectionfrom apoptosis as DN-cdk2 (42,43), opening the possibility thatthese dominant negative mutants could inhibit other kinases.It also was possible that these other mutants bound and therebylowered the availability of cdk2-interacting cyclins, whichare required for cdk2 activity. Second, neither geneticallyremoving the cdk2 gene (22,28) nor inhibiting cdk2 with siRNAor antisense oligonucleotides (40) arrests the cell cycle, butDN-cdk2 prevents growth of several cell types in culture (25,45).Growth inhibition by DN-cdk2 could arise by inhibition of activitiesother than cdk2. Because of these uncertainties, we have complementedour results in which DN-cdk2 protected from cisplatin apoptosisby examining cdk2 knockout cells for cisplatin sensitivity.These cells were protected from apoptosis in the presence ofcisplatin that causes death in wild-type cells (Figures 2 and3). The dependence of the apoptotic pathway on cdk2 was establishedfurther by adenoviral transduction of the knockout cells withwild-type cdk2, which converted them to cisplatin sensitive.Furthermore, we demonstrated that wild-type cdk2 and not adenoviralinfection was stimulating the cisplatin-induced cell death bytreating the cells with cdk2 inhibitor, which protected themfrom cisplatin cytotoxicity (Figures 2 and 3).
These experiments provided definitive proof of the dependenceof the cisplatin-induced apoptosis pathway on cdk2 in vitro.However, most of the cell death in vivo in kidney after cisplatinadministration is morphologically characterized as necrosis.Recent hypotheses have proposed that necrosis could proceedby pathways similar to those described for apoptosis (46–48)and that the type of cell death that is induced by DNA alkylatingagents is a "necrotic form of programmed cell death" (49). Wetherefore extended our investigations to determine whether cdk2inhibition would protect from cisplatin nephrotoxicity in vivo.As shown in Figure 3, the loss of kidney function that is associatedwith cisplatin administration and results in an increase inserum creatinine was almost completely abolished; creatininelevels in mice that were treated with purvalanol and cisplatinwere not significantly elevated from levels in untreated controlmice. Also, the cdk2 inhibitor protected from the morphologicdamage that was caused by cisplatin (Figures 6 and 7) in whichthere was a readily apparent decrease in cellular necrosis inkidneys of treated mice. It therefore is possible that cdk2activity is necessary for many different pathways of apoptosisin vitro and also for similar pathways of necrosis in vivo.
We report that treatment with purvalanol prevented elevationof serum creatinine after cisplatin treatment, so these levelswere statistically indistinguishable from untreated controlvalues. At the same time, BUN levels were elevated, althoughnot to the extent in mice that did not receive purvalanol. Thereare several possible explanations for this discrepancy. First,cisplatin causes systemic toxicity in animals that is not limitedto nephrotoxicity (50), which may affect blood urea levels morethan GFR. Organs and/or cells types other than kidney couldbe less protected by purvalanol from cisplatin toxicity. Second,creatinine is more of a reliable estimate of GFR than is BUN(51), which can be affected by hydration, diet, and other typesof nonrenal tissue trauma.
Many different cancers are susceptible to cisplatin chemotherapy,and cdk inhibitors currently are undergoing clinical trialsas anticancer agents (52,53). Because of the beneficial effectsof cdk inhibitors on the nephrotoxic adverse effects of cisplatinuse, combined with their intrinsic chemotherapeutic potential,cisplatin and cdk2 inhibitors should naturally complement eachother clinically and possibly augment each other’s beneficialeffects as chemotherapeutics. In addition, the use of a cdk2inhibitor to ameliorate the effects of acute renal failure invivo mimics the activity of the p21 protein, which is naturallyinduced in kidney cells as a protective response to renal injuryand stress. The sensitivity of cdk2 to inactivation in vitroby purvalanol likely is much greater than cdk2 inactivationin vivo. This is evident in comparing Figure 1A, lane 9 (invitro inactivation), with Figure 1B, lane 2, 4, or 6 (in vivoinactivation), even though background in vitro activity (Figure 1A,lane 1) is greater than background in vivo activity (Figure 1B,lane 1 or 7). This probably is a result of the efficiency ofdelivery of the drug to cells in the kidney and/or partial inactivation/bindingof the drug before it can be effective. The cytotoxicity ofcisplatin in vitro can be controlled effectively by cdk2 inhibition,by drugs, proteins, or genetic manipulation. Development ofmore efficient means of cdk2 inhibition in vivo is likely toprovide effective therapy from cisplatin nephrotoxicity.
Acknowledgments
This work was supported in part by research grants from theNational Institutes of Health (R01 DK54471 and P01 DK058324)and the Biomedical Research Foundation and with resources andthe use of facilities at the John L. McClennan Memorial Veterans’Hospital (Little Rock, AR).
We thank Dr. Wafik El-Deiry (University of Pennsylvania MedicalSchool) for initially providing the p21-adenovirus, Dr. BertVogelstein (Johns Hopkins University School of Medicine) forproviding the clone of human p21 cDNA and adenoviral constructionmaterials and protocols, Drs. Sander van den Heuvel and Ed Harlow(Massachusetts General Hospital) for providing clones of humanwild-type and DN-cdk2 cDNA, Dr. Elsa Bello-Reuss (Universityof Texas Medical Branch) for providing the TKPTS, and CyrilBerthet for discussion and support. We also thank Kimberly Henningand Wasson Snow for valuable assistance during the in vivo druginjections.
Footnotes
Published online ahead of print. Publication date availableonline at www.jasn.org.
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Abstract
Introduction
apoptotic pathways (10). However, it is unclear whether cisplatin nephrotoxicity depends on any of these pathways or these apoptotic cascades merely amplify more proximal initiated cell death signals. 
-32P-ATP and incubated for 30 min at 30°C. Samples were boiled and electrophoresed by PAGE as described above and autoradiographed. 
