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Originally published In Press as doi:10.1074/jbc.M203891200 on August 26, 2002

J. Biol. Chem., Vol. 277, Issue 44, 42144-42150, November 1, 2002
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Constitutively Active NFkappa B Is Required for the Survival of S-type Neuroblastoma*

Xin BianDagger §, Anthony W. Opipari Jr.§, Anthony B. RatanaproeksaDagger , Anthony E. Boitano||, Peter C. Lucas**, and Valerie P. CastleDagger DaggerDagger

From the Departments of Dagger  Pediatrics,  Obstetrics and Gynecology, || Chemistry, and ** Pathology, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, April 22, 2002, and in revised form, August 9, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The NFkappa B transcription factors can both promote cell survival and induce apoptosis depending on cell type and context. Neuroblastoma (NB) cells display two predominant culture phenotypes identified as N- and S-types. Malignant S-type cells express neither high levels of MYCN nor Bcl-2, suggesting that other survival mechanisms are important. We characterized NFkappa B activity in S-type cells and determined its role in their survival. S-type lines (SH-EP1 and SK-N-AS) were treated with pyrrolidine dithiocarbamate (PDTC), a NFkappa B inhibitor, or L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a serine protease inhibitor that blocks Ikappa Balpha degradation. Both agents induced cell death, suggesting that constitutive NFkappa B activity is required for survival. The transient expression of a super-repressor Ikappa Balpha mutant killed S-type cells. The inhibition of NFkappa B produced an apoptotic response characterized by the collapse of the mitochondrial transmembrane electrochemical gradient, caspase-9 activation, and apoptotic DNA changes. Constitutive NFkappa B DNA binding activity specifically involving p65 and p50 was demonstrated in S- but not N-type cells by electromobility supershift and gene reporter assays. This study demonstrates a role for NFkappa B in the survival of S-type NB tumor cells and suggests that NFkappa B activity and function differ according to NB tumor cell phenotype.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear factor-kappa B (NFkappa B)1 is a transcription factor comprised of heterodimers and homodimers of a family of related proteins including NFkappa B1/p50, NFkappa B2/p52, RelA/p65, RelB, and c-Rel (1, 2). In the absence of appropriate signals, NFkappa B is sequestered in the cytoplasm by association with one of several inhibitory Ikappa B species (1). Its release from Ikappa B and translocation to the nucleus results from activation of Ikappa B kinase (IKK) (3). NFkappa B transcriptionally regulates an array of genetic responses, many of which determine the balance between cell survival and death. Its activity is particularly important for cells to resist apoptosis triggered by surface death receptors (4). As an illustration, mice lacking p65 die at embryonic day 12 as a result of massive liver apoptosis triggered by TNF-alpha (5). However, the nature of actions of NFkappa B on the balance between life and death is complex because NFkappa B also mediates cell death. For example, wild-type p53 induces activation of NFkappa B in response to genotoxic stress, and inhibition of NFkappa B activity under these circumstances abrogates p53-mediated apoptosis (6).

NB is a malignant tumor that develops in children, arising from neoplastic cells of neural crest origin (7). Unfortunately, the cure rate of children with high risk NB remains at <20%, providing a compelling reason to better understand the molecular mechanisms that can be targeted to treat this disease (8, 9). The study of this disease is further complicated by the fact that NB tumors are composed of a mixture of histologically distinct malignant cell types identified as neuroblastic and stromal cells (10). In culture, tumor cell explants reflect this heterogeneity in that cells demonstrate one of the three phenotypes: neuronal (N)-, stromal (S)-, or intermediate (I)-type. N-type cells contain enzymes involved in neurotransmitter synthesis, grow as poorly adherent aggregates of small round cells with neuritic processes, and express cell surface receptors characteristic of embryonic neuronal precursors. S-type cells grow flattened and adherent to substrate and are devoid of the synthetic machinery for neurotransmitter synthesis (11). I-type cells share certain characteristics with S- and N-type cells and can be induced to further differentiate along either lineage (12).

Compared with S-type cells, N-type cells are more tumorigenic in immunodeficient mice and express higher levels of the tumor marker MYCN and the anti-apoptotic protein Bcl-2 (13). In human NB, disease prognosis worsens as the proportion of neuroblastic cells in a tumor increases. These characteristics have made N-type cells the primary focus of studies of NB chemotherapy responsiveness. In previous work, we reported that N-type cells respond to doxorubicin, an agent used to treat this disease, by inducing NFkappa B activation and that NFkappa B is necessary for the cytotoxic effects of this drug (14).

However, the heterogeneity of NB tumors presents the possibility that malignant behavior (including development, persistence, and treatment response) is influenced by the non-neuroblastic components that correspond to S- and I-type cells in culture. Despite their low tumorigenicity in xenograft models, S-type cells in particular have characteristics that suggest an important role in determining disease outcome. Most importantly, recent data show that S- and N-type cells are derived from neoplastic clones that are genetically identical (15). Secondly, stromal tumor cells appear more able to persist after treatment with cytotoxic drugs. Following cytotoxic therapy, residual tumor deposits are primarily composed of mature atypical ganglion cells and stromal components and lack neuroblastic elements that predominate in specimens prior to treatment (16). Chemotherapy-induced differentiation does not correlate with more favorable outcomes, further suggesting that stromal cells have malignant capability. These observations may be partly explained by the increased expression of membrane-bound complement inhibitors such as CD59 on S-type cells (17). By resisting complement-mediated cell lysis, S-type cells evade an arm of immune surveillance as well as a mechanism that eliminates tumor cells following cytotoxic therapy (18). Because S-type cells are capable of trans-differentiating into N-type cells in vitro (19), a model of NB tumorigenesis has been proposed in which S-type cells provide a reserve pool of tumor cells that survive even when N-type cells are successfully targeted (15).

Bcl-2 and MYCN are highly expressed in N-type cells and appear to primarily account for their survival advantage and tumorigenic phenotype (20). S-type cells express at most low levels of Bcl-2 and are rarely MYCN-amplified. Consequently, their survival must depend on other molecular mechanisms that could be important targets for therapy. This report describes experiments performed to test the hypothesis that NFkappa B supports S-type cell survival. Results show that NFkappa B is constitutively active in two S-type NB cell lines and that this activity is necessary for their survival. Pharmacological or molecular inhibition of NFkappa B induced apoptosis that was associated with the collapse of the mitochondrial transmembrane gradient and caspase-9 activation. NFkappa B species comprised of p50/p50 homodimers and p65/p50 heterodimers were present in the nuclei of S-type cells but not in N-type cells. Consistent with these results, constitutive NFkappa B-dependent gene transcription was detected in S-type cells. These findings add to the understanding of the mechanistic importance of NFkappa B in NB and define a significant difference between S- and N-type cells with respect to NFkappa B. Future therapeutic strategies should consider the complex behavior of NFkappa B as an arbiter of NB cell survival.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All tissue culture supplies and reagents including minimal essential medium, fetal bovine serum, Opti-MEM I, and LipofectAMINE Plus were purchased from Invitrogen. Pyrrolidine dithiocarbamate (PDTC), L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), carbonyl cyanide p-chlorophenylhydrazone (CCCP) were purchased from Sigma. TNF-alpha was purchased from BD Biosciences. 4,6-diamidino-2-pheylindole, Hoechst-33258 and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) were purchased from Molecular Probes (Eugene, OR). Rabbit polyclonal anti-Ikappa Balpha , anti-NFkappa B p65, and anti-NFkappa B p50 antibodies were purchased from Santa Cruz Biochemicals (Santa Cruz, CA). Anti-FLAG antibody was purchased from Sigma. X-Gal (5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside) was purchased from Roche Molecular Biochemicals. The pEGFP-C1 expression plasmid encoding green fluorescent protein (GFP) was purchased from Clontech (Palo Alto, CA). The reporter plasmid pBVIx-Luc containing six NFkappa B recognition sites within the promoter sequence linked to the luciferase reporter gene and the expression plasmid pCDNA3-p35 (21) were provided by Dr. Gabriel Nuñez (University of Michigan, Ann Arbor, MI). The expression plasmid pCMV4-SR-Ikappa Balpha -FLAG was provided by Dr. Cun-Yu Wang (University of Michigan).

Cell Culture and Transfections-- The S-type cell lines, SH-EP1 and SK-N-AS, were cultured under 5% CO2 at 37 °C in minimal essential medium supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, and 100 units/ml streptomycin. NB cells were transiently transfected with the indicated expression plasmids using LipofectAMINE Plus according to the manufacturer's instructions. Cells were seeded in tissue culture plates to achieve 50% confluence. 24 h later, cells were transfected using a mixture of DNA and LipofectAMINE Plus in Opti-MEM I. 8 h following transfection, cells were supplemented with serum at a final concentration of 10%. Cells were then treated as indicated.

MTT Cytotoxicity Assay-- NB cells were plated in triplicates at 104 cells/well in 96-well culture plates in minimal essential medium. For consecutive days following treatment, cells were incubated with MTT dye (5 mg/ml) at 37 °C for 4 h and lysed in buffer containing 20% (w/v) SDS, 50% (v/v) N,N-dimethylformamide (pH 4.5). Absorbance at 600 nm (A600) was determined for each well using an automated microplate reader (Biotek Instruments, Winnoski, VT). After subtraction of the background absorbance, the absorbance of treated cells was divided by that of the untreated cells to obtain the percent viability. The Student's t test was used to test the significance of the difference in cell viability for these assays.

beta -Galactosidase Viability Assay-- Cells were co-transfected with 1.75 µg of control vector pCMV4 or pCMV4-SR-Ikappa Balpha -FLAG along with 0.25 µg of beta -galactosidase reporter plasmid (pEF-1-Bos/beta -galactosidase). 24 h following transfection, cells were stained with X-gal and visualized by light microscopy.

Immunoblot Analysis-- Following treatment, cells were washed in phosphate-buffered saline and lysed in a buffer containing 50 mM Tris-HCL, 100 mM dithiothreitol, 2% (w/v) SDS, 0.1% (w/v) bromphenol blue, 10% (v/v) glycerol. Aliquots of cell lysates containing 30 µg of total protein were resolved on 12% SDS-PAGE and transferred to Hybond-P membranes (Amersham Biosciences). Membranes were incubated with anti-Ikappa Balpha or anti-glyceraldehyde-3-phosphate dehydrogenase. Immunoreactivity was detected using the ECL detection system (Amersham Biosciences).

Determination of NFkappa B-dependent Reporter Gene Activation-- S-type NB cells were transfected with 1 µg of the reporter plasmid pBVIx-Luc as described. To assure identical transfection efficiency in control and treated cells, cells were replated 12 h after transfection into 12-well plates, and after attachment they were treated as indicated. After cells were harvested, luciferase activity was determined according to the manufacturer's instructions using the luciferase reagent kit (Promega, Madison, WI) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). An aliquot of the same samples was subjected to protein concentration determination (Bio-Rad). Luciferase activity was then normalized to protein concentration.

Measurement of NFkappa B Activation by Electromobility Shift Assay (EMSA)-- Nuclear extracts were prepared as described previously (22) and incubated in EMSA reaction buffer containing 500 mM NaCl, 2 µg/ml poly(dI·dC), 100 mM Tris (pH 7.6), 10 mM DTT with a 32P-labeled oligonucleotide encompassing the consensus NFkappa B binding site (5'-AGTTGAGGGGACTTTCCCAGGC-3'). In the antibody supershift assays, nuclear extracts were incubated with 0.2 µg of anti-p65 or anti-p50 polyclonal antibody for 15 min at room temperature prior to the addition of the 32P-labeled oligonucleotides. After incubation, samples were resolved on 6% polyacrylamide gels, dried, and exposed to BioMax MS film (Eastman Kodak Co.).

Flow Cytometric Analysis of the Mitochondrial Transmembrane Gradient (Delta psi M)-- Cells were plated in duplicate at a density of 2 × 105 cells/well and treated as indicated. Cells were then incubated at 37 °C for 20 min with JC-1 (1 µM) and harvested by trypsinization. Delta psi M was determined for 10,000 cells by flow cytometry, and the data were analyzed using CellQuest software (BD Biosciences).

Fluorescence Microscopy-- NB cells were seeded on 2-chamber slides (Lab-Tek, Naperville, IL) at 104 cells/well. After attachment, cells were transiently transfected with pCMV4-SR-Ikappa Balpha and pEGFP at a ratio of 5:1 as described above. 24 h following transfection, cells were washed with phosphate-buffered saline and fixed in 4% paraformaldehyde. Nuclei were stained with 4,6-diamidino-2-pheylindole (2 µg/ml). Epifluorescence was detected using a Nikon eclipse E600 (Nikon, Melville, NY).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemical Inhibitors of NFkappa B Induce Apoptosis of S-type Cells-- To explore the role of NFkappa B in S-type cell survival, two S-type cells lines were treated with small molecules known to inhibit NFkappa B activation. PDTC is an antioxidant and chelator of heavy metals that blocks NFkappa B activity by suppressing the release of Ikappa Balpha from NFkappa B (23). TPCK inhibits NFkappa B by blocking Ikappa Balpha degradation (24). Both compounds dose-dependently kill SH-EP1 and SK-N-AS cells after 24 h of treatment (Fig. 1A). When visualized by interference contrast microscopy after treatment with PDTC or TPCK, cells demonstrated an apoptotic appearance including plasma membrane blebbing, nuclear condensation, and detachment from the culture flask (Fig. 1B). These findings were in sharp contrast with our previous results that showed that PDTC blocks cell death of N-type cells, suggesting that NFkappa B activity and function might differ dramatically according to NB cell phenotype (14).


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  		Fig. 1.   PDTC and TPCK kill S-type NB. A, viability determined by MTT for SH-EP1 (black-square) and SK-N-AS (black-diamond ) cells following treatment for 24 h with the indicated concentrations of PDTC or TPCK. Data presented as the mean ± S.D. from three separate experiments. B, SH-EP1 and SK-N-AS cells were treated with control medium (0.1% Me2SO (DMSO), PDTC (100 µM), or TPCK (50 µM) for 24 h. Representative examples of cell morphology were photographed (×400). Arrows indicate cells displaying apoptotic features.

The mechanism and time course of cell death was further characterized with respect to mitochondria dysfunction and caspase activation. JC-1, a fluorescent potentiometric indicator, selectively accumulates in mitochondria and was used to monitor the mitochondrial transmembrane gradient (Delta psi M) by flow cytometry. As shown in Fig. 2A, the treatment of SH-EP1 cells with PDTC or TPCK causes Delta psi M collapse beginning within 4 h of treatment. Caspase-9 activation, which depends on apoptosome formation that is in turn triggered by cytochrome c release from mitochondria (25), was also detected in treated cells. Both drugs the induced processing of pro-caspase-9 to the active 35-kDa form between 8 and 12 h after treatment (Fig. 2B). Thus, its activation is slightly delayed relative to Delta psi M collapse. Of note, caspase-9 is processed into two forms in the cells treated with TPCK, corresponding to molecular masses of 35 and 37 kDa. In PDTC-treated cells, only the 35-kDa form is detected. This difference may be related to serine protease inhibition by TPCK that can result in altered caspase processing (26). Together, these data are consistent with an apoptotic response induced by PDTC and TPCK that involves mitochondria-associated signals beginning within 4 h following treatment. Identical changes in Delta psi M and in caspase processing were detected in SK-N-AS cells after these treatments (data not shown).


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  		Fig. 2.   PDTC induces collapse of Delta psi M and caspase-9 activation. A, SH-EP1 cells were treated with control medium (0.1% Me2SO) or PDTC (100 µM) for 12 h or CCCP (10 µM) for 30 min, and after the indicated times, medium was incubated with JC-1 for 15 min, trypsinized, and then subjected to flow cytometry. Delta psi M is indicated by fluorescence at 590 nm (red) where complete collapse is identified by treatment with the protonophore CCCP. Percentages indicate cells with intact Delta psi M. B, SH-EP1 cells were treated with PDTC (100 µM) or TPCK (50 µM) for the indicated times and followed to determine Delta psi M as noted above and by immunoblotting to detect pro-caspase-9 (46 kDa) and processed caspase-9 (37 and 35 kDa). The fraction of cells with collapsed Delta psi M is presented. ND indicates time points where it was not determined. Caspase-9 processing (detected by 8 h) lags behind the collapse of Delta psi M.

NFkappa B Inactivation Is Associated with the S-type Response to PDTC or TPCK-- Because Ikappa Balpha levels inversely correlate with NFkappa B activity, its levels were measured in SH-EP1 cells during treatment to directly correlate the death response with the inhibition of NFkappa B. As expected, increased Ikappa Balpha levels in SH-EP1 cells were detected by 8 h of treatment with PDTC and 4 h of treatment with TPCK (Fig. 3A). Similar increases were also observed in SK-N-AS cells after treatment with these agents (data not shown). To confirm the increase in Ikappa Balpha reflects diminished NFkappa B activity, NFkappa B-dependent gene expression was measured using a NFkappa B-dependent luciferase reporter construct. SH-EP1 and SK-N-AS cells were transiently transfected with pBVIx-Luc, which contains six tandemly placed NFkappa B consensus binding sites within the promoter region upstream of sequence encoding firefly luciferase. Using this construct, constitutive NFkappa B-dependent luciferase activity was detectable in both S-type cell lines in the absence of any treatment (Fig. 3B). When cells were treated with PDTC or TPCK, luciferase activity decreased in a time-dependent fashion beginning within 4 h of treatment. With PDTC, decreased transcriptional activity occurred prior to the detectable increase in Ikappa Balpha . This difference may be attributed to the relative insensitivity of immunoblotting to detect early small increases in the protein. On the basis of measurements using the NFkappa B-dependent luciferase assay, NFkappa B transcriptional activity decreases within the time frame during which Delta psi M is disrupted and prior to pro-caspase-9 processing or morphological evidence of cell death. These results are consistent with the possibility that changes in NFkappa B activity underlie the apoptotic response to PDTC and TPCK and suggest that constitutive NFkappa B activity is necessary for SH-EP1 and SK-N-AS cell survival.


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  		Fig. 3.   PDTC and TPCK inhibit Ikappa Balpha degradation and block NFkappa B-dependent transcription. A, SH-EP1 cells were treated with PDTC (100 µM) or TPCK (50 µM) for the indicated times. Cell lysates (30 µg) were immunoblotted to detect Ikappa Balpha . The same blots were probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to confirm equivalent protein loading. B, SH-EP1 cells transfected with pBVIx-Luc were treated in the presence or absence of PDTC (100 µM) and TPCK (50 µM) for the indicated times before luciferase activity was determined. Luciferase activity (normalized to protein concentration) is reported as arbitrary relative light units (mean ± S.D.) based on three experiments.

NFkappa B Is Constitutively Active in S-type Cells-- The reporter gene assays above, which showed basal activity of the NFkappa B promoter construct in untreated cells, provided initial direct evidence that NFkappa B is indeed activated in untreated S-type cells. To more firmly establish that this is indeed the case, we directly measured NFkappa B sequence-specific DNA binding. Nuclear extracts prepared from untreated S-type cells (SH-EP1 and SK-N-AS) were incubated with a 32P end-labeled double-stranded DNA oligonucleotide with the sequence 5'-AGTTGAGGGGACTTTCCCAGGC-3'. As seen in Fig. 4A, extracts of both S-type lines shifted the labeled NFkappa B oligonucleotide during electrophoresis, resulting in three distinct complexes. Additional experiments were performed to determine which shifted complexes correspond to NFkappa B-specific binding. First, nuclear extracts were prepared from cells treated with TNF-alpha , a potent inducer of NFkappa B activity (27). Upon comparison with the EMSA results from untreated cells, the intensity of two bands were increased by TNF-alpha treatment, consistent with their identity as NFkappa B·DNA complexes (Fig. 4A, asterisk). Second, confirmation was obtained by antibody supershift experiments in which antibodies specific for either p50 or p65 were added to the mixture of nuclear extract and labeled oligonucleotide. As seen in Fig. 4B, each of the two putative NFkappa B·DNA complexes was supershifted by the p50-specific antibody, whereas the larger complex (upper band) was also supershifted by anti-p65. Hence, constitutively active NFkappa B is detected in S-type cells and consists of both p50/p50 and p50/p65 dimers.


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  		Fig. 4.   NFkappa B is constitutively active in S-type NB cells. A, nuclear extracts prepared from SH-EP1 and SK-N-AS cells cultured with or without TNF-alpha (100 ng/ml) for 20 min were used in EMSAs with a NFkappa B consensus sequence probe. NFkappa B-specific bands (indicated by asterisk) were identified on the basis of their increase in response to TNF-alpha . B, EMSA performed with supershift analysis using anti-p65 or anti-p50 antibodies. The NFkappa B probe and nuclear extracts from untreated SH-EP1 and SK-N-AS cells confirm NFkappa B proteins are involved in binding activity. C, EMSA using nuclear extracts from SH-EP1 and N-type cell lines (SH-SY5Y, IMR32) indicates that NFkappa B-specific binding is not detected in nuclear extracts from untreated N-type cells. D, EMSA using nuclear extracts from SH-EP1 cells treated with PDTC (50-150 µM, 4 h), TNF-alpha (100 ng/ml, 20 min), or control medium indicates PDTC dose-dependently reduces NFkappa B-specific binding activity.

N-type cells were expected to have little or no basal NFkappa B DNA binding activity, because prior experiments showed the absence of constitutive NFkappa B reporter gene activation in N-type cells (14). We tested this possibility and directly compared S-type and N-type cells with respect to constitutive NFkappa B DNA binding activity. As expected, when the EMSA was performed with nuclear extracts prepared from untreated N-type cells (SH-SY5Y and IMR32), neither of the two complexes specific for NFkappa B·DNA binding was detected (Fig. 4C). We confirmed that the cytotoxic action of PDTC in S-type cells is related to a reduction of NFkappa B·DNA binding. Treating SH-EP1 cells with PDTC resulted in a dose-dependent reduction in DNA binding activity (Fig. 4D). Similar results were obtained when cells were treated instead with TPCK (data not shown). These results confirm the constitutive NFkappa B activity in S-type cells and show that the inhibition of NFkappa B is associated with the response of S-type cells to small molecule inhibitors of NFkappa B signaling that induce apoptosis.

SR-Ikappa Balpha Induces Apoptosis of S-type Cells-- Experiments were next performed to test whether the specific inhibition of NFkappa B is sufficient to induce S-type cell death. The super-repressor mutant form of Ikappa Balpha (SR-Ikappa Balpha ) binds NFkappa B but can not be phosphorylated on the basis of alanine substitution for serines 32 and 36 (28). SH-EP1 and SK-N-AS cells were co-transfected with pCMV4-SR-Ikappa Balpha -FLAG along with the NFkappa B-responsive luciferase reporter (pBVIx-Luc) and the expression plasmid pCDNA3-p35 (21). pCDNA3-p35 encodes a dominant negative inhibitor of caspase-9 and was included in these experiments to block the expected cytotoxic effect of inhibiting NFkappa B so that changes in transcriptional activity could be detected. SR-Ikappa Balpha expression was first confirmed by immunoblotting (Fig. 5A). Additionally, cells transfected in this manner showed no evidence of increased cell death compared with cells transfected with SR-Ikappa Balpha vector-only plasmid (data not shown). In a dose-dependent manner, SR-Ikappa Balpha expression decreased NFkappa B-dependent luciferase activity in both cell lines, confirming the activity of the super-repressor against NFkappa B transcriptional activation in these cells (Fig. 5A).


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  		Fig. 5.   SR-Ikappa Balpha inhibits NFkappa B-dependent transcription and decreases survival of S-type cells. A, SH-EP1 and SK-N-AS cells were co-transfected with pCMV4-SR-Ikappa Balpha -FLAG (0-500 ng/well) and the reporter pBIVx-Luc (50 ng/well). SR-Ikappa Balpha -FLAG expression was confirmed by immunoblotting. Luciferase activity was normalized to protein concentration and expressed as arbitrary relative light units. Mean ± S.D. from three experiments is shown and indicates that SR-Ikappa Balpha expectedly decreases NFkappa B-dependent luciferase activity. B, SH-EP1 cells were co-transfected with the pEGFP-C1 (as a marker of transfection, 20 ng/well) and either pCMV4-SR-Ikappa Balpha -FLAG or the control plasmid (100 ng/well). 24 h after transfection, cells were stained with 4,6-diamidino-2-pheylindole. Fluorescent microscopy (×400) enumerated surviving transfected clones (cells with green fluorescence). Cells with SR-Ikappa Balpha had reduced survival and increased DNA condensation (fragmented nuclear staining), consistent with apoptosis.

To determine whether the inhibition of NFkappa B caused cell death, SH-EP1 cells were transiently co-transfected to express SR-Ikappa Balpha and GFP (as a transfection marker) without the expression plasmid pCDNA3-p35. Control-transfected cells demonstrated normal morphology including intact nuclei (Fig. 5B, upper panels). In contrast, 24 h following transfection, cells expressing SR-Ikappa Balpha demonstrated apoptotic morphology: rounding up, detachment, and nuclear condensation (Fig. 5B, lower panels). This cytotoxic effect was further quantified by enumerating cell survival following transfection on the basis of expression of a co-transfected marker gene, beta -galactosidase. SH-EP1 and SK-N-AS cells were transfected with efficiencies of 5.5 and 8.0%, respectively, using the beta -galactosidase expression vector. beta -Galactosidase-positive cells per high power field were counted 24 h after six independent co-transfections with pEF-1-Bos/beta -galactosidase and either pCMV4 control or pCMV4-SR-Ikappa Balpha -FLAG. For SH-EP1 cells, beta -galactosidase positivity decreased from 50 ± 1.9 cells/high power field with control to 17 ± 1.1 following pCMV4-SR-Ikappa Balpha -FLAG transfection, revealing a 60% reduction in survival (p < 0.01). For SK-N-AS cells, 84 ± 10 beta -galactosidase-positive cells/high power field were counted for control compared with 45 ± 5.2 with pCMV4-SR-Ikappa Balpha -FLAG, a 40% reduction in survival (p < 0.01). Together, these results confirm that specific molecular inhibition of NFkappa B changes NFkappa B-dependent gene expression and concomitantly reduces the survival of S-type cells in culture.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the absence of appropriate stimuli, NFkappa B is sequestered in the cytoplasm by physical association with Ikappa B, which prevents exposure of nuclear localization signals. As an immediate response to stimuli including pro-inflammatory cytokines, lipopolysaccharides, and phorbol esters, Ikappa B is phosphorylated, ubiquitinated, and degraded by proteasomes (29). The cytoplasmic to nuclear translocation of NFkappa B and transcriptional activation of NFkappa B-responsive genes follow (30). In turn, NFkappa B induces the synthesis of Ikappa Balpha , which results in negative feedback, limiting the magnitude and duration of the response (31).

Despite tight coupling of NFkappa B to specific signals as outlined above, constitutive NFkappa B activation can occur. Constitutive NFkappa B activation appears to have an important role in tumorigenesis. For example, persistent nuclear NFkappa B localization and NFkappa B-dependent transcription is detected in breast (32), ovarian (33, 34), colon (34), thyroid (35), prostate (36), and melanoma (37) tumors. In breast and prostate tumor cells, constitutive NFkappa B activity is associated with reduced levels of Ikappa Balpha that appears related to increased degradation of Ikappa B proteins in these cells (38, 39). Constitutive NFkappa B activity is also associated with the tumorigenic properties of viruses. The viral homologue of c-rel (v-rel) is transduced in the reticuloendotheliosis virus and is responsible for the acute-transforming properties of this virus in mammalian cells (40). Constitutive activation of cellular gene-encoded NFkappa B mediates transformation by human T-cell lymphotrophic virus, type I. The human T-cell lymphotrophic virus, type I-encoded Tax protein activates NFkappa B by stably activating IKK, providing an ongoing signal for Ikappa Balpha degradation (41).

Ikappa Balpha degradation is primarily regulated through the phosphorylation of N-terminal serine residues 32 and 36 by the IKK complex. Phosphorylation at these residues leads to dissociation from NFkappa B and targets the protein for ubiquitination and proteasomal degradation. Alternatively, the phosphorylation of C-terminal serine residues by casein kinase 2, phosphorylation at tyrosine 42 under conditions of anoxia, and treatment with UV radiation can result in NFkappa B activation independent of IKK (42). Our results are most consistent with the two N-terminal serine residues determining constitutive NFkappa B activity in S-type cells. This conclusion is based on the inhibitory activity of the mutant SR Ikappa Balpha that derives its dominant-negative action solely on the basis of alanine substitutions at serine 32 and 36. However, to completely exclude a contribution of the C-terminal region or tyrosine 42, additional mutants involving these residues would need to be studied.

Because the IKK complex is the only kinase recognized to directly phosphorylate serine 32 and 36 on Ikappa Balpha (3), constitutive IKK activity is suspected to underlie constitutive NFkappa B activation in this model. The regulation of IKK is complex, and input converges from multiple signaling cascades including those coupled to TrkA (the high affinity receptor for nerve growth factor) and tumor necrosis receptor-1. Additionally, other signals that activate Ras, phosphatidylinositol 3-kinase, and MEKK1 also increase IKK activity. Because S-type cells neither express nerve growth factor receptors nor demonstrate nerve growth factor responsiveness (43, 44), the activity of TrkA or the low affinity nerve growth factor receptor is unlikely to sustain NFkappa B activation. The autocrine activity of TNF-alpha seems a more probable possibility. S-type cells are responsive to this cytokine (see Fig. 2), and cells derived from the neural crest (including sympathetic neurons) are known to synthesize both TNF-alpha and TNFR1 (45).

NFkappa B supports neoplastic growth by preventing cell death. In this study, we found that inhibition of constitutive NFkappa B activity engages an apoptotic response that specifically involves the collapse of the mitochondrial transmembrane gradient and caspase-9 activation, both responses associated with the mitochondrial permeability transition. The anti-apoptotic function of NFkappa B in these cells could be mediated by transcriptional up-regulation of any one of the array of NFkappa B-responsive genes including TRAF1, TRAF2, c-IAP-1, c-IAP-2, A20, and Bcl-xL (46-48). With the involvement of the mitochondria and caspase-dependent processes in the death response, it is possible that c-IAP-1, c-IAP-2 (inhibitors of caspase activation), and Bcl-xL (stabilizer of the mitochondrial permeability transition pore) are potentially important. Indeed, S-type cells have previously been shown to constitutively express Bcl-xL (49).

In preliminary experiments using an activation-specific antibody against p65, we have uncovered evidence of NFkappa B activation in a NB tumor specimen (data not shown). From hereon, it will be important to firmly establish whether NFkappa B is indeed activated specifically within the stromal components of NB tumor specimens and whether differences in activation are associated with stage and histopathologic grade. Efforts to directly measure transcription factor activation are best combined with experiments to quantify the expression of NFkappa B target genes to more strongly support conclusions regarding NFkappa B activation. Along these lines, a recent study has elegantly used gene expression profiling to show that diffuse large B cell lymphoma consists of two molecularly distinct diseases that are distinguished on the basis of their constitutive expression of NFkappa B-induced genes (50). Similar experimental strategies are planned to address the question of in vivo NFkappa B activation in NB.

In conclusion, NFkappa B is constitutively active in S-type NB cells. Moreover, because it is necessary for S-type cell survival in culture, it has the potential to contribute to cellular transformation, tumor progression and resistance to chemotherapy. Given its opposing, pro-apoptotic activity in N-type NB cells, therapeutic strategies designed to inhibit NFkappa B must be approached cautiously.

    ACKNOWLEDGEMENTS

We acknowledge Alexander Arlt (University of Kiel, Kiel, Germany), Alan Porter (Molecular Institute of Singapore, Singapore, Singapore), Robert Ross, and Barbara Spengler (Fordham University, New York, NY) for technical advice.

    FOOTNOTES

* This work was supported in part by Grant CA697276-04 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.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

Dagger Dagger To whom correspondence should be addressed: Comprehensive Cancer Center, University of Michigan, Rm. 4302, 1500 E. Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-763-0019; Fax: 734-647-9654; E-mail: vcastle@umich.edu.

Published, JBC Papers in Press, August 26, 2002, DOI 10.1074/jbc.M203891200

    ABBREVIATIONS

The abbreviations used are: NFkappa B, nuclear factor-kappa B; Ikappa B, inhibitor of kappa B; NB, neuroblastoma; S-type, stromal-type; N-type, neuroblastic-type; I-type, intermediate-type; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PDTC, pyrrolidine dithiocarbamate; SR-Ikappa Balpha , super-repressor Ikappa Balpha ; IKK, Ikappa B kinase; IAPs, inhibitors of apoptosis proteins; MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CCCP, carbonyl cyanide m-chlorophenylhydrazone; Delta psi M, mitochondrial transmembrane gradient; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide; GFP, green fluorescent protein; EMSA, electromobility shift assay; X-gal, 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside; TNF, tumor necrosis factor alpha ; Luc, luciferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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