Homeodomain-interacting protein kinase-2 activity and p53 phosphorylation are critical events for cisplatin-mediated apoptosis.

Homeodomain-interacting protein kinase-2 activity and p53

phosphorylation are critical events for cisplatin-mediated apoptosis

Valeria Di Stefano,

Cinzia Rinaldo,

Ada Sacchi,

Silvia Soddu,

and Gabriella D’Orazi

*

a

Deparment of Experimental Oncology, Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, 00158 Rome, Italy

b

Department of Oncology and Neurosciences, University ‘‘G. D’Annunzio’’, 66013 Chieti, Italy Received 15 May 2003, revised version received 10 August 2003

Abstract

HIPK2 is a member of a novel family of nuclear serine – threonine kinases identified through their ability to interact with the Nkx-1.2 homeoprotein. The physiological role of these kinases is largely unknown, but we have recently reported on the involvement of HIPK2 in the induction of apoptosis of tumor cells after UV stress through p53 phosphorylation and transcriptional activation. Here, we demonstrate that the chemotherapeutic drug cisplatin increases HIPK2 protein expression and its kinase activity, and that HIPK2 is involved in cisplatin-dependent apoptosis. Indeed, induction of HIPK2 and of cell death by cisplatin are efficiently inhibited by the serine – threonine kinase inhibitor SB203580 or the transduction of HIPK2-specific RNA-interfering molecules. HIPK2 gene silencing efficiently reduces the p53-mediated transcriptional activation of apoptotic gene promoters as well as apoptotic cell death after treatment with cisplatin. These findings, along with the involvement of p53 phosphorylation at serine 46 (Ser46) in the transcriptional activation of apoptotic gene promoters, suggest a critical role for HIPK2 in triggering p53-dependent apoptosis in response to the antineoplastic drug cisplatin.

D 2003 Elsevier Inc. All rights reserved.

Keywords: HIPK2; Serine – threonine kinase; Kinase inhibitor SB203580; RNAi; Cisplatin; Antineoplastic drug; p53; p53 phosphorylation; Apoptosis

Introduction

HIPK2 is a newly identified nuclear serine – threonine kinase that interacts with homeodomain transcription factors and is well conserved in various organisms [1,2]. HIPK family members can act as corepressors for homeodomain transcription factors, but unlike other transcriptional core-pressors, they have protein kinase activity[3,4]. The phys-iological role of these kinases is largely unknown, but we and others have recently identified a function for HIPK2 in p53 activation[5,6]. The p53 tumor suppressor protein plays an essential role in orchestrating many of the biological responses elicited by the exposure of cells to genotoxic stress[7]. The function of this nuclear phosphoprotein now appears to be controlled by a complex regulatory network

[8]. Moreover, p53 activation occurs at multiple levels including protein stabilization, nuclear accumulation, and posttranslational modifications[9,10].

We have recently found that HIPK2 physically and functionally interacts with the p53 oncosuppressor. In par-ticular, after high doses of UV light, HIPK2 binds and phosphorylates p53 at residue serine 46 (Ser46) with induc-tion of p53-mediated apoptosis [5]. Phosphorylation at Ser46 is a late event after severe DNA damage and regulates p53-induced apoptosis [5,6,11,12]. As far as it is known, only UV irradiation strongly activates HIPK2. Thus, since HIPK2 may play an important role in the regulation of p53 apoptotic function, these observations raise the possibility that additional stress stimuli may induce the HIPK2/p53 pathway.

Apoptotic cell death is the unique outcome that may lead to a successful cancer therapy. Indeed, several investigations have indicated that the failure to die in response to several genotoxic and/or chemotherapeutic agents is a determinant of tumor cell resistance to antineoplastic treatments [13 – 16]. Anticancer treatment using cytotoxic drugs is consid-ered to mediate cell death by activating essential elements of the apoptosis program and the cellular stress response. Cancer chemotherapeutic agents such as cisplatin (cis-dia-mminedichloroplatinum) exert their cytotoxic effect by inducing DNA damage and activating apoptosis in most

0014-4827/$ - see front matterD 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2003.09.032

* Corresponding author. Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, via delle Messi d’Oro 156, 00158 Rome, Italy, Fax: +39-06-52662505.

E-mail address: dorazi@ifo.it (G. D’Orazi).

cell types. The type and dose of stress appear to dictate the outcome of the cellular response, which is linked to com-plex pathways mediating cell-cycle control or cell death. Apoptosis seems to be induced if damage exceeds the capacity of repair mechanisms[17].

On the basis of these observations, we wanted to explore whether DNA-damaging stimuli such as chemotherapeutic agents could induce the HIPK2/p53 apoptotic pathway to uncover new signaling pathways to suppress tumor growth or restore sensitivity to anticancer therapeutics. We find that HIPK2 is induced and activated by cisplatin and that HIPK2 function is critical for p53-mediated apoptosis. Indeed, in response to cisplatin, HIPK2 is induced concomitantly with p53-mediated transcriptional activation of apoptotic gene promoters.

Silencing of endogenous human genes has recently been achieved by RNA interference (RNAi)[18], a powerful tool for manipulating gene expression and investigating gene function. Interference of RNA expression causes efficient and specific gene down-regulation, resulting in functional inactivation of the targets [19,20]. On the basis of these observations, we set out to target HIPK2 by inhibiting its kinase function or abrogating the endogenous HIPK2 with RNAi, in the attempt to clarify the role of HIPK2 in response to cisplatin. We find that inhibition of HIPK2 diminishes the effects of cisplatin, increasing the survival of the treated cells and efficiently reducing the p53-mediated transcriptional activation of the bax gene promoter. The failure of cisplatin to elicit a successful apoptotic response in HIPK2-interfered cells indicates that impaired HIPK2 function could contribute to the development of chemo-resistance. In addition, we also show that the involvement of p53 phosphorylation at Ser46, specifically targeted by HIPK2, is a critical event in the transcriptional activation of apoptotic gene promoters after cisplatin treatment. In-deed, reduction of cisplatin-induced apoptosis in HIPK2-interfered cells is accompanied by reduction of Ser46 phosphorylation, along with that of Bax expression, further underlying the critical role played by the activation of the HIPK2/p53 pathway in the induction of apoptosis by the genotoxic agent cisplatin.

Material and methods

Cell culture, kinase inhibitor, and cisplatin treatment Human cells lines RKO (colon cancer, wtp53), 293 (adenovirus-transformed embryo kidney, wtp53), and H1299 (lung cancer, p53-null) were maintained in RPMI-1640 or DMEM (GIBCO-BRL, Life Technology, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO-BRL), glutamine, and anti-biotics in humidified atmosphere with 5% CO2at 37jC.

Kinase inhibitor SB203580 (Calbiochem) was prepared as 20 mM stock in DMSO, stored at 20jC, and diluted

into the medium at the indicated concentrations; DMSO was used as mock control in the kinase assay, cell viability, and Western blot analyses.

For cisplatin treatment, 5  105 exponentially prolifer-ating cells were seeded on 60-mm plates and 24 h later, exposed to 5 Ag/ml of cisplatin (TEVA PHARMA ITALIA). At different time points, both floating and adherent cells were collected for analysis.

Cell proliferation and viabilty

Exponentially proliferating, 5  104, cells were plated into 60-mm plates and treated with cisplatin as indicated above. Cells numbers were determined in duplicate at different time points. Both floating and adherent cells were collected, washed in PBS, and resuspended in culture medium. Cells were counted in hemocytometer after addi-tion of trypan blue. The percentage of viable cells, that is, blue/total cells, was determined by scoring 100 cells per chamber for three times.

For TUNEL assays, cells were treated with cisplatin or kinase inhibitor as above, and fixed in 4% paraformalde-hyde for 30 min at room temperature at different time points. After rinsing with PBS, the samples were permea-bilized in a solution of 0.1% Triton X-100 in sodium citrate for 2 min. Samples, washed with PBS, were then incubated in the TUNEL reaction mix for 1 h at 37jC, according to the manufacturer’s manual (Boehringer Mannheim, Germany). Cells were counterstained with Hoechst 33342 before anal-ysis with a fluorescent microscope (Zeiss).

Plasmid construction for RNAi and cells transfection To generate the vector to suppress the endogenous HIPK2, the pSuper plasmid [20] was digested with BglII and HindIII, and the annealed oligos (5VgatccccGAA AGT ACA TTT TCA ACT GttcaagagaCAG TTG AAA ATG TAC TTT Ctttttggaaa3Vand 5VagcttttccaaaaaGAA AGT ACATTT TCA ACT GtctcttgaaCAG TTG AAA ATG TAC TTT Cggg3V), specific for both human and mouse HIPK2, were ligated to it. The 19nt-HIPK2 target sequences are indicated in capitals in the oligonucleotide sequence. To generate pSuper and pSuper-HIPK2 transfectants, RKO cells were co-transfected with 0.25 Ag of a vector containing the puromycin-resistance marker and 2.5 Ag of both pSuper and pSuper-HIPK2 vectors, using the lipofectaminePlus method (Invitrogen). Cells were subsequently selected with puromycin, and after 4 weeks, resistant clonal and polyclonal populations were analyzed for HIPK2 interference by West-ern blotting. Two clonal and one polyclonal populations were selected for following experiments. Interfered cells will be referred as RKO-pSuper and RKO-pSuper-HIPK2.

Transient transfection assays were performed with the BES or the lipofectaminePlus (Invitrogen) methods, as described in Ref. [5]. Amounts of plasmid DNA in each sample were equalized by supplementing with empty

plas-mid. Expression plasmids used were: pCAG3.1, pCAG3.1-wtp53, and pCAG3.1-S46A (kindly provided by E. Appella, NIH, Bethesda, MD, USA).

Colony-formation assay

Approximately 1  105 RKO-HIPK2-interfered cells (pSuper and pSuper-HIPK2) were plated on 60-mm dishes and 24 h later treated with cisplatin (5 Ag/ml). After a further 24 h, the medium was replaced. Death-resistant colonies were stained with crystal violet 14 days later.

RNA extraction and RT-PCR analysis

Cells were plated in 60-mm dishes and 24 h later treated with cisplatin or transfected with wtp53 expression vector. At the indicated times, cells were collected and total messenger RNA was extracted using the RNeasy mini kit (Qiagen S.P.A., Milano, Italia). Reverse-transcription reac-tions and PCR for HIPK2 and GAPDH were performed using the MuLV reverse transcriptase and the AmpliTaq DNA Polymerase (Gene Amp RNA PCR kit, Perkin Elmer, Roche Molecular System, Brachburg, NJ, USA). The se-quence of the primers were as follow: human HIPK2 (2539 – 2559) (21), TA= 59jC: 5V > 3V-AGG AAG AGT AAG CAG CAC CAG-; 3V > 5V-TGC TGA TGG TGA TGA CAC TGA-; and human GAPDH (24), TA= 60jC: 5V > 3V-TCC ATG ACA ACT TTG GCA TCG TGG-; 3V > 5V-GTT GCT 5V-GTT GAA GTC ACA GGA GAC-. After 28 cycles of amplification, DNA products were run on 2% agarose gels, visualized by ethidium bromide, and analyzed by NIH Image 1.62 program, following gel scanning using Epson Perfection 1240 scanner, to quantitate bands density and normalize the amount of HIPK2 to that of GAPDH. Western blot analysis

Total cell lysates were prepared by incubating cell pellets for 30 min on ice in lysis buffer as previously described[5]. Equal amounts of proteins were separated by SDS-PAGE, blotted onto nitrocellulose membrane (Bio-Rad, Hercules, CA) and incubated with anti-p53 (FL393), and anti-Bax polyclonal antibodies (Santa Cruz Biotechnology), anti-phospho-p53-Ser15, and anti-phospho-p53-Ser46 rabbit polyclonal antibodies (Cell Signaling), and anti-tubulin monoclonal antibody (SIGMA, BIO-Sciences, St. Louis, MO), or blotted onto PVDF membrane (Millipore) and incubated with anti-HIPK2 rabbit antiserum (kindly provid-ed by M.L. Schmitz, University of Bern, Switzerland). Immunoreactivity was detected by the ECL chemilumines-cence reaction kit (Amersham Corp., Arlington Heights, IL). Anti-HIPK2 serum

Polyclonal antibodies recognizing HIPK2 were pre-pared by injecting 100 Ag of pL-HIPK2-SP plasmid [5]

into the leg muscle of rats. Plasmid injections were repeated once a week for four times, and 30 days later, rats were bled and the sera were tested on HIPK2-Flag and HIPK2 proteins.

Immunoprecipitation and kinase assays

Total cell extracts were prepared from drug-treated cells or cells transiently transfected with HIPK2-Flag expression vector [5]; equal amounts of proteins were immunopreci-pitated with both anti-HIPK2 rat-polyclonal antibody and monoclonal anti-Flag (SIGMA) antibody. Immunocom-plexes were incubated in kinase buffer (20 mM HEPES, pH 7.4; 10 mM MgCl2; 200 AM sodium orthovanadate) with or without kinase inhibitor in the presence of 5 ACi [g-32P]ATP, 50 mM unlabeled ATP, and 10 Ag MBP (SIGMA) as substrate, for 30 min at 30jC. Reaction products were resolved by SDS-PAGE and [32P]-labeled proteins were detected by autoradiography.

Transactivation assay

Cells were transiently transfected with the BES [21] or lipofectaminePlus (Invitrogen) methods using luciferase reporter genes driven by the p53-dependent promoters of bax and PIG3 genes[22,23]. Transfection efficiencies were normalized with the use of a co-transfected h-galactosidase construct as previously described [24]. Transcriptional activity was evaluated after cisplatin treatment and co-transfection with wtp53 expression vector, and p53S46A mutant. Luciferase activity was assayed as previously described [24].

Results

Cisplatin treatment of wtp53-cancer cells induces apoptosis Given the key role of p53 in apoptosis in response to genotoxic stress [16], we first analyzed the effect of the alkylating agent cisplatin that causes formation of DNA adducts [25] on cell growth and viability of tumor cells carrying an endogenous wtp53 gene. We used cisplatin because of its broad spectrum of anti-tumor activity and wide use in the treatment of solid tumors [26]. As shown in Fig. 1A (upper panel), proliferation of RKO cells was completely suppressed by exposure to cisplatin (5 Ag/ml) compared to mock-treated control cells. Concomitantly, cisplatin decreased cell viability to 20%, by 72 h of treatment (Fig. 1A, lower panel). We next tested by TUNEL staining whether the observed cell death was due to apoptosis. As shown inFig. 1B, cisplatin treatment resulted in up to 35% apoptosis over a period of 48 h. Taken together, these results show that the dose of cisplatin used (5 Ag/ml) is able to induce apoptotic cell death.

Fig. 1. (A) Analyses of proliferation rate (upper panel) and cell viability (lower panel) of RKO cells during 24, 48, and 72 h of treatment with cisplatin (5 Ag/ml; open squares); black circles represent mock-treated cells. Numbers are mean of three different experiments F standard deviations (SD). (B) Time-course analysis of cisplatin treatment on cell apoptosis. TUNEL staining of RKO cells, mock (black bars) or treated with cisplatin (5 Ag/ml) (gray bars), to quantitate apoptotic cells. TUNEL positivity was shown as percentage of stained cells F SD.

Fig. 2. (A) Western blot analyses of HIPK2, p53, and Bax proteins levels, and Ser15 and Ser46 phosphorylations of p53 on total cell extracts from RKO cells treated with cisplatin (5 Ag/ml) for 24 and 48 h. Equal amounts of proteins were separated on a denaturing SDS-PAGE and immunoblot analyses were performed using specific antibodies. Blots were routinely reprobed with anti-tubulin to ensure equivalence of loading. (B) RT-PCR analysis of RKO cells treated with cisplatin for 8, 12, and 24 h, showing comparable expression of HIPK2, as assessed by densitometric analysis. HIPK2/GAPDH ratio is shown below the picture. (C) Kinase assay for HIPK2 activation upon cisplatin treatment. Equal amounts of total cell lysates from RKO cells treated with cisplatin (5 Ag/ml) were collected at the indicated times, immunoprecipitated with anti-HIPK2 polyclonal rat antiserum, and assayed for kinase activity in the presence of [g-32P]ATP and MBP as substrate.

HIPK2 induction and activation in response to cisplatin Because activation of HIPK2 and phosphorylation of the substrate MBP have been reported after UV treatment[5,6], we explored whether cisplatin too could activate HIPK2. As shown inFig. 2A, exposure of RKO cells to cisplatin caused a persistent increase in HIPK2 protein expression during 24 and 48 h of treatment. Moreover, p53 and Bax protein expression increased concomitantly to HIPK2 up-regula-tion. Analysis of the phosphorylation of the p53 residues Ser15 and Ser46 revealed that phosphorylation of Ser15 was detected almost at the same time as p53 accumulation, while initiation of phosphorylation of Ser46 was delayed, as already suggested by Kazuyasu et al. [27]. To gain some mechanistic understanding, we next investigated whether the increase of HIPK2 protein was also seen at the mRNA level. To this end, RT-PCR analyses were carried out using cDNAs derived from RKO cells treated with cisplatin. As shown inFig. 2B, HIPK2 does not appear to be regulated at the mRNA level during treatment with cisplatin.

Next we assessed HIPK2 enzymatic activity after treat-ment with cisplatin. Total protein extracts from RKO cells

treated with cisplatin for 24 and 48 h were immunoprecipi-tated with specific anti-HIPK2 polyclonal rat antiserum, and immunocomplexes were analyzed in a kinase assay by the addition of [g-32P]ATP and the MBP as substrate. As shown in Fig. 2C, HIPK2 efficiently phosphorylates the MBP substrate after treatment of cells with cisplatin. Taken together, these data indicate that cisplatin treatment increases HIPK2 protein levels that result in an increased HIPK2 enzymatic activity.

HIPK2 is involved in apoptosis induced by cisplatin As an approach to investigate the role of HIPK2 following cisplatin treatment, we took advantage of the availability of the p38-specific inhibitor SB203580, since HIPK2 was shown to have features of p38-like MAP kinase [28]. We first used this inhibitor to test the suppression of HIPK2 activity in vitro. To this end, 293 cells were transiently transfected with HIPK2-Flag expression vector; 36 h after transfection, equal amounts of total cell proteins were immunoprecipitated with monoclonal anti-Flag antibody and assayed for kinase activity in the absence or presence

Fig. 3. (A) For in vitro kinase assay, 5  105293 cells were plated in 60-mm plates and 24 h later, transiently transfected with 7 Ag of HIPK2-Flag expression vector, as described inRef. [5]. Cells were collected in lysis buffer 36 h after transfection and immunoprecipitated with monoclonal anti-Flag antibody. Anti-Flag immunocomplexes were assayed for kinase activity in the absence (DMSO treatment as mock-control) or presence of SB203580 (5 AM). (B) Western blot analyses of HIPK2 protein expression and the relative loading control (l.c.) in RKO cells treated with cisplatin (5 Ag/ml) for 24 h, in the absence (DMSO) or presence of SB203580 (30 AM) (upper panels). Equal amounts of total cell lysates were immunoprecipitated with anti-HIPK2 polyclonal rat antiserum and assayed for kinase activity in the presence of [g-32P]ATP and MBP substrate (lower panel). (C) Cell viability of RKO cells treated with cisplatin (5 Ag/ml) for 12, 24, and 48 h, in the absence (DMSO) or presence of SB203580 (30 AM). Cell numbers were determined by trypan blue exclusion; the results shown represent the mean of two independent experiments F SD. (D) Time course analysis of apoptotic response. TUNEL staining of RKO cells treated with cisplatin in the absence (DMSO) (gray bar) or presence of SB203580 (black bar) (as previously described). TUNEL positivity is shown as percentage of stained cells F SD.

of SB203580 (5 AM). As shown in Fig. 3A, when the SB203580 inhibitor was added to the kinase assay reaction, phosphorylation of MBP was nearly abolished, compared to the DMSO control, indicating that this p38-kinase inhibitor can also impede the activity of the p38-like HIPK2. Subse-quently, we used SB203580 to test the suppression of HIPK2 activity in cultured cells. To this end, RKO cells were treated with cisplatin for 24 h, in the presence or absence of SB203580 (30 AM). Equal amounts of total cell proteins were immunoprecipitated with anti-HIPK2 rat antiserum and assayed for kinase activity. As shown inFig. 3B, treatment of RKO cells with SB203580 and cisplatin markedly reduced the activity of HIPK2 immunocomplexes on MBP phos-phorylation. Moreover, the accumulation of HIPK2 protein was also slightly reduced compared to the DMSO control

(Fig. 3B), suggesting that both protein stabilization and activation are involved in HIPK2 function.

We then tested the involvement of HIPK2 in cisplatin-mediated cell death. RKO cells were treated with cisplatin in the absence or presence of SB203580, and cell viability was analyzed by trypan blue exclusion during 12, 24, and 48 h of treatment. As shown inFig. 3C, treatment of cells with the inhibitor resulted in a marked reduction in cell death induced by cisplatin, compared to the DMSO control cells. In particular, SB203580 rescued cells from apoptotic cell death, as shown by TUNEL staining(Fig. 3D).

In the attempt to specifically identify a role for HIPK2 in the cellular response to cisplatin, we also took advantage of the recently developed strategy of specific gene silencing by RNA interference (RNAi)[20]. When analyzed by Western blotting, RKO-pSuper-HIPK2 cells, stably transduced with the pSuper-HIPK2 vector, to suppress the endogenous HIPK2, showed significant reduction in HIPK2 protein expression, compared to the RKO-pSuper control cells

(Fig. 4A). Two clonal and one polyclonal population were selected. Viability of RKO-pSuper or-pSuper-HIPK2 poly-clonal cells, in the presence or absence of cisplatin, was then determined by trypan blue exclusion at 12, 24, and 48 h of cisplatin treatment. As shown in Fig. 4B, RKO-pSuper-HIPK2 polyclonal cells showed increased cell survival in response to cisplatin compared to the RKO-pSuper control cells (with an intact endogenous HIPK2); such survival level was comparable to that of pSuper and RKO-pSuper-HIPK2 cells in absence of drug. Moreover, as shown in Fig. 4C, colony-forming ability, after cisplatin treatment, was strongly reduced in cells expressing endog-enous HIPK2 (RKO-pSuper), compared to the HIPK2-interfered cells (RKO-pSuper-HIPK2). Similar results were obtained with the two clonal populations selected (data not shown). Taken together, these data suggest that HIPK2 is involved in cisplatin-induced apoptosis and that its inhibi-tion can exert a protective effect.

Fig. 4. (A) Interference with HIPK2 protein expression by pSuper-HIPK2 vector. RKO-pSuper (empty vector as control: C) and pSuper-HIPK2 cells (with interference of endogenous HIPK2: Int) were lysed, separated on 9% SDS-PAGE, and immunoblotted to detect HIPK2 protein levels. An immunoblot with antibody against tubulin was used as loading control. A representative image of endogenous HIPK2 and the densitometric analysis, compared to tubulin expression and relative to the HIPK2 expression in the pSuper control (fixed as 100%), is shown in two clonal populations (1, and 2) and one polyclonal population (mix). (B) Cell viability of RKO-pSuper and -pSuper-HIPK2 cells, with or without cisplatin treatment (5 Ag/ml). Cell numbers were determined by trypan blue exclusion at the indicated times. Results shown represent the mean of three independent experiments F SD. (C) Colony-forming ability of RKO-pSuper and -RKO-pSuper-HIPK2 cells treated with cisplatin (5 Ag/ml) for 24 h and soon after refeeding with fresh medium; death-resistant colonies were stained with crystal violet 14 days later.

Fig. 5. (A) Induction of luciferase activity after cisplatin treatment (5 Ag/ml for 24 h) on the bax- (left histogram) and PIG3-reporter vectors (right histogram), in H1299 cells, transfected with 50 ng of wtp53, or S46A mutant vectors. The graph shows a representative experiment, normalized to h-gal activity, and presented as arbitrary unit of relative luciferase activity. All transfections were performed in duplicates. (B) Analysis of H1299 cell viability 24 and 48 h after treatment with cisplatin (open circles), in the presence of wtp53 (open triangle) or S46A mutant (open squares) proteins, transiently transfected with expression vectors; black symbols represent samples without cisplatin. Cells were then washed with PBS, trypsinized, and replated at 2  104

density into 12-well plate. Cell numbers were determined by trypan blue exclusion 24 and 48 h after transfection, and represented as the mean of two independent experiments F SD. (C) (Upper panel) RKO-pSuper and -pSuper-HIPK2 cells were transfected with bax-luciferase reporter gene and soon after were treated with cisplatin (5 Ag/ml). Twenty-four hours after treatment, cells were lysed and processed for luciferase activity. Data are expressed as the fold of induction with respect to control cells without cisplatin. Western blot analysis (lower panel) of Bax, p53, S15, and S46 protein levels. Tubulin shows protein-loading equivalence. (D) TUNEL assay showing apoptotic cell death of RKO-pSuper and -pSuper-HIPK2 cells 48 h after cisplatin treatment. Hoechst staining shows the typical features of apoptosis such as condensation of the nuclei, shrinkage of the cytoplasm, and membrane blebbing only in cells where the endogenous HIPK2 is still present (RKO-pSuper).

HIPK2-mediated p53 phosphorylation at Ser46 is involved in apoptosis induced by cisplatin

To elucidate the role that the p53 oncosuppressor plays during apoptosis following cisplatin treatment, we first performed a luciferase assay. H1299 cells were transfected with reporter vectors containing the luciferase gene under the control of bax or PIG3 apoptotic gene promoters along with small amounts of wtp53 expression vector, or S46A mutant expression vector. Soon after transfection, cells were treated with cisplatin and 24 h later were harvested and luciferase activity determined. As expected, wtp53 efficient-ly induced luciferase activity relative to the empty plasmid

(Fig. 5A). With the p53S46A mutant, transactivation of the bax-luc reporter was comparable to that of wtp53, whereas transactivation of PIG3-luc was reduced. Treatment with cisplatin resulted in increased transactivation of both report-ers by wtp53, but it did not alter, or slightly reduced, transactivation of both reporters by p53S46A. Cell viability was then analyzed on H1299 cells transfected with wtp53 or S46A mutant expression vectors and soon after transfection treated with cisplatin. Viable cells were identified by trypan blue exclusion at 12, 24, and 48 h of treatment. As shown in

Fig. 5B, cells expressing the S46A mutant showed increased survival in response to cisplatin compared to the wtp53 transfectants.

To elucidate the role played by the p53 oncosuppressor during HIPK2-mediated apoptosis following cisplatin treat-ment, we analyzed p53-mediated transcriptional activity in a HIPK2-dependent or independent manner. RKO-pSuper and-pSuper-HIPK2 cells were transfected with a reporter construct containing the luciferase gene under the control of the bax promoter and soon after transfection cells were treated with cisplatin. Twenty-four hours later, cells were harvested and luciferase activity determined.Fig. 5C(upper panel) shows that HIPK2 gene silencing by pSuper-HIPK2 vector strongly reduced p53-mediated transcriptional activ-ity of the bax-luc reporter gene upon cisplatin treatment, compared to the pSuper control cells. Western blot analyses, performed 48 h after treatment with cisplatin (Fig. 5C, lower panel), showed, in addition, a strong reduction of Bax protein expression along with reduction of Ser46 phosphorylation in HIPK2-silenced cells, compared to the pSuper control cells. On the contrary, Ser15 phosphoryla-tion was not altered in both RKO-pSuper and -pSuper HIPK2 cells treated with cisplatin, confirming that HIPK2 can specifically phosphorylate Ser46 and that this function is important for a successful p53-dependent transcriptional activation of apoptotic genes. Apoptosis was measured 48 h after cisplatin treatment with the TUNEL assay. As shown inFig. 5D, cisplatin treatment of RKO-pSuper and-pSuper-HIPK2 cells induced strong TUNEL positivity along with the typical features of apoptosis such as condensation of nuclei, shrinkage of cytoplasm, and membrane blebbing, only in cells where the endogenous HIPK2 was still present (RKO-pSuper).

Taken together, these data suggest that HIPK2 is in-volved in apoptosis induced by cisplatin, in the presence of wtp53 through its specific phosphorylation at residue Ser46, which is important for a correct induction of p53 transcrip-tional activity. Moreover, HIPK2 inhibition can exert a protective effect against apoptotic cell death induced by cisplatin.

Discussion

In this paper, we show that treatment of RKO cells with cisplatin, a chemotherapeutic drug frequently used in cancer therapy, can induce the HIPK2 protein, which in turn is responsible for the induction of the p53-Ser46-mediated apoptotic pathway.

Anticancer drugs inhibit proliferation and induce apopto-sis in sensitive tumor cells. The common underlying mech-anism for chemotherapy-induced apoptosis might be damaged to DNA with imbalance of cellular homeostasis commonly defined as cellular stress. Apoptosis occurs when damage exceeds the capacity of repair mechanisms [17]. Moreover, recent investigations have indicated that the failure to die in response to several genotoxic and/or chemotherapeutic agents is a determinant of tumor cell resistance [13 – 15]. Thus, the discovery of new molecules involved in chemotherapeutic response is potentially useful in anticancer strategies.

The serine – threonine kinase HIPK2 is involved in in-ducing apoptotic cell death after DNA damage, as recently demonstrated, by activating the oncosuppressor protein p53 through its direct binding and phosphorylation at residue Ser46[5,6]. The tumor suppressor p53 is believed to play a central role in sensing DNA damage and in dictating the nature of the subsequent cell responses[29]. However, very distinct molecular mechanisms appear to participate in p53 activation by different stresses [30,31]. Phosphorylation of p53 in response to DNA damage is one mechanism by which p53 activity may be modulated[7,30,32,33]. Among many kinases implicated in such phosphorylation is p38 a, a member of the MAPK superfamily of proline-targeted serine – threonine protein kinases that can be activated by cisplatin and is involved in p53-mediated apoptosis through phosphorylation of Serine 33 [31]. Of note, it has been suggested that HIPK2 has features of p38-like MAP kinase

[28]. Interestingly, we found that HIPK2 can be activated by cisplatin and is involved in p53-mediated apoptosis through phosphorylation of Serine 46. Until now, it was shown that HIPK2 was activated only in response to high doses of UV

[5,6], whereas for instance, g-irradiation failed to trigger HIPK2 activity[6], indicating that specific stress stimuli can activate HIPK2 function. The finding that HIPK2 can be induced and activated also by the antineoplastic drug cisplatin, inducing p53-mediated apoptosis through specific phosphorylation of Ser46, was of particular interest, in light of its potential implication in cancer treatment. The

molec-ular mechanisms responsible for this selective activation are still unclear and need to be further studied. Nonetheless, the availability of the p38-specific inhibitor SB203580 and of the recently identified strategy of specific gene silencing by RNA interference (RNAi)[20]afforded us the possibility of exploring HIPK2 contribution to the cellular response to genotoxic stress. Indeed, inhibition of HIPK2 revealed that this kinase is required for the activation of p53-dependent transcription in response to cisplatin, as judged by the significantly lower levels of the apoptotic protein Bax and reduced phosphorylation of p53-residue Ser46. In contrast, HIPK2 silencing by RNAi did not reduce phosphorylation of p53-residue Ser15, supporting the specificity of HIPK2 activity. Taken together, these results support a role for HIPK2 in the response to genotoxic stress by the chemo-therapeutic agent cisplatin through activation of p53-Ser46-mediated apoptosis.

A series of different phosphorylation sites in the p53 protein are presumed to be involved in p53 oncosuppressing functions [30]. The involvement of Ser46 phosphorylation in specifically regulating the ability of p53 to induce apoptosis has recently become evident. In particular, it has been shown that p53 phosphorylation on Ser46 induces a subtle change of p53 conformation and a stronger affinity to promoters of apoptosis-related genes [12]. To a larger extent, p53 triggers apoptosis by direct transcriptional activation of an array of pro-apoptotic genes, including Bax and PIG3 [22,23]. To confirm the relevance of these observations, we performed experiments using HIPK2-si-lenced cells where we observed reduced phosphorylation of p53-residue Ser46, while the phosphorylation status of residue Ser15 was unchanged. Moreover, the serine 46 mutant (S46A) of p53 failed to respond transcriptionally to cisplatin treatment. Taken together, these data strongly suggest that the Ser46 residue of p53 is a biologically relevant target of the enzymatic activity of HIPK2. In addition, these findings are in agreement with the model proposed by Oda et al.[12], where Ser15, Ser20, and some other sites are phosphorylated after DNA damage at an early stage, whereas Ser46 is phosphorylated as a later event and when DNA repair is impossible.

A role of HIPK2 activation after genotoxic stress has been recently suggested[34]. In that case, the authors reported that HIPK2-U2OS cells, stably overexpressing HIPK2, exhibited increased apoptosis after cisplatin or adriamycin treatment, suggesting that HIPK2 may be a critical mediator for the cellular functions of p53, such as apoptosis in response to chemotherapeutic drugs. Those findings are in great agree-ment with the results of the present paper where the endog-enous HIPK2 is shown to be involved in the p53-Ser46-dependent apoptosis induced by cisplatin. HIPK2 has been recently found to interact with other members of the p53 family, named p63 and p73, suggesting a p53-independent function of HIPK2 that may reflect the presence of various cellular phosphorylation targets of HIPK2 that are involved in growth regulation independent of p53[34]. This is supported

by the existence of other targets of HIPK2 such as NK homeodomain transcription factor [1], interferon type I-induced MxA [35], fas (CD95) [36], STAT3 [37], and HMGI(Y)[38]. However, it has been shown that both p73 and p63 are necessary for a correct p53-mediated apoptosis

[39]. Thus, the net output of this network depends on the status of the main player p53: in fact, if p53 is in a wild-type conformation, the p53/p63/p73 protein complexes can max-imize drug-induced cell death, even though the exact mech-anism is largely unknown. However, if mutant p53 is present, p73/p63-mediated apoptosis is severely impaired[40,41].

In summary, our work demonstrates that the induction and activation of HIPK2 and the subsequent phosphoryla-tion of p53 at residue Ser46 are critical events in the response of p53 to genotoxic stress with apoptotic outcome. These findings are in line with the pivotal role of p53 in the cytotoxic response to drugs such as cisplatin, and further support an essential role for HIPK2 in the stimulation of p53 in response to this DNA-damaging agent. Interestingly, these results also suggest that the HIPK2 pathway should be explored as a potential mechanism to explain chemo-resistance. Further work will be necessary to fully elucidate the molecular mechanisms leading to the activation of HIPK2 by genotoxic stress and to investigate the likely clinical consequences of these findings in the search for novel approaches to improve cancer therapy.

Acknowledgments

We gratefully acknowledge constructive criticisms and helpful discussion by G. Blandino and S. Bacchetti; we thank G. Citro and E. Spugnini for helpful advise on antibody generation; we also greatly appreciate the cooperative and enjoyable interaction with P. D’Avenia and I. Manni, and the help and encouragement of A. Porrello throughout this project. This work was supported by grants from FIRB-MIUR, Ministero della Salute, and MURST Fondi Ateneo.

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