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(2011) 30, 346–355 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE does not protect cells from induced by nongenotoxic activation

M Xia, D Knezevic and LT Vassilev

Discovery Oncology, Roche Research Center, Hoffmann-La Roche Inc, Nutley, NJ, USA p21Waf1/Cip1 is a p53 target implicated in both complexes (for example, CDK1, 2 and 4), thus major functions of the tumor suppressor— arrest effectively blocking cell-cycle progression (Xiong et al., and apoptosis. It is a potent inhibitor of the key - 1993). Forced expression of p21 has been shown to dependent (CDK1–4), and has been thought to be arrest the cell cycle at the G1/S or G2/M phase border the main mediator of p53-dependent G1 and G2 arrest. (Bunz et al., 1999, Dulic et al., 1998). As a cell-cycle However, an increasing body of information suggests that inhibitor, p21 has been assigned a major role in the in addition to its cell-cycle inhibitory activity, p21 can growth arrest after DNA damage and is thought to affect p53-dependent apoptosis. These data have been be a key mediator of p53 tumor suppressor function obtained from experiments in which p53 is activated (Xiong et al., 1993; Bunz et al., 1999). Although p21 is a primarily by genotoxic stress. In this study, we use the transcription target of p53 and its expression is mainly selective antagonist, nutlin-3a, as a nongenotoxic regulated by the tumor suppressor, p53-independent p53 activator and show that the cell-cycle arrest function mechanisms have also been described in response to of p21 is dependent on the cellular context. In most cancer variety of stresses including DNA damage (Gartel and cell lines, p53-dependent p21 induction is essential for cell- Tyner, 1999). cycle arrest, but in some, p21 is dispensable. Depletion of In addition to its cell-cycle inhibitor function, p21 has p21 did not increase the apoptotic response to nutlin-3a in been reported to affect either negatively or positively all seven lines tested and p21 overexpression another major p53 function, induction of apoptosis did not protect apoptosis-sensitive lines from . p21 (Abbas and Dutta, 2009). In response to DNA damage was found to mediate nutlin-induced p53-dependent and subsequent p53 activation, cells can either undergo downregulation of another antiapoptotic , , cell-cycle arrest or apoptosis depending on the extend of without significantly affecting the apoptotic outcome. damage and other still unknown factors (Vousden and Taken together our results suggest that p21 induction does Lu, 2002). Although the mechanisms underlying this life not affect the apoptotic response to nongenotoxic p53 or death decision remain mostly elusive, p21 has been activation. suggested to have a role by inhibiting apoptosis (Gartel Oncogene (2011) 30, 346–355; doi:10.1038/onc.2010.413; and Tyner, 2002; Janicke et al., 2007). HCT116 human published online 27 September 2010 colon carcinoma cells have been shown to undergo cell- cycle arrest following DNA damage in the presence of Keywords: p53; MDM2; p21; apoptosis; cell cycle , but their p21-null variant undergoes apoptosis (Bunz et al., 1999; Chan et al., 2000). Similarly, p21-specific antisense oligonucleotides in- Introduction crease the sensitivity of HCT116 cells to chemo- and radiotherapy in vitro and HCT116 tumor xenografts in The tumor-suppressor p53 is a potent transcription nude mice (Tian et al., 2000). Other experiments have factor, which is activated in response to diverse forms of suggested that repression of p21 transcription or defects oncogenic stress and induces cell-cycle arrest, apoptosis in p53-dependent induction of p21 may also lead to or senescence to protect us from cancer (Vogelstein increased apoptosis. The c- oncogene can switch et al., 2000). p53 protein accumulates in cell nuclei by a p53-dependent response to DNA damage from cell-cycle posttranslational mechanism and activates multiple arrest to apoptosis by inhibiting p21 transactivation target genes that mediate its tumor suppressor functions (Seoane et al., 2002). Similarly, p53 point mutants (Harris and Levine, 2005). One of these genes, p21Waf1/Cip1 unable to transactivate p21 have been found to be more (el-Deiry et al., 1993, Harper et al., 1993), a member of potent inducers of apoptosis than wild-type p53 the Cip/Kip family of cyclin-dependent (CDK) (Bissonnette and Hunting, 1998; Kokontis et al., 2001). inhibitors, binds and inhibits the activity of cyclin/CDK The proposed mechanisms by which p21 could protect cancer cells from apoptosis include direct binding to procaspase-3 and blocking its activation (Suzuki et al., Correspondence: Dr LT Vassilev, Discovery Oncology, Hoffmann-La 1998), inhibition of apoptosis signal-regulating kinase 1 Roche Inc, 340 Kingsland Street, Nutley, NJ 07110, USA. E-mail: [email protected] (Asada et al., 1999; Huang et al., 2003), inhibition of Received 28 February 2010; revised 11 July 2010; accepted 19 July 2010; CDKs required for the apoptotic process (Lu et al., published online 27 September 2010 1998; Sohn et al., 2006) and regulation of gene p21 and nutlin-induced apoptosis MXiaet al 347 expression through direct interaction with several (HCT116), partially sensitive (RKO) and highly sensi- transcription factors and cofactors (Perkins 2002). tive (SJSA1) (Tovar et al., 2006). Incubation of Furthermore, -3-mediated cleavage of p21 has exponentially growing cells with the active enantiomer, also been reported to contribute to the antiapoptotic nutlin-3a, for 24 h led to accumulation of p53 protein activity of p21 (Levkau et al., 1998; Zhang et al., 1999). and induction of p21 mRNA and protein (Figures 1a The bulk of data in support of the proposed anti- and c). This was accompanied by upregulation of apoptotic role of p21 has been derived from experiments another p53 target, MDM2, in all three cell lines in which p53 is induced by genotoxic insults. However, (Figure 1c). The inactive enantiomer, nutlin-3b, which p53 response to genotoxic agents is known to involve is approximately 200-fold less potent in binding to multiple mechanisms, both p53-dependent and p53- MDM2, was used as a negative control. Although independent. Therefore, it is of interest to know if p21 p21 induction compared with controls was in close induced by nongenotoxic activation of the p53 pathway range between the cell lines (four to five fold), the has a role in the apoptotic outcome. relative mRNA levels varied approximately three fold Given its powerful growth suppressive and proapop- (Figure 1a). Consistent with our previous data (Tovar totic potential, p53 activation has been considered an et al., 2006), all three cell lines were effectively arrested attractive therapeutic strategy in cancer (Brown et al., in G1 and G2/M phase (Figure 1d). As previously 2009). One p53 activation strategy targets the interac- reported (Thompson et al., 2004) nutlin treatment did tion of p53 with its negative regulator MDM2 (Vassilev not induce Ser15 phosphorylation on p53, confirming its 2007). Recently developed small-molecule p53-MDM2 nongenotoxic mechanism of p53 activation (Supplemen- binding inhibitors, the nutlins, stabilize p53 and activate tary Figure 1). the p53 pathway only in cells with wild-type but not We then used RNA interference to inhibit p21 mutant p53 (Vassilev et al., 2004). They are highly expression. Our p21-specific small interfering RNAs selective to their target in cells, and thus provide a (siRNAs) effectively reduced both the basal and induced unique tool to study the activation of the p53 pathway mRNA levels greater than 85% of the control levels without DNA damage or other stress-induced signaling in RKO and 490% in HCT116 and SJSA cells events (Thompson et al., 2004; Tovar et al., 2006). In (Figure 1b). Western analysis showed that p21 protein this study, we use the MDM2 antagonist, nutlin-3a, to levels are below the control uninduced level of the investigate the role of p21 in cell-cycle arrest and protein in the presence of nutlin-3a in all three lines apoptosis induced by nongenotoxic p53 activation. We (Figure 1c). Depletion of p21 either completely (RKO) find that although p21 is highly upregulated following or partially (HCT116) restored cell-cycle progression, nutlin-induced p53 accumulation, it does not affect p53- suggesting that p21 is required for nutlin-induced p53- dependent apoptosis. Furthermore, whereas the growth dependent cell-cycle arrest in these lines. However, inhibitory function of p21 is preserved in most tumor knockdown of p21 in SJSA cells did not release them cell lines, p21 is dispensable for p53-dependent cell-cycle from the cell-cycle block (Figure 1d), indicating that arrest in some cell lines. Our data suggest that p21 p21 is dispensable for nutlin-induced cell-cycle arrest in induced by pharmacological p53 activation, unlike these cells. stress-induced p53 activation, is not antiapoptotic.

p21 does not affect the apoptotic outcome Results of nutlin-induced p53 activation In contrast to cell-cycle arrest, tumor cell lines with wild- Dependency of p53-induced cell-cycle arrest on p21 type p53 have shown a wide range of sensitivity to Nutlin-3 is a potent and selective small molecule that nutlin-induced apoptosis (Tovar et al., 2006). HCT116 binds MDM2 in the p53-binding pocket, thus prevent- has been found to be resistant, whereas SJSA is highly ing p53 degradation, leading to stabilization and sensitive and RKO has shown intermediate apoptotic activation of the tumor suppressor (Vassilev et al., index. As p21 is universally upregulated on p53 2004). Nutlin treatment of cancer cells effectively activation by nutlin, it may influence the apoptotic activates both main functions of p53, cell-cycle arrest response as previously shown in experiments with and apoptosis. We have shown previously that p53- genotoxic agents (Gartel and Tyner, 2002; Janicke dependent apoptosis is compromised in many cancer cell et al., 2007). Because of the different dependency on lines, but cell-cycle arrest function of p53 is preserved in p21 for cell-cycle arrest, the selected cancer cell lines also all tested tumor-derived cell lines expressing wild-type offer the opportunity to test the hypothesis that cell- p53 (Tovar et al., 2006; Xia et al., 2008). The immediate cycle state can affect p53-dependent apoptotic response. downstream p53 target, p21, has been thought to be the RKO cells, in particular, represent a good model as main mediator of p53-dependent cell-cycle arrest (el- depending on the presence or absence of p21, they can Deiry et al., 1993; Bunz et al., 1999). To examine the role be either completely arrested in G1 and or of p21 in nutlin-induced cell-cycle block, we chose three actively cycling in the presence of the same nutlin tumor cell lines expressing wild-type p53, the colon concentration. Thus, one can distinguish the direct effect cancer RKO and HCT116 cells, and SJSA1 osteosarco- of p21 on apoptosis from the indirect effect of p21- ma. These cell lines were primarily selected to cover the induced cell-cycle arrest. To this end, RKO, HCT116 spectrum of apoptotic sensitivity to nutlin-3a: resistant and SJSA cells were first depleted of endogenous p21 by

Oncogene p21 and nutlin-induced apoptosis MXiaet al 348 siRNA and then exposed to nutlin-3a for 24 or 48 h (in the apoptotic fraction remained low from 24 to 96 h of the apoptosis-sensitive SJSA cells) or up to 96 h in RKO treatment (Figure 2a). Another indicator of apoptosis, and HCT116 cells. The fraction of dead/apoptotic cells PARP, showed similar degree of cleavage in the was measured using the V and poly (ADP- presence of p21 siRNA compared with the nontargeting ribose) polymerase (PARP) cleavage assays. Using control siRNA (Figure 2b) at all time points of exposure both methods, we were unable to detect any signs to nutlin-3a. Doxorubicin (1 mM), used as a positive of enhanced (Figures 2a and b). Instead, the control, produced a pronounced PARP cleavage in both Annexin V registered a slight, but consistent RKO and HCT116 cells (Figure 2b). Propidium iodide decrease in the apoptotic cell fraction in all three lines in and fluorescence-activated cell sorting analysis the absence of p21 (Figure 2a). In RKO and HCT116, revealed that the percentage of subG1 fraction remained unaltered on p21 depletion (data not shown). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) experiments yielded comparable a 7 Nutlin3b 25 Nutlin3b Nutlin3a positivity in SJSA1 and slightly reduced terminal 6 Nutlin3a 20 deoxynucleotidyl transferase-mediated dUTP nick end 5 labeling-positive fraction in HCT116 and RKO cells 4 15 (data not shown). To minimize the possibility for off- 3 10 target siRNA effects, key experiments were repeated 2 using an independent siRNA from Santa Cruz Biotech- 5 p21 Fold induction 1 nology (Santa Cruz, CA, USA), as well as p21-specific 0 p21 Relative expression 0 small hairpin RNA and the results were identical RKO HCT116 SJSA RKO HCT116 SJSA (Supplementary Figure 2).

siCtrl-3a sip21-3a We then tested if exogenous p21 could block the p53- b 120 siCtrl-3b sip21-3b 120 dependent apoptotic response induced by nutlin. Full- 100 length human p21 was transiently expressed in SJSA 100 cells and nutlin-3a was added for 48 h. Western analysis 80 80 showed substantial elevation of the basal p21 levels in 60 60 the cells transfected with p21 expression construct compared with the vector controls (Figure 2c). Total 40 40 p21 protein levels were further increased in the presence p21 expression (%)

p21 expression (%) 20 20 of nutlin-3a. However, no significant changes in the apoptotic fraction were registered by the Annexin V or 0 0 RKO HCT116 SJSA RKO HCT116 SJSA PARP cleavage assays (Figure 2d). Basal Induced We expanded our cell panel by including additional four solid tumor-derived cell lines: A549 and H460 (lung c C p21 C p21 C p21 siRNA cancer), MCF7 (breast) cancer and MHM (osteosarco- -++ -+ + -+ + -+ + -+ + -+ + Nutlin-3a ma). These tumor cell lines had shown low (A549, p53 H460), intermediate (MCF7) and high (MHM) apopto- tic index in response to nutlin-3a (Tovar et al., 2006). MDM2 The activation of the p53 pathway by nutlin-3a was p21 confirmed by the accumulation of p53, MDM2 and p21 (Figure 3a). The p21-specific siRNA effectively depleted p21, bringing the nutlin-induced protein levels RKO HCT116 SJSA d Nutlin-3b Nutlin-3a Nutlin-3b Nutlin-3a Figure 1 Cancer cell lines with wild-type p53 differ in their 42.4% 1.9% 47.5% 43.9% dependency on p21 for cell-cycle arrest. (a) p21 is upregulated by nutlin treatment of cancer cell lines. Exponentially growing RKO, HCT116 and SJSA1 cells were incubated with 10 mM nutlin-3a for RKO 24 h and the relative level of p21 mRNA was determined by qPCR and calculated as fold induction or relative expression normalized to RKO cells treated with the inactive enantiomer nutlin-3b. 39.8% 0.6% 43.1% 28.9% (b) p21 mRNA is effectively depleted by siRNA. Exponentially proliferating cells were transfected with gene-specific p21 siRNA or nontargeted control siRNA and after 24 h were incubated with

HCT116 10 mM nutlin-3a or 3b for 24 h. The levels of p21 mRNA were determined by qPCR and expressed as percent of control. (c) p21 26.4% 0.5% 21.4% 0.9% protein levels after treatment with p21 siRNA. Cells were transfected and treated as in (b) and their protein content was analyzed by western blotting. (d) Effect of p21 knockdown on cell- SJSA cycle distribution. Cell-cycle profiles were analyzed by BrdU labeling and flow cytometry. Cells in each phase were calculated siRNA-Control siRNA-p21 from the flow cytograms and expressed as percentage of the total cell population.

Oncogene p21 and nutlin-induced apoptosis MXiaet al 349

a 90 RKO 90 HCT116 90 SJSA

80 siRNA-Control-3b 80 80 siRNA-Control-3a 70 siRNA-p21-3b 70 70 60 siRNA-p21-3a 60 60 50 50 50 40 40 40 30 30 30

Annexin V+ cells (%) 20 20 20 10 10 10 0 0 0 24h 48h 72h 96h 24h 48h 72h 96h 24h 48h b 48h 72h 96h 0h 24h 48h C C p21p 21 C C p21 C p21 siRNA C p21 C p21 C p21 siRNA - Dox -+-+ - ++++ Nutlin-3a --++++Nutlin-3a PARP PARP Cleaved PARP Cleaved PARP

Actin SJSA Actin HCT116 PARP Cleaved PARP

RKO Actin

c --++p21 d 80 Vector-NTC -+-+Nutlin-3a 60 p53 Vector-3a 40 p21-NTC MDM2

cells (%) 20 p21-3a p21 Annexin V+ 0 Actin --++p21 -+-+Nutlin-3a PARP Cleaved PARP

Actin

Figure 2 Effect of p21 modulation on nutlin-induced apoptosis. (a) Proliferating RKO, HCT116, and SJSA1 cells were transfected with targeted p21 siRNA or control siRNA for 24 h followed by incubation with 10 mM nutlin-3a or 3b for indicated times. Annexin V positive cells were measured by the Guava Nexin assay and expressed as percentage of the total population. (b) Western analysis was performed to detect the full length and cleaved PARP. (c) SJSA1 cells were transfected with pReceiver-p21 or control vector as indicated. 24 h posttransfection, cells were incubated with 10 mM nutlin-3a for additional 24 h and cell lysates were collected for western analysis. (d) Cells were exposed to 10 mM nutlin-3a for 48 h following transfection with p21 expression plasmids and their apoptotic fraction was determined by the Annexin V assay and western analysis for PARP cleavage. at or below their uninduced level in all four cell lines Treatment of RKO and HCT116 cells with 300 nM (Figure 3a). Depletion of p21 followed by 48 h of doxorubicin gave rise to a moderate fraction of exposure to nutlin-3a did not significantly alter the apoptotic cells. Targeted p21 siRNA dramatically apoptotic response in any of these cells, confirming that elevated the apoptotic rate revealed by the increase of p21 does not have apoptosis-inhibitory function on p53 subG1 cell fraction from 17 to 52% in RKO and 9 to activation by nutlin. Taken together, our data derived 37% in HCT116 cells (Figure 4a). Western analysis using multiple cancer cell lines and alternative siRNA showed a drop in full-length PARP and reciprocal species does not support an antiapoptotic role of p21 in increase in cleaved PARP in the higher apoptotic the response to p53-dependent apoptosis induced by fraction (Figure 4b). These results indicate that p21 nutlin-3 in these lines. can function as an induced in response to DNA damage as previously reported by Elevated p21 protects cancer cells from others. doxorubicin-induced apoptosis We next checked whether p21 can have an antiapoptotic p21 mediates survivin downregulation in response role in the response to DNA damage as previously to nutlin-3a reported (Bunz et al., 1999; Tian et al., 2000; Gartel and Disruption of the p53–MDM2 interaction by nutlin Tyner, 2002). We chose the genotoxic drug, doxorubi- leads to stabilization and activation of p53, followed by cin, frequently used to activate the p53 response. profound changes in (Tovar et al.,

Oncogene p21 and nutlin-induced apoptosis MXiaet al 350 a C p21 C p21 C p21 C p21 siRNA - + - + - + - + - + - + - + - + Nutlin-3a p53

MDM2

p21

Actin A549 H460 MCF7 MHM

b 80 siCtrl-3b 70 siCtrl-3a 60 sip21-3b 50 sip21-3a 40 30 20

Annexin V+ cells (%) 10 0 A549 H460 MCF7 MHM Figure 3 Knockdown of p21 does not change sensitivity of cancer cell lines with wild-type p53 to nutlin-induced apoptosis. (a) Cells were transfected with p21 siRNA or control siRNA for 24 h and then incubated with 10 mM nutlin-3a for additional 24 h for western analysis (a) or 48 h for apoptosis analysis by the Annexin V assay (b).

a RKO HCT116 NTC Dox NTC Dox

3.04% 17.74% 3.14% 9.22% siRNA-Control

2.53% 52.08% 3.31% 35.96% siRNA-p21

b C p21 C p21 siRNA PARP Cleaved PARP

Actin

RKO HCT116 Figure 4 p21 protects cancer cells from doxorubicin-induced apoptosis in RKO and HCT116. RKO and HCT116 were transfected with p21 siRNA or control siRNA for 24 h and 300 nM doxorubicin or DMSO solvent were added for additional 96 h. The effect of p21 knockdown on apoptosis was detected by the increase of subG1 cell fraction measured by flow cytometry (a) or appearance of cleaved PARP by western analysis (b).

2006). One of the most prominently downregulated in human cancer (Altieri 2003). Owing to its antiapop- genes was BIRC5 encoding the survivin protein (Tovar totic activity, survivin overexpression has been thought et al., 2006). Survivin is a member of the inhibitors of to have an important role in tumor development, apoptosis protein family frequently found overexpressed although the precise molecular mechanisms have not

Oncogene p21 and nutlin-induced apoptosis MXiaet al 351 a RKO SJSA

Survivin Survivin c H1299 Nutlin-3b 1.2 5 1.4 Nutlin-3a 1.2 1.0 4 1.0 0.8 3 Vector 0.8 0.6 p21 0.6 2 0.4 0.4 1 0.2 0.2 Relative expression Relative expression 0.0 0.0 0 siR-C siR-p21 siR-C siR-p21 p21 Survivin

p21 p21 7 30 d 8 H1299 6 25 7 5 20 6 4 15 5 NTC 3 Dox 10 4 2 3 5 1 2 Relative expression 0 0 1 siR-C siR-p21 siR-C siR-p21 Relative expression 0 p21 Survivin

b C p21 C p21 siRNA e H1299 H1299 − + − + − + − + Nutlin-3a C p21 − Dox Survivin Survivin

Actin Actin RKO SJSA

fgSJSA MHM CT Birc5 CT Birc5 70 80 C Birc5 C Birc5 60 70 − + − + − + − + Nutlin-3a 50 60 50 40 40 Survivin 30 30 20 20 10 10 Actin Annexin V+ cells (%) 0 0 NTC Nut-3a NTC Nut-3a Figure 5 p21 mediates the suppression of survivin expression in response to nutlin-3a. RKO and SJSA cells were transfected with p21 siRNA or control siRNA for 24 h. Nutlin-3a (10 mM) was added to the culture 24 h later and cells were collected for qPCR analysis of survivin and p21 mRNA (a) and western blotting (b). Expression levels of mRNA were normalized to siRNA controls. (c) H1299 cells were stably transfected with pReceiver-p21 or control plasmid and G418-resistant cells were pooled. Survivin and p21 mRNA were measured by qPCR and expressed as relative levels normalized to vector-transfected controls. (d) H1299 cells were incubated with 1 mM doxorubicin or equivalent DMSO solvent (NTC) for 24 h before qRCR analysis and expressed as relative values normalized to no- treatment controls. (e) Survivin protein levels in the p21-overexpressing or doxorubicin-treated H1299 cells were analyzed by western blotting. (f) SJSA and MHM cells were transfected with human full-length survivin expression plasmid or vector control (pEZ-M02 from GeneCopoeia) for 24 h and then treated with 10 mM nutlin-3a for additional 24 h. The expression of survivin was analyzed by western blotting. (g) Cells were transfected as in (f) and then incubated with nutlin-3a for 48 h. Apoptosis was measured by the Annexin V assay. been elucidated (Altieri, 2008). Treatment of RKO and If cells are prevented from entering they may SJSA cells with nutlin-3a for 24 h led to a strong not reach the peak of survivin expression leading to an inhibition of survivin expression (Figure 5a). However, artificial lowering of expression in arrested vs asynchro- as survivin expression is cell cycle dependent with the nous cell populations. To test this possibility, we used bulk of mRNA accumulating in cells before entry into siRNA to deplete p21 in RKO and SJSA cells. As mitosis (Li et al., 1998), this suppression could result previously shown (Figure 1d), in the presence of nutlin from cell-cycle arrest in G1 and G2 phase after nutlin and depletion of p21, SJSA cells remain arrested in G1 treatment (Vassilev et al., 2004; Tovar et al., 2006). and G2 phase, whereas RKO cells restored cell-cycle

Oncogene p21 and nutlin-induced apoptosis MXiaet al 352 progression. Therefore, RKO cells offered a model for Tyner, 2002; Abbas and Dutta, 2009). Most of these testing the role of nutlin-induced p53 activation on studies have used DNA damage to activate p53 that is survivin expression in both asynchronous and G1- and known to induce both p53-dependent and p53-indepen- G2-arrested cells. Knockdown of p21 restored most of dent signaling events, making the data difficult to the lost survivin expression in both SJSA and RKO interpret and sometimes confusing. In this study, we cells, suggesting that survivin downregulation is not a use the p53-MDM2 binding inhibitor, nutlin-3, to consequence of nutlin-induced cell-cycle block, but selectively activate p53 without DNA damage and mediated by cell-cycle-independent activity of p21 investigate the role of p21 in cell-cycle arrest and (Figures 5a and b). apoptosis in multiple cancer cell lines. We find that the Next, we tested if ectopic expression of p21 in the cell-cycle arrest function of p21 depends on the cellular absence of p53 is sufficient for suppression of survivin. context. p21 induction by p53 is essential for cell-cycle To this end, p21 was overexpressed in the p53-null arrest in the colon cancer lines, RKO and HCT116. H1299 cell line. The cellular levels of p21 However, p21 is completely dispensable for cell-cycle measured 24 h after transfection (Figure 5c) were arrest in the osteosarcoma cell line SJSA1. The reasons comparable to the levels produced by nutlin-induced behind p21 independency are not clear yet, but one can p53 activation in RKO cells (Figure 5a) and other speculate that other p53-dependent cell-cycle regulators cancer cells lines (Tovar et al., 2006). However, the can substitute for p21 to assure effective cell-cycle survivin mRNA and protein levels remained the same as arrest. Three possible players (SFN, Gadd45A and in the control vector-transfected cells (Figures 5c and e). BTG2) were investigated, but no evidence for role in It has been shown that p21 expression is not entirely p21-independent cell-cycle arrest was found (data p53-dependent and can be induced in p53-null or not shown). mutant cancer cells in response to genotoxic stress Interestingly, nutlin-induced apoptotic response was (Gartel and Tyner, 1999; Yang et al., 2003), Therefore, independent of p21 upregulation by p53 in all seven we used the genotoxic drug doxorubicin to upregulate cancer lines tested in this study. These results strongly p21. Treatment of H1299 cells with 1 mM doxorubicin suggest that p21 does not have an important role in resulted in p53-independent upregulation of p21 tran- modulating the apoptotic response to nongenotoxic p53 scription (Figure 5d) and protein levels (data not activation. This conclusion can be extended to the cell- shown). However, the survivin expression remained at cycle state as an alleged modulator of the apoptotic comparable levels as in the no-drug control cells response. By depleting p21, we generated a population (Figures 5d and e). These experiments suggest that of RKO cells that can proliferate in the presence of enhanced expression of p21 alone is insufficient to nutlin-induced p53 with rates identical to untreated suppress survivin transcription that requires concomi- controls. Careful comparison of the apoptotic rate in tant p53 activation. these p21 knockdown cells and p21 ‘normal’ RKO cells Owing to the potential antiapoptotic role of survivin, blocked in G1 and G2 phase in the presence of the same we asked whether overexpression of exogenous survivin nutlin concentration did not reveal any differences. would protect cancer cells from nutlin-induced apopto- These results clearly indicate that cell-cycle arrest does sis. SJSA and MHM cells were transfected with the not affect the apoptotic response to nongenotoxic p53 survivin-expression plasmids and survivin protein was activation. In contrast, ablation of p21 by siRNA in the analyzed by western blotting. Despite the massive colon cancer lines RKO and HCT116, sensitized these increase in survivin protein levels, sensitivity of both cells to doxorubicin, confirming previous observations cell lines to nutlin-induced apoptosis remained un- that p21 can function as an apoptosis modulator in the changed (Figures 5f and g). These results suggest that response to genotoxic stress. survivin is unlikely to have an antiapoptotic role and its We found that the induction of p21 by nutlin-3a, downregulation by MDM2 antagonists does not affect strongly suppresses the expression of survivin that has the apoptotic response. been shown to act as an inhibitor of apoptosis. It has been reported previously that the survivin gene can be inhibited transcriptionally by wild-type p53 protein that binds in its promoter region and recruits a repressor Discussion complex (Hoffman et al., 2002). However, later studies have not confirmed the direct binding of p53 to the Since its discovery as the first p53-regulated gene survivin promoter (Lohr et al., 2003). Furthermore, they (Harper et al., 1993; el-Deiry et al., 1993), p21 has been provided experimental evidence that p21 functions as a extensively studied and its important role in p53- mediator of survivin suppression (Lohr et al., 2003). Our mediated tumor suppression as a potent cell-cycle results are consistent with a critical role of p21 in inhibitor has been well documented (Bunz et al., 1999; mediating negative regulation of survivin expression by Harris and Levine, 2005). However, the proposed p21 p53. However, we do not find significant effect of functions outside the cell cycle are poorly defined and ectopic p21 expression on survivin transcription in the somewhat controversial. A number of studies have absence of p53, suggesting that p53 expression/activa- presented evidence that p21 can protect cells from tion is necessary for the observed inhibitory effect. The apoptosis induced by genotoxic insults or chemother- molecular mechanism of survivin suppression by apeutics both in vitro and in animal models (Gartel and p21/p53 needs further investigation. The fact that p21

Oncogene p21 and nutlin-induced apoptosis MXiaet al 353 induction by nutlin leads to downregulation of a known nutlin induces robust apoptotic response and there is no antiapoptotic protein also argues against antiapoptotic correlation between p21 induction and response (Tovar role of p21 in the response to nongenotoxic p53 et al., 2006). This study presents direct evidence that p21 activation. does not have a role in the switch from cell-cycle arrest However, besides SJSA and RKO cells, we have to apoptosis induced by selective nongenotoxic p53 observed strong downregulation of survivin by nutlin-3a activation. In fact, the cell line with the strongest in several other cancer cell lines, but no correlation apoptotic response, SJSA, has the most pronounced between the expression of survivin and their apoptotic cell-cycle arrest that persists even in the absence of p21 index (data not shown). Our experiments directly (Figure 1d). The molecular determinants of apoptotic addressing the role of survivin in SJSA and MHM cells response may differ between tumor types and stages, also did not show any apoptosis-inhibitory effect and there are likely multiple ways cancer cells can use to (Figures 5f and g). The role of survivin in modulation disable p53 downstream signaling. However, p21 does of apoptosis seems to be much more complex than not seem to be a factor in the resistance to selective initially thought. Its ability to antagonize cell death nongenotoxic p53 activation. relies on multiple factors such as mitochondrial localiza- tion, phosphorylation status and physical interaction with other cofactors (Dohi et al., 2004, 2007; Qi et al., Materials and methods 2009). In fact, studies using RNA interference to deplete survivin have not been able to show a substantial effect Cells, constructs and drug treatment MHM osteosarcoma cells were provided by the Norwegian on apoptosis (Carvalho et al., 2003; Lens et al., 2003). Radium Hospital, Oslo, Norway. All other cell lines were Our results also do not support an antiapoptotic role of purchased from the American Type Culture Collection (ATCC, survivin in p53-dependent apoptosis induced by MDM2 Manassas, VA, USA) and maintained in the recommended antagonists. media supplemented with 10% heat-inactivated fetal bovine A recent study published in the course of this study serum (Invitrogen, Carlsbad, CA, USA) in a humidified has reported that p21 downregulation by siRNA incubator with 5% CO2. Human p21-expressing construct, enhances the apoptotic response to nutlin-3 (Enge pReceiver-p21, was purchased from GeneCopoeia, Rockville, et al., 2009). This conclusion was based on limited MD, USA. Nutlin-3a and 3b were synthesized at Hoffmann-La experiments with a single cell line, HCT116. Our results Roche, Nutley, NJ, USA. Doxorubicin was purchased from using multiple cancer cell lines (including HCT116) and Sigma (St Louis, MO, USA). Drugs were dissolved in dimethyl sulfoxide and kept at 20 1Cas10mM stock solutions. readouts do not support such a conclusion. In contrast, À we were not able to find a single cellular system in which elevation of p21 levels have a significant role in the Immunoblotting Cells were grown in 75 cm2 flasks (106 cells per flasks) or 6-well apoptotic response to nutlin. plates (2 Â 105 cells per well), lysed in 0.1–0.2 ml RIPA buffer, Although a substantial number of reports claim an and western blotting was performed as previously described antiapoptotic role of p21 in the p53 pathway, many of (Tovar et al., 2006). Primary used are as follows: these studies have used as a model system HCT116 mouse monoclonal anti-p53 (sc-263) and mouse monoclonal (p21À/À) cells, generated by homologous disruption of anti-MDM2 (sc-965) were from Santa Cruz Biotechno- the p21 gene (Bunz et al., 1999). A recent study on the logy, mouse monoclonal anti-p21 (OP64) from Calbiochem role of p21 in the response to have shown that (Gibbstown, NJ, USA), rabbit polyclonal anti-PARP from the hypersensitivity of HCT116 (p21À/À) cells is p53- (Danvers, MA, USA) (9542), rabbit polyclonal dependent but p21-independent (Ferrandiz et al., 2009). anti-survivin (NB500–201) from Novus Biologicals (Littleton, Ferrandiz et al. have demonstrated that inducible p21 CO, USA) and b-actin (AC-74) from Sigma. The secondary antibodies, anti-mouse IgG-HRP (sc-2302) and anti-rabbit expression in HCT116 (p21À/À) cannot rescue the IgG-HRP (sc-2301) were from Santa Cruz Biotechnology. hypersensitivity of these cells to the tyrosine kinase inhibitor. In addition, downregulation of p21 expression by RNA interference or by forced expression of c-myc siRNA experiments Cells were seeded in 6-well plates at 50–60% confluence and did not sensitize parental HCT116 cells. These observa- transfected with 100 nM siRNAs using Lipofectamine 2000 tions suggest that the role of p21 in apoptosis should be (Invitrogen). The two human p21 siRNAs were from reexamined using additional cellular models. Dharmacon, Chicago, IL, USA (sense: 50-GAUGGAACUUC Our results that p21 does not inhibit the apoptotic GACUUUGUUU-30; antisense: 50-ACAAAGUCGAAGUU response to nongenotoxic p53 activation have important CCAUCUU-30) or Santa Cruz Biotechnology (A pool of two implications for the development of MDM2 antagonists target-specific siRNAs) respectively. After 24 h incubation and other modalities directly targeting p53 for activa- with lipofectamine/siRNA mix, the medium was replaced with tion. As p21 is universally upregulated by activated p53, 3 ml fresh medium containing appropriate concentrations of its inhibitory role will be detrimental to achieving drugs and incubated for additional time intervals as described. effective therapeutic response in any cancer expressing Cells were then collected and processed for , Quantitative RT–PCR and apoptosis analysis as previously wild-type p53. Our previous results suggested that this is described (Xia et al., 2008). For small hairpin RNA , an unlikely reason for lack of apoptotic response (Tovar cells were seeded in 6-well plates at 50–60% confluence and et al., 2006). Despite the fact that some cancer cell lines infected with SMARTvector shRNA-p21 lentiviral particles have shown minimal apoptosis in response to p53 (Dharmacon) at multiplicity of infection 10. At 48 h post- activation by nutlin, there are cancer cell lines in which infection, the cells were selected with 5 mg/ml puromycin.

Oncogene p21 and nutlin-induced apoptosis MXiaet al 354 Quantitative RT–PCR calculated as the total fraction Annexin V-positive cells as Cells were seeded in 6-well plates and total RNA was isolated described (Thompson et al., 2004). using RNeasy kit (Qiagen, Valencia, CA, USA). cDNA was synthesized from 4 mg of total RNA from each sample using Cell-cycle analysis Superscript II reverse transcriptase (Applied Biosystems, Changes in cell-cycle distrubution were assessed by flow Foster City, CA, USA) according to manufacturer’s protocol. cytometry. Cells were collected and fixed in 70% ethanol at The relative mRNA levels of the human p21 were determined À20 1C for 1 h and kept frozen. After thawing, cells were by Taqman, using gene-specific primer/probe sets and 18S washed twice in cold phosphate-buffered saline and resus- RNA as a normalization control. The sequence of the human 0 pended in 0.5 ml PI/RNase staining buffer (BD Pharmingen, p21 primers and probe were as follows: forward: 5 -CTGGA- San Diego, CA, USA), incubated for 15 min at 37 1C and GACTCTCAGGGTCGAA-30; reverse: 30-CGGCGTTTGGA 0 0 analyzed using FACSCalibur cytometer (Becton Dickson, GTGGTAGAA-5 ; probe: 5 -TTGGCTCACTGCAAGCTCG Franklin Lakes, NJ, USA) using CellQuest software (BD CCCTT-30. The survivin Taqman gene expression assay was Biosciences, San Jose, CA, USA). In total, 20 mM BrdU purchased from Applied Biosystems. (Sigma) was added 1 h before collecting the cells. Cells were fixed in 70% ethanol, permeablized with 2N HCl and 0.5% TritonX-100 for 30 min at room temperature, and neutralized Annexin V assay with 0.1 M sodium tetraborate (pH 8.5). After washing, cells Cells were seeded in 6-well tissue culture plates the night before were labeled with anti-BrdU FITC-conjugated siRNA transfections. At 24 h post-siRNA addition, the cells (Becton Dickinson) in 0.5% Tween 20/phosphate-buffered were treated with nutlin-3b or 3a for additional 24–96 h. saline for 1 h before analysis. Culture media were collected and combined with the trypsinized-attached cells. Cell pellets were collected by centrifugation at 2000 r.p.m. for 10 min at 4 1C. The percentage of apoptotic cells was determined by Annexin V/7-AAD Conflict of interest staining using the Guava Nexin kit and the Guava personal cell analyzer (Guava Technologies, Hayward, CA, USA), and The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene