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

The protein product of the p53 tumor suppressor gene plays a key role in orchestrating many of the biological responses elicited by the exposure of cells to genotoxic stress, including those caused by chemotherapeutic agents or radiation (1, 2). The function of this nuclear phosphoprotein now appears to be controlled by a complex regulatory network (3). Indeed, a number of proteins participate in the regulation of p53 by increasing or decreasing the expression of p53 messages or the stability of newly synthesized p53 protein (4), by controlling its subcellular localization (5), or by posttranslational modification of p53, including acetylation and phosphorylation(6, 7, 8). For example, genotoxic stress leads to the rapid accumulation of p53 by decreasing its ubiquitin-dependent degradation(9) and stimulates p53 function by the phosphorylation of a number of NH2-terminal and COOH-terminal serine and threonine residues. This leads to the activation of several transcriptional targets of p53, including the cyclin-dependent kinase inhibitor p21WAF(10), which is implicated in the blockade of the cell cycle (11), thus allowing the cells to assess the extent of the DNA damage and initiate the repair mechanism or, if the damage is extensive, to trigger an apoptotic response (12). The latter has been shown to include p53-dependent as well as p53-independent processes(13).

In addition, genotoxic stress stimulates the activity of a family of protein kinases closely related to MAPKs4termed stress-activated protein kinases, which includes JNK and p38 family members. Perhaps the most studied members among them have been the JNKs, which can be potently activated by genotoxic stress induced by UV, γ-radiation, and chemotherapeutic drugs (14). Of interest, it has been recently shown that JNK is able to phosphorylate p53 (15), thereby enhancing its protein stability(16, 17). However, there is no clear evidence with regard to whether JNK is the only mediator of p53 activation in response to genotoxic stress (1). Moreover, recently available reports suggest that JNK and p53 are independently activated by genotoxic stress (18) and that JNK activation does not always lead to an increase in the activity of p53 (19). Thus, although JNK may play an important role in the regulation of p53 stability,these observations raised the possibility that additional signaling molecules may participate in the enhanced transcriptional activity of p53 in response to genotoxic stress.

In this study, we found that p38 plays a key role in the activation of p53 by genotoxic stress when provoked by DNA-damaging chemotherapeutic agents. We demonstrate that in NIH 3T3 cells, p38 can be potently activated by drugs that cause DNA damage by either promoting the formation of DNA adducts or inhibiting topoisomerase II, but not by therapeutically relevant doses of γ-radiation, and that the blockade of p38 by chemical inhibitors prevent the transcriptional activation of p53 by these anticancer drugs. This effect was found not to be related to changes in the amount or localization of the p53 protein. Instead,we found that p38 phosphorylates p53 in its NH2-terminal transactivating domain at serine 33,one of the residues previously described to be phosphorylated in response to DNA damage. We also found that activation of p38 by upstream molecules is sufficient to stimulate p53 and that this response requires the phosphorylation of p53 in serine 33. Moreover, we observed that the blockade of p38 diminishes the apoptotic response to anticancer agents, thus increasing the survival of the treated cells. Taken together, these findings suggest a critical role for p38 in p53 activation through the phosphorylation of an NH2-terminal regulatory residue, serine 33, and in the apoptotic response to genotoxic stress when elicited by chemotherapeutic agents.

Drugs and Treatments.

CDDP and DOX (Sigma) were dissolved in distilled water and used immediately, and the p38 inhibitors SB 203580 and SKF 86002(Calbiochem) were dissolved in DMSO and stored at −20°C as a 1000×concentrated stock solution. For γ-irradiation and UV irradiation, we used a gamma cell (dose rate, 2.5 Gy/min) and a stratalinker(Stratagene).

Cell Lines.

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

Reporter Gene Assays.

NIH 3T3 cells and Saos-2 cells were transfected in triplicates either by the calcium-phosphate precipitation technique or with LipofectAMINE(Life Technologies, Inc.), with the indicated expression and reporter plasmids, together with pCDNAIII β-gal as a control for transfection efficiency. After 24 h, cells were left untreated or treated with either DOX or CDDP for the indicated times and doses in the presence or absence of p38 inhibitors, which were added 40 min before drug or physical treatment. Cells were then lysed using reporter lysis buffer(Promega) and processed for CAT, luciferase, and β-gal activity, as reported previously (20). CAT and luciferase activity were normalized by the β-gal activity in each sample and expressed as the fold increase with respect to control cells.

Kinase Assays.

Cells were transfected by LipofectAMINE Plus Reagent according to the manufacturer’s instructions (Life Technologies, Inc.) with the different expression plasmids. The phosphorylating activity of an epitope-tagged p38α MAPK or its mutant, T106M, was assessed as described previously (20), using 1.5 μg/μl myelin basic protein (Sigma) or 5 μg of purified, bacterially expressed GST-p53 fusion proteins as substrates, as indicated. Samples were analyzed by SDS-gel electrophoresis on acrylamide gels, and autoradiography was performed with the aid of an intensifying screen.

Western Blot Analysis and Antibodies.

Cells were analyzed by Western blotting after SDS-PAGE using different antibodies. Rabbit polyclonal antisera to phospho-p38 protein was purchased from New England Biolabs, and anti-p38 and p21WAF serums were from Santa Cruz Biotechnology. Monoclonal antibodies 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 from Babco (clone HA 11). Phospho-specific purified antibody to serine 33 of p53 has been described previously (21). All antibodies were used according to the manufacturer’s instructions. Immunocomplexes were visualized by enhanced chemiluminescence detection(Amersham Corp.) using goat antimouse IgGs or antirabbit IgGs coupled to horseradish peroxidase as secondary antibodies (Cappel).

Immunofluorescence.

NIH 3T3 cells were treated with different stimuli for the indicated times, washed twice with PBS, fixed with 4% formaldehyde and 5%sucrose in PBS for 10 min, and permeabilized with 0.5% Triton X-100 in 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 incubated with a 1:100 dilution of fluorescein-conjugated goat F(ab′)2 IgG antimouse antibody (Jackson ImmunoResearch Laboratories, Inc.). Coverslips were mounted in Gel-mount (Biomeda Corp., Foster City, CA) and then examined using a Carl-Zeiss Confocal microscope.

Plasmids.

pG13 CAT, pG13 Luc, and pCMVp53 were kindly supplied by B. Vogelstein(Howard Hughes Medical Institute, The Johns Hopkins University,Baltimore, MD). p38α tagged with HA and MKK6 has been described previously (20). HA-p38 in the expression vector pCEFL was used as a template to generate a mutant p38 in which Thr106 was changed to Met using oligonucleotides by the PCR overlapping extension technique. The PCR product was then cloned into pCEFL as a HA tag. GST-fusion proteins including the NH2-terminal 86 or 126 amino acids of p53 were generated by subcloning the corresponding coding region amplified by PCR from pCMVp53 into the pGEX expression vector (Pharmacia). GST-p53 1–86(1–86) mutants in residues 15, 33, and 46 were generated using specific primers in which serine or threonine was substituted for alanine, using the Quickchange site-directed mutagenesis kit (Stratagene). Bacterially expressed proteins were purified using standard techniques. Expression plasmids for 6 His-tagged p53 were generated by subcloning the coding region of p53 obtained by PCR amplification into the pEF1/His C expression vector(Invitrogen). For the serine 33 mutant of p53, the mutated fragment from pGEX-p53 1–86(1–86) A33 was used to replace the corresponding sequence in wt p53 in pEF1/His C, using a BamHI site upstream from the ATG initiation codon and an internal SgrI site in position 241 of the p53 coding sequence. All mutations were confirmed by sequencing.

Flow Cytometry and Viability Assay.

Attached and nonadherent cells were collected, fixed in 70% ethanol,washed in PBS, and stained with propidium iodide (25 μg/ml). Samples were analyzed on a FACScan (Becton Dickinson). Apoptosis was evaluated as the population of cells in the sub-G0-G1 peak. Viability was evaluated by the crystal violet method (22).

JNK has been shown to regulate the stability of the p53 protein in MCF-7 cells (16, 17). However, in preliminary experiments,we observed that in NIH 3T3 cells, which express low levels of wt p53,the direct stimulation of JNK by the expression of activated upstream molecules, such as MAPK kinase kinase 1 (23), did not result in the activation of the transcriptional activity of p53 when assessed using a tandem of 13 p53-responsive elements linked to the CAT gene as a reporter plasmid (Ref. 24; data not shown). Similarly, we did not observe a decrease in the transcriptional response of p53 in response to genotoxic stress when JNK activity was blocked by overexpression of JNK-inhibitory protein, which sequesters JNK in the cytosolic compartment, although JNK-inhibitory protein abolishes the activation of JNK-specific responses, as we and others have shown previously (Refs. 20 and25; data not shown). Thus, these observations prompted us to explore whether additional stress-activated protein kinases could participate in signaling p53 activation in response to genotoxic stress in this cellular system. We began by exploring whether distinct DNA-damaging agents can activate p38. For these experiments, we used chemotherapeutic drugs that act by causing the formation of DNA adducts, such as CDDP (26), or inhibiting topoisomerase II, such as DOX (27), and other agents such asγ-radiation that cause DNA damage by promoting the formation of DNA double-strand breaks (28). As shown in Fig. 1, exposure of NIH 3T3 cells to two distinct chemotherapeutic drugs, CDDP and DOX, caused a dramatic increase in the level of active p38, which was detectable 2 and 1 h after treatment, respectively. In contrast, lethal doses of γ-radiation (20 Gy) did not cause a consistent activation of p38 (<2-fold induction), as has been described previously (29). These observations were further confirmed by assessing the enzymatic activity of p38 using bacterially expressed GST-activating transcription factor 2 as a substrate(data not shown).

Because these stimuli are known to activate p53, we examined the expression of the cyclin-dependent kinase inhibitor p21WAF, one of the best-characterized targets of p53 (10), as an approach to evaluate the functional activity of this tumor suppressor gene under these experimental conditions. As shown in Fig. 1,A, both CDDP and DOX provoked a detectable increase in the level of expression of p21WAF 3–4 h after treatment, which remained elevated even after 24 h (data not shown). Similarly,γ-radiation induced the expression of p21WAF,which was detectable as early as 2 h after treatment (Fig. 1 A). In contrast, the total amount of p38 did not change with any of the treatments and also served as a loading control. Thus, these results suggest that p38 is activated in NIH 3T3 cells in response to chemotherapeutic drugs, such as CDDP and DOX, and that this activation precedes the enhanced expression of p21WAF. However, the lack of activation of p38 byγ-radiation indicates that this kinase is not a universal sensor for genotoxic stress.

As an approach to investigate whether p38 affects p53 function, we took advantage of the availability of two p38-specific inhibitors, SB 253080 and SKF 86002 (30, 31). As shown in Fig. 1,B,the incubation of the cells in the presence SB 253080 prevented the increase of p21WAF in response to CDDP, and very similar results were obtained with SKF 86002 (data not shown). In contrast, the treatment with SB 253080 did not affect the elevation of p21WAF expression provoked by γ-radiation (Fig. 1,C), suggesting that p38 may participate in signaling to p53 in response to chemotherapeutic DNA-damaging agents, but not when DNA damage is caused by γ-radiation. Interestingly, when Western blots for p53 were performed, we observed that the blockade of p38 did not cause any demonstrable effect on the accumulation of p53 protein elicited by these treatments (Fig. 1 B and the text below), thus indicating that p38 does not affect the protein levels of p53.

We next set out to investigate whether p38 affects the transcriptional activity of p53 in response to chemotherapeutic agents, using as a reporter system the pG13 CAT plasmid, whose expression is controlled by a tandem of p53-responsive elements (24). As shown in Fig. 2,A, exposure of cells to CDDP and DOX resulted in a remarkable increase in the transcriptional activity of p53 (Fig. 2,A),with DOX demonstrating a greater response. When the p38 inhibitor SB 203580 was added, we observed that the transcriptional activation of p53 by CDDP was nearly abolished, whereas the response to DOX was largely diminished (Fig. 2,B). To control for the specificity of this approach and to exclude the possibility that the p38 inhibitor affects additional molecules under our assay conditions, we took advantage of the observation that the replacement of threonine 106 with methionine renders p38α insensitive to SB 253080(32). We engineered such an epitope-tagged mutant of p38α and confirmed that SB 253080 prevents the in vitroactivity of p38, but not that of the p38 Met106 mutant(Fig. 2,C). Furthermore, as shown in Fig. 2,D,whereas SB 253080 abolished the p53 response in CDDP-treated NIH 3T3 cells, this response was nearly restored by expression of the inhibitor-insensitive form of p38α (Fig. 2 D). Thus, taken together, these data indicate that p38α participates in the activation of p53-dependent pathways in response to chemotherapeutic agents. Furthermore, because SB 253080 did not abolish the response to DOX, it is also possible that this drug might stimulate additional SB 253080-insensitive isoforms of p38, such as p38γ and p38δ(32), a possibility that is under current investigation.

We next decided to examine the mechanism by which p38 affects the function of p53. p38 does not appear to regulate the protein levels of p53 because the accumulation of p53 in response to CDDP and DOX was not affected by the presence of p38 inhibitors (Fig. 1,B). Thus,one possible mechanism might involve the control of the nuclear translocation of p53, a process that is essential for the transcriptional activity of p53 (33). To address this possibility, we performed immunofluorescence analysis of p53 using confocal microscopy in cells treated with CDDP or DOX in the presence or absence of p38 inhibitors. As shown in Fig. 3, the p38 inhibitors did not affect the accumulation and localization of the p53 tumor suppressor gene product, thus indicating that p38 or its downstream targets do not participate in the biochemical route regulating the translocation of p53 to the nucleus.

In search of a putative mechanism by which p38 participates in the activation of p53 in response to genotoxic stress induced by chemotherapeutic drugs, we explored whether these proteins interact physically. For these experiments, we immunoprecipitated p38 from NIH 3T3 cells transfected with an epitope-tagged p38 and performed a Western blot analysis using anti-p53 antibodies. As shown in Fig. 4 A, p53 coimmunoprecipitates with p38, but not when p38 is activated by CDDP or UV radiation (control). Thus, inactive p38 can bind to p53, but activation of p38 appears to cause the release of p53 from the complex. Similar results have been reported for JNK(16). The functional significance of this event is under investigation. Nonetheless, our previous results using p38 inhibitors indicated that p38 does not regulate the level and localization of p53,thus suggesting that an additional mechanism might participate in the regulation of p53 by p38.

Because our results indicated that p38 affects the transcriptional activity of p53, we asked whether p53 could be phosphorylated by p38,using as a substrate a GST-fusion protein containing the first 86 or 126 amino acids of p53, an area including the p53 transactivation domain (34). As shown in Fig. 4,B, activated p38 phosphorylates both GST-fusion proteins with very high efficiency. These results prompted us to investigate which residue(s) are phosphorylated by p38 in the most NH2-terminal 86 amino acids. Serine 15, which has been recently shown to be phosphorylated by the AT gene (35, 36), was used as a control. Because p38 is a proline-targeted kinase, we mutated all serines or threonines adjacent to prolines in this region. Three residues, serine 33, serine 46, and threonine 81, represent such potential targets. When using these mutant GST-p53 proteins as substrates, we observed that the replacement of serine 33 with alanine abolishes the ability of the NH2-terminal domain of p53 to serve as an in vitro substrate for p38 (Fig. 4,C). All other mutant proteins behaved as did the wt p53. Thus, the serine 33 residue in p53 represents a likely candidate as a phosphoacceptor for the enzymatic activity of p38. On the basis of these results, we generated a full-length p53 cDNA including a mutation in serine 33 (p53 S33A). wt p53 and its S33A mutant were then subcloned into a tagging expression vector (pECF/6His). As shown in Fig. 4,D, both wt p53 and p53 S33A were detectably expressed, as judged by Western blotting with antibodies against the 6His epitope. Furthermore, an anti-phospho-serine 33-specific antibody detected the wt 6His-tagged p53 but not its S33A mutant, thus indicating that serine 33 of p53 can be phosphorylated in vivo and further supporting the specificity of this antibody. However, mutation of p53 in serine 33 did not affect its basal transcriptional activity when expressed in p53-null cells, such as Saos-2 cells (Fig. 4 E).

To explore whether serine 33 participates in the transcriptional activation of p53 by genotoxic chemotherapy, we transfected both wt and S33A p53 constructs into NIH 3T3 cells. As shown in Fig. 4 F,expression of wt p53 increased the basal p53-dependent transcriptional activity in NIH 3T3 cells and caused a remarkable increase in the reporter activity in response to CDDP. In contrast, the S33A mutant form of p53 also enhanced the basal activity in these cells but displayed only a limited response to CDDP when compared with the wt p53. These results support the importance of serine 33 in the activation of p53 by genotoxic agents such as CDDP.

To examine whether p38α activation is sufficient to stimulate p53 function, we transfected NIH 3T3 cells with an increasing amount of its upstream activator, MKK6 (37, 38). A clear dose-response effect was observed on the activity of p53, thus indicating that the stimulation of p38α is sufficient to enhance the activity of the endogenous p53 (Fig. 5,A). Using a similar approach, we examined the role of serine 33 in the activation of p53 by the MKK6-p38 pathway. To avoid the background response due to endogenous p53, we chose to use Saos-2 cells for these experiments. As shown in Fig. 5,B, cotransfection of p38α and MKK6 induced a remarkable increase in the transcriptional response to p53. However, activation of the p38 pathway provoked a very limited activation of the p53 S33A mutant. Similar results were obtained in NIH 3T3 cells (data not shown). Furthermore, the use of the p53 phospho-serine 33-specific antibody revealed that the in vivo phosphorylation of this p53 residue increases on CDDP treatment and that this response can be inhibited by the use of p38 blockers (Fig. 5 C). These findings strongly suggest that p38α can activate p53 directly and that transcriptional activation of p53 by p38α involves the phosphorylation of p53 in serine 33.

Because of the key role of p53 in apoptosis in response to genotoxic stress (2, 12), we analyzed the role of p38 in the apoptotic response of NIH 3T3 cells to CDDP. Cells were treated with CDDP for 24 h, in the presence or absence of SB 203580 or SKF 86002 for 12 h, and cell cycle was analyzed by flow cytometry. As shown in Fig. 6,A, control cells exposed to CDDP displayed an apoptotic fraction of 20–25%, whereas this fraction was reduced to 10–13% by treatment with p38 inhibitors. These results were further supported by a cell viability assay, which helped demonstrate that the treatment with p38α inhibitors resulted in a 2-fold increase in the IC50 of CDDP (Fig. 6 B), a remarkable increase that can be regarded as significant in terms chemoresistance. This decrease in the effectiveness of CDDP suggests that p38α is necessary for the correct execution of the apoptotic program initiated by p53 after genotoxic stress induced by CDDP.

The tumor suppressor p53 is believed to play a central role in sensing damaged DNA and in dictating the nature of the consequent cellular responses. As such, a variety of genotoxic stresses result in the 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 molecular mechanisms appear to participate in p53 activation by each of these DNA-damaging agents. The activation of p53 by phosphorylation in response to γ-radiation and UV radiation has been extensively investigated among these agents. For example, serines 15, 33, and 37 have been 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 only phosphorylated in response to UV light (40, 41). Much less is understood about the role of p53 phosphorylation in the activation of the p53 pathway on genotoxic stress when induced by other classes of DNA-damaging agents,including the many widely used chemotherapeutic drugs.

Interestingly, we found that treatment of cells with CDDP and DOX,which are frequently used for the treatment of cancer patients, can cause the sustained activation of p38α, a member of MAPK superfamily of proline-targeted serine/threonine protein kinases. In contrast,lethal doses of γ-radiation failed to stimulate p38α activity. Thus, activation of p38α appears not to result from DNA damage but to be triggered in response to genotoxic stress provoked specifically by the formation of DNA adducts and by inhibition of topoisomerase II. The molecular mechanisms responsible for this selective activation are still unclear and are being actively investigated. Nonetheless, the availability of specific p38α inhibitors (30, 31)afforded the possibility of exploring its contribution to the cellular responses to genotoxic stress. Indeed, blockade of p38 revealed that this kinase is necessary for the activation of p53-dependent transcription in response to chemotherapeutic agents, as judged by the remarkable inhibition of the accumulation of p21WAF and the activation of reporter systems by the treatment with p38α inhibitors. In contrast, blockade of p38αdid not prevent the increase in p21WAF expression provoked by γ-radiation, thus further supporting the specificity of this approach. Together, these results support a role for p38α in the response to genotoxic stress caused by chemotherapeutic agents, likely by activating p53 function.

A role in p53 activation has been proposed recently for another stress-activated kinase family member, JNK (16, 17). In this case, the inactive form of JNK was found to bind p53 and to diminish the cellular pool of p53 by targeting its degradation(16, 17). On activation, JNK appears to dissociate from p53, thus enhancing the stability of the newly synthesized p53 protein. However, activation of JNK alone does not appear to be sufficient to increase p53 activity because no increase was detected in the transcriptional activity of p53 on expression of molecules such as MAPK kinase kinase 1 that effectively stimulate JNK-dependent transcription in our cellular system (20). In contrast, activation of p38α is itself sufficient to increase the activity of the endogenous p53 in cells expressing wt p53, such as NIH 3T3 cells, and on expression of wt p53 in a p53-null background, such as that seen in Saos-2 cells (42). p38α was also found to coimmunoprecipitate with p53; however, in this case, we did not obtain any evidence that p38α can affect the level of p53 protein or its intracellular distribution or that blockade of p38α affects the accumulation of p53 in the nucleus on genotoxic stress. In contrast, we obtained evidence that p53 could be a relevant substrate for p38α.

Transcriptional activation of the tumor suppressor p53 is often achieved by phosphorylation of key regulatory residues (6, 43). Indeed, extensive phosphorylation on the NH2-terminal transactivating domain of p53 has been reported in response to physical or chemically induced damage to the DNA, thus providing a mechanism by which p53 can act as a universal sensor for DNA damage (1). For instance, serine 15 is phosphorylated in response to γ-radiation by the product of the AT gene (35, 36) and by others members of the AT-related protein family (44), thus stimulating p53. Similarly, DNA-protein kinase (PK) may be involved in the transcriptional activation of p53 through phosphorylation in serine 15(45), although this issue still remains unclear (46, 47). Serine 37 may be also targeted for phosphorylation, and several candidate kinases have been proposed, including ATR or DNA-PK (44, 48). Serine 33 has also been described as a target for kinase activity after DNA damage, and although this residue can be phosphorylated in vitro by JNK and the cyclin H-CDK7-p36 MAT complex (21, 49, 50), the identity of the actual kinase acting on p53 serine 33 in response to DNA damage remains elusive (8, 51). In this regard, several lines of evidence suggest that p38 can phosphorylate the transactivating domain of p53 in serine 33. In vitro, p38 can phosphorylate GST-fusion protein containing the NH2-terminal,transactivating domain of p53, and mutational analysis revealed that among all candidate residues, serine 33 was the phosphoacceptor site. To confirm the relevance of these in vitro data, we reconstituted a p53 with a mutation in residue serine 33. Under basal conditions, we did not detect any significant difference between this mutant form and the wt p53 in expression level or ability to stimulate expression from reporter plasmids, as reported previously(52). However, the serine 33 mutant of p53 failed to respond transcriptionally to CDDP treatment and to its direct activation of p38 in both wt p53 and p53-null cellular backgrounds. Taken together, these data strongly suggest that the serine 33 residue of p53 is a biologically relevant target for the enzymatic activity of p38.

Another residue, serine 392, has also been described recently as a site for p38α phosphorylation (53, 54). However, this site is not adjacent to a proline residue, thus representing an unlikely candidate for direct phosphorylation by proline-targeted kinases,including p38. Furthermore, phosphorylation of this site was only observed after UV irradiation of cells (40, 41), a condition that might activate a number of additional kinases, including the double-stranded RNA activated protein kinase (PKR)(55), which has previously been described as a candidate to phosphorylate this particular residue. Thus, although we cannot exclude the possibility that p38 may also directly or indirectly phosphorylate additional residues in p53, including serine 392, the available evidence suggests that these putative events might not be sufficient to activate p53 in the absence of serine 33.

In summary, our work demonstrates that the activation of p38α and the subsequent phosphorylation, at least in residue 33, is a critical event in the response of p53 to genotoxic stress and provides the first evidence that p38 might represent the highly sought after DNA damage-induced p53 serine 33 kinase. Furthermore, the use of specific p38α inhibitors revealed that interfering with this kinase diminishes apoptosis and enhances the viability of cells exposed to chemotherapeutic agents. These findings are in line with the pivotal role of p53 in the cytotoxic response to drugs such as CDDP or DOX and further support a key role for p38α in the stimulation of p53 in response to these DNA-damaging agents. Interestingly, these results also suggest that p38α should be explored as a putative mechanism to explain chemoresistance. Further work will be necessary to fully elucidate the molecular mechanism leading to the activation of p38αby genotoxic stress and to investigate the likely clinical consequences of these findings in the search for novel approaches to improve cancer therapy.

Fig. 1.

Activation of p38 by DNA-damaging therapeutic agents. A, subconfluent plates of NIH 3T3 cells were treated with CDDP (15 μg/ml), DOX (1 μg/ml), or exposed to γ-radiation(20 Gy; dose rate, 2.5 Gy/min) and lysed at the indicated times. In B and C, cells were pretreated with 0.1%DMSO (control) or SB 203580 (20 μm) 40 min before treatment with CDDP or γ-radiation and every 4 h. In all cases, total cell lysates (100 μg) were resolved by SDS-PAGE and immunoblotted with anti-P-p38, p38, p21WAF, and p53 serum or antibodies, as indicated. Autoradiograms are from a representative experiment that was repeated three times with nearly identical results.

Fig. 1.

Activation of p38 by DNA-damaging therapeutic agents. A, subconfluent plates of NIH 3T3 cells were treated with CDDP (15 μg/ml), DOX (1 μg/ml), or exposed to γ-radiation(20 Gy; dose rate, 2.5 Gy/min) and lysed at the indicated times. In B and C, cells were pretreated with 0.1%DMSO (control) or SB 203580 (20 μm) 40 min before treatment with CDDP or γ-radiation and every 4 h. In all cases, total cell lysates (100 μg) were resolved by SDS-PAGE and immunoblotted with anti-P-p38, p38, p21WAF, and p53 serum or antibodies, as indicated. Autoradiograms are from a representative experiment that was repeated three times with nearly identical results.

Close modal
Fig. 2.

p38 inhibitors decrease the p53-dependent transcriptional response to chemotherapeutic drugs. A, NIH 3T3 cells were transfected with 0.5 μg of pG13 CAT and 1 μg of pCDNAIIIβ-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 CAT activity. B, cells transfected with 0.5 μg of pG13 CAT and 1 μg of pCDNAIII β-gal were left untreated (control) or incubated with CDDP (5μg/ml) or DOX (0.5 μg/ml) for 12 h, in the absence (c) or presence of SB 203580 (20 μm; SB-80). SB 203580 was added 40 min before treatment with CDDP or DOX and every 4 h. CAT activities were normalized by the β-gal activity in each sample and expressed as the fold induction with respect to control, untreated cells. Data represent the mean ± SE of triplicate samples from a representative experiment that was repeated four independent times. C, 293T cells were transfected with wt HA-p38 (p38; 2 μg) or with HA-p38T106M (p38T106M; 2μg). Thirty min after UV stimulation (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 (5μ m), as indicated. D, NIH 3T3 cells were transfected with 0.25 μg of pG13 CAT and 1 μg of pCDNAIIIβ-gal plus 0.5 μg/plate of wt p38 or p38T106M. Cells were treated with CDDP in the presence or absence of SB 203580, as described in B. Fold induction was calculated with respect to the corresponding untreated controls. Similar results were obtained in three additional experiments.

Fig. 2.

p38 inhibitors decrease the p53-dependent transcriptional response to chemotherapeutic drugs. A, NIH 3T3 cells were transfected with 0.5 μg of pG13 CAT and 1 μg of pCDNAIIIβ-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 CAT activity. B, cells transfected with 0.5 μg of pG13 CAT and 1 μg of pCDNAIII β-gal were left untreated (control) or incubated with CDDP (5μg/ml) or DOX (0.5 μg/ml) for 12 h, in the absence (c) or presence of SB 203580 (20 μm; SB-80). SB 203580 was added 40 min before treatment with CDDP or DOX and every 4 h. CAT activities were normalized by the β-gal activity in each sample and expressed as the fold induction with respect to control, untreated cells. Data represent the mean ± SE of triplicate samples from a representative experiment that was repeated four independent times. C, 293T cells were transfected with wt HA-p38 (p38; 2 μg) or with HA-p38T106M (p38T106M; 2μg). Thirty min after UV stimulation (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 (5μ m), as indicated. D, NIH 3T3 cells were transfected with 0.25 μg of pG13 CAT and 1 μg of pCDNAIIIβ-gal plus 0.5 μg/plate of wt p38 or p38T106M. Cells were treated with CDDP in the presence or absence of SB 203580, as described in B. Fold induction was calculated with respect to the corresponding untreated controls. Similar results were obtained in three additional experiments.

Close modal
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 with CDDP (15 μg/ml)or DOX (1 μg/ml) for 8 or 4 h, respectively, in the presence or absence of SB 203580 or SKF 86002 (20 μm). The immunofluorescence analysis was performed using a monoclonal antibody against p53 (Ab-1), and samples were analyzed using a confocal microscope. Bar, 10 μm.

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 with CDDP (15 μg/ml)or DOX (1 μg/ml) for 8 or 4 h, respectively, in the presence or absence of SB 203580 or SKF 86002 (20 μm). The immunofluorescence analysis was performed using a monoclonal antibody against p53 (Ab-1), and samples were analyzed using a confocal microscope. Bar, 10 μm.

Close modal
Fig. 4.

Phosphorylation of the transactivating domain of p53 in serine 33 by p38: evidence for a role in p53 activation by DNA-damaging agents. A, NIH 3T3 cells were transfected by the LipofectAMINE method with expression plasmids for HA-p38 (1 μg) or green fluorescent protein (1 μg), as indicated, and treated with CDDP(15 μg/ml) or UV (40 J/m2). Four h later, cells were lysed. Anti-HA immunoprecipitates were immunoblotted against p53 or HA, as indicated. B, 293T cells were transfected with plasmids encoding HA-p38 (2 μg) with or without MKK6 (2 μg). Samples were collected and processed for kinase assays using as a substrate 5 μg of GST-p53 fusion proteins or myelin basic protein. C, GST-p53 (1–86) proteins, wt or mutated in residues 15, 33, and 46, were purified and used as a substrate for p38 kinase assays. Western blot anti-GST confirmed the equal loading of the samples. D, expression of 6His-tagged p53 and its serine 33 mutant was confirmed by Western blotting using an anti-epitope antibody after transfection of 293T cells. The status of phosphorylation in serine 33 was confirmed using a p53 phospho-serine 33-specific antibody. Similar results were obtained in a number of cell types (data not shown). E, plasmids for epitope-tagged p53 and its serine 33 mutant or green fluorescent protein were transfected into Saos-2 cells (1 μg) together with 0.5 μg of pG13 Luc and 1 μg of pCDNAIII β-gal. Luciferase activity was normalized for the β-gal activity in each sample and represented as the fold induction with respect to control cells transfected with an empty vector. F, NIH 3T3 cells were transfected with plasmids for the wt or S33A mutant of p53 (0.5 μg), pG13CAT (0.25 μg), andβ-gal (1 μg). The transcriptional response to CDDP (5 μg/ml) was evaluated as indicated. Values are expressed as the ratio between CAT activity (cpm) and β-gal activity(A420 nm) in triplicate samples to depict the transcriptional response under each experimental condition. Similar results were obtained in five independent experiments.

Fig. 4.

Phosphorylation of the transactivating domain of p53 in serine 33 by p38: evidence for a role in p53 activation by DNA-damaging agents. A, NIH 3T3 cells were transfected by the LipofectAMINE method with expression plasmids for HA-p38 (1 μg) or green fluorescent protein (1 μg), as indicated, and treated with CDDP(15 μg/ml) or UV (40 J/m2). Four h later, cells were lysed. Anti-HA immunoprecipitates were immunoblotted against p53 or HA, as indicated. B, 293T cells were transfected with plasmids encoding HA-p38 (2 μg) with or without MKK6 (2 μg). Samples were collected and processed for kinase assays using as a substrate 5 μg of GST-p53 fusion proteins or myelin basic protein. C, GST-p53 (1–86) proteins, wt or mutated in residues 15, 33, and 46, were purified and used as a substrate for p38 kinase assays. Western blot anti-GST confirmed the equal loading of the samples. D, expression of 6His-tagged p53 and its serine 33 mutant was confirmed by Western blotting using an anti-epitope antibody after transfection of 293T cells. The status of phosphorylation in serine 33 was confirmed using a p53 phospho-serine 33-specific antibody. Similar results were obtained in a number of cell types (data not shown). E, plasmids for epitope-tagged p53 and its serine 33 mutant or green fluorescent protein were transfected into Saos-2 cells (1 μg) together with 0.5 μg of pG13 Luc and 1 μg of pCDNAIII β-gal. Luciferase activity was normalized for the β-gal activity in each sample and represented as the fold induction with respect to control cells transfected with an empty vector. F, NIH 3T3 cells were transfected with plasmids for the wt or S33A mutant of p53 (0.5 μg), pG13CAT (0.25 μg), andβ-gal (1 μg). The transcriptional response to CDDP (5 μg/ml) was evaluated as indicated. Values are expressed as the ratio between CAT activity (cpm) and β-gal activity(A420 nm) in triplicate samples to depict the transcriptional response under each experimental condition. Similar results were obtained in five independent experiments.

Close modal
Fig. 5.

Activation of p38 is sufficient to stimulate p53: a role for serine 33. A, NIH 3T3 cells were transfected with increasing amounts of a 1:1 mixture of plasmids encoding p38 and MKK6(ranging from 0.05–0.5 μg) plus 0.1 μg of pG13 CAT and 0.5 μg ofβ-gal per plate. After 24 h, cells were lysed, and CAT activity was measured. Values are expressed as the ratio between CAT activity(cpm) and β-gal activity (A420 nm) in triplicate samples. B, Saos-2 cells were transfected with 0.1 μg of plasmid for wt p53 (WT) or the mutant p53 (S33A) plus 0.1 μg of pG13 Luc and 0.5 μg ofβ-gal plasmids and MKK6 and p38 (0.1 μg each; +) or with empty vector as a control (−). Twenty-four h after transfection, cells were lysed and processed for luciferase assays. Data are expressed as the fold induction with respect to control (−) transfected cells. C, 293T cells were treated with 50 μg/ml CDDP for the indicated times in the presence or absence of 20 μm SKF 86002. Cell extracts were collected and analyzed by Western blot using a p53 phospho-serine 33-specific antibody.

Fig. 5.

Activation of p38 is sufficient to stimulate p53: a role for serine 33. A, NIH 3T3 cells were transfected with increasing amounts of a 1:1 mixture of plasmids encoding p38 and MKK6(ranging from 0.05–0.5 μg) plus 0.1 μg of pG13 CAT and 0.5 μg ofβ-gal per plate. After 24 h, cells were lysed, and CAT activity was measured. Values are expressed as the ratio between CAT activity(cpm) and β-gal activity (A420 nm) in triplicate samples. B, Saos-2 cells were transfected with 0.1 μg of plasmid for wt p53 (WT) or the mutant p53 (S33A) plus 0.1 μg of pG13 Luc and 0.5 μg ofβ-gal plasmids and MKK6 and p38 (0.1 μg each; +) or with empty vector as a control (−). Twenty-four h after transfection, cells were lysed and processed for luciferase assays. Data are expressed as the fold induction with respect to control (−) transfected cells. C, 293T cells were treated with 50 μg/ml CDDP for the indicated times in the presence or absence of 20 μm SKF 86002. Cell extracts were collected and analyzed by Western blot using a p53 phospho-serine 33-specific antibody.

Close modal
Fig. 6.

Blockade of p38 diminishes the apoptotic response to chemotherapeutic agents and increases cell survival. A,NIH 3T3 cells were left untreated (−) or treated with CDDP (10 μg;+) for 24 h in the absence (control) or presence of the p38 inhibitors SB 203580 (SB-80; 20μ m) or SKF 86002 (SKF; 20μ m). The fraction of cells undergoing apoptosis was evaluated by flow cytometry and expressed as a percentage of total cells. Similar results were obtained in two additional experiments. B, cells were treated with CDDP (dose, 1–15 μg/ml) in the absence (NIH 3T3) or presence of SB 203580(NIH 3T3 +SB8O; 20 μm) or SKF 86002 (NIH 3T3 + SKF; 20μ m), as indicated. Cell viability was assayed after 2 days using the crystal violet method and expressed as a percentage of control, untreated cells. The p38 inhibitors alone did not display any significant difference with respect to control cells. The SEs in triplicate samples were lower than the symbol size. Similar results were obtained in three independent experiments.

Fig. 6.

Blockade of p38 diminishes the apoptotic response to chemotherapeutic agents and increases cell survival. A,NIH 3T3 cells were left untreated (−) or treated with CDDP (10 μg;+) for 24 h in the absence (control) or presence of the p38 inhibitors SB 203580 (SB-80; 20μ m) or SKF 86002 (SKF; 20μ m). The fraction of cells undergoing apoptosis was evaluated by flow cytometry and expressed as a percentage of total cells. Similar results were obtained in two additional experiments. B, cells were treated with CDDP (dose, 1–15 μg/ml) in the absence (NIH 3T3) or presence of SB 203580(NIH 3T3 +SB8O; 20 μm) or SKF 86002 (NIH 3T3 + SKF; 20μ m), as indicated. Cell viability was assayed after 2 days using the crystal violet method and expressed as a percentage of control, untreated cells. The p38 inhibitors alone did not display any significant difference with respect to control cells. The SEs in triplicate samples were lower than the symbol size. Similar results were obtained in three independent experiments.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

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

4

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

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

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