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J. Biol. Chem., Vol. 281, Issue 34, 24182-24192, August 25, 2006

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Tumor Necrosis Factor Induces Spermidine/Spermine N¹-Acetyltransferase through Nuclear Factor Bin Non-small Cell Lung Cancer Cells^*

From the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

Received for publication, February 27, 2006 , and in revised form, May 16, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Tumor necrosis factor (TNF) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNF are due to its ability to activate multiple signal transduction pathways, including nuclear factor B (NFB), which plays critical roles in cell proliferation and survival. TNF displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNF can lead to the induction of NFB signaling with a concomitant increase in spermidine/spermine N¹-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IB protein led to the suppression of SSAT induction by TNF in these cells, thereby substantiating a role of NFB in the induction of SSAT by TNF. SSAT promoter deletion constructs led to the identification of three potential NFB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFB response elements play an important role in the regulation of SSAT in response to TNF. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFB signaling pathway in non-small cell lung cancer cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Polyamines are aliphatic cations present in all cells, whose levels are intricately controlled by their transport and metabolic enzymes. Spermidine/spermine N¹-acetyltransferase (SSAT)² is a rate-limiting step in polyamine catabolism, which catalyzes the transfer of the acetyl group from acetyl-CoA to the N¹ position of spermidine or spermine and has a predominant role in the regulation of intracellular polyamine concentrations in mammalian cells (1, 2). Decreases in polyamines have been shown to promote decreased growth or apoptosis (3–6), depending on the cell type and the particular stimulus, suggesting a complex interaction between polyamines, cell growth, and cell death. Therefore, although polyamines are required for cell growth and differentiation, SSAT is thought to prevent overaccumulation of the higher polyamines from becoming toxic to the cell and may play a role in reducing the growth rate by decreasing intracellular polyamines.

Recently, considerable attention has been paid to SSAT as a target for cancer chemotherapy. SSAT activity is highly regulated and is induced rapidly in response to a number of stimuli, including polyamines, polyamine analogues, hormones, physiological stimuli, drugs, and toxic agents (1). It has been shown that the regulation of SSAT by the natural polyamines and the anti-tumor polyamine analogues is through the polyamine response element (7). Further, the superinduction of SSAT by polyamine analogues has been implicated in the cell type-specific cytotoxic response of several important human tumors (8–13). Various nonsteroidal anti-inflammatory drugs like aspirin, sulindac, and indomethacin and chemotherapy drugs like 5-flurouracil and oxaliplatin have been shown to induce SSAT expression in various cancer cell types (14–17). Sulindac induces SSAT by inducing peroxisomal proliferator-activated receptors, which, once activated, can bind to the peroxisomal proliferator-activated receptor response elements in the SSAT 5' promoter region (18). Recently, we have shown that aspirin can also induce SSAT expression in colon cancer cells, partly by activating the NFB signaling pathway, which leads to the binding of NFB complexes to the NFB response elements in the SSAT 5' promoter region (19).

Tumor necrosis factor (TNF) is a potent pleiotropic cytokine and is a major mediator of inflammation with multiple biological functions (20). One of the molecular mechanisms responsible for the biological activities of TNF is the ability to activate nuclear factor B (NFB), which plays critical roles in cell proliferation and survival (21, 22). In mammalian cells, five members of the NFB/Rel family are known: NFB1 (p50 or its precursor p105), NFB2 (p52 or its precursor p100), c-Rel, RelA (p65), and RelB (23, 24). Heterodimers composed of p65-p50 are the most abundant active form of NFB in most cell types. However, NFB can consist of other homo- and heterodimers that have different abilities to activate target genes (25, 26). In quiescent cells, NFB resides in the cytosol in a latent form bound to IB. Stimulation of the cell with TNF triggers a series of signaling events that ultimately leads to the phosphorylation and the proteolytic degradation of IB and activation of NFB. The phosphorylation of IB is elicited by an IB kinase (IKK), which can be activated by mitogen-activated protein kinase (27, 28), whereas the proteolysis of IB is mediated by the ubiquitin-proteasome pathway of protein degradation. The degradation of IB triggers the translocation of NFB from the cytoplasm to the nucleus, where it regulates the expression of multiple genes.

In this study, we examined the effects of TNF treatment on SSAT expression in representative human non-small cell lung cancer cells and identified the specific NFB response elements responsible for the regulation of SSAT by TNF.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—All cell culture reagents, DNA-modifying enzymes, TRIzol® reagent (total RNA isolation reagent), and Lipofectamine reagent were purchased from Invitrogen (Carlsbad, CA). All primers and oligonucleotides were custom made by Invitrogen. Recombinant human TNF was obtained from R&D Systems, Inc. (Minneapolis, MN). IB antibody is from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and p65 antibody is from Upstate (Waltham, MA). Paclitaxel (Taxol®) was obtained from Sigma-Aldrich.

Cell Culture—The non-small cell cancer lines NCI-A549 (adenocarcinoma) and NCI-H157 (squamous) were purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% (v/v) bovine calf serum and 100 units/ml each of penicillin and streptomycin. Cultures were maintained at 37 °C in humidified atmosphere of 5% CO₂.

Plasmids—Full-SSAT-luc, having a 3.493-kb-long 5'-flanking sequence of the human SSAT gene, was cloned into a promoterless pGL2-basic (Promega, Madison, WI) as previously reported (29). A series of smaller SSAT promoter constructs were made from Full-SSAT-luc, using PCR and subcloned into pGL2-basic vector. 197-SSAT-luc, having 283 nucleotides of the 5'-flanking region of the SSAT promoter; 358-SSAT-luc, having 441 nucleotides of the 5'-flanking region of the SSAT promoter; and 659-SSAT-luc, having 740 nucleotides of the 5'-flanking region of the SSAT promoter, were made from Full-SSAT-luc using PCR and subcloned into pGL2-basic vector. NFB₂-Luc reporter, having two NFB response elements; dNFB₂-Luc reporter, in which both the NFB response elements have been deleted; and dominant negative-IB (DN/IB) and its control (Ctrl/IB) plasmids were obtained from Dr. Nancy Davidson (Johns Hopkins University, Baltimore, MD). The DN/IB plasmid has a deletion of 36 NH₂-terminal amino acids containing Ser³² and Ser³⁶ phosphorylation sites, which was cloned into the pcDNA3.1 mammalian expression plasmid vector (Invitrogen). mNFB1-SSAT-luc, mNFB3-SSAT-luc, and dmNFB1/3-SSAT-luc were made by mutating the NFB-1, NFB-3, and both the NFB-1 and -3 sites, respectively, in the Full-SSAT-luc using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutations were done by placing four base substitutions in the respective NFB sites to achieve the same mutated sequences as the probes used for the electromobility shift assays.

Stable Transfection—Exponentially growing cells were plated at 1 x 10⁶ cells/100-mm plate and cultured in normal medium for 24 h and then transfected using 50 µl of Lipofectamine reagent with 10 µg of the DNA plasmid. After 6 h of incubation, with Lipofectamine-DNA complex, cells were supplemented with complete medium having 20% bovine calf serum and 2% penicillin and streptomycin and grown overnight, after which the medium was removed, cells were refed complete medium, and cells were allowed to recover for 24 h prior to selection in 0.4 mg/ml G418. Cells were maintained in selection medium until a stable population was achieved. Individual colonies were then isolated, expanded, and used for screening and experiments.

Transient Transfections—Transient transfections were performed using Lipofectamine reagent according to the supplied protocol. Briefly, 5 x 10⁵ cells were seeded in a 6-well plate and cultured in normal medium for 24 h. Cells in each well were transfected with 1 µg of firefly luciferase reporter construct along with 0.2 µg of pCMV--galalactosidase expression plasmid, used as a control for transfectional efficiency. After 6 h, cells were supplemented with complete medium having 20% bovine calf serum and 2% penicillin and streptomycin and grown overnight. 15 h later, the medium was removed, and cells were refed with the medium containing 10 ng/ml TNF for the indicated amount of time. All transfections were performed in triplicates unless otherwise indicated. Transfected cells were washed once with phosphate-buffered saline and lysed, and luciferase activities were measured using 10 µl of cell extract and 50 µl of luciferase reagent (Promega). -Galactosidase activity was measured using the -galalactosidase assay kit (Invitrogen) according to the supplied protocol.

Quantitative Real Time PCR—Cells were seeded, grown overnight, and then treated as indicated. Total cellular RNA was extracted using TRIzol® reagent and RNA isolation according to the method developed by Chomczynski and Sacchi (30). 3 µg of total RNA was treated with RNase-free DNase I (Roche Applied Science) for 30 min at 37 °C before reverse transcription with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) as described by the manufacturer. The forward and reverse primers were used at 10 µM. The resulting cDNA was subjected to quantitative real time PCR. The DNA-intercalating SyBr green reagent (Quantitect; Qiagen) and melting temperature profiles were used for detection of the PCR product. Agarose gel electrophoresis confirmed the presence of a single PCR product. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. The experimental and GAPDH PCRs were done in separate tubes in triplicates in the MyiQ single color real-time PCR machine (Bio-Rad), and the average threshold cycle (C_T) for the triplicate was used in subsequent calculations. SSAT cDNA (180 base pairs) was amplified using the following primers (Invitrogen): forward, 5'-GGATCAAAATTCTGAAGAAT-3'; reverse, 5'-ACCCTCTTCACTGGACAGATC-3'. As a loading control, GAPDH cDNA (188 base pairs) was also amplified using the following primers: forward, 5'-GAAGGTGAAGGTCGGAGTC-3'; reverse, 5'-GAAGATGGTGATGGGATTTC-3'.

Immunoblotting—Total cell extracts were obtained by lysing cells on ice in radioimmune precipitation buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 30 µg/ml aprotinin, 100 µM sodium orthovanadate, 10 µg/ml phenylmethylsulfonyl fluoride) and centrifuging for 20 min at 4 °C. 30 µg of total protein was loaded per lane and separated on a 10% SDS-polyacrylamide gels for IB immunoblotting. Nuclear protein was isolated, using the NE-PER nuclear extraction reagents (Pierce). 50 µg of nuclear protein was loaded per lane and separated on 12% SDS-polyacrylamide gels for p65 immunoblot. The proteins were transferred electrophoretically to immunoblot polyvinylidene difluoride membrane for 1 h. Blots were blocked in Blotto A (5% w/v nonfat dry milk in 20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween 20) and probed for IB and p65 proteins. Blots were stripped and redetected with -actin (Santa Cruz Biotechnology) antibody as a loading control. Western blot analyses were repeated at least three times, and a representative blot was chosen for presentation.

In Taxol experiments, Western blot results were quantified using the LICOR immunofluorescence system (LI-COR Biosciences, Lincoln, NE). Briefly, membranes were blocked for 1 h in Odyssey blocking buffer, per the manufacturer's instructions. Rabbit anti-IB and mouse anti-actin (loading control) (Santa Cruz Biotechnology) primary antibodies were then added together at dilutions of 1:1000 and 1:1500, respectively, with 0.1% Tween 20 in blocking buffer for 1 h at room temperature. Following washes with PBS-Tween, blots were incubated with appropriate fluorescent dye-conjugated secondary antibodies (1:4000 each, 0.1% Tween 20, in blocking buffer, protected from light, for 45 min), which allowed detection and quantification of each protein using an Odyssey infrared detection system and software (LI-COR).

Gel Electromobility Shift Assays—Nuclear extracts were prepared from A549 cells as described previously (31). To study the binding of NFB complexes to the putative NFB sites, double-stranded oligonucleotides for each of the three putative NFB sites in the SSAT 5' sequence were ³²P-labeled with polynucleotide kinase (Promega, Madison, WI). The oligonucleotide containing the first putative NFB site spanned from –304 to –280 of the SSAT 5' sequence and had the sequence NFB-wild-1 (w) (5'-GCTGCAGAGGGAATTACCTTCTTT-3'), whereas the corresponding mutant NFB-mut-1 (m) had the sequence 5'-GCTGCAGAtaGAATgcCCTTCTTT-3'. The oligonucleotide spanning the third putative NFB site spanned from –1751 to –1728 of the SSAT 5' sequence and had the sequence NFB-wild-3 (w₃) (5'-CCTAGGGGGATTCCACGGATCCT-3', whereas the corresponding mutant NFB-mut-3 (m₃) had the sequence 5'-CCTAGGtGATTCagCGGATCCT-3'. The putative NFB sequence is underlined, and the mutated bases are shown in lowercase letters. A 10-µl reaction containing 10 µg of nuclear extract was incubated for 15 min at room temperature in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2.5 mM EDTA, 250 mM NaCl, 2.5 mM dithiothreitol, 20% glycerol, and 1 µg of poly(dI-dC). Following this, 30,000 cpm of the labeled probe was added, and the reaction mixture was incubated for 30 min at room temperature. The DNA-protein complexes were resolved from the free probe by electrophoresis at 4 °C on a 5% polyacrylamide gel in 0.75x TBE buffer, pH 8. Band density was quantified using the Typhoon 8600 PhosphorImager and ImageQuant software (Amersham Biosciences).

Chromatin Immunoprecipitation (ChIP)—Chromatin immunoprecipitation assays were performed using the commercially available ChIP kit (Upstate Cell Signaling) with some modifications. A549 cells were seeded at 2 x 10⁶/culture dish and grown overnight. On the following day, they were treated with 10 ng/ml TNF for increasing times. After treatment, the cells were treated with 1% formaldehyde by adding 0.4 ml of 37% formaldehyde directly to 15 ml of culture medium for 15 min at 37 °C. The cells were washed twice with cold phosphate-buffered saline containing protease inhibitors (leupeptin, phenylmethylsulfonyl fluoride, and aprotinin) and then suspended in 0.7 ml of SDS-lysis buffer (50 mM Tris-Cl, pH 8.1, containing 1% SDS and 10 mM EDTA) plus protease inhibitors. Twenty 10-s sonication pulses with 30-s intervals were done using the Branson Sonifier 250 (cycle 40%, output = 1.5/2) to shear chromatin to <1000-bp fragments. The effectiveness of shearing was confirmed by incubating a 100-µl aliquot of the extract with 10 µl of 5 M NaCl at 65 °C for 4 h (to reverse cross-links) and subsequently subjecting it to electrophoresis on a 1% agarose gel. The sonicated sample was centrifuged at 13,000 rpm for 10 min. 0.2 ml of the cell supernatant was diluted by adding 1.8 ml of ChIP dilution buffer (16.7 mM Tris-Cl, pH 8.1, containing 0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, and 1.2 mM EDTA) plus protease inhibitors. 50 µl of this sample was stored for later PCR analysis as the input sample control. To reduce nonspecific background, the remaining amount (1950 µl) was precleared with 75 µl of salmon sperm DNA/protein-agarose 50% slurry (Upstate Cell Signaling, NY) for 1 h at 4°C with agitation. The cleared supernatant was divided into 0.4-ml aliquots and incubated with either no antibody, a nonspecific antibody (rabbit IgG), 4 µl of anti-p50, or 4 µl of anti-p65 and incubated overnight at 4 °C with rotation, followed by the addition of 30 µl of salmon sperm DNA/protein-agarose 50% slurry and incubated for an additional 3 h.

The agarose was pelleted by centrifugation, and the pellets were washed consecutively with 0.5 ml of low salt buffer (20 mM Tris-Cl, pH 8.1, containing 0.1% SDS, 1% Triton X-100, 150 mM NaCl, and 2 mM EDTA), 0.5 ml of high salt buffer (20 mM Tris-Cl, pH 8.1, containing 0.1% SDS, 1% Triton X-100, 500 mM NaCl, and 2 mM EDTA), 0.5 ml of LiCl wash buffer (10 mM Tris-Cl, pH 8.1, containing 0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, and 1 mM EDTA), and 0.5 ml of 1x Tris/EDTA (10 mM Tris-Cl, pH 8.1 containing 1 mM EDTA). DNA and protein were eluted from the pellets by incubating the pellets twice in 0.25 ml of elution buffer (0.1 M NaHCO₃ with 1% SDS), and protein-DNA cross-links were reversed by incubating with 20 µl of 5 M NaCl at 65 °C for 4 h. Proteins were then degraded by incubating the eluant with 10 µl of 0.5 M EDTA, 20 µl of 1 M Tris-Cl, pH 6.5, and 2 µl of 10 mg/ml Proteinase K for 1 h at 45 °C. DNA was recovered by phenol/chloroform extraction and ethanol-precipitated overnight at –20 °C. Samples were pelleted, washed with 70% ethanol, and dissolved in 100 µl of sterile water. 2-µl aliquots were used for each PCR to determine binding of either p65 or p50 to the co-immunoprecipitated promoter fragments. The primers used to probe for the NFB-1 site are forward (5'-TCCTGAGTTGCTTCCCACT-3') and reverse (5'-GGTGTGTCCCAGTAACAT-3'), primers for the NFB-2 site are forward (5'-GGGCAGAAGGAAAAGTGTCA-3') and reverse (5'-CCAAAGGAATGTGAGCTCGT-3'), and primers for the NFB-3 site are forward (5'-GACCACCCCTCACATTCAAC-3') and reverse (5'-TGGCAGTTGAGAATCAGTGG-3').

SSAT Enzyme Activity Determination—For enzyme activities, cells were grown overnight and then treated with TNF for different times. Cells were harvested after treatment and washed in cold phosphate-buffered saline. The radiochemical assay of the N¹-SSAT activity was performed by estimation of labeled N¹-acetylspermidine synthesized from [¹⁴C]acetyl-coenzyme A and unlabeled spermidine, as previously described (8). Fold change was calculated by dividing the enzyme activity for the sample by the vehicle. The enzyme assays were performed in triplicate.

Polyamine Analysis—Cell extracts were prepared in 0.1 N HCl (4 x 10⁷ cells/900 µl). After sonication, the preparation was adjusted to 0.2 N HClO₄, and the supernatant was analyzed by reverse-phase high performance liquid chromatography with 1,7-diaminoheptane as an internal standard (32). Protein was determined by the BCA assay (33).

Statistical Analysis—All transient transfection experiments were performed in triplicates and were repeated at least three times. Quantitative real time PCR and ChIP assays were done at least three times. Representative experiments or mean values ± S.D. are shown. Statistical differences were determined by Student's t test. A p value of <0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TNF Induces SSAT mRNA and SSAT Enzyme Activity and Decreases Intracellular Polyamine Contents in Non-small Cell Lung Cancer (NSCLC) Cells—We have previously demonstrated the presence of functional NFB response elements in the SSAT gene (34). Since SSAT has been implicated as a stress response gene (1), we sought to determine whether TNF, acting through NFB, altered the expression of SSAT in the NSCLC cells. TNF, at 10 ng/ml, induces SSAT mRNA expression in both A549 and H157 NSCLC cells within 30 min of treatment (Fig. 1A). SSAT is an enzyme that acetylates both spermidine and spermine. The acetylated products can then be either exported or degraded by the action of polyamine oxidase, thereby leading to a decrease in intracellular polyamine content. Therefore, we next determined whether the increase in SSAT mRNA by TNF treatment was accompanied by an increase in SSAT enzyme activity and a decrease in the polyamine content. TNF treatment led to an increase in SSAT enzyme activity in these NSCLC cells (Fig. 1B) and a modest reduction in the intracellular polyamine content (Fig. 2). The decrease in polyamine content is consistent with the -fold induction in the SSAT enzyme activity by TNF in the NSCLC cells.

TNF Activates NFB Signaling in NSCLC Cells—One of the major signaling pathways by which TNF is known to work is by activating NFB signaling in the cell. In unstimulated cells, NFB resides in the cytosol in a latent form bound to IB. Stimulation of the cell with TNF triggers a series of signaling events that ultimately leads to the phosphorylation of IB by IKK, which then becomes a target of ubiquitin-proteosomal degradation. The phosphorylation and subsequent degradation of IB triggers the translocation of NFB complex (p65-p50) protein from the cytoplasm to the nucleus, where it regulates the expression of multiple genes. The finding that TNF induces SSAT expression and the fact that TNF functions predominantly via NFB signaling led us to determine the functionality of the NFB signaling in these NSCLC cells. Treatment of A549 cells with TNF produced a rapid reduction in the IB protein (Fig. 3A), with a concomitant rapid increase in nuclear translocation of the p65 protein in the A549 cells (Fig. 3B). Similar effects on IB and p65 protein were observed in the H157 cells (data not shown). Transient transfection experiments were performed with an NFB reporter construct to determine whether translocation of p65 into the cell nucleus was accompanied by a functional increase in the NFB-responsive genes. As shown in Fig. 3C, TNF treatment led to an increase in the luciferase activity from the NFB₂-luc plasmid in both A549 and H157 cells but had no effect on the control plasmid lacking NFB response elements, suggesting a functional NFB signaling mechanism in these NSCLC cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. TNF induces SSAT expression in NSCLC cells. A, A549 (black bars) and H157 (gray bars) cells were treated with TNF or its vehicle (V, open bar) for 15, 30, 60, and 180 min and harvested, and total RNA was extracted and used for quantitative reverse transcription-PCR. SSAT expression was normalized to the GAPDH expression in these treatments. -Fold induction was calculated by dividing normalized values of the treated sample by the normalized control. The results represent the mean of three different experiments ± S.D. *, a significance with p < 0.05. B, A549 and H157 cells were grown overnight and then treated with either vehicle (V, open bar) or 10 ng/ml TNF (black and gray bars) for 1 or 3 h. Cells were then harvested, and SSAT enzyme activity was measured as described under "Experimental Procedures." SSAT enzyme activity values are given as pmol/min/mg of protein. The result is the mean of three different experiments ± S.D. *, statistical significance of p < 0.05.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. TNF decreases intracellular polyamines in NSCLC cells. A549 and H157 cells were seeded and grown for 24 h, after which they were treated with either vehicle (white bars) or TNF (black and gray bars) for 1 h. Putrescine (A), spermidine (B), and spermine (C) were detected by high pressure liquid chromatography. The polyamine levels were normalized to the protein in the samples and plotted. The result is the mean of two different experiments performed in duplicate ± S.D.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. TNF activates NFB signaling in NSCLC cells. A549 cells were treated with vehicle (V) or TNF (T) for the indicated times. Total protein was extracted, and immunoblot analysis was performed for IB (A). Nuclear protein was also extracted for immunoblot analysis of p65 (B). Actin protein was used as a loading control. C, A549 and H157 cells were transfected with either the NFB2-Luc reporter construct or the control dNFB2-Luc reporter construct along with the -galactosidase plasmid. The cells were then treated for 1 h with either vehicle (open bar) or 10 ng/ml TNF (black and gray bars). Relative light units (RLU) were calculated after normalizing to the protein and -galactosidase activities in the cell lysates. Results representative of three experiments are expressed as -fold induction relative to the activity in the presence of vehicle alone. Normalized luciferase activities are shown as mean ± S.D. *, a statistical significance of p < 0.05.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Dominant negative IB suppresses SSAT mRNA induction by TNF in NSCLC cells. A, Western blot for IB overexpression was done using total protein extracted from stably transfected DN/IB-A549 (clones 1 and 2) and Ctrl/IB-A549 (clones 1c and 2c) cells. B, Western blot for p65 protein was done using nuclear extracts from DN/IB-A549 clones and Ctrl/IB-A549 clones treated with TNF for 1 h. The DN/IB-A549 (gray bars) and Ctrl/IB-A549 (black bars) cells were treated for 1 or 3 h with either vehicle (V, open bar) or 10 ng/ml TNF (C), and the DN/IB-H157 (gray bars) and Ctrl/IB-H157 (black bars) cells were treated for 30 or 60 min with either vehicle (open bar) or 10 ng/ml TNF (D). Total RNA was extracted and used for quantitative reverse transcription-PCR. SSAT expression was normalized to the GAPDH expression in these treatments. -Fold induction was calculated by dividing normalized values of the treated sample by the normalized control. The results represent the mean of three different experiments ± S.D. **, a significance with p < 0.05 between the DN/IB and Ctrl/IB cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Taxol induces SSAT by an NFB-dependent pathway in A549 cells. The DN/IB-A549 (gray bars) and Ctrl/IB-A549 (black bars) cells were treated for 24 h with either vehicle (V, open bar) or different concentrations of Taxol. Total RNA was extracted and used for quantitative real time-PCR. SSAT expression was normalized to the GAPDH expression in these treatments. Fold induction was calculated by dividing normalized values of the treated sample by the normalized control. The results represent the mean of two different experiments done in triplicate ± S.D. *, a statistical significance of p < 0.05 between the vehicle and the Taxol-treated Ctrl/IB-A549 cells; **, a statistical significance of p < 0.05 between the DN/IB and Ctrl/IB cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Various SSAT promoter reporter constructs. A, a schematic representation of SSAT promoter reporter constructs containing three putative NFB elements used in this study. B, A549 cells were transfected with the indicated SSAT deletion reporter constructs and treated for 1 h with either vehicle (V, open bar) or 10 ng/ml TNF (black bars). Relative light units (RLU) were calculated after normalizing to the protein and -galactosidase activities in the cell lysates. Results representative of three experiments are expressed as -fold induction relative to the activity in the presence of vehicle alone. Normalized luciferase activities are shown as mean ± S.D. *, a statistical significance of p < 0.05.

View larger version (71K): [in this window] [in a new window]   FIGURE 7. Increased binding of NFB complexes to the NFB-1 and NFB-3 sequences in the SSAT promoter. A, 30,000 cpm of the 32P-labeled oligonucleotides containing the NFB-1 wild-type (w) or mutant (m) sequence were incubated with 10 µg of A549 nuclear extract from cells treated with either water or TNF for 15 min, 30 min, and 1 h. The cold wild-type probes were used at a 100-fold molar excess for the competition reaction. The supershift control reaction was done with a p50 antibody and labeled probe. The shifted and the supershifted (SS) bands are labeled. B, 30,000 cpm of the 32P-labeled oligonucleotides containing the NFB wild-type (w3) or mutant (m3) sequence were incubated with 10 µg of A549 nuclear extract from cells treated with either water or TNF for 15 min, 30 min, and 1 h. The cold wild-type probes were used at a 100-fold molar excess for the competition reaction. The supershift control reaction was done with a p50 antibody and labeled probe. The shifted and the supershifted bands are labeled.

View larger version (60K): [in this window] [in a new window]   FIGURE 8. In vivo binding of p50 and p65 to the NFB sequences in SSAT promoter. A, chromatin immunoprecipitation assays were performed using A549 cells treated with either water or TNF for 15 min, 30 min, and 1 h. After treatment, cells were cross-linked and sonicated, and the DNA-protein complexes were incubated with either p50 or p65 antibody overnight. The immunoprecipitated DNA was analyzed for the binding of NFB proteins at the NFB-1 (A), NFB-2 (B), and NFB-3 (C) sites in the SSAT promoter by PCR. Input DNA (Inp) served as a positive control, whereas no antibody (No) and nonspecific antibody (IgG) served as negative controls.

View larger version (15K): [in this window] [in a new window]   FIGURE 9. Both NFB-1 and NFB-3, but not NFB-2 reporter sequences, modulate the SSAT promoter response to TNF. A549 cells were transfected with either Full-SSAT-luc, mNFB1-SSAT-luc, mNFB3-SSAT-luc, or dmNFB1/3-SSAT-luc reporter construct along with the -galactosidase plasmid. The cells were then treated for 1 h with either vehicle (open bar) or 10 ng/ml TNF (black bars). Relative light units (RLU) were calculated after normalizing to the protein and -galactosidase activities in the cell lysates. Representative results of three experiments are expressed as -fold induction relative to the activity in the presence of vehicle alone. Normalized luciferase activities are shown as mean ± S.D. *, a statistical significance of p < 0.05 as compared with the Full-SSAT-luc promoter; **, a statistical significance of p < 0.001 between the mNFB1-SSAT-luc and mNFB3-SSAT-luc promoter constructs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TNF is a potent pleiotropic proinflammatory cytokine produced by many different cells in response to infection and inflammatory stress. TNF has been shown to have paradoxical roles in the evolution and treatment of malignant disease (41, 42). Depending on the source, cell type, and level of TNF produced, it can lead to either cell death or cell survival. Activation of NFB has been associated with inflammation, increased cellular proliferation, and decreased programmed cell death, but there is also evidence for the role of NFB activation in leading to cell death and apoptosis (43, 44). The exact action of the NFB activation in a cell depends on the cell type, the type of activating stimuli, and the ratio of different genes induced. Aspirin, a nonsteroidal anti-inflammatory drug, can lead to the activation of NFB in Caco-2 cells, which leads to SSAT induction in these cells (34).

It is demonstrated here that SSAT is one of the genes regulated by TNF in non-small cell lung cancer cells. Treatment of two non-small cell lung cancer cell lines, A549 and H157, with TNF leads to a rapid increase in the SSAT mRNA expression and enzyme activity and to a decrease in the polyamine content. SSAT activity leads to increased acetylation of spermidine and spermine, which can be then be back-converted via the polyamine oxidase pathway or exported from the cells. SSAT has been implicated as a stress response gene whose expression can be induced to a substantial level by endogenous and environmental factors, including a variety of hormones and growth factors, toxic agents, and the polyamines themselves (1). SSAT overexpression has also been implicated in adaptive responses to environmental stress (9), hypoxia and nutrient depletion (13), polyamine analogues (8, 10–12, 46), and exposure to nonsteroidal anti-inflammatory drugs (16, 48). Our data suggest that induction of SSAT by TNF may provide a mechanism by which cells respond to inflammatory stress by reducing intracellular polyamines and slowing growth.

Superinduction of SSAT by specific agents has been shown to be associated with anti-tumor activity in several important human solid tumor models (12, 49), with the NSCLCs typically responding to the polyamine analogue treatment with a significant induction of SSAT and subsequent cell death (8, 11, 50, 51). In specific instances, the superinduction of SSAT results in production H₂O₂ from the constitutive activity of the acetylpolyamine oxidase, polyamine oxidase. The H₂O₂ thus produced can lead to DNA damage and ultimately cell death (52), suggesting another mechanism by which TNF can lead to apoptosis in lung cancer cells.

Interestingly, it has been proposed that polyamines can directly modulate NFB binding in various cell types. Currently, the data are conflicting as to the precise role of polyamines in regulating NFB activation and DNA binding (53–57). Our results suggest that a stress stimulus, like TNF, leads to a rapid induction in SSAT expression via NFB activation, leading to a reduction in polyamine content. It is possible that this rapid polyamine depletion inhibits NFB DNA binding activity, thereby directly providing feedback regulation of SSAT expression. More experiments will be necessary to determine whether such a feedback mechanism exists in NSCLC cells.

The molecular mechanisms responsible for the multiple biological activities of TNF are due to their ability to activate multiple signal transduction pathways, including those involving extracellular signal-regulated kinase, other mitogen-activated protein kinases, and NFB (21, 22). In the systems reported here, the data are most consistent with TNF acting through NFB. Exposure of the NSCLC lines to TNF reduces IB protein, increases nuclear p65 protein, and induces the expression of reporter constructs containing specific NFB response elements. Additional evidence that TNF is acting through NFB is provided by the observed significant reduction in the ability of TNF to induce SSAT in the DN/IB-A549 and DN/IB-H157 cells. It should be noted, however, that the expression of the dominant negative IB does not completely block SSAT induction by TNF in DN/IB-A549 cells, suggesting that TNF may also function through NFB-independent signaling pathways (58–62).

Taxol has been shown to activate NFB signaling by increasing the phosphorylation of IB in various cell types. This activation of NFB has also been associated with resistance to Taxol in these cells (35, 38). Taxol was used here as another NFB activator to study the role of NFB activation in the induction of SSAT in these cells, since Taxol has been specifically demonstrated to activate NFB in the A549 cells (40). We found that Taxol treatment produced an approximately 50% decrease in the IB protein, thereby leading to the activation of NFB and induced SSAT expression in A549 cells. Overexpression of DN/IB significantly attenuated the induction of SSAT observed in the Ctrl/IB-A549 cells. Although Taxol treatment does not result in complete degradation of IB, the suppression of SSAT induction in the presence of DN/IB indicates a central role for NFB activation in the induction of SSAT in response to Taxol.

NFB complexes are present as homo- or heterodimers of p50 and p65 proteins, which can then bind to specific NFB response elements in NFB-regulated genes. Three NFB response elements in the SSAT promoter have previously been identified (34). The results presented here from electrophoretic mobility shift assays, reporter constructs, and ChIP experiments indicate that two sites, NFB-1 at –286 and NFB-3 at –1735, but not NFB-2 at –594, are responsible for the TNF response. Mutating either the NFB-1 or NFB-3 response elements decreased the induction of SSAT reporter constructs by TNF, and mutations in both of the NFB response elements were required to completely abolish the induction of SSAT reporter constructs by TNF in A549 cells. Further, constructs having mutant NFB-1 but wild type NFB-3 showed higher SSAT promoter activity after TNF treatment than the constructs having mutant NFB-3 but wild type NFB-1 sequences. These data suggest that NFB-3 may play a greater role than NFB-1 in regulation of SSAT by TNF in A549 cells. This preference for NFB-3 over the NFB-1 could be due to the 5-base difference between the two response elements or due to the different contacts made by the transcriptional activation complex because of the 1.5-kb distance between them. Further experiments will be required to characterize the exact role of NFB-3 and NFB-1 in the actions of TNF on the SSAT promoter.

It is important to note that the chromatin immunoprecipitation experiments were particularly informative, demonstrating that p50 was bound to the NFB-1 and NFB-2 sequences with or without TNF treatment. Of the NFB/Rel proteins, only p65 and c-Rel have potent transcriptional activation domains, whereas p50 protein lacks these domains. Hence it is thought that the binding of p50 homodimers functions as a transcriptional repressor (47, 63, 64), as compared with the p50-p65 heterodimers, which can function as transcriptional activators. The data presented here suggest that in the basal uninduced state, NFB-1 and NFB-2 are bound by p50 homodimers, thereby keeping the SSAT expression level low, but after TNF treatment (or stress stimuli), there is a change in the promoter-bound NFB complexes such that the p50 homodimers are replaced by p50-p65 heterodimers at the NFB-1 site and a recruitment of p50-p65 heterodimers at the NFB-3 site, which together lead to the induction of SSAT expression.

The discovery that the expression of SSAT is regulated by a common mediator of inflammation, TNF, is highly significant both in the context of understanding the regulation of polyamine homeostasis and in understanding tumor cell responsiveness to various anti-tumor strategies. Depending on the extent, the induction of SSAT has been linked to both growth inhibition and cell death (10, 11, 46). Thus, TNF stimulation of SSAT may, in some cases, allow cells to reduce their growth rate when exposed to a stressful environment, and the observed cytotoxic drug responses may be an exaggerated extension of this normal response. Although more studies will be necessary to test this possibility, it is intriguing to consider the potential cross-talk that may occur between the polyamine response element (7, 45), which is thought to mediate SSAT induction produced by cytotoxic polyamine analogues, and NFB and how such cross-talk might be exploited for therapeutic advantage.

	FOOTNOTES

 
^* The research was supported by National Institutes of Health Grants CA 51085 and CA 98454. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University School of Medicine, Baltimore, MD 21231. Tel.: 410-955-8580; Fax: 410-614-9884; E-mail: rcasero{at}jhmi.edu.

² The abbreviations used are: SSAT, spermidine/spermine N¹-acetyltransferase; TNF, tumor necrosis factor ; NSCLC, non-small cell lung cancer; NFB, nuclear factor B; IB, inhibitor B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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