Cross talk between stimulated NF-κB and the tumor suppressor p53


Nuclear factor-κB (NF-κB) and p53 critically determine cancer development and progression. Defining the cross talk between these transcription factors can expand our knowledge on molecular mechanisms of tumorigenesis. Here, we show that induction of replicational stress activates NF-κB p65 and triggers its interaction with p53 in the nucleus. Experiments with knockout cells show that p65 and p53 are both required for enhanced NF-κB activity during S-phase checkpoint activation involving ataxia-telangiectasia mutated and checkpoint kinase-1. Accordingly, the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) also triggers formation of a transcriptionally active complex containing nuclear p65 and p53 on κB response elements. Gene expression analyses revealed that, independent of NF-κB activation in the cytosol, TNF-induced NF-κB-directed gene expression relies on p53. Hence, p53 is unexpectedly necessary for NF-κB-mediated gene expression induced by atypical and classical stimuli. Remarkably, data from gain- and loss-of function approaches argue that anti-apoptotic NF-κB p65 activity is constitutively evoked by a p53 hot-spot mutant frequently found in tumors. Our observations suggest explanations for the outstanding question why p53 mutations rather than p53 deletions arise in tumors of various origins.


Homeostasis crucially depends on the regulation of cell proliferation. This is prominently evident in cancer, which is caused by aberrant proliferation due to defective cell-cycle checkpoints. Among transcription factors, the contribution of nuclear factor-κB (NF-κB) and p53 to cancer development and progression is well documented (Hanahan and Weinberg, 2000; Karin, 2006; Finkel et al., 2007).

Aberrant NF-κB activity is linked to disorders of the immune system, cancer development and resistance against chemotherapy. The NF-κB family consists of p65 (Rel A), Rel B, c-Rel, NF-κB1/p50 and NF-κB2/p52, which form homo- or heterodimers. Inhibitory factor κB (IκBs; IκBα-ɛ, BCL3, p105 and p100) sequester NF-κB in the cytosol and mask the transcriptional competence of NF-κB. Induction of NF-κB is mediated by the IκB kinase complex by the so-called ‘classical’ and ‘alternative’ pathways, which both involve degradation of IκBs (Yilmaz et al., 2003; Perkins, 2007; Wietek and O’Neill, 2007; Renner and Schmitz, 2009). In addition, a heterogeneous group of ‘atypical’ NF-κB activators, for example, UV light or genotoxic stress, result in NF-κB activation characterized by slow kinetics (Janssens and Tschopp, 2006; Scheidereit, 2006; Tergaonkar, 2006). Low levels of basally active nuclear NF-κB appear to be independent of cytoplasmic NF-κB regulation (Wu and Lozano, 1994; Royds et al., 1998; Krämer et al., 2006).

In contrast to NF-κB, the transcription factor p53 is a first-line tumor suppressor induced by stimuli endangering genome integrity. The p53 protein activates genes promoting apoptosis, senescence and cell-cycle control. The exact regulation of p53-mediated cell-cycle arrest or apoptosis is complex and depends on the cellular context and specific stress stimuli (Vogelstein et al., 2000; Moll et al., 2005; Vousden and Lane, 2007). Inactivation of the p53 pathway is observed in most human cancers, with mutations in p53 occurring in at least 50% of all tumors (Soussi and Wiman, 2007). Interestingly, in addition to the lack of tumor suppressive functions, p53 mutants gain oncogenic activities contributing to carcinogenesis and drug resistance (Moll et al., 2005; Deppert, 2007; Vousden and Lane, 2007).

Considering the deregulation of NF-κB and p53 pathways in numerous cancers, it is not surprising that an extensive cross talk between both pathways exists at various levels (Perkins, 2007). For example, NF-κB attenuates p53 protein stability by inducing the E3 ubiquitin ligase MDM2 (Tergaonkar et al., 2002; Kashatus et al., 2006). Furthermore, the NF-κ B2 gene promoter is activated by p53 mutants, and p52 can modulate the promoter activity of p53 target genes (Scian et al., 2005; Schumm et al., 2006). Moreover, p65 and p53 compete for co-activators, for example, the histone acetyltransferases p300 and CBP (Ravi et al., 1998; Wadgaonkar et al., 1999; Webster and Perkins, 1999; Huang et al., 2007).

Replicational arrest leads to activation of p53 and NF-κB and can therefore be used as a model to study their cross-signaling (Ansari et al., 2001; Gottifredi et al., 2001; Wu and Miyamoto, 2008). We now show that p53 is indispensable for enhanced NF-κB p65 transcriptional activity induced by S-phase arrest. Remarkably, p53 also promotes NF-κB functions after induction of classical NF-κB signaling with the cytokine tumor necrosis factor-α (TNF-α). Furthermore, mutant p53 augments tumor-promoting anti-apoptotic activities of NF-κB p65.


Regulation of NF-κB and p53 during S-phase arrest

Hydroxyurea (HU) arrests cells in S phase by inhibition of ribonucleotide reductase, the enzyme providing cells with deoxynucleotides for DNA replication. HU is used to treat hematological malignancies and also to inhibit the growth of tumor cells from other entities (Schrell et al., 1996; Bug et al., 2005; Krämer et al., 2008). We found that a therapeutically achievable dose of HU (Bug et al., 2005) stalled up to 70% of MCF7 cells in S-phase without notable signs of excessive cell death. Therefore, we chose these Caspase-3 null breast cancer cells as a model for signaling induced during replicational arrest (Figure 1a).

Figure 1

S-phase arrest induces nuclear factor-κB (NF-κB)-dependent gene expression. (a) MCF7 cells were treated for 48 h with hydroxyurea (HU; 1.5 mM) or were left untreated (Ctl). Cell-cycle analysis was performed with PI-FACS-analysis (sub-G1, apoptotic cells with a DNA content <2n; G0/G1, cells in G0 or G1 phase; S, cells in S-phase; G2/M, cells in interphase or mitosis; *P<0.05). (b) MCF7 cells were treated with HU (1.5 mM, 24 h) or left untreated (Ctl). Expression of NF-κB-regulated genes, p53, NF-κB members and Tubulin (loading control) was analyzed by western blot analysis. (c) MCF7 cells were treated with HU (1.5 mM) for 6–24 h, or left untreated (0). Expression of NF-κB-regulated genes and NF-κB members was analyzed by western blot analysis of cytosolic and nuclear fractions. HDAC1 and Tubulin are markers for the purity of the fractions. (d) Avidin-biotin-coupled DNA (ABCD) assay to detect the binding of NF-κB family members and p53 to a κB consensus site. MCF7 cells were treated as stated in (b). Input (IN) represents 10% of whole cell extract used for the assay. The specificity and purity of the assay used were tested by probing for IκB p105, which cannot bind an NF-κB consensus oligonucleotide (FasLm, mutant oligonucleotide from the FAS ligand promoter). (e) MCF7 cells were treated for 0–24 h with 1.5 mM HU. NF-κB p65 was immunoprecipitated from nuclear fractions. Western blot analysis was probed for precipitated p65 and co-bound 53 (pre, precipitation with pre-immune serum; IgG, IgG heavy chain).

Stalling of replication forks activates both pro- and anti-apoptotic signaling by p53 and NF-κB (Ansari et al., 2001; Gottifredi et al., 2001; Wu and Miyamoto, 2008). We analyzed whether treatment of MCF7 cells with HU induces NF-κB target genes. Indeed, S-phase arrest enhanced the expression of several genes under control of this transcription factor, BCL-XL, FasL, MnSOD, TRAF1 and Survivin (Figure 1b). These are, for example, important for signaling, apoptosis, immune functions and the elimination of reactive oxygen species. In contrast to a strong accumulation of p53 in HU-treated cells, the levels of NF-κB p65, p50/p105 and p52/p100 remained constant under such conditions (Figure 1b and data not shown). Hence, NF-κB target gene activation during S-phase arrest cannot simply be explained by increased NF-κB expression. Analyzing cytosolic and nuclear fractions obtained from MCF7 cells, we found a time-dependent accumulation of NF-κB p65/p50 together with p53 in the nuclei of cells arrested in S phase (Figure 1c). Hence, nuclear accumulation of p65/p50 and p53 induction correlates with NF-κB target gene expression on DNA replication arrest.

To further verify the induction of NF-κB-dependent gene expression in HU-treated cells, we analyzed lysates of MCF7 cells with avidin-biotin-coupled DNA (ABCD) assay. Increased amounts of NF-κB p65/p50 were recovered with a cognate κB consensus oligonucleotide (Krämer et al., 2006), but not with a control oligonucleotide. Thus, treatment of MCF7 cells with HU augments the affinity of NF-κB for DNA (Figure 1d). This finding can explain the enhanced expression of its target genes during S-phase arrest. Of note, a markedly increased amount of p53 specifically associated with p65/p50 on κB DNA after cells had been treated with HU. Direct immunoprecipitations in the absence of exogenously added oligonucleotides confirmed that in response to HU nuclear p65 interacts with p53 in a time-dependent manner (Figure 1e).

These observations show that p53 expression, complex formation of p53 with p65 and NF-κB target gene expression are induced during replicational stress.

HU induces NF-κB p65

We tested our results from ABCD assays with an additional method. We chose electrophoretic mobility shift assays (EMSAs) using a κB site different from the one for ABCD assays and cell lysates from murine embryonic fibroblasts (MEFs). These experiments revealed that HU also induced an NF-κB complex on this κB consensus sequence. This complex, which was also induced by TNF-α, was supershifted strongly with an antibody specific for p65 (Figure 2a). To further validate this result, we tested Rel A (p65)-deficient MEFs side by side with wild-type MEFs in both ABCD and EMSA. HU did not induce a detectable complex between κB consensus DNA in Rel A−/− MEFs and p53 was subsequently not recovered (Figure 2b). We deduce that p53 is recruited to κB oligonucleotides by p65.

Figure 2

Hydroxyurea (HU) induces nuclear factor-κB (NF-κB) p65. (a) Wild-type murine embryonic fibroblasts (MEFs) were treated with HU (1.5 mM, 24 h) or remained untreated (−). Electrophoretic mobility shift assay (EMSA) was performed to detect the binding of NF-κB to a κB consensus site. DNA binding of p65 was confirmed with p65 antibodies allowing supershift (SS) of the DNA/p65 complex (pre, pre-immune serum control). Lysates from the same MEFs treated with murine TNF-α (20 ng/ml; 1 h) served as positive control in EMSA. (b) Wild-type and Rel A−/− MEFs were treated as in (a). EMSA did not detect the p65/κB complex in knockout MEFs (upper; non-spec., unspecific signal). ABCD assay detected that binding of p53 to κb consensus DNA is p65 dependent (FasLm, mutant oligonucleotide from the FAS ligand promoter). (c) The viability of MEF cells (WT, wild-type; Rel A, NF-κB p65 knockout) was determined by MTT test after exposure to HU (48 h, 5 mM, *P<0.05). (d) MEF cells were treated with 600 ng/ml doxorubicin (DOX; adriamycin) for 16 h. Status of p65 and accumulation of p53 and p21 were tested with western blot analysis of whole-cell lysates. Actin and HMG1 served as independent loading controls.

NF-κB proteins can promote anti-apoptotic gene expression and Rel A−/− MEFs are more susceptible to TNF-induced apoptosis (Gapuzan et al., 2005). We asked whether a lack of Rel A correlates with an increased cytotoxicity in response to HU. Using MTT assays, we noted that Rel A-deficient MEFs were more sensitive toward HU than their wild-type counterparts (Figure 2c). A recent report (Wu and Miyamoto, 2008) also suggested that NF-κB p65 is a key member during replicational arrest.

It was shown that different isolates of Rel A-deficient MEFs frequently harbor mutated p53 that could generate different reactions of wild-type and such knockout MEFs toward HU (Gapuzan et al., 2005). Following this publication, we treated wild-type and Rel A-deficient MEFs with the topoisomerase II inhibitor doxorubicin and analyzed cells for p53 and its target gene p21. We observed accumulation of p53 and p21 arguing that p53 is wild-type in both cell lines used (Figure 2d).

These findings indicate that NF-κB p65 is as a major and functional component in the HU-induced protein/DNA complex.

NF-κB target gene induction in response to replicational stress requires p53

Next, we evaluated whether p53 is indeed necessary for NF-κB target gene expression during S-phase arrest. We silenced p53 with shRNA and analyzed the expression of the NF-κB target genes FasL and MCL1. Basal levels of these proteins were enhanced in cells with silenced p53 (Figure 3a), which is consistent with a negative cross-regulation between NF-κB and p53 in unstimulated cells (Hoffman et al., 2002; Kawauchi et al., 2008a, 2008b). In contrast, products of genes controlled by NF-κB did not accumulate in DNA replication-arrested cells with low p53 levels (Figure 3a). Moreover, this dependency on p53 could not be overcome with higher dose of HU (Figure 3b). Concordant observations were made for p53-positive and p53-negative mammary epithelial cells (Nayak and Das, 2002) and other matched cells with different p53 background (Szoltysek et al., 2008). Moreover, HU was reported to activate NF-κB in p53-positive 70Z/3 pre-B cells but not in HEK293 cells (Wu and Miyamoto, 2008) harboring aberrations in the p53 pathway (Lowe and Ruley, 1993).

Figure 3

p53 controls nuclear factor-κB (NF-κB) target gene expression accentuated by S-phase arrest. (a) MCF7 cells were electroporated with shRNA against p53 or a nonspecific shRNA. At 48 h later, cells were treated with hydroxyurea (HU) (1.5 mM, 24 h) or left untreated (Ctl). Expression of NF-κB-regulated genes, p53, and Tubulin, GAPDH and Actin as three independent loading controls was analyzed by western blot analysis. (b) Same as in a, except that cells were incubated with up to 3 mM HU for 6 h. Western blot analysis was carried out as indicated. (c) HCT-116 cells (p53+, wild-type; p53−, p53 knockout) were treated with 1.5 mM HU for 24 h. Expression of NF-κB-regulated genes, p53, NF-κB members and Tubulin was analyzed by western blot analysis. (d) HCT-116 cells (p53+/p53−) were treated with HU (1.5 mM, 0–24 h). Expression of p53, the anti-apoptotic NF-κB target genes Survivin and BCL-XL, and the pro-apoptotic p53 target gene BAX were determined by western blot analysis. (e) HCT-116 cells (p53+/p53−) were treated with HU (1.5 mM, 2 h). Protein expression analyses were carried out by western blot as specified. (f) RKO and H1299 cells were treated with HU (1 or 1.5 mM, respectively, for 24 h). Expression levels of Survivin, FasL, p53 and Actin were detected by western blot analysis. (g) Wild-type (WT) and p53−/− murine embryonic fibroblasts (MEFs) were treated with HU (0.5 or 1.5 mM, 16 h). Levels of p53, Survivin and p65 were determined by western blot analysis.

To further prove the contribution of p53 to NF-κB activation after HU treatment, we used HCT-116 cells and engineered p53 knockout cells (HCT-116p53− (Bunz et al., 1998)). These allowed us to analyze p53-NF-κB cross-signaling in similar genetic backgrounds with wild-type p53 or no p53 at all. Basal expression of several independent NF-κB target genes was enhanced in p53 null cells (Figures 3c–e). Like in MCF7 cells, p53 was again indispensable for the induction NF-κB target genes in colon cancer cells treated with HU (Figure 3c). This effect was time dependent with different NF-κB target genes responding to a various extent and with different kinetics (Figure 3d). This has similarly been detected in other cellular systems treated with HU (Wu and Miyamoto, 2008). We additionally asked whether treatment with HU generates pro-apoptotic p53-dependent gene expression. In HCT-116 cells, S-phase checkpoint activation promoted expression of BAX dependent on p53 (Figure 3d). These data support previous observations (Nayak and Das, 2002) showing that HU does not globally inhibit p53-dependent gene expression.

To exclude indirect mechanisms contributing to the upregulation of NF-κB target genes on HU treatment, we additionally investigated early HU-induced events. As shown in Figure 3e, already after 2 h of HU treatment, we observed upregulation of the NF-κB target genes BCL2 and Survivin in HCT-116p53+ cells. These data argue for a direct p53-mediated effect on NF-κB target genes.

To substantiate the contribution of p53 toward the replication stress-dependent induction of NF-κB target genes, we used further model systems. Whereas p53 is induced concomitantly with the NF-κB target genes Survivin and FasL on HU treatment in p53 wild-type RKO colon cancer cells, high expression and a lack of HU-mediated induction was observed in H1299 lung cancer cells harboring a p53 deletion (Szoltysek et al., 2008) (Figure 3f). Furthermore, replication fork arrest induced expression of p53 and Survivin in wild-type MEFs, but not in p53-null MEFs. Again basal expression of Survivin was increased (Figure 3g).

We summarize that immediate and delayed NF-κB-mediated gene expression in cells with stalled S phase is under control of p53.

NF-κB and p53 are required for replication stress signaling involving ATM and CHK1

To test direct recruitment of p53 to NF-κB target gene promoters, we performed chromatin immunoprecipitation assays in HCT-116 and HCT-116p53− cells. Indeed, replicational stress induced recruitment of p65 together with p53 to NF-κB target genes. In HCT-116p53− cells we observed increased basal binding of p65 to the NF-κB promoter investigated, which was even decreased on HU treatment (Figure 4a). These data are consistent with the basal and HU-induced expression levels of NF-κB target genes in this cellular model. Likewise, a negative impact of p53 toward NF-κB-dependent gene expression under basal conditions was reported by others (Hoffman et al., 2002; Perkins, 2007; Kawauchi et al., 2008a, 2008b).

Figure 4

Hydroxyurea (HU)-induced nuclear factor-κB (NF-κB) target gene expression relies on p53 and the DNA binding of p65. (a) Chromatin immunoprecipitation (ChIP) analysis of the FasL and MnSOD genes. HCT-116 and HCT-116p53− cells were treated with 1.5 mM HU for 24 h or were left as vehicle-treated control. Sheared chromatin of these cells was immunoprecipitated with p65- or p53-specific antibodies, or IgG control antibodies as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for the FasL, MnSOD or GAPDH promoters. (b) HCT-116p53+ cells were treated with caffeic acid phenethyl ester (CAPE) (12.5 μM; 16.5 h) or/and HU (1.5 mM, 16 h). Expression levels of p53, Survivin, MnSOD and Actin were determined by western blot analysis. The panel also shows a shorter film exposure for detection of Survivin for comparison. (c) Lysates from HCT-116 (p53+/p53−) cells treated with HU (1.5 mM; 4 h) were analyzed by western blot analysis for p-CHK1, human double minute 2 (HDM2), and Tubulin (n.sp., nonspecific band). (d) HCT-116 cells (p53+/p53−) were treated with HU (1.5 mM, 1–4 h) or etoposide (10 μM, 1 h) as a positive control. Phosphorylation of ATM and Tubulin levels was determined by western blot analysis.

We asked if DNA binding is an absolute requirement for HU-induced NF-κB-dependent target gene expression. Caffeic acid phenethyl ester (CAPE) specifically inhibits DNA binding of NF-κB in cancer cells (Natarajan et al., 1996; Gilmore and Herscovitch, 2006; Ha et al., 2009). Experiments conducted with CAPE revealed that replicational arrest induced NF-κB target genes strictly dependent on the ability of NF-κB to attach to target DNA (Figure 4b). Consistent with previous reports (Hung et al., 2003; Lee et al., 2003), we found that CAPE induced p53. Nevertheless, CAPE suppressed expression of the NF-κB target genes Survivin and MnSOD even in the presence of HU. Thus, NF-κB-dependent gene induction in HU-treated cells is only possible when p65 can bind to its target DNA. These findings agree with those shown in Figure 2b.

Recent experiments showed that the DNA-damage modules ataxia-telangiectasia mutated (ATM)/checkpoint kinase-2 (CHK2) and ataxia-telangiectasia and Rad3-related (ATR)/CHK1 are activated by single-stranded DNA exposed at replication forks (Ho et al., 2006; Grallert and Boye, 2008; Wu and Miyamoto, 2008). Therefore, we tested if the p53 effects we observed could be explained by different effects on checkpoint kinase signaling. We detected HU-induced phosphorylation of CHK1 (Figure 4c) and ATM (Figure 4d) in p53-positive and p53-negative HCT-116 cells at different treatment time points. Similar results were obtained for p53-deficient and p53-proficient MEFs (data not shown). These results disfavor that a lack of S-phase kinase activation accounts for the lack of NF-κB target gene induction in cells without p53. Consistent with a previous report (Gottifredi et al., 2001), we noted that ATM phosphorylation not necessarily promotes degradation of the human double minute 2 E3 ubiquitin ligase (Figure 4c). Other E3s may degrade p53 ATM-dependently (Dornan et al., 2006) or alternative mechanisms mediate p53 stabilization during S-phase arrest.

On the basis of these results, we conclude that the p65/p53 complex formed on κB DNA on replicational stress translates into productive NF-κB target gene expression. Formation of this complex still permits p53-mediated signaling.

TNF-α-induced NF-κB target gene expression requires p53

Having assessed the critical function of p53 for NF-κB induction, we determined whether p53 also has an impact on NF-κB induction by the physiological stimulus TNF-α. We treated MCF7 and HCT-116 cells with this pro-inflammatory cytokine and analyzed NF-κB p65/p50 DNA binding with ABCD assays. Independent of p53 expression, NF-κB binding to a cognate oligonucleotide peaked at 20 min after TNF stimulation (Figures 5a and b). Similar to HU-induced NF-κB activation, we observed a specific recruitment of p53 to the NF-κB consensus oligonucleotide, peaking 10 min after stimulation with TNF-α (Figures 5a and b). Nuclear p53 is though not necessary for nuclear translocation of NF-κB (Figure 5c). These findings are in agreement with the well-established model of cytokine-mediated activation of NF-κB signaling in the cytosol.

Figure 5

p53 controls nuclear factor-κB (NF-κB) target gene expression in response to the cytokine tumor necrosis factor-α (TNF-α). (a) Avidin-biotin-coupled DNA (ABCD)-assay was used to detect the binding of NF-κB p65 and p53 to a κB consensus site. MCF7 cells were stimulated with TNF-α (50 ng/ml; 0–60 min). Input (IN) represents 10% of whole-cell extract used. FasLm is a mutant oligonucleotide from the FAS ligand promoter. (b) Same as in a, with p53-positive and p53-negative HCT-116 cells stimulated with TNF-α (50 ng/ml; 0–20 min). (c) Cytosolic (Cyt) and nuclear (Nuc) fractions of untreated (0) and TNF-α-treated (+; 50 ng/ml; 20 min) HCT-116 cells (p53 +/−) were analyzed by western blot analysis for the localization of the NF-κB subunits p65 and p50, and of p53. The western blot analysis also shows a shorter film exposure for detection of p65. (d) Quantitative RT–PCR was used to detect expression of the NF-κB target genes TRAF1 and BCL-XL. HCT-116 cells expressing or lacking p53 (p53 +/−) were stimulated with TNF-α (+; 50 ng/ml; 4 h). The mRNA levels measured in untreated p53-positive HCT-116 cells were set as 1. Data show a representative experiment and are normalized to 18S RNA. (e) HCT-116 cells (p53 +/−) were treated with TNF-α (+; 5–50 ng/ml; 4 h). Expression levels of BCL-XL, MnSOD and Tubulin were determined by western blot analysis.

To investigate whether the TNF-evoked binding of p53 to NF-κB consensus DNA is functionally required for enhanced NF-κB target gene expression, we performed quantitative RT–PCR and western blot analyses. We stimulated HCT-116 and HCT-116p53− cells with TNF-α and analyzed the expression of NF-κB-regulated genes. We found that TNF-α induced these genes only in p53-positive HCT-116 cells on mRNA and protein levels (Figures 5d and e). However, there was no induction of p53 expression in cells exposed to 5–50 ng/ml TNF-α (Figure 5e).

These findings suggest that this cytokine activates nuclear functions of NF-κB dependent on basal p53 expression levels.

TNF-α and HU induce formation of an NF-κB/p53 complex on DNA

Immunoprecipitation experiments disclosed that similar to HU, TNF-α induced the formation of a complex between nuclear p65 and p53 (Figure 6a). Furthermore, EMSAs showed that HU as well as TNF-α induced NF-κB complexes that could be supershifted with antibodies specific for p53 in wild-type MEFs (Figure 6b; compare Figures 2a and b). To control specificity of the p53 supershift, we used wild-type and p53-deficient MEFs. As shown in Figure 6c, the HU- and TNF-α-evoked NF-κB complex built on NF-κB consensus DNA was not supershifted in p53-deficient MEFs. Again, the HU-induced NF-κB complex was supershifted by p53 antibodies in wild-type MEFs (Figure 6c). We tested this finding further with ABCD assays probing for p53 and could confirm that, consistent with Figures 5a and b, p53 bound to this κB nucleotide in wild-type MEFs (Figure 6d).

Figure 6

The hydroxyurea (HU)/tumor necrosis factor (TNF)-inducible nuclear factor-κB (NF-κB) p65 complex contains p53. (a) HCT-116 cells were treated with TNF-α (+; 50 ng/ml; 20 min). Nuclear fractions of these cells were subjected to p65-immunoprecipitation (IP) and detection of p53 and p65 in western blot analysis (IN, 10% of material used for IP). (b) The binding of NF-κB to a κB consensus site was analyzed with electrophoretic mobility shift assay (EMSA). Wild-type murine embryonic fibroblasts (MEFs) were treated with HU (1.5 mM; 24 h) or murine TNF-α (20 ng/ml; 1 h). NF-κB complexes formed under these conditions were supershifted (SS) with p53 antibodies showing the presence of p53 in NF-κB complexes on DNA (pre, pre-immune serum was used as a control). (c) Same as in a, with matched wild-type and p53−/− MEFs for direct comparison giving affirmation for antibody and complex specificity. (d) Wild-type (WT) and p53−/− MEFs were treated with HU (1.5 mM, 6 h). Levels of p53 bound to the κB consensus site were assessed by avidin-biotin-coupled DNA (ABCD) assay. (e) Wild-type (WT) and matched p65−/− MEFs were treated with TNF-α (20 ng/ml; 1–10 h). Levels and specificity of the p65 complex were detected with supershift (SS) and comparison of signals obtained for the two MEF cell lines. (f) Wild-type (WT) and matched p65−/− MEFs were treated as indicated. Real-time PCRs were carried out for three independent genes (relative induction is in relation to Actin mRNA as an internal reference gene).

Similar to that in Figure 2a, we tested for the specificity of this complex with wild-type and Rel A-deficient MEFs proficient for p53 (Figure 2d). When we added TNF-α for 1–10 h to such cells, we only detected the NF-κB complex in wild-type MEFs (Figure 6e). Thus, the possibility that a cryptic p53 site within the nucleotide exists can once more be ruled out. Likewise, only wild-type MEFs induced NF-κB-controlled pro-inflammatory genes and a cell adhesion molecule (Figure 6f).

Hence, halted replication fork signaling and classical activation by TNF-α stimulate p65/p53 complex formation on κB consensus DNA and subsequent gene induction in various cell types.

Mutant p53R172H activates tumor-promoting NF-κB target gene expression

Next, we analyzed whether mutant p53 contributes to NF-κB activity, we established new cellular models with genetically defined p53 backgrounds and the possibility to express p53 from its endogenous promoter. We isolated cell lines from primary pancreatic ductal adenocarcinomas of mice with homozygous deletions of p53 (W22 and 6554 cells) or homozygous expression p53R172H (5436 cells) (Jonkers et al., 2001; Hingorani et al., 2005; Seidler et al., 2008). The p53R172H protein bears a so-called ‘hot-spot’ mutation corresponding to p53R175H frequently found in human tumors.

When we compared NF-κB-dependent reporter gene expression in W22 and 5436 cells, we detected significantly higher activities of a specifically NF-κB-dependent luciferase reporter in 5436 cells (Figure 7a). NF-κB target genes regulating signaling and cell survival were consistently more strongly expressed in 5436 cells (Figure 7b).

Figure 7

Mutant p53 confers constitutive anti-apoptotic nuclear factor-κB (NF-κB) signaling. (a) An HIV-derived NF-κB-dependent luciferase reporter was transfected into W22 and 5436 cells. Relative luciferase values (RLU) are luciferase/β-Gal. Western blot analysis verifies the p53 null status of W22 cells and expression of p53R172H in 5436 cells (*P<0.05). (b) Expression levels of NF-κB target genes, NF-κB subunits, p53R172H and Actin in W22 and 5436 cell lysates were analyzed by western blot analysis. (c) Increasing amounts of a luciferase reporter construct from the Survivin promoter (Rep) were transfected into W22 and 5436 cells. RLUs are luciferase/β-Gal (*P<0.05). (d) Microscopical analysis showing the localization and expression of NF-κB p65, p53R172H and Survivin (Topro, unspecific DNA dye). (e) 5436 and W22 cells were treated with hydroxyurea (HU) (1.5 mM, 24 h). Viability of the cells was determined by MTT assay, untreated cells (−) are set as 100% (left). Cells were treated with murine TNF-α for 24 h as indicated. Caspase activation was measured by Caspase-3/7 assay (middle) (*P<0.05). Expression and localization of Caspase-3 (Casp3) was analyzed by cellular fractionation followed by western blot analysis (C, cytosol; N, nucleus). SIRT1 and Actin serve as markers for the purity of the fractions and equal loading (right).

Because Survivin reveals the most strikingly divergent expression in W22 and 5436 cells, we compared the activities of a reporter construct containing the Survivin promoter in these cells. Increased expression of Survivin paralleled increased transcriptional activity of the Survivin promoter in 5436 cells positive for p53R172H. In contrast, an SP1-dependent UAS-TK reporter was equally active in both cell lines (Figure 7c and data not shown).

Western blot and microscopical analyses strengthened this observation and in addition revealed that NF-κB p65 was expressed and localized equally in both cell lines (Figure 7d and data not shown). Furthermore, the transcriptional repressors HDAC1-4, HDAC7, SIRT1, mSIN3, and the histone acetyltransferase CBP were also expressed at similar levels in W22 and 5436 cells (data not shown). These findings disfavor the possibility that variations in the expression of these factors account for the differential NF-κB transcriptional activities in our models.

Corresponding to the low expression of anti-apoptotic NF-κB target genes in W22 cells, we also observed increased sensitivity of these cells toward HU (Figure 7e). TNF-α treatment also enforced stronger caspase activation in W22 cells, although Caspase-3 was expressed at the same level as in 5436 cells (Figure 7e). These data are consistent with a recent report showing that H1299 lung cancer cells overexpressing mutant p53R175H are less sensitive to TNF-α (Weisz et al., 2007).

These data elucidate that mutant p53 significantly contributes to the productivity of classical NF-κB signaling at the level of gene induction.

Mutant p53R172H forms a transcriptionally relevant complex with NF-κB on its target DNA

Of note, even in unstimulated 5436 cells p53R172H, which itself cannot bind to DNA, could be co-precipitated with active, nuclear p65 in solution (Figure 8a). Mutant p53R172H was also recovered with a κB oligonucleotide (Figure 8b). Binding of p65 was also detected in W22 cells arguing that these pancreatic ductal adenocarcinoma tumor cells have active NF-κB and that p53R172H potently enhances NF-κB-dependent anti-apoptotic gene expression. Thus, regarding complex formation with p65 and induction of anti-apoptotic gene expression, the p53R172H mutant behaves as wild-type p53 under conditions of replicational stress and cytokine stimulation.

Figure 8

Binding of nuclear factor-κB (NF-κB) p65 with mutant p53 is functionally relevant. (a) NF-κB p65 or pre-immune serum immunoprecipitates (IPs) were formed from nuclear fractions of 5436 (p53R172H) and W22 (p53−/−) cells. Western blots of IPs were probed with antibodies against p53 and p65. Amounts of lysates used for IP were normalized to nuclear p65 levels. (b) DNA binding of p65 and co-binding of p53R172H to κB consensus DNA was tested with avidin-biotin-coupled DNA (ABCD) assay. (c) 5436 cells were treated with caffeic acid phenethyl ester (CAPE) (12.5 μM; 16 h). Expression levels of BCL-XL, Survivin and Actin were determined by western blot analysis. (d) Expression levels of NF-κB p65 or p53R172H were decreased with siRNAs against these transcription factors (si Ctl, irrelevant siRNA sequence). Western blot analysis shows efficacy of knock-downs and levels of Survivin and Actin. (e) MTT assays were carried out for W22 p53−/−, 6554 p53−/− and 5436 p53R172H cells left untreated (−) or placed in the presence of 1.5 mM for 24 h (+; *P<0.05). (f) p53R172H was stably expressed in 6554 cells lacking endogenous p53 (UT, untransduced; RCAS Ctl, empty RCAS vector; RCAS p53R172H#1/2, independently generated 6554 cell lines reconstituted with p53R172H). Western blot analysis shows p53, Survivin and Actin levels. (g) Model for the p53/p65 cross talk. In response to replicational arrest or stimulation with tumor necrosis factor-α (TNF-α), p53 binds p65 and is necessary for NF-κB p65 target gene expression, for example, of Survivin (left). Mutant p53 mimics NF-κB activation, which constitutively induces expression of anti-apoptotic NF-κB target genes (right).

To show the dependency of Survivin and BCL-XL on NF-κB in 5436 cells, we again used CAPE. As shown in Figure 8c, treatment of 5436 cells with CAPE leads to a distinct decrease of Survivin and BCL-XL expression. To further validate whether the regulation of Survivin depends on p53R172H and p65 expression, we used RNAi. Similar to the decrease of Survivin in p65 siRNA-transfected 5436 cells, levels of Survivin decreased on attenuation of p53R172H (Figure 8d). To independently validate the p53R172H-dependent expression of Survivin, we reconstituted p53R172H expression in p53-deficient 6554 cells, which are equally sensitive to HU as W22 cells (Figure 8e). Compared to parental and vector control 6554 cells, 6554 cells expressing p53R172H presented increased Survivin levels (Figure 8f). This finding confirms that p65 and mutant p53 induce expression of Survivin.


Cross talk between p53 and p65 is appreciated as an important regulator of tumorigenesis. We used biochemical and genetic assays to analyze how wild-type and mutant p53 influence NF-κB signaling. Our analyses rely on several cancer cell models and MEFs, in which p53 and p65 are modulated in highly similar genetic backgrounds. Moreover, we use a newly established cellular system in which a p53 mutant is expressed from its endogenous promoter. We show the novel and unexpected finding that p53 significantly contributes to nuclear NF-κB activity induced during S-phase arrest and remarkably also in response to TNF-α. Furthermore, we show that mutant p53 activates endogenous NF-κB p65-dependent gene expression counteracting apoptosis induced by HU and TNF-α. Our results, summarized in Figure 8g, contribute to a deeper understanding of the cross talk between NF-κB and p53.

At first glance, a requirement of p53 for gene expression dependent on the tumor promoter NF-κB contradicts the well-established tumor suppressor function of p53. However, our results reveal a regulatory circuit placing stress-dependent expression of pro-survival NF-κB target genes, such as Survivin, under control of p53. Cells with p53 levels insufficient to promote transcriptional activities of NF-κB are consequently disfavored. Pronounced stress and damage can dynamically switch to a p53-inducible apoptotic program (Zhang et al., 2009) ensuring genomic fidelity. Thus, our findings suggest that p53 dictates cell fate decisions not only at the level of cell-cycle arrest and induction of pro-apoptotic genes but also at the level anti-apoptotic gene expression by Rel A/p65. Given that checkpoint proteins have functions in normal S phase (Grallert and Boye, 2008; Levine, 2009), our conclusions might as well apply for regular S-phase entry.

An additional layer controlling apoptosis on strong induction of the intra-S checkpoint could rely on pro-apoptotic functions of NF-κB (Ryan et al., 2000; Karl et al., 2009) and p53/NF-κB-induced pro-apoptotic signaling, for example, by the Fas/FasL system for immunological tumor surveillance (O’Brien et al., 2005). Such indirect p53-dependent anti-tumor effects would be antagonized when tumors block p53 functions of normal tissue. Indeed, p53 null lung cancer cells secrete factors suppressing p53 in adjacent stromal cells (Bar et al., 2009).

The dependence of TNF-α-induced NF-κB activation on p53 shows a previously unknown control function for p53 in cytokine signaling. Similar as in replicational stress, linking p53 to NF-κB signaling allows anti-apoptotic gene expression in cells where the tumor suppressor can execute apoptosis if necessary. Such situations arise, for example, when TNF promotes inflammation-related tumorigenesis by NF-κB activation (Pikarsky et al., 2004; Hayashi et al., 2007) or by TNF-mediated induction of reactive oxygen species bearing mutagenic potential (Yan et al., 2006; Li et al., 2007; Yazdanpanah et al., 2009). Of note, p53-positive cells can protect themselves from such cyto- and genotoxic molecules by inducing MnSOD, an anti-oxidizing enzyme known to balance TNF signaling (Gilmore and Herscovitch, 2006).

Other NF-κB activating stimuli might equally require p53 for full NF-κB activation. For example, it has been shown that p53 crucially contributes to NF-κB induction in response to the bacterial endotoxin lipopolysaccharide (Schäfer et al., 2003). In contrast, double-strand DNA break inducers cause apoptosis dependent on NF-κB but independent of p53 (Karl et al., 2009). Hence, the interplay between p53 and NF-κB we describe here is activated by particular stimuli. Thus, cross talk between these transcription factors represents a molecularly defined therapeutic target.

Therefore, modulators of NF-κB, p53 and their associated regulators could be therapeutically useful for the treatment of cancer and inflammation (Gilmore and Herscovitch, 2006; Wang and El-Deiry, 2008; Spange et al., 2009). Valid targets could equally be the candidate kinase(s) modulating physical and functional interactions between p53 and NF-κB. For example, checkpoint pathways involving ATM, ATR and p38 MAPK are operational on DNA damage and replicational stress (Reinhardt et al., 2007; Grallert and Boye, 2008). Although we could not detect a lack of ATM or CHK1 activation in HU-treated HCT-116p53− cells, these kinases could regulate p53-dependent NF-κB actions, possibly collateral with RSK1 and MEK1 that govern the nuclear p53/p65 cross talk (Armstrong et al., 1995; Ryan et al., 2000; Bohuslav et al., 2004; Yoshida et al., 2008). Our data and a recent report (Kawauchi et al., 2008a) reveal physical interactions between p65 and p53, which may allow an IκB kinase α-dependent transit of CBP (Huang et al., 2007). The fact that p53 binding to a region in the N-terminal part of p65 does not disrupt IκB kinase α binding to the C-terminal transactivation domain is furthermore compatible with a transcriptionally active p53/p65 complex (Kawauchi et al., 2008a). Future studies will clarify the exact composition of this protein assembly likely comprising additional proteins. Strikingly, TNF-induced p53 binding to κB consensus DNA peaks before p65 recruitment. Perhaps, this complex is required for opening of chromatin at NF-κB loci and for the persistence of p65 at such sites. TP53 might equally replace NF-κB members in this complex and other p53 family members could be involved (Alsafadi et al., 2009). Furthermore, corepressor/coactivator binding to transcription factors changes in response to physiological and pharmacological stimulation (Krämer, 2009), any putative interaction partner might exert hub functions (Tsai et al., 2009) and the p53 N-terminal transcriptional activation domain is a multifunctional module for dynamic interactions (Kaustov et al., 2006).

In agreement with such complex regulation of NF-κB and p53 at several steps, these transcription factors can functionally antagonize, cooperate or exhibit independence (Scian et al., 2005; Schumm et al., 2006; Huang et al., 2007; Weisz et al., 2007; Nesic et al., 2008; Szoltysek et al., 2008). Whereas uninduced wild-type p53 attenuates NF-κB signaling directly or indirectly (Webster and Perkins, 1999; Hoffman et al., 2002; Perkins, 2007; Kawauchi et al., 2008a, 2008b), p53 regulates NF-κB positively under conditions of cellular stress (Ryan et al., 2000; Bohuslav et al., 2004; Armstrong et al., 2006). Our analyses elucidate that the p53 hot-spot mutant p53R172H promotes anti-apoptotic activities of NF-κB p65. Weisz et al. (2007) accordingly showed that p53R175H corresponding to murine p53R172H prolongs nuclear localization and activity of induced NF-κB p65. Moreover, expression of mutated p53 positively correlates with nuclear p65 expression in head and neck squamous cell carcinomas and non-small cell lung cancers (Weisz et al., 2007).

Mutant p53 cannot induce pro-apoptotic gene expression but allows productive NF-κB signaling linked to tumor progression, survival and therapeutic resistance. Hence, our observations provide an explanation for the important question why p53 mutations rather than p53 deletions arise in tumors (Deppert, 2007; Soussi and Wiman, 2007). Consistent with this, it was found that chemotherapeutics, hypoxia and oncogene activation induce checkpoint activation, replicational arrest and selection for aberrant p53 (Hanahan and Weinberg, 2000; Janssens and Tschopp, 2006; Finkel et al., 2007; Marusyk and DeGregori, 2007; Krämer, 2009; Viale et al., 2009).

Materials and methods

These are described in Supplementary Materials and Methods and in recent publications (Schneider et al., 2006; Knauer et al., 2007; Seidler et al., 2008; Fritsche et al., 2009; Krämer et al., 2009).


  1. Alsafadi S, Tourpin S, Andre F, Vassal G, Ahomadegbe JC . (2009). P53 family: at the crossroads in cancer therapy. Curr Med Chem 16: 4328–4344.

  2. Ansari SA, Safak M, Del Valle L, Enam S, Amini S, Khalili K . (2001). Cell cycle regulation of NF-kappa b-binding activity in cells from human glioblastomas. Exp Cell Res 265: 221–233.

  3. Armstrong JF, Kaufman MH, Harrison DJ, Clarke AR . (1995). High-frequency developmental abnormalities in p53-deficient mice. Curr Biol 5: 931–936.

  4. Armstrong MB, Bian X, Liu Y, Subramanian C, Ratanaproeksa AB, Shao F et al. (2006). Signaling from p53 to NF-kappaB determines the chemotherapy responsiveness of neuroblastoma. Neoplasia 8: 967–977.

  5. Bar J, Feniger-Barish R, Lukashchuk N, Shaham H, Moskovits N, Goldfinger N et al. (2009). Cancer cells suppress p53 in adjacent fibroblasts. Oncogene 28: 933–936.

  6. Bohuslav J, Chen LF, Kwon H, Mu Y, Greene WC . (2004). p53 induces NF-kappaB activation by an IkappaB kinase-independent mechanism involving phosphorylation of p65 by ribosomal S6 kinase 1. J Biol Chem 279: 26115–26125.

  7. Bug G, Ritter M, Wassmann B, Schoch C, Heinzel T, Schwarz K et al. (2005). Clinical trial of valproic acid and all-trans retinoic acid in patients with poor-risk acute myeloid leukemia. Cancer 104: 2717–2725.

  8. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP et al. (1998). Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282: 1497–1501.

  9. Deppert W . (2007). Mutant p53: from guardian to fallen angel? Oncogene 26: 2142–2144.

  10. Dornan D, Shimizu H, Mah A, Dudhela T, Eby M, O'Rourke K et al. (2006). ATM engages autodegradation of the E3 ubiquitin ligase COP1 after DNA damage. Science 313: 1122–1126.

  11. Finkel T, Serrano M, Blasco MA . (2007). The common biology of cancer and ageing. Nature 448: 767–774.

  12. Fritsche P, Seidler B, Schuler S, Schnieke A, Göttlicher M, Schmid RM et al. (2009). HDAC2 mediates therapeutic resistance of pancreatic cancer cells via the BH3-only protein NOXA. Gut 58: 1399–1409.

  13. Gapuzan ME, Schmah O, Pollock AD, Hoffmann A, Gilmore TD . (2005). Immortalized fibroblasts from NF-kappaB RelA knockout mice show phenotypic heterogeneity and maintain increased sensitivity to tumor necrosis factor alpha after transformation by v-Ras. Oncogene 24: 6574–6583.

  14. Gilmore TD, Herscovitch M . (2006). Inhibitors of NF-kappaB signaling: 785 and counting. Oncogene 25: 6887–6899.

  15. Gottifredi V, Shieh S, Taya Y, Prives C . (2001). p53 accumulates but is functionally impaired when DNA synthesis is blocked. Proc Natl Acad Sci USA 98: 1036–1041.

  16. Grallert B, Boye E . (2008). The multiple facets of the intra-S checkpoint. Cell Cycle 7: 2315–2320.

  17. Ha J, Choi HS, Lee Y, Lee ZH, Kim HH . (2009). Caffeic acid phenethyl ester inhibits osteoclastogenesis by suppressing NF kappaB and downregulating NFATc1 and c-Fos. Int Immunopharmacol 9: 774–780.

  18. Hanahan D, Weinberg RA . (2000). The hallmarks of cancer. Cell 100: 57–70.

  19. Hayashi T, Ishida Y, Kimura A, Iwakura Y, Mukaida N, Kondo T . (2007). IFN-gamma protects cerulein-induced acute pancreatitis by repressing NF-kappa B activation. J Immunol 178: 7385–7394.

  20. Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, Hruban RH et al. (2005). Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7: 469–483.

  21. Ho CC, Siu WY, Lau A, Chan WM, Arooz T, Poon RY . (2006). Stalled replication induces p53 accumulation through distinct mechanisms from DNA damage checkpoint pathways. Cancer Res 66: 2233–2241.

  22. Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M . (2002). Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 277: 3247–3257.

  23. Huang WC, Ju TK, Hung MC, Chen CC . (2007). Phosphorylation of CBP by IKKalpha promotes cell growth by switching the binding preference of CBP from p53 to NF-kappaB. Mol Cell 26: 75–87.

  24. Hung MW, Shiao MS, Tsai LC, Chang GG, Chang TC . (2003). Apoptotic effect of caffeic acid phenethyl ester and its ester and amide analogues in human cervical cancer ME180 cells. Anticancer Res 23: 4773–4780.

  25. Janssens S, Tschopp J . (2006). Signals from within: the DNA-damage-induced NF-kappaB response. Cell Death Differ 13: 773–784.

  26. Jonkers J, Meuwissen R, van der Gulden H, Peterse H, van der Valk M, Berns A . (2001). Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat Genet 29: 418–425.

  27. Karin M . (2006). Nuclear factor-kappaB in cancer development and progression. Nature 441: 431–436.

  28. Karl S, Pritschow Y, Volcic M, Hacker S, Baumann B, Wiesmuller L et al. (2009). Identificationof a novel pro-apopotic function of NF-kappaB in the DNA damage response. J Cell Mol Med (in press; doi:10.1111/j.1582-4934.2009.00888.x).

  29. Kashatus D, Cogswell P, Baldwin AS . (2006). Expression of the Bcl-3 proto-oncogene suppresses p53 activation. Genes Dev 20: 225–235.

  30. Kaustov L, Yi GS, Ayed A, Bochkareva E, Bochkarev A, Arrowsmith CH . (2006). p53 transcriptional activation domain: a molecular chameleon? Cell Cycle 5: 489–494.

  31. Kawauchi K, Araki K, Tobiume K, Tanaka N . (2008a). Activated p53 induces NF-kappaB DNA binding but suppresses its transcriptional activation. Biochem Biophys Res Commun 372: 137–141.

  32. Kawauchi K, Araki K, Tobiume K, Tanaka N . (2008b). p53 regulates glucose metabolism through an IKK-NF-kappaB pathway and inhibits cell transformation. Nat Cell Biol 10: 611–618.

  33. Knauer SK, Krämer OH, Knosel T, Engels K, Rodel F, Kovacs AF et al. (2007). Nuclear export is essential for the tumor-promoting activity of survivin. FASEB J 21: 207–216.

  34. Krämer OH . (2009). HDAC2: a critical factor in health and disease. Trends Pharmacol Sci 30: 647–655.

  35. Krämer OH, Baus D, Knauer SK, Stein S, Jager E, Stauber RH et al. (2006). Acetylation of Stat1 modulates NF-kappaB activity. Genes Dev 20: 473–485.

  36. Krämer OH, Knauer SK, Zimmermann D, Stauber RH, Heinzel T . (2008). Histone deacetylase inhibitors and hydroxyurea modulate the cell cycle and cooperatively induce apoptosis. Oncogene 27: 732–740.

  37. Krämer OH, Knauer SK, Greiner G, Jandt E, Reichardt S, Gührs KH et al. (2009). A phosphorylation-acetylation switch regulates STAT1 signaling. Genes Dev 23: 223–235.

  38. Lee YJ, Kuo HC, Chu CY, Wang CJ, Lin WC, Tseng TH . (2003). Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells. Biochem Pharmacol 66: 2281–2289.

  39. Levine AJ . (2009). The common mechanisms of transformation by the small DNA tumor viruses: the inactivation of tumor suppressor gene products: p53. Virology 384: 285–293.

  40. Li J, Sejas DP, Zhang X, Qiu Y, Nattamai KJ, Rani R et al. (2007). TNF-alpha induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells. J Clin Invest 117: 3283–3295.

  41. Lowe SW, Ruley HE . (1993). Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes Dev 7: 535–545.

  42. Marusyk A, DeGregori J . (2007). Replicational stress selects for p53 mutation. Cell Cycle 6: 2148–2151.

  43. Moll UM, Wolff S, Speidel D, Deppert W . (2005). Transcription-independent pro-apoptotic functions of p53. Curr Opin Cell Biol 17: 631–636.

  44. Natarajan K, Singh S, Burke Jr TR., Grunberger D, Aggarwal BB . (1996). Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. Proc Natl Acad Sci USA 93: 9090–9095.

  45. Nayak BK, Das GM . (2002). Stabilization of p53 and transactivation of its target genes in response to replication blockade. Oncogene 21: 7226–7229.

  46. Nesic D, Grumont R, Gerondakis S . (2008). The nuclear factor-kappaB and p53 pathways function independently in primary cells and transformed fibroblasts responding to genotoxic damage. Mol Cancer Res 6: 1193–1203.

  47. O'Brien DI, Nally K, Kelly RG, O'Connor TM, Shanahan F, O'Connell J . (2005). Targeting the Fas/Fas ligand pathway in cancer. Expert Opin Ther Targets 9: 1031–1044.

  48. Perkins ND . (2007). Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 8: 49–62.

  49. Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S et al. (2004). NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 431: 461–466.

  50. Ravi R, Mookerjee B, van Hensbergen Y, Bedi GC, Giordano A, El-Deiry WS et al. (1998). p53-mediated repression of nuclear factor-kappaB RelA via the transcriptional integrator p300. Cancer Res 58: 4531–4536.

  51. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB . (2007). p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11: 175–189.

  52. Renner F, Schmitz ML . (2009). Autoregulatory feedback loops terminating the NF-kappaB response. Trends Biochem Sci 34: 128–135.

  53. Royds JA, Dower SK, Qwarnstrom EE, Lewis CE . (1998). Response of tumour cells to hypoxia: role of p53 and NFkB. Mol Pathol 51: 55–61.

  54. Ryan KM, Ernst MK, Rice NR, Vousden KH . (2000). Role of NF-kappaB in p53-mediated programmed cell death. Nature 404: 892–897.

  55. Schäfer T, Scheuer C, Roemer K, Menger MD, Vollmar B . (2003). Inhibition of p53 protects liver tissue against endotoxin-induced apoptotic and necrotic cell death. FASEB J 17: 660–667.

  56. Scheidereit C . (2006). IkappaB kinase complexes: gateways to NF-kappaB activation and transcription. Oncogene 25: 6685–6705.

  57. Schneider G, Saur D, Siveke JT, Fritsch R, Greten FR, Schmid RM . (2006). IKKalpha controls p52/RelB at the skp2 gene promoter to regulate G1- to S-phase progression. EMBO J 25: 3801–3812.

  58. Schrell UM, Rittig MG, Koch U, Marschalek R, Anders M . (1996). Hydroxyurea for treatment of unresectable meningiomas. Lancet 348: 888–889.

  59. Schumm K, Rocha S, Caamano J, Perkins ND . (2006). Regulation of p53 tumour suppressor target gene expression by the p52 NF-kappaB subunit. EMBO J 25: 4820–4832.

  60. Scian MJ, Stagliano KE, Anderson MA, Hassan S, Bowman M, Miles MF et al. (2005). Tumor-derived p53 mutants induce NF-kappaB2 gene expression. Mol Cell Biol 25: 10097–10110.

  61. Seidler B, Schmidt A, Mayr U, Nakhai H, Schmid RM, Schneider G et al. (2008). A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors. Proc Natl Acad Sci USA 105: 10137–10142.

  62. Soussi T, Wiman KG . (2007). Shaping genetic alterations in human cancer: the p53 mutation paradigm. Cancer Cell 12: 303–312.

  63. Spange S, Wagner T, Heinzel T, Krämer OH . (2009). Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol 41: 185–198.

  64. Szoltysek K, Pietranek K, Kalinowska-Herok M, Pietrowska M, Kimmel M, Widlak P . (2008). TNFalpha-induced activation of NFkappaB protects against UV-induced apoptosis specifically in p53-proficient cells. Acta Biochim Pol 55: 741–748.

  65. Tergaonkar V . (2006). NFkappaB pathway: a good signaling paradigm and therapeutic target. Int J Biochem Cell Biol 38: 1647–1653.

  66. Tergaonkar V, Pando M, Vafa O, Wahl G, Verma I . (2002). p53 stabilization is decreased upon NFkappaB activation: a role for NFkappaB in acquisition of resistance to chemotherapy. Cancer Cell 1: 493–503.

  67. Tsai CJ, Ma B, Nussinov R . (2009). Protein-protein interaction networks: how can a hub protein bind so many different partners? Trends Biochem Sci 34: 594–600.

  68. Viale A, De Franco F, Orleth A, Cambiaghi V, Giuliani V, Bossi D et al. (2009). Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells. Nature 457: 51–56.

  69. Vogelstein B, Lane D, Levine AJ . (2000). Surfing the p53 network. Nature 408: 307–310.

  70. Vousden KH, Lane DP . (2007). p53 in health and disease. Nat Rev Mol Cell Biol 8: 275–283.

  71. Wadgaonkar R, Phelps KM, Haque Z, Williams AJ, Silverman ES, Collins T . (1999). CREB-binding protein is a nuclear integrator of nuclear factor-kappaB and p53 signaling. J Biol Chem 274: 1879–1882.

  72. Wang W, El-Deiry WS . (2008). Restoration of p53 to limit tumor growth. Curr Opin Oncol 20: 90–96.

  73. Webster GA, Perkins ND . (1999). Transcriptional cross talk between NF-kappaB and p53. Mol Cell Biol 19: 3485–3495.

  74. Weisz L, Damalas A, Liontos M, Karakaidos P, Fontemaggi G, Maor-Aloni R et al. (2007). Mutant p53 enhances nuclear factor kappaB activation by tumor necrosis factor alpha in cancer cells. Cancer Res 67: 2396–2401.

  75. Wietek C, O′Neill LA . (2007). Diversity and regulation in the NF-kappaB system. Trends Biochem Sci 32: 311–319.

  76. Wu H, Lozano G . (1994). NF-kappa B activation of p53. A potential mechanism for suppressing cell growth in response to stress. J Biol Chem 269: 20067–20074.

  77. Wu ZH, Miyamoto S . (2008). Induction of a pro-apoptotic ATM-NF-kappaB pathway and its repression by ATR in response to replication stress. EMBO J 27: 1963–1973.

  78. Yan B, Wang H, Rabbani ZN, Zhao Y, Li W, Yuan Y et al. (2006). Tumor necrosis factor-alpha is a potent endogenous mutagen that promotes cellular transformation. Cancer Res 66: 11565–11570.

  79. Yazdanpanah B, Wiegmann K, Tchikov V, Krut O, Pongratz C, Schramm M et al. (2009). Riboflavin kinase couples TNF receptor 1 to NADPH oxidase. Nature 460: 1159–1163.

  80. Yilmaz ZB, Weih DS, Sivakumar V, Weih F . (2003). RelB is required for Peyer's patch development: differential regulation of p52-RelB by lymphotoxin and TNF. EMBO J 22: 121–130.

  81. Yoshida K, Ozaki T, Furuya K, Nakanishi M, Kikuchi H, Yamamoto H et al. (2008). ATM-dependent nuclear accumulation of IKK-alpha plays an important role in the regulation of p73-mediated apoptosis in response to cisplatin. Oncogene 27: 1183–1188.

  82. Zhang XP, Liu F, Cheng Z, Wang W . (2009). Cell fate decision mediated by p53 pulses. Proc Natl Acad Sci USA 106: 12245–12250.

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We thank Dr B Vogelstein for HCT-116 cells, Dr A Hoffmann for wild-type and Rel A−/− MEFs, Dr T Jacks and Dr D Tuveson for LSL-KRASG12D and LSL-p53R172H mice, Dr A Berns for TP53lox/lox mice, Dr H Nakhai for Ptf1a/p48ex1Cre/+ mice, Dr R Bernards and Dr R Agami for shRNA against p53, M Buchwald for help with electroporation, Dr A Licht for help with EMSAs, Dr Z-Q Wang and Dr W-K Min for wild-type and p53−/− MEFs and very helpful discussions and suggestions. This study was supported by DFG (SCHN 959/1-2) and SFB456 grants to GS, Landesprogramm ‘ProExzellenz’ (PE 123-2-1) to OHK, and a grant from Deutsche Krebshilfe to GS and OHK.

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Schneider, G., Henrich, A., Greiner, G. et al. Cross talk between stimulated NF-κB and the tumor suppressor p53. Oncogene 29, 2795–2806 (2010).

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  • NF-κB
  • p53
  • S-phase
  • hydroxyurea
  • TNF-α
  • apoptosis

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