Induction of p21 by p65 in p53 null cells treated with Doxorubicin

Induction of p21by p65in p53null cells treated with DoxorubicinShenglin Ma b ,Juanjuan Tang a ,Jianguo Feng b ,Yaping Xu b ,Xinmin Yu b ,Qinghua Deng b ,Yanjun Lu a,⁎aLaboratory of Biochemistry and Molecular biology,Tongji University,1239Siping Road,Shanghai 200092,ChinabZhejiang Tumor Hospital,38Guangji Road,Banshanqiao,Hangzhou 310022,Zhejiang Province,ChinaReceived 6September 2007;received in revised form 10January 2008;accepted 11January 2008Available online 26January 2008AbstractNF κB/p65is a transcription factor that can protect or contribute to cell death.Here we show that knockdown of p65by I κBSR or p65siRNA decreased the cytotoxic effect of DOX on HCT116(p53+/+)cells,correlating with increased induction of p21.In previous work,we demonstrated that p21suppressed cell death via its CDK-inhibitory activity.Thus,we propose that the p65activity is required for p53-dependent cell death through limitation of p53-induced p21expression.In HCT116(p53−/−)cells,downregulation of p65expression enhanced the cytotoxic effect of DOX,due to decreased p21expression levels.We present evidence that in p53-null tumor cells treated with DOX,p65was involved in induction of p21expression by directly binding to the p21promoter.Our data suggested that both p53and p65limited each other's ability to stimulate p21induction and this mutual repression mechanism was consistent with a model in which both factors were competing for limiting pool of p300/CBP coactivator protein complexes.These findings indicate an association between p21expression and resistance to cell death through p65,a novel regulatory mechanism in which p21bridges a transcriptional crosstalk between p53and p65.©2008Elsevier B.V .All rights reserved.Keywords:p21;NF κB/p65;p53;CBP/p300;Cell death1.IntroductionIn response to DNA damage induced by cytotoxic agents,the tumor suppressor p53accumulates and functions as a sequence-specific DNA-binding protein,which positively regulates ex-pression of several genes,including p21(waf1/cip1/sdl1)[1].P21is an important cellular checkpoint protein for G1and G2arrest.Recently,contribution of p21to suppression of DNA damage agents induced cell death has been widely accepted [2–7].Nuclear factor-κB (NF κB)is a transcriptional factor involved in the response to various stimuli and plays a central role in inflammatory reactions.NF κB generally exists as an inactive dimer sequestered in the cytoplasm by an inhibitor protein termed I κB.Activation of NF κB involves phosphorylation of I κB on two critical serine residues at codon 32and 36.Oncephosphorylated,I κB is subjected to ubquitination at two lysine residues and subsequent degradation by the 26S proteasome.Unmasking I κB allows NF κB/p65to translocate into the nuc-leus,where it binds to the target genes,and promotes trans-cription [8].It is widely accepted that activation of NF κB may function to protect cells from apoptosis in response to TNF-αtreatment.NF κB can also be implicated in cell cycle arrest.It has been reported that a novel potential NF κB binding site is at position −2008of the p21promoter and the binding of NF κB/p65protein to this κB site results in transactivation of p21promoter by p65[9].In this study,we used HCT116(p53+/+)cells and their matched HCT116(p53−/−)cells in which the p53gene has been deleted by homologous recombination.We showed that the reduction of p65remarkably decreased the cytotoxic effect of Doxorubicin (DOX)on HCT116(p53+/+)cells,correlating with the increasing of induction for p21level by p53after transfection with I κBSR (I κB αsuper-repressor)or p65small interferingAvailable online at Biochimica et Biophysica Acta 1783(2008)935–940/locate/bbamcr⁎Corresponding author.Tel.:+862165984636;fax:+862165983793.E-mail address:yanjunlu@ (Y .Lu).0167-4889/$-see front matter ©2008Elsevier B.V .All rights reserved.doi:10.1016/j.bbamcr.2008.01.008RNA.In contrast,on HCT116(p53−/−)cells the reduction of p65enhanced the cytotoxic effect of DOX treatment due to the decrease of p21level.We also documented that in the absence of p53,p65was able to directly bind to the p21promoter by Chromatin Immunoprecipitation(ChIP).Our data suggested that both p53and p65inhibited each other's ability to stimulate p21 expression and this mutual repression mechanism is consistent with a model in which both factors are competing for limiting pool of p300/CBP coactivator protein complexes.Thus we presented evidence that p21may be involved in transcriptional cross talk between p53and p65.2.Materials and methods2.1.Cells and transfectionHuman colorectal cancer cell lines HCT116(p53+/+)and HCT116(p53−/−) were generous gifts of Dr.B.V ogelstein.Cells were cultured in DMEM medium supplemented with10%FBS(Hyclone,logan,UT).The plasmid expressing the IκBSR was provided by Dr.Yamagishi(Kyoto University,Japan).IκBSR containing the mutations at Ser32,Ser36phosphorylation residues which cannot be phosphorylated.P65small interfering RNA(psiRNA-p65),was constructed according to the procedure of psiRNA-hH1zeo kit(Invivogen,San Diego, USA).Sense and antisense RNA oligonucleotides,5′-CAAAAAAAGGT-CATGGACGGTCTATCTCTTGAATAGACCGTCCATGACCTTT-3′and5′-TCCCAAAGGTCATGGACGGTCTATTCAAGAGATAGACCGTCCAT-GACCTTTT-3′,which contained the loop sequence were synthesized by Sangon Ltd.(Shanghai,China).The plasmids of pRc/RSV-mCBP-HA and pRc/ RSV-p300were kindly provided by Dr.Goodman(Oregon Health&Science University,US).HCT116cells were transiently transfected with expression constructs for IκBSR,psiRNA-p65,pRc/RSV-mCBP-HA or pRc/RSV-p300, using GeneJammer transfection reagents(Stratagene,La Jolla,CA).2.2.Measurement of cell deathCells were seeded into96-well microtiter plates and transiently transfected 12h with expression constructs for IκBSR,psiRNA-p65,pRc/RSV-mCBP-HA or pRc/RSV-p300,followed by DOX treatment(2μg/ml)for24h.Then10μl of the CCK-8solution was added to each well of the plates and then incubated for 2h in the incubator(37°C and5%CO2).Cytotoxicity assay could be used with the CCK-8solution according to the procedure of Cell Counting Kit-8(Dojindo Laboratories,Tokyo,Japan).The absorbency was measured at450nm using a microplate reader(BioTeK).2.3.Western blotting and immunoprecipitationThe nuclear and cytoplasmic extract from the cells were prepared by the Nuclear Extract Kit(Active Motif,Carlsbad,CA).The protein content of the cell lysate was determined by using the Bradford calorimetric assay method (Bio-Rad,Richmond,CA).The40μg aliquot of cytoplasmic or nuclear lysate were resolved by12%polyacrylamide-sodium lauryl sulfate gel electrophoresis and transferred to a Hybond-C Super membrane(Amersham,Buckinghamshire, UK).P65and p21were detected with an antibody against p65(Upstate,U.S.A.) and an antibody against p21(Santa Cruz Biotechnology,CA),respectively. Then the blot was incubated with a secondary antibody,IRDye800conjuga-ted affinity purified anti-mouse or anti-rabbit IgG(Rockland Immunochemi-cals,Inc.,Gilbertsville,PA)and detected with Odyssey Infrared Imaging System (LI-COR Bioscienceces,Nebraska,USA).For immunoprecipitation,a500μg aliquot of cell lysate was incubated for1h at4°C with the anti-CBP antibody (Santa Cruz Biotechnology,CA)or anti-p300antibody(Santa Cruz Biotech-nology,CA).Immune complexes were captured by incubation with80μl protein G-plus-agarose(Santa Cruz Biotechnology,Santa Cruz,CA)for10h,then washed several times with NP-40buffer and lysed with loading buffer to do Western blotting analysis.P65was detected with an antibody against p65 (Upstate,U.S.A.).2.4.Cell growth analysisCells were washed twice with PBS,trypsinized and resuspended in PBS containing0.1%Triton X-100and RNase(1mg/ml)(Sigma,St Louis,MO).The cell suspension was incubated at37°C for30min.Propidium iodide(Molecular Probes,Inc.Eugene,OR)was added at a final concentration of50μg/ml and the cell suspension was kept at4°C for1h.The cells were filtered and the cell cycle was analysed by flow cytometry with the FACScan system(Becton Dickinson, Franklin Lakes,NJ).2.5.ChIPHCT116cells(5×108)were treated with or without DOX(2μg/ml)for24h. According to Chromatin Immunoprecipitation(ChIP)Assay Kit(Upstate),for-maldehyde was added at1%to the culture media and cells were incubated at room temperature for10min with mild shaking.Protein-DNA complexes were immunoprecipitated using the p65antibody(Upstate)or mouse IgG control.DNAs were purified and used for PCR with primers specific for the p21gene promoter region encompassing p65binding sites.The primers of p21were:5′-TA TTTGG-GACTCCCCAGTCTCT-3′and5′-GGCTCTCTGCTTGTCA TCCTTT-3′.The primers of GAPDH were:5′-TACTAGCGGTTTTACGGGCG-3′and5′-TCGAA-CAGGAGGAGCAGAGAGCGA-3′.2.6.Electrophoretic mobility shift assaysA30μl of annealing mixture containing2μl of eachκB site oligonucleotide (sense:5′-AGT TGA GGG GAC TTT CCC AGG C-3′;antisense:5′-GCC TGG GAA AGT CCC CTC AAC T-3′)in0.5M NaCl was incubated2min at80°C then stopped the heating and maintained in water bath until35°C.The above 8μl of solution,2μl of10×labeling Buffer and2μl IR Dye label solution (IRDye™800labeling kit,Odyssey,U.S.A.)were mixed,and the reaction proceeded for5min at70°C.The cells were washed with ice-cold PBS,and then collected.The nuclear extracts were prepared by the Nuclear Extract Kit(Active Motif,Carlsbad,CA), and5μg aliquots of the extracts were preincubated with1.5μl of above labeled oligonucleotide on ice for30min.The complexes were resolved on a5% polyacrylamide gel.The gel was scanned by Odyssey infrared imaging system (LI-COR Biosciences).2.7.Northern blot analysisNorthern blotting was performed using total RNA following the protocol provided with the Odyssey Infrared Imaging System(LI-COR Biosciences).Briefly, 5μg total RNA per lane isolated from cells using TRIzol reagent(Invitrogen)were electrophoresed and transferred onto Biodyne®B Nylon Membrane(Pall)using the NorthernMax Kit(Ambion).RNA was cross-linked to the membrane the blot was pre-hybridized at42°C with ULTRAhyb™-Oligo Buffer(Ambion).Blot was hybridized overnight at42°C with biotinylated probes which had been denatured and then washed with NorthernMax Low and High Stringency Washes.Blot was blocked for30min with Odyssey®Blocking Buffer plus1%SDS and then incubated for 30min with Streptavidin IRDye800CW conjugate in blocking buffer plus1%SDS (LI-COR Biosciences).Blot was washed,then scanned by an Odyssey Infrared Imaging System(LI-COR Biosciences).HCT116cell DNA was used as a template for the PCR amplification in the presence of biotin-16-dUTP(2:3biotin-dUTP:TTP,Roche)to generate bio-tinylated probes.The probe forβ-actin mRNA is a PCR-amplified0.5kb coding sequence,while the probe for p21mRNA is0.49kb coding sequence.3.Results3.1.Requirement of p65for DOX-induced cell death inp53-dependent wayP53tumor suppressor is known to play an important role in mediating DNA damage agents induced-cell death through a936S.Ma et al./Biochimica et Biophysica Acta1783(2008)935–940variety of mechanisms [10–12].To confirm this point,we treated HCT116(p53+/+)cells and HCT116(p53−/−)cells with DOX for 24h.As shown in Fig.1A,DOX-induced cell death was higher in p53+/+cells than in p53−/−cells.This result suggests a requirement of p53for DOX-induced cell death.P65is involved in the regulation of cell death in several systems.To examine the effect of p65in this study,a vector was generated to synthesize p65small interfering RNA by the human H1polymerize III promoter.The p65small interfering RNA (psiRNA-p65)and the unphosphorylatable I κB αsuper-repressor (I κBSR)were used to inhibit the activity of endogenous NF κB/p65in cells.The rate of cell survival was shown in Fig.1A.When the HCT116(p53+/+)cells were forced to express I κBSR or p65small interfering RNA,the percen-tage of DOX-induced cell death were remarkably reduced as compared to the control cells.In contrast,expression of I κBSR or p65small interfering RNA significantly sensitized to DOX-induced cell death in HCT116(P53−/−)cells.The effects ofpsiRNA-p65and I κBSR on cell death of HCT116(p53+/+)and HCT116(P53−/−)cells were also shown in Fig.1A.No effect was seen after transfection of the empty vector.The levels of nuclear activated and cytoplasmic inactivated p65were assessed by immunoblotting in HCT116(P53+/+)and HCT116(P53−/−)cells.A clear reduction of nuclear activated p65was seen after expression of I κBSR or p65small interfering RNA(Fig.1B).Electrophoretic mobility shift assays suggested that over-expressing of I κBSR and psiRNA-p65significantly decreased the induction of NF-κB DNA-binding activity in HCT116(p53+/+)and HCT116(p53−/−)cells treated with DOX (Fig.1C).These data led us to suggest that loss of NF κB/p65activity specifically abrogated the p53-mediated cell death res-ponse to DOX.On the other hand,these observation also determined that in p53null or defective tumors,targeting in inhibition of NF κB/p65activation was useful,leading to the potency ofDOX-treatment.Fig.1.Role of down-regulation of p65in DOX-induced cell death in HCT116(p53+/+)and HCT116(p53−/−)cells.Cells were transiently transfected with or without I κBSR and psiRNA-p65followed by DOX treatment (2μg/ml)for 24h.(A)Cell survival was examined by CCK8kit.Error bar indicates the standard error of the mean of three independent experiments.(B)The nuclear and cytoplasmic p65protein were detected by Western blotting in HCT116(p53+/+)cells (above)and HCT116(p53−/−)cells (bottom).β-actin and PCNAwere used as controls.(C)Nuclear extracts from the cells were subjected to electrophoretic mobility shift assay (EMSA)using IR Dye ™labeled κB oligonucleotide in HCT116(p53+/+)cells (above)and HCT116(p53−/−)cells(bottom).Fig.2.Regulation of p21expression by down-regulation of p65.HCT116(p53+/+)cells and HCT116(p53−/−)cells were transiently transfected with or without I κBSR and psiRNA-p65followed by DOX treatment (2μg/ml)for 24h.(A)P21protein expression was detected by Western blotting in HCT116(p53+/+)cells (above)and HCT116(p53−/−)cells (bottom).β-actin was used as control.(B)Northern blot of total RNA from HCT116(p53+/+)or HCT116(p53−/−)cells.Hybridization with biotinylated p21-specific probe (above)or biotinylated β-actin-specific probe (bottom).(C)Cell cycle pattern of HCT116(p53+/+)cells ()and transfected with psiRNA-p65(□)or HCT116(p53−/−)cells ()and transfected with psiRNA-p65(■)followed by DOX treatment (2μg/ml)for 24h.Error bar indicates the standard error of the mean of three independent experiments.937S.Ma et al./Biochimica et Biophysica Acta 1783(2008)935–9403.2.Regulation of induction for p21by p65In previous studies,we showed that DOX is a cell cycle-dependent anti-tumor drug and p21suppresses DOX-induced cell death by arresting cells at G1or G2phase [5,6].The expression level of p21increased in p53-dependent or p53-independent pathway.To investigate the role of p21in this study,we assessed the expression levels of p21protein and mRNA in both HCT116(p53+/+)and HCT116(p53−/−)cells.As shown in Fig.2A,the p21protein expression level in the cell extract was elevated in HCT116(p53+/+)cells after transfec-tion with I κBSR or p65small interfering RNA followed by DOX treatment (2μg/ml)for 24h.Interestingly,under identical conditions to HCT116(p53−/−)cells,the p21level was down regulated,as compared to the control cells.Moreover,the expression levels of p21mRNA corresponded to their pro-teins pattern in HCT116(p53+/+)and HCT116(p53−/−)cells (Fig.2B).To examine the change in the cell cycle pattern of these cells after regulating of p21expression by p65,the flow cytometry was carried out (Fig.2C).As expected,in HCT116(p53+/+)cells a fraction of G1-phase cells was increased from 67to 81%,indicating the cell cycle is arrested at G1-phase due to increasing of p21expression.On the other hand,in the HCT116(p53−/−)cells the fraction of G1-phase cells was decreased from 61to 35%by reducing of p21expression.These data led us to suggest that p65limited p53-induced p21expression in p53+/+cells and was involved in inducing p21expression in p53−/−cells after DOX treatment.In com-bination with our previous data that over expression of p21prevents cell death due to its ability to stop cell cycle,we could suggest that p65may be necessary for DOX-induced cell death in p53-dependent way.To further determine whether p65was involved in induction of p21expression,we preformed the Chromatin Immunopre-cipitation assay to examine the ability of p65to bind to thep21promoter.After cross-linking and immunoprecipitation with anti-p65antibody,PCR using a pair of primers to the fragment that contains κB site at position −2008of the p21promoter showed a DNA band in HCT116(p53−/−)cells but not in HCT116(p53+/+)cells with DOX treatment.Moreover,the DNA band was found in HCT116(p53+/+)cells after transiently over-expressing transcriptional coactivator pro-teins p300and CREB-binding protein (CBP)(Fig.3A above),thereby suggesting that the competition between p53and p65for limiting quantities of endogenous transcriptional coactivator protein CBP/p300.For negative control,we performed PCR using a pair of primers to the fragment of GAPDH promoter,showed no DNA band in the presence of p65antibody (Fig.3A bottom).To further prove that p53competition with p65for limiting pool of CBP/p300coactivator protein complexes,we performed the coimmunoprecipitation with anti-p300antibody and then immunoblotted with anti-p65antibody.We observed the DOX treatment increased binding of p300to p65in HCT116(p53−/−)cells as compared to HCT116(p53+/+)cells,which shown in the Fig.3B.These data suggested that in tumor cells that are p53-null or defective,NF κB/p65was involved in induction of p21expression after DOX treatment by directly binding to the p21promoter.It may need further to elucidate the association between p21expression and resistance to cell death by NF κBactivation.Fig.3.Binding of P65protein to the p21promoter in HCT116(p53−/−)cells.(A)Cells were transiently transfected with or without pRc/RSV-mCBP-HA and pRc/RSV-p300followed by DOX treatment (2μg/ml)for 24h.The protein –DNA complexes were cross-linked and immunoprecipitated by the indicated antibodies.The precipitated DNA samples were analyzed by PCR using primers of p21and GAPDH.(B)Whole cell extract of HCT116(p53+/+)cells (above)and HCT116(p53−/−)cells (bottom),treated as indicated was subjected to immunoprecipitation with anti-300antibody and then immunoblotted with anti-p65antibody.Fig.4.Effects of over-expression of CBP/p300on HCT116(p53+/+)cells treated with DOX.Cells were transiently transfected with or without pRc/RSV-mCBP-HA and pRc/RSV-p300followed by DOX treatment (2μg/ml)for 24h.(A)Whole cell extract was subjected to immunoprecipitation with anti-CBP antibody or anti-p300antibody and then immunoblotted with anti-p65antibody.(B)P21expression was determined by Western blotting analysis.β-actin was used as control.(C)Cell death was examined by CCK8kit.Error bar indicates the standard error of the mean of three independent experiments.938S.Ma et al./Biochimica et Biophysica Acta 1783(2008)935–9403.3.Regulation of transcriptional activaties of p65and p53by CBP/p300These observations above suggested that both p53and p65 inhibited each other's ability to stimulate their gene expression and this mutual repression mechanism is consistent with a model in which both factors are competing for limiting pool of CBP/p300coactivator protein complexes.This idea has been reinforced by several reports[13–18].Here,in an attempt to understand how CBP/p300-dependent crosstalk between p65 and p53is regulated in this study,we performed the immu-noprecipitation with anti-CBP antibody or anti-p300anti-body and then immunoblotted with anti-p65antibody.As shown in Fig.4A,after over expressing of CBP and p300 protein,the binding of CBP and p300to p65was increased in HCT116(p53+/+)cells with DOX treatment.In parallel with these results,we also observed that p21protein levels were elevated(Fig.4B).These data suggested that high level of CBP or p300proteins increased their binding to p65and then restored the p65transcriptional activity of p21-induction in HCT116(p53+/+)cells.Next we examined the direct effect of CBP/p300over-expression on the HCT116(p53+/+)cells treated with DOX(2μg/ml)for24h(Fig.4C).As expected, upon over-expression of CBP/p300,cell death rate decreased in comparison to that in the cells transfected with a control vector. It indicated that suppression of p53transactivation through induction of p21by p65might result from limiting quantities of endogenous levels of this transcription factors CBP/p300.The present data suggested the regulation of the transcriptional decision-making mechanism that may be a key player in cellu-lar processes such as cell death.4.DiscussionIn this study,we showed that the down-regulation of p65 remarkably decreased the cytotoxic effect of DOX on HCT116 (p53+/+)cells.That correlated with the increasing of induction for p21level by p53after transfection with IκBSR or p65small interfering RNA.In contrast,on HCT116(p53−/−)cells the down-regulation of p65enhanced the cytotoxic effect to DOX treatment due to the decrease of p21level.We documented that in the absence of p53,p65was able to directly bind to the p21 promoter.Our data suggested that both p53and p65inhibited each other's ability to stimulate p21induction and this mutual repression mechanism is consistent with a model in which both factors are competing for limiting pool of p300/CBP.Thus we presented evidence that p21may be involved in transcriptional cross talk between p53and p65.In response to DOX,p21is regulated transcriptionally by p53.Our previous data suggested that expression of p21im-pedes DOX-induced cell death by inactivation of cyclin-dependent kinase(CDK)activity,which in turn blocks the cell cycle at the G1and G2phases[6].The present findings here reinforced this idea by showing p21's ability of abrogating DOX-induced cell death correlated with its inhibition of cell cycle progression after reducing p65in p53+/+cells(Fig.1A and Fig.2).Recent studies have shown the existence of p21induction in a p53-independent pathway.Here we observed p21induction in NFκB/p65-dependent mechanism.Our data indicated that DOX-induced NFκB/p65complexes were able to bind the p21promoter to activate its transactivation in HCT116(p53−/−) cells but not HCT116(p53+/+)cells(Fig.3).It suggested the competition between p53and p65in induction of p21expres-sion in response to DOX.Many cellular stimuli result in the induction of both p53and NFκB/p65[19–21].The tumor suppressor p53inhibits cell growth through activation of cell-cycle arrest and apoptosis. NFκB/p65is a transcription factor that can protect or contri-bute to apoptosis[22,23].These observations suggest that a regulatory mechanism must exist to integrate and coordinate this critical cellular decision-making event.Webester's group found that the transcriptional activities of p53and p65are governed by their relative levels of expression:p65inhibits p53-dependent transactivation,while p53expression can also suppress p65transcriptional activity[13].Growing evidences show that both p53and p65interact with the transcriptional coactivator protein p300and CBP.It is important to notice that reducing50% of the level of expression of both p300and CBP in mice,which result in profound developmental defects in mice,confirming that these coactivator proteins are at limiting concentrations in vivo [24,25].In present study,we found that p53transactivation of inducing p21was suppressed by p65,as evidenced by a clear elevation of p21level induced by p53after knockdown of p65by IκBSR or p65small interfering RNA(Fig.2A).Moreover,DOX treatment increased binding of p300to p65in HCT116(p53−/−) cells as compared to HCT116(p53+/+)cells.Then p65was able to bind the p21promoter to activate its transactivation in HCT116 (p53−/−)cells but not HCT116(p53+/+)cells(Fig.3).Over expression of transcriptional coactivator proteins p300and CBP increased the binding to p65and then restored p65transcriptional activity of inducing p21in HCT116(p53+/+)cells(Fig.4). Lately,Huang and coworkers clearly established a role of nuclear IKKαcan phosphorylate CBP thus to switch the binding from p53to p65[26].Thus,together with other reports we could favor a model in which p53can also limit p65-mediated transactivation and this mutual repression mechanism is due to their limiting common coactivators of transcription.It has been shown that loss of NFκB/p65activity specifi-cally abrogated the p53-mediated cell death response to DOX (Fig.1A and[27]).The present findings indicate that p65limiting p53-induced p21expression by competition with endogenous transcriptional coactivators CBP/p300are at least in part respon-sible for that the p65activity was required for p53-induced cell death.It has been reported that NFκB/p65can mediate to stop cell cycle and to protect cell apoptosis[28–30].Here we suggested that in tumor cells that are p53-null,NFκB/p65was involved in induction of p21expression by directly binding to the p21 promoter.Our previous data suggested that NFκB/p65-induced anti-apoptotic gene expressions are higher in p53−/−cells than in p53+/+cells[31].Thus,we could speculate that NFκB/p65mostly promotes to induce anti-apoptotic gene expressions without competing with p53in p53−/−cells.939S.Ma et al./Biochimica et Biophysica Acta1783(2008)935–940NFκB/p65has been reported to be aberrantly constitutively active in some diseases,such as ataxia telangiectasia,Hodgkin's lymphoma and breast cancer[32,33].It could speculate that transactivation by p65might contribute to p65role in tumo-rigensis.It is worth noting that in p53null or defective tumors, targeting in inhibition of NFκB/p65activation may well be useful,leading to the potentiality of chemotherapeutic drugs. However,in tumors that retain wild-type p53,such targeting may be counter productive,because it is likely to result in the repression of p53-mediated tumor cell death.In the present study,we observed that the p21expression level was a key player in the transcriptional cross talk between p53and NFκB/p65.The clear elevation of p21expression after reducing p65may provide a good rationale for resistance to DOX treatment in HCT116(p53+/+)cells.In our previous work reported that a combined treatment DOX-TNF-αis an effective chemotherapeutic strategy for cancer due to that p21's negative regulation on CDK2is blocked by DOX-TNF-αtreatment[34]. 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