[CANCER RESEARCH 60, 2464–2472, May 1, 2000]

A Role for the p38 Mitogen-activated Protein Kinase Pathway in theTranscriptional Activation of p53 on Genotoxic Stress byChemotherapeutic Agents1

Ricardo Sanchez-Prieto, Jose M. Rojas,2 Yoichi Taya, and J. Silvio Gutkind3

Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland 20892-4330 [R. S-P., J. M. R., J. S. G.], and NationalCancer Center Research Institute, Tokyo 104, Japan [Y. T.]

ABSTRACT

The tumor suppressor p53 plays a central role in sensing damaged DNAand orchestrating the consequent cellular responses. However, how DNAdamage leads to the activation of p53 is still poorly understood. In thisstudy, we have found that the p38 mitogen-activated protein kinase(MAPK) plays a key role in the activation of p53 by genotoxic stress whenprovoked by chemotherapeutic agents. Indeed, we found that blockade ofp38 prevents stimulation of the transcriptional activity of p53 and thatactivation of the p38 pathway is sufficient to stimulate p53 function.Furthermore, we observed that p38 does not affect the accumulation ofp53 in response to DNA damage or its nuclear localization. In contrast, weobserved that p38 associates physically with p53, and we provide evidencethat this MAPK phosphorylates the NH2-terminal transactivation domainof p53 in serine 33, thereby stimulating its functional activity. Moreover,inhibition of the p38 MAPK diminished the apoptotic fraction of cellsexposed to chemotherapeutic agents and increased cell survival, thussuggesting a role for p38 activation in the apoptotic response to genotoxicstress when elicited by drugs used in cancer therapy.

INTRODUCTION

The protein product of thep53 tumor suppressor gene plays a keyrole in orchestrating many of the biological responses elicited by theexposure of cells to genotoxic stress, including those caused bychemotherapeutic agents or radiation (1, 2). The function of thisnuclear phosphoprotein now appears to be controlled by a complexregulatory network (3). Indeed, a number of proteins participate in theregulation of p53 by increasing or decreasing the expression of p53messages or the stability of newly synthesized p53 protein (4), bycontrolling its subcellular localization (5), or by posttranslationalmodification of p53, including acetylation and phosphorylation (6–8).For example, genotoxic stress leads to the rapid accumulation of p53by decreasing its ubiquitin-dependent degradation (9) and stimulatesp53 function by the phosphorylation of a number of NH2-terminal andCOOH-terminal serine and threonine residues. This leads to the acti-vation of several transcriptional targets of p53, including the cyclin-dependent kinase inhibitor p21WAF (10), which is implicated in theblockade of the cell cycle (11), thus allowing the cells to assessthe extent of the DNA damage and initiate the repair mechanism or,if the damage is extensive, to trigger an apoptotic response (12). Thelatter has been shown to include p53-dependent as well as p53-independent processes (13).

In addition, genotoxic stress stimulates the activity of a family ofprotein kinases closely related to MAPKs4 termed stress-activatedprotein kinases, which includes JNK and p38 family members. Per-haps the most studied members among them have been the JNKs,which can be potently activated by genotoxic stress induced by UV,g-radiation, and chemotherapeutic drugs (14). Of interest, it has beenrecently shown that JNK is able to phosphorylate p53 (15), therebyenhancing its protein stability (16, 17). However, there is no clearevidence with regard to whether JNK is the only mediator of p53activation in response to genotoxic stress (1). Moreover, recentlyavailable reports suggest that JNK and p53 are independently acti-vated by genotoxic stress (18) and that JNK activation does notalways lead to an increase in the activity of p53 (19). Thus, althoughJNK may play an important role in the regulation of p53 stability,these observations raised the possibility that additional signalingmolecules may participate in the enhanced transcriptional activity ofp53 in response to genotoxic stress.

In this study, we found that p38 plays a key role in the activationof p53 by genotoxic stress when provoked by DNA-damaging che-motherapeutic agents. We demonstrate that in NIH 3T3 cells, p38 canbe potently activated by drugs that cause DNA damage by eitherpromoting the formation of DNA adducts or inhibiting topoisomeraseII, but not by therapeutically relevant doses ofg-radiation, and that theblockade of p38 by chemical inhibitors prevent the transcriptionalactivation of p53 by these anticancer drugs. This effect was found notto be related to changes in the amount or localization of the p53protein. Instead, we found that p38 phosphorylates p53 in its NH2-terminal transactivating domain at serine 33, one of the residuespreviously described to be phosphorylated in response to DNA dam-age. We also found that activation of p38 by upstream molecules issufficient to stimulate p53 and that this response requires the phos-phorylation of p53 in serine 33. Moreover, we observed that theblockade of p38 diminishes the apoptotic response to anticanceragents, thus increasing the survival of the treated cells. Taken to-gether, these findings suggest a critical role for p38 in p53 activationthrough the phosphorylation of an NH2-terminal regulatory residue,serine 33, and in the apoptotic response to genotoxic stress whenelicited by chemotherapeutic agents.

MATERIALS AND METHODS

Drugs and Treatments. CDDP and DOX (Sigma) were dissolved indistilled water and used immediately, and the p38 inhibitors SB 203580 andSKF 86002 (Calbiochem) were dissolved in DMSO and stored at220°C as a10003concentrated stock solution. Forg-irradiation and UV irradiation, weused a gamma cell (dose rate, 2.5 Gy/min) and a stratalinker (Stratagene).

Cell Lines. NIH 3T3 fibroblasts were maintained in DMEM (Life Tech-nologies, Inc.) supplemented with 10% calf serum. 293T and Saos-2 cells weremaintained in DMEM supplemented with 10% fetal bovine serum.

Received 11/2/99; accepted 3/6/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 R. S-P. was partially supported by the North Atlantic Treated Organization ScienceProgram. J. M. R. was supported by Grants FIS-BAE 98/5093 and FIS 98/1336 from theInstituto de Salud Carlos III, Spain.

2 Present address: Unidad de Biologı́a Celular, Centro Nacional de Biologı́a Funda-mental, Instituto de Salud, Carlos III carretera Majadahonda-Pozuelo, Km 2 Majada-honda, 28220 Madrid, Spain.

3 To whom requests for reprints should be addressed, at Oral and Pharyngeal CancerBranch, National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive,Building 30, Room 211, Bethesda, MD 20892-4330. Phone: (301) 496-6259; Fax: (301)402-0823; E-mail: sg39v@nih.gov.

4 The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, c-Jun-NH2-terminal kinase;b-gal, b-galactosidase; DOX, doxorubicin; CDDP, cisplatin; CAT,chloramphenicol acetyltransferase; GST, glutathioneS-transferase; HA, hemagglutinin;wt, wild-type; MKK6, MAPK kinase 6.

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Reporter Gene Assays.NIH 3T3 cells and Saos-2 cells were transfected intriplicates either by the calcium-phosphate precipitation technique or withLipofectAMINE (Life Technologies, Inc.), with the indicated expression andreporter plasmids, together with pCDNAIIIb-gal as a control for transfectionefficiency. After 24 h, cells were left untreated or treated with either DOX orCDDP for the indicated times and doses in the presence or absence of p38inhibitors, which were added 40 min before drug or physical treatment. Cellswere then lysed using reporter lysis buffer (Promega) and processed for CAT,luciferase, andb-gal activity, as reported previously (20). CAT and luciferaseactivity were normalized by theb-gal activity in each sample and expressed asthe fold increase with respect to control cells.

Kinase Assays.Cells were transfected by LipofectAMINE Plus Rea-gent according to the manufacturer’s instructions (Life Technologies, Inc.)with the different expression plasmids. The phosphorylating activity of anepitope-tagged p38a MAPK or its mutant, T106M, was assessed as de-scribed previously (20), using 1.5mg/ml myelin basic protein (Sigma) or 5mg of purified, bacterially expressed GST-p53 fusion proteins as substrates,as indicated. Samples were analyzed by SDS-gel electrophoresis on acryl-amide gels, and autoradiography was performed with the aid of an inten-sifying screen.

Western Blot Analysis and Antibodies. Cells were analyzed by Westernblotting after SDS-PAGE using different antibodies. Rabbit polyclonal antiserato phospho-p38 protein was purchased from New England Biolabs, and anti-p38 and p21WAF serums were from Santa Cruz Biotechnology. Monoclonalantibodies against p53 were from Oncogene Science (Ab-1) or Chemicon(UCN-1), the anti-6-His tag was from Sigma, and the anti-HA tag was fromBabco (clone HA 11). Phospho-specific purified antibody to serine 33 of p53has been described previously (21). All antibodies were used according to themanufacturer’s instructions. Immunocomplexes were visualized by enhancedchemiluminescence detection (Amersham Corp.) using goat antimouse IgGs orantirabbit IgGs coupled to horseradish peroxidase as secondary antibodies(Cappel).

Immunofluorescence.NIH 3T3 cells were treated with different stimulifor the indicated times, washed twice with PBS, fixed with 4% formaldehydeand 5% sucrose in PBS for 10 min, and permeabilized with 0.5% Triton X-100in PBS for 10 min. The cells were incubated with Ab-1 anti-p53 antibody(Oncogene Science) for 2 h, washed three times with PBS, and then incubatedwith a 1:100 dilution of fluorescein-conjugated goat F(ab9)2 IgG antimouseantibody (Jackson ImmunoResearch Laboratories, Inc.). Coverslips weremounted in Gel-mount (Biomeda Corp., Foster City, CA) and then examinedusing a Carl-Zeiss Confocal microscope.

Plasmids. pG13 CAT, pG13 Luc, and pCMVp53 were kindly supplied byB. Vogelstein (Howard Hughes Medical Institute, The Johns Hopkins Univer-sity, Baltimore, MD). p38a tagged with HA and MKK6 has been describedpreviously (20). HA-p38 in the expression vector pCEFL was used as atemplate to generate a mutant p38 in which Thr106 was changed to Met usingoligonucleotides by the PCR overlapping extension technique. The PCR prod-uct was then cloned into pCEFL as a HA tag. GST-fusion proteins includingthe NH2-terminal 86 or 126 amino acids of p53 were generated by subcloningthe corresponding coding region amplified by PCR from pCMVp53 into thepGEX expression vector (Pharmacia). GST-p53 (1–86) mutants in residues 15,33, and 46 were generated using specific primers in which serine or threoninewas substituted for alanine, using the Quickchange site-directed mutagenesiskit (Stratagene). Bacterially expressed proteins were purified using standardtechniques. Expression plasmids for 6 His-tagged p53 were generated bysubcloning the coding region of p53 obtained by PCR amplification into thepEF1/His C expression vector (Invitrogen). For the serine 33 mutant of p53,the mutated fragment from pGEX-p53 (1–86) A33 was used to replace thecorresponding sequence in wt p53 in pEF1/His C, using aBamHI site upstreamfrom the ATG initiation codon and an internalSgrI site in position 241 of thep53 coding sequence. All mutations were confirmed by sequencing.

Flow Cytometry and Viability Assay. Attached and nonadherent cellswere collected, fixed in 70% ethanol, washed in PBS, and stained withpropidium iodide (25mg/ml). Samples were analyzed on a FACScan (BectonDickinson). Apoptosis was evaluated as the population of cells in the sub-G0-G1 peak. Viability was evaluated by the crystal violet method (22).

RESULTS

JNK has been shown to regulate the stability of the p53 protein inMCF-7 cells (16, 17). However, in preliminary experiments, weobserved that in NIH 3T3 cells, which express low levels of wt p53,the direct stimulation of JNK by the expression of activated upstreammolecules, such as MAPK kinase kinase 1 (23), did not result in theactivation of the transcriptional activity of p53 when assessed using atandem of 13 p53-responsive elements linked to the CAT gene as areporter plasmid (Ref. 24; data not shown). Similarly, we did notobserve a decrease in the transcriptional response of p53 in responseto genotoxic stress when JNK activity was blocked by overexpressionof JNK-inhibitory protein, which sequesters JNK in the cytosoliccompartment, although JNK-inhibitory protein abolishes the activa-tion of JNK-specific responses, as we and others have shown previ-ously (Refs. 20 and 25; data not shown). Thus, these observationsprompted us to explore whether additional stress-activated proteinkinases could participate in signaling p53 activation in response togenotoxic stress in this cellular system. We began by exploringwhether distinct DNA-damaging agents can activate p38. For theseexperiments, we used chemotherapeutic drugs that act by causing theformation of DNA adducts, such as CDDP (26), or inhibiting topoi-somerase II, such as DOX (27), and other agents such asg-radiationthat cause DNA damage by promoting the formation of DNA double-strand breaks (28). As shown in Fig. 1, exposure of NIH 3T3 cells totwo distinct chemotherapeutic drugs, CDDP and DOX, caused adramatic increase in the level of active p38, which was detectable 2and 1 h after treatment, respectively. In contrast, lethal doses ofg-radiation (20 Gy) did not cause a consistent activation of p38(,2-fold induction), as has been described previously (29). Theseobservations were further confirmed by assessing the enzymatic ac-tivity of p38 using bacterially expressed GST-activating transcriptionfactor 2 as a substrate (data not shown).

Because these stimuli are known to activate p53, we examined theexpression of the cyclin-dependent kinase inhibitor p21WAF, one ofthe best-characterized targets of p53 (10), as an approach to evaluatethe functional activity of this tumor suppressor gene under theseexperimental conditions. As shown in Fig. 1A, both CDDP and DOXprovoked a detectable increase in the level of expression of p21WAF

3–4 h after treatment, which remained elevated even after 24 h (datanot shown). Similarly,g-radiation induced the expression of p21WAF,which was detectable as early as 2 h after treatment (Fig. 1A). Incontrast, the total amount of p38 did not change with any of thetreatments and also served as a loading control. Thus, these resultssuggest that p38 is activated in NIH 3T3 cells in response to chemo-therapeutic drugs, such as CDDP and DOX, and that this activationprecedes the enhanced expression of p21WAF. However, the lack ofactivation of p38 byg-radiation indicates that this kinase is not auniversal sensor for genotoxic stress.

As an approach to investigate whether p38 affects p53 function, wetook advantage of the availability of two p38-specific inhibitors, SB253080 and SKF 86002 (30, 31). As shown in Fig. 1B, the incubationof the cells in the presence SB 253080 prevented the increase ofp21WAF in response to CDDP, and very similar results were obtainedwith SKF 86002 (data not shown). In contrast, the treatment with SB253080 did not affect the elevation of p21WAF expression provokedby g-radiation (Fig. 1C), suggesting that p38 may participate insignaling to p53 in response to chemotherapeutic DNA-damagingagents, but not when DNA damage is caused byg-radiation. Interest-ingly, when Western blots for p53 were performed, we observed thatthe blockade of p38 did not cause any demonstrable effect on theaccumulation of p53 protein elicited by these treatments (Fig. 1B and

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the text below), thus indicating that p38 does not affect the proteinlevels of p53.

We next set out to investigate whether p38 affects the transcrip-tional activity of p53 in response to chemotherapeutic agents, using asa reporter system the pG13 CAT plasmid, whose expression is con-trolled by a tandem of p53-responsive elements (24). As shown in Fig.2A, exposure of cells to CDDP and DOX resulted in a remarkableincrease in the transcriptional activity of p53 (Fig. 2A), with DOXdemonstrating a greater response. When the p38 inhibitor SB 203580was added, we observed that the transcriptional activation of p53 byCDDP was nearly abolished, whereas the response to DOX waslargely diminished (Fig. 2B). To control for the specificity of thisapproach and to exclude the possibility that the p38 inhibitor affectsadditional molecules under our assay conditions, we took advantageof the observation that the replacement of threonine 106 with methi-onine renders p38a insensitive to SB 253080 (32). We engineeredsuch an epitope-tagged mutant of p38a and confirmed that SB 253080prevents thein vitro activity of p38, but not that of the p38 Met106

mutant (Fig. 2C). Furthermore, as shown in Fig. 2D, whereas SB253080 abolished the p53 response in CDDP-treated NIH 3T3 cells,this response was nearly restored by expression of the inhibitor-insensitive form of p38a (Fig. 2D). Thus, taken together, these dataindicate that p38a participates in the activation of p53-dependentpathways in response to chemotherapeutic agents. Furthermore, be-cause SB 253080 did not abolish the response to DOX, it is alsopossible that this drug might stimulate additional SB 253080-insen-sitive isoforms of p38, such as p38g and p38d (32), a possibility thatis under current investigation.

We next decided to examine the mechanism by which p38 affects thefunction of p53. p38 does not appear to regulate the protein levels of p53because the accumulation of p53 in response to CDDP and DOX was notaffected by the presence of p38 inhibitors (Fig. 1B). Thus, one possiblemechanism might involve the control of the nuclear translocation of p53,a process that is essential for the transcriptional activity of p53 (33). Toaddress this possibility, we performed immunofluorescence analysis ofp53 using confocal microscopy in cells treated with CDDP or DOX in the

Fig. 1. Activation of p38 by DNA-damagingtherapeutic agents.A, subconfluent plates of NIH3T3 cells were treated with CDDP (15mg/ml), DOX(1 mg/ml), or exposed tog-radiation (20 Gy; doserate, 2.5 Gy/min) and lysed at the indicated times. InB and C, cells were pretreated with 0.1% DMSO(control) or SB 203580 (20mM) 40 min beforetreatment with CDDP org-radiation and every 4 h.In all cases, total cell lysates (100mg) were resolvedby SDS-PAGE and immunoblotted with anti-P-p38,p38, p21WAF, and p53 serum or antibodies, as indi-cated. Autoradiograms are from a representative ex-periment that was repeated three times with nearlyidentical results.

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presence or absence of p38 inhibitors. As shown in Fig. 3, the p38inhibitors did not affect the accumulation and localization of the p53tumor suppressor gene product, thus indicating that p38 or its down-stream targets do not participate in the biochemical route regulating thetranslocation of p53 to the nucleus.

In search of a putative mechanism by which p38 participates in theactivation of p53 in response to genotoxic stress induced by chemo-therapeutic drugs, we explored whether these proteins interact phys-ically. For these experiments, we immunoprecipitated p38 from NIH3T3 cells transfected with an epitope-tagged p38 and performed aWestern blot analysis using anti-p53 antibodies. As shown in Fig. 4A,p53 coimmunoprecipitates with p38, but not when p38 is activated byCDDP or UV radiation (control). Thus, inactive p38 can bind to p53,but activation of p38 appears to cause the release of p53 from thecomplex. Similar results have been reported for JNK (16). The func-tional significance of this event is under investigation. Nonetheless,our previous results using p38 inhibitors indicated that p38 does notregulate the level and localization of p53, thus suggesting that anadditional mechanism might participate in the regulation of p53by p38.

Because our results indicated that p38 affects the transcriptionalactivity of p53, we asked whether p53 could be phosphorylated byp38, using as a substrate a GST-fusion protein containing the first 86or 126 amino acids of p53, an area including the p53 transactivationdomain (34). As shown in Fig. 4B, activated p38 phosphorylates bothGST-fusion proteins with very high efficiency. These resultsprompted us to investigate which residue(s) are phosphorylated byp38 in the most NH2-terminal 86 amino acids. Serine 15, which hasbeen recently shown to be phosphorylated by the AT gene (35, 36),was used as a control. Because p38 is a proline-targeted kinase, wemutated all serines or threonines adjacent to prolines in this region.Three residues, serine 33, serine 46, and threonine 81, represent suchpotential targets. When using these mutant GST-p53 proteins assubstrates, we observed that the replacement of serine 33 with alanineabolishes the ability of the NH2-terminal domain of p53 to serve as anin vitro substrate for p38 (Fig. 4C). All other mutant proteins behavedas did the wt p53. Thus, the serine 33 residue in p53 represents a likelycandidate as a phosphoacceptor for the enzymatic activity of p38. Onthe basis of these results, we generated a full-length p53 cDNAincluding a mutation in serine 33 (p53 S33A). wt p53 and its S33A

Fig. 2. p38 inhibitors decrease the p53-dependent transcriptional response to chemotherapeutic drugs.A, NIH 3T3 cells were transfected with 0.5mg of pG13 CAT and 1mg ofpCDNAIII b-gal by using the calcium-phosphate precipitation technique. The cells were then incubated with the indicated doses of CDDP or DOX for 16 h and assayed for CATactivity. B, cells transfected with 0.5mg of pG13 CAT and 1mg of pCDNAIII b-gal were left untreated (control) or incubated with CDDP (5mg/ml) or DOX (0.5mg/ml) for 12 h,in the absence (c) or presence of SB 203580 (20mM; SB-80). SB 203580 was added 40 min before treatment with CDDP or DOX and every 4 h. CAT activities were normalized bytheb-gal activity in each sample and expressed as the fold induction with respect to control, untreated cells. Data represent the mean6 SE of triplicate samples from a representativeexperiment that was repeated four independent times.C, 293T cells were transfected with wt HA-p38 (p38; 2mg) or with HA-p38T106M (p38T106M; 2 mg). Thirty min after UVstimulation (12 J/m2 ), cells were collected in lysis buffer, and anti-HA immunoprecipitates were assayed for kinase activity in the absence (control) or presence of SB 203580 (5mM),as indicated.D, NIH 3T3 cells were transfected with 0.25mg of pG13 CAT and 1mg of pCDNAIII b-gal plus 0.5mg/plate of wt p38 or p38T106M. Cells were treated with CDDPin the presence or absence of SB 203580, as described inB. Fold induction was calculated with respect to the corresponding untreated controls. Similar results were obtained in threeadditional experiments.

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mutant were then subcloned into a tagging expression vector (pECF/6His). As shown in Fig. 4D, both wt p53 and p53 S33A weredetectably expressed, as judged by Western blotting with antibodiesagainst the 6His epitope. Furthermore, an anti-phospho-serine 33-specific antibody detected the wt 6His-tagged p53 but not its S33Amutant, thus indicating that serine 33 of p53 can be phosphorylatedinvivo and further supporting the specificity of this antibody. However,mutation of p53 in serine 33 did not affect its basal transcriptionalactivity when expressed in p53-null cells, such as Saos-2 cells(Fig. 4E).

To explore whether serine 33 participates in the transcriptionalactivation of p53 by genotoxic chemotherapy, we transfected both wtand S33A p53 constructs into NIH 3T3 cells. As shown in Fig. 4F,expression of wt p53 increased the basal p53-dependent transcrip-tional activity in NIH 3T3 cells and caused a remarkable increase inthe reporter activity in response to CDDP. In contrast, the S33A

mutant form of p53 also enhanced the basal activity in these cells butdisplayed only a limited response to CDDP when compared with thewt p53. These results support the importance of serine 33 in theactivation of p53 by genotoxic agents such as CDDP.

To examine whether p38a activation is sufficient to stimulate p53function, we transfected NIH 3T3 cells with an increasing amount ofits upstream activator, MKK6 (37, 38). A clear dose-response effectwas observed on the activity of p53, thus indicating that the stimula-tion of p38a is sufficient to enhance the activity of the endogenousp53 (Fig. 5A). Using a similar approach, we examined the role ofserine 33 in the activation of p53 by the MKK6-p38 pathway. Toavoid the background response due to endogenous p53, we chose touse Saos-2 cells for these experiments. As shown in Fig. 5B, cotrans-fection of p38a and MKK6 induced a remarkable increase in thetranscriptional response to p53. However, activation of the p38 path-way provoked a very limited activation of the p53 S33A mutant.

Fig. 3. Blockade of p38 does not affect the accumulation of p53 in the nucleus after genotoxic stress. NIH 3T3 cells were plated on sterile cover slides 24 h before treatment withCDDP (15mg/ml) or DOX (1 mg/ml) for 8 or 4 h, respectively, in the presence or absence of SB 203580 or SKF 86002 (20mM). The immunofluorescence analysis was performedusing a monoclonal antibody against p53 (Ab-1), and samples were analyzed using a confocal microscope.Bar, 10 mM.

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Similar results were obtained in NIH 3T3 cells (data not shown).Furthermore, the use of the p53 phospho-serine 33-specific antibodyrevealed that thein vivo phosphorylation of this p53 residue increaseson CDDP treatment and that this response can be inhibited by the useof p38 blockers (Fig. 5C). These findings strongly suggest that p38acan activate p53 directly and that transcriptional activation of p53 byp38a involves the phosphorylation of p53 in serine 33.

Because of the key role of p53 in apoptosis in response to genotoxicstress (2, 12), we analyzed the role of p38 in the apoptotic response ofNIH 3T3 cells to CDDP. Cells were treated with CDDP for 24 h, inthe presence or absence of SB 203580 or SKF 86002 for 12 h, and cell

cycle was analyzed by flow cytometry. As shown in Fig. 6A,control cells exposed to CDDP displayed an apoptotic fraction of20 –25%, whereas this fraction was reduced to 10 –13% by treat-ment with p38 inhibitors. These results were further supported bya cell viability assay, which helped demonstrate that the treatmentwith p38a inhibitors resulted in a 2-fold increase in the IC50 ofCDDP (Fig. 6B), a remarkable increase that can be regarded assignificant in terms chemoresistance. This decrease in the effec-tiveness of CDDP suggests that p38a is necessary for the correctexecution of the apoptotic program initiated by p53 after genotoxicstress induced by CDDP.

Fig. 4. Phosphorylation of the transactivatingdomain of p53 in serine 33 by p38: evidence for arole in p53 activation by DNA-damaging agents.A,NIH 3T3 cells were transfected by the Lipo-fectAMINE method with expression plasmids forHA-p38 (1mg) or green fluorescent protein (1mg),as indicated, and treated with CDDP (15mg/ml) orUV (40 J/m2). Four h later, cells were lysed. An-ti-HA immunoprecipitates were immunoblottedagainst p53 or HA, as indicated.B, 293T cells weretransfected with plasmids encoding HA-p38 (2mg)with or without MKK6 (2 mg). Samples were col-lected and processed for kinase assays using as asubstrate 5mg of GST-p53 fusion proteins or my-elin basic protein.C, GST-p53 (1–86) proteins, wtor mutated in residues 15, 33, and 46, were purifiedand used as a substrate for p38 kinase assays.Western blot anti-GST confirmed the equal loadingof the samples.D, expression of 6His-tagged p53and its serine 33 mutant was confirmed by Westernblotting using an anti-epitope antibody after trans-fection of 293T cells. The status of phosphorylationin serine 33 was confirmed using a p53 phospho-serine 33-specific antibody. Similar results wereobtained in a number of cell types (data not shown).E, plasmids for epitope-tagged p53 and its serine 33mutant or green fluorescent protein were trans-fected into Saos-2 cells (1mg) together with 0.5mgof pG13 Luc and 1mg of pCDNAIII b-gal. Lucif-erase activity was normalized for theb-gal activityin each sample and represented as the fold induc-tion with respect to control cells transfected with anempty vector.F, NIH 3T3 cells were transfectedwith plasmids for the wt or S33A mutant of p53(0.5 mg), pG13CAT (0.25mg), andb-gal (1 mg).The transcriptional response to CDDP (5mg/ml)was evaluated as indicated. Values are expressed asthe ratio between CAT activity (cpm) andb-galactivity (A420 nm) in triplicate samples to depict thetranscriptional response under each experimentalcondition. Similar results were obtained in fiveindependent experiments.

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DISCUSSION

The tumor suppressor p53 is believed to play a central role insensing damaged DNA and in dictating the nature of the consequentcellular responses. As such, a variety of genotoxic stresses result inthe rapid activation of p53. As shown in this study and in other studies(1, 2, 39), these stresses include those provoked by DNA double-strand breaks, by the formation of thymidine dimers or DNA adducts,or by inhibition of topoisomerase II. However, very distinct molecularmechanisms appear to participate in p53 activation by each of theseDNA-damaging agents. The activation of p53 by phosphorylation inresponse tog-radiation and UV radiation has been extensively inves-tigated among these agents. For example, serines 15, 33, and 37 havebeen shown to be phosphorylated by both stimuli (6, 7). However,there are some differences in the phosphorylation pattern of p53,including the status of phosphorylation on serine 392, which is onlyphosphorylated in response to UV light (40, 41). Much less is under-stood about the role of p53 phosphorylation in the activation of thep53 pathway on genotoxic stress when induced by other classes of

DNA-damaging agents, including the many widely used chemother-apeutic drugs.

Interestingly, we found that treatment of cells with CDDP andDOX, which are frequently used for the treatment of cancer patients,can cause the sustained activation of p38a, a member of MAPKsuperfamily of proline-targeted serine/threonine protein kinases. Incontrast, lethal doses ofg-radiation failed to stimulate p38a activity.Thus, activation of p38a appears not to result from DNA damage butto be triggered in response to genotoxic stress provoked specificallyby the formation of DNA adducts and by inhibition of topoisomeraseII. The molecular mechanisms responsible for this selective activationare still unclear and are being actively investigated. Nonetheless, theavailability of specific p38a inhibitors (30, 31) afforded the possibil-ity of exploring its contribution to the cellular responses to genotoxicstress. Indeed, blockade of p38 revealed that this kinase is necessaryfor the activation of p53-dependent transcription in response to che-motherapeutic agents, as judged by the remarkable inhibition of theaccumulation of p21WAF and the activation of reporter systems by thetreatment with p38a inhibitors. In contrast, blockade of p38a did notprevent the increase in p21WAF expression provoked byg-radiation,thus further supporting the specificity of this approach. Together,

Fig. 5. Activation of p38 is sufficient to stimulate p53: a role for serine 33.A, NIH 3T3cells were transfected with increasing amounts of a 1:1 mixture of plasmids encoding p38and MKK6 (ranging from 0.05–0.5mg) plus 0.1mg of pG13 CAT and 0.5mg of b-galper plate. After 24 h, cells were lysed, and CAT activity was measured. Values areexpressed as the ratio between CAT activity (cpm) andb-gal activity (A420 nm) intriplicate samples.B, Saos-2 cells were transfected with 0.1mg of plasmid for wt p53 (WT)or the mutant p53 (S33A) plus 0.1mg of pG13 Luc and 0.5mg of b-gal plasmids andMKK6 and p38 (0.1mg each;1) or with empty vector as a control (2). Twenty-four hafter transfection, cells were lysed and processed for luciferase assays. Data are expressedas the fold induction with respect to control (2) transfected cells.C, 293T cells weretreated with 50mg/ml CDDP for the indicated times in the presence or absence of 20mM

SKF 86002. Cell extracts were collected and analyzed by Western blot using a p53phospho-serine 33-specific antibody.

Fig. 6. Blockade of p38 diminishes the apoptotic response to chemotherapeutic agentsand increases cell survival.A, NIH 3T3 cells were left untreated (2) or treated with CDDP(10 mg; 1) for 24 h in the absence (control) or presence of the p38 inhibitors SB 203580(SB-80; 20mM) or SKF 86002 (SKF; 20mM). The fraction of cells undergoing apoptosiswas evaluated by flow cytometry and expressed as a percentage of total cells. Similarresults were obtained in two additional experiments.B, cells were treated with CDDP(dose, 1–15mg/ml) in the absence (NIH 3T3) or presence of SB 203580 (NIH 3T31SB8O; 20mM) or SKF 86002 (NIH 3T31 SKF; 20mM), as indicated. Cell viability wasassayed after 2 days using the crystal violet method and expressed as a percentage ofcontrol, untreated cells. The p38 inhibitors alone did not display any significant differencewith respect to control cells. The SEs in triplicate samples were lower than the symbolsize. Similar results were obtained in three independent experiments.

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these results support a role for p38a in the response to genotoxicstress caused by chemotherapeutic agents, likely by activating p53function.

A role in p53 activation has been proposed recently for anotherstress-activated kinase family member, JNK (16, 17). In this case, theinactive form of JNK was found to bind p53 and to diminish thecellular pool of p53 by targeting its degradation (16, 17). On activa-tion, JNK appears to dissociate from p53, thus enhancing the stabilityof the newly synthesized p53 protein. However, activation of JNKalone does not appear to be sufficient to increase p53 activity becauseno increase was detected in the transcriptional activity of p53 onexpression of molecules such as MAPK kinase kinase 1 that effec-tively stimulate JNK-dependent transcription in our cellular system(20). In contrast, activation of p38a is itself sufficient to increase theactivity of the endogenous p53 in cells expressing wt p53, such asNIH 3T3 cells, and on expression of wt p53 in a p53-null background,such as that seen in Saos-2 cells (42). p38a was also found tocoimmunoprecipitate with p53; however, in this case, we did notobtain any evidence that p38a can affect the level of p53 protein or itsintracellular distribution or that blockade of p38a affects the accu-mulation of p53 in the nucleus on genotoxic stress. In contrast, weobtained evidence that p53 could be a relevant substrate for p38a.

Transcriptional activation of the tumor suppressor p53 is oftenachieved by phosphorylation of key regulatory residues (6, 43). In-deed, extensive phosphorylation on the NH2-terminal transactivatingdomain of p53 has been reported in response to physical or chemicallyinduced damage to the DNA, thus providing a mechanism by whichp53 can act as a universal sensor for DNA damage (1). For instance,serine 15 is phosphorylated in response tog-radiation by the productof the AT gene (35, 36) and by others members of the AT-relatedprotein family (44), thus stimulating p53. Similarly, DNA-proteinkinase (PK) may be involved in the transcriptional activation of p53through phosphorylation in serine 15 (45), although this issue stillremains unclear (46, 47). Serine 37 may be also targeted for phos-phorylation, and several candidate kinases have been proposed, in-cluding ATR or DNA-PK (44, 48). Serine 33 has also been describedas a target for kinase activity after DNA damage, and although thisresidue can be phosphorylatedin vitro by JNK and the cyclinH-CDK7-p36 MAT complex (21, 49, 50), the identity of the actualkinase acting on p53 serine 33 in response to DNA damage remainselusive (8, 51). In this regard, several lines of evidence suggest thatp38 can phosphorylate the transactivating domain of p53 in serine 33.In vitro, p38 can phosphorylate GST-fusion protein containing theNH2-terminal, transactivating domain of p53, and mutational analysisrevealed that among all candidate residues, serine 33 was the phos-phoacceptor site. To confirm the relevance of thesein vitro data, wereconstituted a p53 with a mutation in residue serine 33. Under basalconditions, we did not detect any significant difference between thismutant form and the wt p53 in expression level or ability to stimulateexpression from reporter plasmids, as reported previously (52). How-ever, the serine 33 mutant of p53 failed to respond transcriptionally toCDDP treatment and to its direct activation of p38 in both wt p53 andp53-null cellular backgrounds. Taken together, these data stronglysuggest that the serine 33 residue of p53 is a biologically relevanttarget for the enzymatic activity of p38.

Another residue, serine 392, has also been described recently as asite for p38a phosphorylation (53, 54). However, this site is notadjacent to a proline residue, thus representing an unlikely candidatefor direct phosphorylation by proline-targeted kinases, including p38.Furthermore, phosphorylation of this site was only observed after UVirradiation of cells (40, 41), a condition that might activate a numberof additional kinases, including the double-stranded RNA activatedprotein kinase (PKR) (55), which has previously been described as a

candidate to phosphorylate this particular residue. Thus, although wecannot exclude the possibility that p38 may also directly or indirectlyphosphorylate additional residues in p53, including serine 392, theavailable evidence suggests that these putative events might not besufficient to activate p53 in the absence of serine 33.

In summary, our work demonstrates that the activation of p38a andthe subsequent phosphorylation, at least in residue 33, is a criticalevent in the response of p53 to genotoxic stress and provides the firstevidence that p38 might represent the highly sought after DNAdamage-induced p53 serine 33 kinase. Furthermore, the use of spe-cific p38a inhibitors revealed that interfering with this kinase dimin-ishes apoptosis and enhances the viability of cells exposed to chemo-therapeutic agents. These findings are in line with the pivotal role ofp53 in the cytotoxic response to drugs such as CDDP or DOX andfurther support a key role for p38a in the stimulation of p53 inresponse to these DNA-damaging agents. Interestingly, these resultsalso suggest that p38a should be explored as a putative mechanism toexplain chemoresistance. Further work will be necessary to fullyelucidate the molecular mechanism leading to the activation of p38aby genotoxic stress and to investigate the likely clinical consequencesof these findings in the search for novel approaches to improve cancertherapy.

ACKNOWLEDGMENTS

We appreciate the comments of C. Murga, V. Patel, S. Fukuhara, M.Chiarello, S. Pece, H. Miyazaki, M. J. Marinisen, S. Montaner, A. Sodhi, andA. Senderowicz. We also appreciate the assistance and advice of L. Vitale-Cross and Dr. B. Swain.

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