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Neoplasia. Nov 2006; 8(11): 964–974.
PMCID: PMC1764827

Signaling from p53 to NF-κB Determines the Chemotherapy Responsiveness of Neuroblastoma1

Abstract

Neuroblastic (N) type neuroblastoma (NB) is the predominant cell type in NB tumors. Previously, we determined that activated nuclear factor κB (NF-κB) is required for doxorubicin and etoposide to kill N-type NB cells. This study was undertaken to determine how NF-κB is activated by these agents. The results show that p53 protein levels increase within 15 to 30 minutes of treatment. This increase occurs before the degradation of inhibitor of NF-κB (I-KB) α and the NF-κB-dependent activation of gene transcription. Moreover, p53 is necessary for NF-κB activation because cells with inactive p53 were resistant to NF-κB-mediated cell death. This pathway was further defined to show that p53 leads to the activation of MAPK/ERK activity kinase (MEK) 1 through a process that depends on protein synthesis and H-Ras. MEK1, in turn, mediates I-κB kinase activation. Together, these results demonstrate for the first time how NF-κB is activated in NB cells in response to conventional drugs. Furthermore, these findings provide an explanation as to why H-Ras expression correlates with a favorable prognosis in NB and identify intermediary signaling molecules that are targets for discovering treatments for NB that is resistant to conventional agents.

Keywords: Chemotherapy, H-Ras, NF-κB, neuroblastoma, p53

Introduction

Neuroblastoma (NB), a malignancy of childhood, arises from the neoplastic transformation of neural crest cells or cells of neural crest origin. Despite intensive therapy with conventional drugs, prognosis remains poor for advance-stage cases and the chance of long-term survival is < 30% [1]. Understanding the pathogenesis of NB is complicated by the heterogeneous nature of cell types within these tumors. When NB cells are grown in vitro, distinct functional phenotypes result, with the most prevalent cell types being neuroblastic (N) type and schwannian/stromal (S) type. N-type cells grow in culture as poorly attached aggregates of small round cells, exhibit neurite-like processes, possess enzymatic activity required for neurotransmitter synthesis, are MYCN-amplified, and are tumorigenic in mice [2–10]. In contrast, S-type cells grow with a flattened and adherent morphology in culture, do not synthesize neurotransmitters, are not MYCN-amplified but can express low levels of protein, and are generally incapable of forming tumors when xenografted into nude mice [2,3,9,10].

In recent studies, we have determined that the levels of nuclear factor κB (NF-κB) basal activity and the consequences of activating NF-κB also differ between S-type and N-type NB cells. In S-type cells, NF-κB is constitutively active and is required for survival [11]. In contrast, NF-κB is not constitutively active in N-type cells, and when its activity is induced [e.g., by treatment with doxorubicin (Dox)], it leads to cell death [12].

NF-κB proteins constitute a family of mammalian transcription factors that bind a consensus DNA motif. There are five identified members of this family: NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel. At baseline, NF-κB associates with one of several inhibitory inhibitor of NF-κB (I-κB) species, which excludes NF-κB from the nucleus. I-κB kinase (IKK) α and β protein serine kinases directly phosphorylate I-κB, triggering its ubiquitination and proteasomal degradation, thereby releasing NF-κB, which then translocates to the nucleus to regulate transcription [13].

In many cell types, NF-κB mediates resistance against cell death by inducing gene expression that blocks apoptotic signals. However, in some cell types, NF-κB triggers apoptosis. Although less frequently observed than prosurvival functions, several examples of NF-κB-induced death have been reported in neuronal cell types. For example, dopamineinduced death of pheochromocytoma cells and neuronal death in response to ischemia require NF-κB [14,15]. Moreover, we have previously demonstrated that, in N-type NB cells, Dox induces I-κBα degradation and increases the DNA binding of NF-κB p65/p50 heterodimers, and specific inhibition of NF-κB renders cells resistant to Dox [12].

Although the phosphorylation of I-κBα is the best-characterized mechanism leading to NF-κB activation, other mechanisms are also known. The 90-kDa ribosomal S6 kinase (pp90/Rsk1) is a signal transactivator and is part of mitogen-activated protein kinase (MAPK) cascade. pp90/Rsk1 directly phosphorylates p65, leading to increased NF-κB activity because phosphorylated p65 does not bind its inhibitor, I-κBα [16]. Interestingly, pp90/Rsk1, as well as other MAPKs including MAPK/ERK activity kinase (MEK) 1/2, directly phosphorylates the IKK complex and therefore has the potential to regulate NF-κB at multiple levels.

p53, a tumor suppressor, inhibits cell growth and induces apoptosis [17]. For example, fibroblasts derived from p53 knockout mice are resistant to a diverse group of anticancer agents, including etoposide (VP16), 5-fluorouracil, and Dox [18]. VP16-induced apoptosis of ovarian cancer cells requires p53, and reintroduction of wild-type p53 into p53-null tumor cells reestablishes chemosensitivity [19,20]. Given that the N-type NB cells used to demonstrate drug-induced NF-κB-mediated cell death contain wild-type p53, we hypothesized a link between p53 and NF-κB activation. Indeed, Ryan et al. [21] demonstrated that induction of wild-type p53 led to activation of NF-κB in Saos-2 osteosarcoma cells.

In this report, we demonstrate the functional importance and interconnection of p53, H-Ras, MEK1, IKKα/β, and NF-κB in chemotherapy-induced N-type cell death. The results indicate that p53 is required for chemotherapy-induced NF-κB activation and cell death. Dox and VP16 trigger cell death through a process that begins with p53 response followed by a p53-dependent sequential activation of MEK1 and IKKα/β, which depends on intervening protein synthesis and H-Ras. These findings are discussed with respect to the significance of implicating H-Ras (a favorable prognostic marker) and alternative therapeutic targets for chemoresistant NB.

Materials and Methods

Cell Lines

Human NB cell lines SH-SY5Y and IMR32 were cultured in minimal essential medium (MEM) supplemented with 10% (vol/vol) fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were maintained in a humidified 5% CO2 incubator at 37°C. The SH-SY5Y cell line stably expressing c90p53 (c90p53/SH-SY5Y) was generated as previously described [22] and maintained in routine MEM with hygromycin (500 mg/ml) (Invitrogen, Carlsbad, CA). SK-N-BE(2) cells were generously provided by Dr. C. Patrick Reynolds (Children's Hospital of Los Angeles, Los Angeles, CA) and maintained in MEM.

Generation of SH-SY5Y Cells Stably Expressing E6

SH-SY5Y cells were transfected with an expression plasmid encoding E6 or vector control. Transfection was carried out using LipofectAMINE PLUS (Invitrogen) according to the manufacturer's instructions. After 48 hours of transfection, cells were cultured in a medium containing G418 (500 µg/ml) (Gibco BRL, Grand Island, NY). Individual colonies were propagated separately, and transfectants expressing high levels of E6 were chosen for subsequent studies.

Cell Viability Assays

NB cells were plated at 1.2 x 104 cells/well in 96-well culture plates. For experiments with dominant-negative (DN) H-Ras, N17, cells were transfected and incubated overnight with the vector. On the following day, the cells were trypsinized and plated in 96-well plates. After being allowed to attach overnight, the cells were treated in triplicate with Dox or VP16 at indicated concentrations. On consecutive days, the cells were incubated with tetrazolium salt [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide] (MTT; 1 mg/ml) at 37°C for 4 hours and then lysed in a buffer containing 20% (wt/vol) sodium dodecyl sulfate (SDS) and 50% (vol/vol) N,N-dimethylformamide (pH 4.5). Absorbance at 600 nM (A600) was determined using an ELX 808 automated microplate reader (Bioteck Instruments, Winnoski, VT). After the subtraction of background absorbance, the A600 of the drug-treated cells was divided by that of untreated cells to obtain the percentage of viable cells. Student's paired t test was used to test the significance of differences.

Determination of NF-κB-Dependent Reporter Gene Activation

SH-SY5Y and IMR32 cells were transfected with 1 mg of the reporter plasmid pBVIx-Luc, which contains six NF-κB recognition sites within the promoter sequence linked to the luciferase reporter gene (gift from Gabriel Nuñez, University of Michigan, Ann Arbor, MI). Briefly, the cells were grown in sixwell tissue culture plates up to 50% confluence. Twenty-four hours later, the cells were transfected using a mixture of DNA and LipofectAMINE PLUS in OPTI-MEM (Invitrogen). To ensure identical transfection efficiency in control and treated cells, the cells were replated 24 hours after transfection into 12-well plates; after attachment, identical wells were treated with Dox (0.5 g/ml), VP16 (10 g/ml), or vehicle control. Cell extracts were harvested by lysis, and luciferase activity was determined using the Luciferase Assay System (Promega, Madison WI) according to the manufacturer's instructions. Light output wasmeasured with aMonolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). Luciferase activity was normalized to protein concentration.

Measurement of NF-κB Activation by Electromobility Shift Assay (EMSA)

Aliquots of nuclear extracts (10 µg of protein), prepared as described [23] from SH-SY5Y cells following treatment, were added to a reaction buffer containing 250 mM NaCl, 50 mM Tris (pH 7.6), 5 mM dithiothreitol, and 1 µg/µl poly(dI-dC) (Amersham Pharmacia Biotech, Buckinghamshire, UK). The mixture was incubated with 5 x 104 cpm of a [α-32P]dATP-labeled oligonucleotide probe containing an NF-κB binding site (5′-AGT TGA GGG GAC TTT CCC AGG C-3′). For antibody supershift assays, 1 µg of monoclonal antibody specific for either the p65 or the p50 subunit of NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA) was added with nuclear proteins, and reactions were incubated at 24°C for 20 minutes. Samples were resolved on 6% polyacrylamide gels in Tris-glycine buffer. Gels were dried and subjected to autoradiography.

Immunoblot Analysis

Cells were harvested and lysed in a buffer containing 50 mM Tris-HCl, 5 mM NaCl, 2% SDS (wt/vol), and 10% glycerol (vol/vol). Protein was quantitated using the DC Protein Assay System (BioRad Laboratories, Hercules, CA). Protein from whole-cell lysates (25–35 µg) was resolved by SDS polyacrylamide gel electrophoresis on 10% to 15% SDS polyacrylamide gels and transferred to Hybond-P PVDF membranes (Amersham Pharmacia Biotech, Peapack, NJ) by electroblotting. Primary antibodies used in this study included the following: p53 (1:1000; CalBiochem, La Jolla, CA); IKKα/β (1:200; Upstate Biotech, Lake Placid, NY); I-κBα, MEK1, pMEK1, pIKKα, p44/42 MAP kinase (ERK) 1/2, and pERK1/2 (1:500; Cell Signaling, Beverly, MA); and β-tubulin (1:2000; Sigma, St. Louis, MO). Antigen-antibody complexes were detected using enhanced chemiluminescence technique (ECL Detection Kit; Amersham International) followed by exposure of membranes to Kodak XAR film (Eastman Kodak Co., Rochester, NY).

MEK1/Rsk1 Kinase Assays

MEK1 and Rsk1 kinase activities were determined in NB cells following Dox or VP16 treatment. Briefly, cells were transiently transfected with pCMV5-MEK1 or pcDNA3-HA-Rsk1 using LipofectAMINE PLUS. Whole-cell lysates (100 µg) were incubated overnight at 4°C with anti-MEK1 antibody (Santa Cruz Biotechnology) to immunoprecipitate MEK1 or with anti-HA antibody (Santa Cruz Biotechnology) to immunoprecipitate Rsk1. Kinase assays were performed using a MEK1 kinase assay kit or an S6 kinase assay kit (Upstate Biotech) according to themanufacturer's instructions.

Inhibition of Protein Synthesis with Cyclohexamide

SH-SY5Y cells were plated as described above. Cells were pretreated for 1 hour with cyclohexamide (CHX; 2 µg/ml) or vehicle control (0.01% DMSO) followed by treatment with Dox (0.5 µg/ml). Cells were incubated for 4 hours, after which cell lysates were collected. Phospho-MEK1 and p21 protein levels were detected by immunoblot analysis, as described above.

Pharmacological Inhibition of the NF-κB Pathway

SH-SY5Y cells were treated with the MEK1/2 inhibitor PD98059 (CalBiochem), the IKK inhibitor BAY 11-7082 (Cal-Biochem), or the H-Ras geranyl-geranyl transferase inhibitor GGTI-287 (CalBiochem). For immunoblot analysis, cells were pretreated with the appropriate inhibitor for 2 hours before the addition of Dox or VP16. Proteins were isolated as described above. For reporter analysis, SH-SY5Y cells were transfected with the NF-κB reporter vector as described above. On the next day, cells were incubated with GGTI-287 or vehicle control in MEM for 24 hours at indicated concentrations before the addition of Dox. After incubation, luciferase activity was measured as described above.

Modulation of H-Ras through Mutant Forms

SH-SY5Y, E6/SH-SY5Y, and SK-N-BE(2) cells were transfected, as described above, with an expression vector for a constitutively active form of H-Ras, V12, or DN mutant N17. For experiments with V12 H-Ras, cells were incubated overnight with the vector cotransfected with the NF-κB luciferase reporter vector. On the next morning, cell lysates were harvested, and luciferase activity was measured as described above. For experiments with DN N17 H-Ras, cells were cotransfected with the NF-κB luciferase reporter vector. On the next day, cells were treated with vehicle or Dox (0.5 µg/ml) for 8 hours. Cell lysates were collected, and luciferase activity was measured.

Quantitation of mRNA Levels by Real-Time Polymerase Chain Reaction (PCR)

Cells were treated with Dox as indicated, then total RNA was isolated with the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's directions. RNA was reverse-transcribed to cDNA using oligo-dTas primer (Superscriptase II; Invitrogen). Message levels were quantitated by real-time PCR using gene-specific primers (Applied Biosystems, Foster City, CA) on the Applied Biosystems 7300 Real-Time PCR System.

Results

VP16, Like Dox, Induces NF-κB Activation

Previously, we have determined that NF-κB transcriptional activity was induced by Dox in NB cells [12]. Because VP16 is able to activate NF-κB in other cell types [24], we postulated that its mechanism in NB is similar to that of Dox. To test this, NF-κB sequence-specific DNA binding was measured following treatment with VP16 (10 mg/ml; 2 hours) or Dox (0.5 µg/ml; 8 hours). Nuclear extracts prepared from treated SH-SY5Y cells were incubated with 32P end-labeled double-stranded DNA containing a consensus NF-κB recognition site, and binding was detected by EMSA. As shown in Figure 1A, untreated N-type cells have no detectable NF-κB DNA-binding activity. Following treatment with Dox or VP16, NF-κB DNA binding is detected as a specific band that, on supershift, is shown to be composed of p65/p50 heterodimers.

Figure 1
Dox and VP16 induce NF-κB activation. (A) Nuclear extracts prepared from SH-SY5Y cells treated with Dox (0.5 µg/ml; 2 hours) or VP16 (10 µg/ml; 8 hours) were used in EMSA and supershift assays. Supershift using anti-p65 or anti-p50 ...

To test NF-κB transcriptional activation after VP16 treatment, we measured NF-κB-dependent gene transcription by transiently transfecting SH-SY5Y and IMR32 cells with a reporter plasmid containing six NF-κB consensus-binding sites tandemly placed within the luciferase promoter [25]. Both Dox and VP16 activated NF-κB transcriptional activity within 8 hours (Figure 1B). These findings suggest that, like Dox, VP16 induces NF-κB heterodimer activation and specific NF-κB transcriptional response.

To determine if endogenous NF-κB-responsive genes are regulated by drug treatment, mRNA levels corresponding to selected genes with (I-κBα, RelB, and Bax) or without (GAPDH) NF-κB consensus elements in their promoters were measured. RNA isolated from SH-SY5Y cells treated with Dox for 6 hours was analyzed by real-time PCR. As shown in Figure 1C, the levels of transcripts for I-κBα and RelB increased in response to Dox. Furthermore, the proapoptotic gene Bax was also upregulated in response to Dox. Transcript levels of the non-NF-κB-responsive gene GAPDH were unchanged. Thus, this experiment demonstrates regulation of transcripts from endogenous NF-κB-responsive genes, further supporting the hypothesis that NF-κB is activated in response to Dox.

IKK Kinase Complex Mediates Dox and VP16-Induced NF-κB Activation

Activation of NF-κB p65/p50 heterodimers typically involves phosphorylation of I-κB at specific serine residues [13]. In Saos-2 cells, NF-κB mediates p53-controlled programmed cell death through a process in which mitogen-activated ribosomal pp90/Rsk1 (S6 kinase) phosphorylates I-κB. To determine the involvement of pp90/Rsk1 in response to Dox and VP16, SH-SY5Y and IMR32 cells were treated with Dox or VP16, and lysates were immunoblotted to detect the active phosphorylated form of pp90/Rsk1. Phosphorylation of pp90/Rsk1 increased within 2 to 4 hours after exposure to these agents (data not shown). Next, we cotransfected pcDNA3-Rsk1-D205N, encoding a DN form of pp90/Rsk1, with a mutated ATP-binding site in the amino-terminal kinase domain, together with the NF-κB transcription reporter plasmid. High-level expression of DN mutant was confirmed by immunoblotting (data not shown). Surprisingly, neither Dox-induced nor VP16-induced NF-κB activation was reduced in cells expressing DN pp90/Rsk1 compared to vector-only control cells (Figure 2A). To ensure that the expression of DN pp90/Rsk1 was sufficient to block wild-type activity, transfected cells were exposed to phorbol 12-myristate 13-acetate (PMA) for 2 hours to induce NF-κB by directly activating pp90/Rsk1, which is essential for NF-κB activation by PMA [26]. As seen in Figure 2A, the expression of DN pp90/Rsk1 effectively blocked PMA-induced NF-κB activity. Collectively, these results suggest that pp90/Rsk1 is not necessary for NF-κB activation by Dox or VP16 in NB cells.

Figure 2
IKK, but not pp90/Rsk1, is required for NF-κB activation in response to Dox and VP16. (A) SH-SY5Y cells were cotransfected with pcDNA3-DNRsk1 and pBVIx-Luc reporter plasmid. Twenty-four hours after transfection, cells were treated with Dox (0.5 ...

Because our previous studies demonstrated that I-κBα levels decrease as part of drug-induced NF-κB activation, we hypothesized that IKK mediates this response by phosphorylating I-κBα. Direct evidence of IKK activation by Dox was detected by immunoblotting with antibodies specific for phospho-IKKα, which showed that the phosphorylated (active) form of IKK was induced within 1 hour of exposure to drugs (Figure 2B). We also tested IKK's involvement by transfecting cells with DN mutant forms of IKKα and IKKβ. SH-SY5Y and IMR32 cells were cotransfected with pcDNA3-DN-IKKα-Flag, pcDNA3-DN-IKKβ-Flag, or control plasmid, along with the NF-κB reporter plasmid. As seen in Figure 2C, expression of DN-IKKα or DN-IKKβ blocked drug-induced NF-κB activation, arguing that IKK activity is necessary. These results indicate that IKK is activated and necessary for Dox-induced and VP16-induced activation of NF-κB. Finally, the presence of IKK activation was verified by measuring I-κB levels. As shown in Figure 2D, treating SH-SY5Y cells with Dox or VP16 results in lower levels of I-κB protein. These results are consistent with Dox and VP16 inducing IKK activation that leads to I-κB degradation and subsequent NF-κB activation.

MEK Mediates Dox and VP16-Induced NF-κB Activation

Because MEK1/2 is associated with NF-κB activation, we next tested whether Dox and VP16 activate MEK1/2, leading to NF-κB response. Consistent with this hypothesis, phosphorylated (active) MEK1/2 was detected in cells following Dox or VP16 treatment (Figure 3A). Phosphorylated MEK1/2 is induced within 15 minutes of Dox or VP16 treatment. To directly confirm MEK activity, we measured MEK1 kinase function in lysates prepared from SH-SY5Y cells treated with Dox or VP16. Based on the phosphorylation of recombinant ERK2 as a substrate in in vitro kinase assays, MEK1 activity increased in response to both Dox and VP16 (Figure 3B).

Figure 3
MEK1 is activated by Dox and VP16, and mediates NF-κB activation. (A) SH-SY5Y cells were treated with Dox (0.5 µg/ml) or VP16 (10 µg/ml) for indicated times. Cell lysates (30 µg) were immunoblotted to detect MEK1 and pMEK1. ...

In subsequent experiments, we evaluated whether MEK1/2 activity is necessary for drug-induced NF-κB activation using the MEK1/2-selective kinase inhibitor PD98059. As shown in Figure 3C, PD98059 dose-dependently inhibited Dox-induced NF-κB activation as assessed by NF-κB-dependent luciferase reporter gene expression. Because PD98059 was noted to have significant cytotoxic effects on NB cells after longer periods of incubation, its ability to block Dox-induced cell death was not determined. Together, these results support the hypothesis that MEK1/2 is activated as part of response to Dox and VP16 and argue that its activity is necessary for NF-κB activation, which is itself necessary for drug-induced killing.

MEK Activates NF-κB through IKK

Next, we examined the relationship between MEK and IKK, testing the hypothesis that MEK activation is independent (and therefore upstream) of IKK. SH-SY5Y cells were pretreated with the MEK inhibitor PD98059 before exposure to Dox. As expected, pretreatment with PD98059 blocked I-κBα degradation, in addition to inhibiting ERK phosphorylation in Dox-treated and VP16-treated cells (Figure 4). This result indicates that IKK is activated downstream of MEK and confirms that IKK is required for I-κBα degradation in response to Dox treatment.

Figure 4
MEK is required for I-κB degradation. SH-SY5Y cells were treated with Dox (0.5 µg/ml) (A) or VP16 (10 µg/ml) (B) in the presence or in the absence of PD98059 (20 nM). Cell lysates (35 µg) were immunoblotted to detect endogenous ...

p53 Mediates NF-κB-Dependent Death

Dox and VP16 cause DNA damage, and both agents have previously been shown to induce p53 expression in carcinoma cells. In fact, Dox treatment of SH-SY5Y cells results in rapid and sustained upregulation of p53 [27]. Because work by Ryan et al. [21] suggested that wild-type p53 is important for drug-induced NF-κB activity and that these NB cell lines express wild-type p53, we tested whether cell death, NF-κB activation, and MEK activation are p53-dependent responses to Dox and VP16 in N-type cells.

p53 levels were assessed by immunoblotting lysates of SH-SY5Y cells after treatment with Dox or VP16. As shown in Figure 5A, p53 protein increases within 10 minutes of VP16 treatment and within 60 minutes of Dox treatment. Moreover, when lysates are immunoblotted to detect I-κBα, the increase in p53 was observed to precede the decrease in I-κBα (Figure 2D). This temporal order is consistent with the hypothesis that p53 mediates NF-κB signaling.

Figure 5
E6/SH-SY5Y cells do not activate NF-κB and are resistant to Dox-induced and VP16-induced cell death. (A) SH-SY5Y cells were treated with Dox (0.5 µg/ml) or VP16 (10 µg/ml) for indicated times, and p53 and β-tubulin protein ...

Next, to assess the functional contribution of p53 to NF-κB response, SH-SY5Y cells were stably transfected to express the E6 protein derived from human papilloma virus type 16. This viral protein binds to p53 and forces its degradation [28]. The increase of p53 in response to drug treatment was prevented in E6-expressing SH-SY5Y cells (Figure 5B). Consistent with the hypothesis that killing by these drugs depends on p53, E6/SH-SY5Y cells were less sensitive to both Dox and VP16 killing than were vector controls (Figure 5B). Using these same cells, we tested whether drug-induced activation of NF-κB was also blocked as a consequence of reducing p53 levels. As expected, when E6/SH-SY5Y cells were transiently transfected with the NF-κB luciferase reporter plasmid, Dox-induced and VP16-induced luciferase expression was suppressed, whereas expected induction was observed in control cells (Figure 5C). Given the possibility that E6 viral protein was acting through a non-p53-specific mechanism, SH-SY5Y cells were transfected to stably express DN mutant p53, which encodes the carboxy-terminal portion (codons 302–393) of human p53 and forms nonfunctional hetero-oligomers with endogenous wild-type p53. This truncated form of p53 specifically interferes with its transcriptional activity [22]. In cotransfection experiments, we found that DN p53/SH-SY5Y cells also failed to increase NF-κB-dependent luciferase activity in response to Dox (data not shown) and, like E6-expressing cells, were less sensitive to Dox killing. Dox (1 µM) killed 89.1% (± 0.15%) of vector control but only 57.7%(± 0.8%) of DN p53-transfected SH-SY5Y cells (P < .001). Together, these results provide firm evidence that Dox-induced and VP16-induced NF-κB activation and cell death are p53-dependent.

Because p53 and MEK1/2 are both necessary for NF-κB-mediated death, experiments were conducted to determine the interrelationship of MEK1/2 activation and p53 response. First, phosphorylated ERK, a marker of MEK1/2 activity, was measured in p53-depleted E6/SH-SY5Y cells following Dox or VP16 treatment (Figure 6A). The results of this experiment showed that reducing p53 attenuated ERK phosphorylation, arguing that MEK1/2 activation is p53-dependent. Then, to further verify that MEK1/2 acts downstream of p53, SH-SY5Y cells were pretreated with PD90589 to inhibit MEK1/2 and then immunoblotted to detect the level of p53 expression following Dox and VP16 treatment. As shown in Figure 6B, although PD98059 completely blocked ERK phosphorylation as expected (top panel), it did not affect the drug-induced increase in p53 (middle panel). From these experiments, we conclude that p53 induction is upstream of MEK activation and that p53 is necessary for subsequent signaling leading to NF-κB activation-associated cell death, including MEK activity.

Figure 6
p53 is an upstream mediator of Dox-induced and VP16-induced MEK activation. (A) E6/SH-SY5Y and vector control cells were treated with Dox (0.5 µg/ml) or VP16 (10 µg/ml) for indicated times. Immunoblotting was performed to detect total ...

We also tested this apoptotic pathway in cells with abnormal p53 expression. SK-N-BE(2) NB cells contain only a single nonfunctional p53 allele with a point mutation that renders the protein transcriptionally inactive [29]. The concentration of Dox required to kill 50% of SK-N-BE(2) cells exceeded 2 µg/ml, which is more than eight times that required for the other N-type lines with wild-type p53 studied here. Consistent with our proposed model, when SK-N-BE(2) NB cells were exposed to either Dox or VP16, there was no increase in NF-κB-dependent luciferase reporter gene expression and there was no evidence of MEK1/2 activation (no phosphorylation of either MEK1 or ERK; Figure 7). These results indicate that wild-type p53 transcriptional activity is required for the activation of NF-κB-induced cell death.

Figure 7
MEK1 and NF-κB activation is suppressed in chemoresistant mutant p53-expressing cells. (A) SK-N-BE(2) cells were transfected with the pBVIx-Luc reporter plasmid and treated with Dox (0.5 µg/ml; 8 hours) or VP16 (10 µg/ml; 8 hours). ...

H-Ras Mediates p53-Induced NF-κB Activation

We next explored the mechanism that links p53 activation to MEK1 activation. Because the DN p53 mutant employed above specifically interferes with p53 transcriptional activation, we hypothesized that p53-induced changes in gene expression were involved. To test this, CHX was used to block protein synthesis during Dox treatment. CHX blocked the Dox-induced increase of p21 (an established p53-responsive gene), the phosphorylation of MEK1, and the increase in the level of p53 in response to Dox treatment (data not shown). Thus, new protein synthesis is required for MEK1 activation. The results, however, do not exclude the possibility that other transcriptional events upstream of p53 may also be necessary. Taken together with the data above showing that transcriptionally inactive p53 mutants fail to engage MEK1 activation, our results point to p53-dependent change in gene expression mediating MEK1 activation.

In other systems, p53-induced gene expression has been linked to MAPK cascade activation through the induced expression of G-protein-coupled receptors such as heparin-binding growth factor-like growth factor receptor and DDR1 [30,31]. Moreover, G proteins such as H-Ras and K-Ras are linked to MAPK activity and NF-κB activation [32–34]. Interestingly, H-Ras expression is correlated with favorable outcomes in NB, suggesting that this protein may facilitate therapy-induced remission in NB [35]. Therefore, we decided to test whether H-Ras mediates response to Dox by specifically coupling p53 to MAPK activity.

SH-SY5Y cells were treated with the geranyl-gernayl transferase inhibitor GGTI-287, which blocks Ras prenylation and inhibits its membrane targeting and activity [36]. As seen in Figure 8A, GGTI-287 blocked Dox-induced NF-κB activation. Next, to confirm the involvement of Ras, we expressed a DN mutant form of H-Ras and tested whether Dox-induced NF-κB activation and cell death were affected. We found that expression of DN H-Ras blocked Dox-induced NF-κB activation (Figure 8B). Moreover, when the Dox-induced death of transfected cells was quantified, we found that, after 24 hours, 0.5 µM Dox killed 65% (± 10%) of vector control-transfected SH-SY5Y cells but only 25% (± 16%) of DN H-Ras-transfected SH-SY5Y cells (P < .01).

Figure 8
H-Ras is necessary for NF-κB activation in response to Dox. (A) After transfection of the pBVIx-Luc reporter vector, SH-SY5Y cells were treated with Dox (0.5 µg/ml) for 8 hours in the absence or in the presence of the geranyl-geranyl transferase ...

Finally, we explored conditions under which H-Ras activation may be sufficient to signal NB cell death. Cells were transiently transfected to express the constitutively active H-Ras mutant V12. As seen in Figure 9A, NF-κB activity was induced in SH-SY5Y cells by V12 H-Ras. Moreover, in E6/SH-SY5Y and SK-N-BE(2) cells, which each lacks p53, NF-κB was activated in response to constitutively active H-Ras (Figure 9, B and C). These results indicate that, in this mechanism, H-Ras acts downstream of p53 and that it is sufficient for activating NF-κB. As predicted, I-κB SR expression, which blocks NF-κB, abrogates V12 H-Ras effect on NF-κB activation (Figure 9D).

Figure 9
H-Ras acts downstream of p53 and upstream of NF-κB. SH-SY5Y (A), E6/SH-SY5Y (B), and SK-N-BE(2) (C) cells were cotransfected with a vector expressing a constitutively active V12 H-Ras at the quantities shown and with the pBVIx-Luc reporter vector. ...

Discussion

p53 is paramount in determining responsiveness to many chemotherapeutic agents. Mutations in p53 or other disruptions of the p53 pathway produce multidrug resistance in vitro and in vivo [22]. Although p53 mutations occur in < 3% of primary NB tumors, p53 mutation or loss of p53 function commonly develops during therapy in association with drug resistance [29,37,38]. Moreover, amplification of the p53 inhibitor MDM2, as well as homozygous deletion of the p53-responsive gene CDKN2A (p16INK4a/p14ARF), has been observed in NB cell lines and tumors [39,40]. Together, these observations tie abnormal p53 responsiveness to the tumorigenic behavior of NB, particularly with respect to treatment resistance and relapse. Here, we provide evidence that p53 is important in NB chemoresponsiveness as a critical mediator of chemotherapy-induced NF-κB activation—a signal that can independently induce apoptosis in N-type NB cells. Dox and VP16 are drugs that kill NB cells through an NF-κB-driven response, and each increases p53 protein levels. Using three independent strategies, our results show that p53 is required for both of these drugs to activate NF-κB and to kill N-type NB cells. As shown in Figure 10, we outline a pathway in which p53 activation leads to H-Ras-dependent MAP kinase activity, resulting in NF-κB activation through the IKK phosphorylation of I-κBα.

Figure 10
Dox and VP16 signaling pathway in NB.

Our results implicate MEK1 downstream of p53. Ryan et al. [21] have also shown that MEK1 is required for p53-induced apoptosis in Saos-2 cells. No direct interaction between p53 and MEK1 is known; therefore, it is likely that intervening signaling is required. In untreated NB cells, p53 is sequestered in the cytoplasm. In this regard, unpublished data from our group show that Dox and VP16 cause not only p53 accumulation but also nuclear translocation. Second, our results with SK-N-BE(2) cells, which express p53 with a point mutation that completely abolishes transcriptional activity, and our experiments with CHX suggest a requirement for p53-mediated gene transcription for MEK activation in this model.

MAPKs consist of three families: ERK, c-Jun NH2-terminal kinases, and p38-MAPKs. Only ERK1 and ERK2 are activated by MEK1. Following activation, ERK translocates to the nucleus and phosphorylates a variety of substrates, including pp90/Rsk1; the cytosolic phospholipase A2; transcription factors c-Myc, Elk-1, NF-IL-6/C/EBP/NF-M, Tal-1, and Ets-2; and STAT proteins [41]. Our results show that ERK1 is present in NB cells and that it is phosphorylated in response to Dox and VP16. Although ERK signaling pathways are usually linked to cell survival, ERK1/2 can also be involved in cell death [42–47]. Interestingly, ERK activation has previously been shown to mediate cell cycle arrest and apoptosis after VP16 or Dox treatment, independently of p53 [48]. A requirement for ERK in NF-κB signaling has also been described in other cell lines. In T cells, macrophages, and melanoma cells, ERK is necessary for NF-κB responses induced by HIV-mediated CD4 stimulation, induced by lipopolysaccharide, or in constitutive baseline activity, respectively [49–51]. Experiments are necessary to next test whether ERK transduces the signal between MEK and IKK in NB cells.

Small GTPases of the Ras family are important signaling molecules in the regulation of a variety of cellular processes, including growth, differentiation, and survival [52]. Growth factors and other external stimuli lead to transient activation of Ras; however, mutations in Ras alleles, which occur in 30% of human tumors, result in a constitutively active protein [53]. Active GTP-bound Ras signals to a variety of downstream effector pathways. The three most extensively characterized Ras effectors are Raf kinase, phosphatidylinositol 3-kinase, and RalGDS [54]. The activation of Ras and its downstream signal transduction cascades ultimately leads to the activation of transcription factors involved in proliferation, differentiation, and apoptosis [55]. Although normally thought of as an oncogenic protein, our results indicate that H-Ras mediates apoptosis in response to chemotherapy agents. We showed that H-Ras is both necessary and sufficient to activate NF-κB and to induce apoptosis in N-type NB cells. It is tempting to speculate that its proapoptotic activity in NB explains the direct correlation between H-Ras expression and favorable outcomes in patients with NB [56]. It is intriguing that H-Ras, NF-κB, MEK, and ERK are each generally understood to promote tumor growth but to possess death-inducing roles in NB. These results support the need to define the function of these signaling proteins according to disease and cell type before applying therapeutic strategies that target these proteins.

With respect to NB, our data suggest the potential to circumvent p53-associated resistance by engaging H-Ras, MEK1, IKK, and, potentially, even ERK. Genotoxic agents, such as Dox and VP16, produce multiple effects in cells, in addition to p53 stabilization and NF-κB activation. Many of these effects, such as DNA adduct formation, oxygen radical formation, and mitochondrial failure, are catastrophic, especially to normal cells. Therefore, therapeutic measures that selectively activate appropriate downstream signal transducers may be effective against this disease independent of p53 status and may be potentially less toxic. Conversely, our results point to the potential risk of potent MAPK or IKK inhibitors, which currently exist as experimental therapeutics, to reduce the response of NB tumors to conventional agents such as Dox or VP16.

Acknowledgements

We thank Jason Jarzembowski for critically reviewing the manuscript and Kai Wang for his technical and graphical assistance.

Abbreviations

DN
dominant-negative
Dox
doxorubicin
ERK
p44/42 MAP kinase
I-κB
inhibitor of NF-κB
IKK
I-κB kinase
MEK
MAPK/ERK activity kinase
MEM
minimal essential medium
MTT
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
NB
neuroblastoma
NF-κB
nuclear factor κB
PMA
phorbol 12-myristate 13-acetate
pp90/Rsk1
90-kDa ribosomal S6 kinase
VP16
etoposide

Footnotes

1This work was supported, in part, by grants CA697276-04 (V.P.C.) and 2T32HL07622 (M.B.A.) from the National Institutes of Health, the Janette Ferrantino Hematology Research Fund (to V.P.C.), and the Ravitz Foundation (to A.W.O and V.P.C).

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