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(The FASEB Journal. 1998;12:1713-1723.)
© 1998 FASEB

RESEARCH COMMUNICATION

Inhibition of AP-1 and NF-{kappa}B by manganese-containing superoxide dismutase in human breast cancer cells

Jian-Jian Lia,1, Larry W. Oberleyb, Ming Fan2,b and Nancy H. Colburna

a Gene Regulation Section, Laboratory of Biochemical Physiology, National Cancer Institute, Frederick, Maryland 21702–1201; USA
b Radiation Research Laboratory, The University of Iowa, Iowa City, Iowa 52242, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the primary antioxidant enzymes, manganese-containing superoxide dismutase (MnSOD), has shown the ability to reverse malignant phenotypes in a variety of human tumor cells that are low or absent in MnSOD expression. We have observed that overexpression of human MnSOD in human breast cancer MCF-7 cells inhibits tumor growth both in vitro and in vivo. The signaling pathway underlying the MnSOD induced tumor suppression is unknown. We demonstrate here that transcriptional and DNA binding ability of AP-1 and NF-{kappa}B, but not SP-1, were inhibited (by 50%) in the MCF-7 cell line overexpressing MnSOD. When transiently expressing, MnSOD inhibited AP-1 but increased NF-{kappa}B transactivation, which can be abolished by sodium pyruvate, a hydrogen peroxide scavenger. To analyze the target genes responsible for MnSOD-induced tumor suppression, genes related to tumor growth and responsive to AP-1 or NF-{kappa}B were analyzed. AP-1 responsive collagenase I, stromelysin I, and NF-{kappa}B responsive IL-1 and IL-6 were down-regulated in the MnSOD stable transfectants compared to the control cell lines. Since TPA induces differentiation in human breast cancer cells and up-regulates MnSOD gene in HeLa cells, MnSOD expression and AP-1 and NF-{kappa}B activity were measured under TPA treatment. The results showed that TPA induced endogenous MnSOD expression and inhibited both AP-1 and NF-{kappa}B. Together, these results suggest that tumor suppression by overexpressing MnSOD is related to a modulation of AP-1 and NF-{kappa}B, which causes a down-regulation of genes responsible for tumor malignant phenotype.—Li, J.-J., Oberley, L. W., Fan, M., Colburn. N. H. Inhibition of AP-1 and NF-{kappa}B by manganese-containing superoxide dismutase in human breast cancer cells. FASEB J. 12, 1713–1723 (1998)


Key Words: tumor suppressor gene • redox • transcription factor • MCF-7 cells • TPA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
101REACTIVE OXYGEN SPECIES (ROS)3 are generated endogenously by all aerobic cells because of the metabolism of oxygen. ROS at high concentrations exert harmful effects on living organisms including damage to DNA and cell membranes. ROS at low concentrations perform essential metabolic functions in regulating signal transduction pathways and transcription factors. Intensive investigations have revealed possible effects of ROS on tumor initiation and transformation (1–6). ROS, such as hydrogen peroxide and superoxide, induce the expression of proto-oncogenes including c-fos, c-myc, and c-jun (7–10). UV light, which increases ROS production and induces oxidant stress, increases DNA binding activity of AP-1 in HeLa cells (9). H2O2 (hydrogen peroxide) induces NF-{kappa}B translocation from the cytoplasm into the nucleus and HIV-LTR transactivation (11) and increases the expression of c-fos, c-jun, and erg-1 genes (10). Subunits of AP-1 and NF-{kappa}B are able to cross talk (12) and both factors may play a role in cell transformation (13). Recent data indicate that both AP-1 and NF-{kappa}B are involved in tumor promoter induced progression in the JB6 mouse skin and human keratinocyte transformation models (14, 15). In the JB6 mouse skin cell model, ROS, especially superoxide, is involved in signaling the transformation response (16).

Superoxide dismutase (SOD) is one of the primary antioxidant enzymes. In eukaryotic cells, there are two main forms of intracellular SOD: manganese-containing superoxide dismutase (MnSOD), and copper- and zinc-containing superoxide dismutase (CuZnSOD). MnSOD is located in the mitochondrial matrix, whereas CuZnSOD is found primarily in the cytoplasm. Both forms of SOD convert superoxide radical to hydrogen peroxide, which is further metabolized to water by the other two primary antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GPX). Several lines of evidence indicate a relationship between SOD and malignant transformation. First, transformation is often accompanied by lowered MnSOD and CuZnSOD activity (1, 3). Second, increased cellular ROS have been shown to be associated with carcinogenesis, whereas antioxidants prevent malignant transformation both in vitro and in vivo (6, 17). Last, several techniques have been used to elevate SOD in malignant cells in culture and all techniques led to a reversion of the malignant phenotype. The techniques are 1) elevation by exposure to a superoxide generator and subsequent isolation of superoxide-resistant cells (18); 2) addition of liposomal CuZnSOD (19); and 3) elevation of MnSOD by sense cDNA transfection (20–22). In MnSOD gene transfection studies, increased expression of human MnSOD cDNA was shown to suppress radiation-induced neoplastic transformation (5), enhance cell differentiation in mouse fibroblasts (23), and inhibit anchorage-independent growth in vitro and tumor growth in vivo (20, 21). Our previous data showed that overexpression of the human MnSOD gene driven by the human ß-actin promoter in MCF-7 cells suppressed the malignant phenotype and inhibited cell proliferation both in vitro and in vivo (21). These findings suggest that MnSOD functions as a tumor suppressor gene in human breast cancer cells. The underlying signaling pathway of the tumor suppressive effects of MnSOD needs to be elucidated. We hypothesized that MnSOD overexpression leads to changes in ROS levels; ROS in turn modulate the transcription factor activity that controls the target genes responsible for tumor phenotype. Since many transcription factors have already been shown to be modulated by ROS, overexpression of MnSOD should affect transcription factor activity. This hypothesis is logical since overexpression of CAT (24, 25) and GPX (26) by cDNA transfection has already been shown to inhibit NF-{kappa}B activation, whereas overexpression of CuZnSOD has been demonstrated to enhance activation of the above transcription factor by tumor necrosis factor (24, 25); all these papers suggest that hydrogen peroxide is the relevant messenger in the activation pathway. Very recently, Amstad et al. (27) demonstrated that MnSOD overexpression JB6 clone 41 mouse epidermal cells inhibited growth rate and the ability to form colonies in soft agar. This clone is sensitive to the promotion of neoplastic transformation by 12-O- tetradecanoylphorbol-13-acetate (TPA). Since the transformation-sensitive phenotype of these cells is associated with increased expression of the transcription factor AP-1, c-jun and c-fos mRNA expression was compared in parental and MnSOD-transfected cells. Overexpression of MnSOD led to a significant decrease in c-fos and c-jun expression in response to TPA or the oxidant superoxide.

The phorbol ester TPA acts as a tumor promoter for preneoplastic cells but exerts a range of inhibitory effects on tumor cells, including reduced cell proliferation and enhanced differentiation (28, 29). Evidence supports that TPA-induced cell differentiation of human breast cancer cells is related to the decrease of estrogen receptor binding activity and down-regulation of gene expression after TPA treatment (30, 31). TPA also causes MnSOD induction in HeLa cells (32).

To understand the molecular mechanism of MnSOD-induced tumor suppression, we analyzed transcription factor AP-1 and NF-{kappa}B and their regulated genes in the MnSOD transfectants and in TPA-treated MCF-7 cells. The results show that activity of transcription factors AP-1 and NF-{kappa}B was inhibited and the target genes were down-regulated in the MnSOD transfectants. TPA up-regulated endogenous MnSOD gene expression and inhibited AP-1 and NF-{kappa}B transactivation. These results suggest that MnSOD-induced tumor phenotype reversion is related to an inhibition of AP-1 and NF-{kappa}B activity and the target gene down-regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fetal bovine serum was purchased from BioWhittaker (Walkersville, Md.); TPA was from Alexis Biochemicals (San Diego, Calif.); [32P]ATP and IEC Western detection kit were purchased from Amersham International (Princeton, N.J.). Nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Pierce (Iselin, N.J.); bovine serum albumin, bovine insulin, goat anti-rabbit IgG [F(ab')2 fragment] antibody conjugated with alkaline phosphatase, and methotrexate were purchased from Sigma Chemical (St. Louis, Mo.). Acrylamide and bisacrylamide and riboflavin were purchased from Bio-Rad Laboratories (Hercules, Calif.); improved minimum Eagle's medium (IMEM) was from Biofluids, Inc. (Rockville, Md.). Nonessential amino acid, sodium pyruvate, IPTI-MEM, and LipofectAMINE were from Gibco BRL Life Technologies (Gaithersburg, Md.). Rabbit polyclonal antibodies against human kidney MnSOD and human placental CuZnSOD were described earlier (33, 34).

Plasmids
A sequence of the collagenase promoter region (-73–+67 containing one AP-1 binding site TGAGTCA) was excised from the collagenase-AP-1 CAT construct and inserted into a luciferase reporter vector pGl2-basic (Promega, Madison, Wis.) to make the AP-1 luciferase reporter construct (35, 36). CMV-neo-selecting plasmid (pBKCMV) was obtained from Invitrogen (San Diego, Calif.). pHIV-luciferase reporter was made by inserting a 196 bp fragment of the long-terminal repeat of human immunodeficiency virus type I (HIV-I), which contains two NF-{kappa}B binding sites, into the pGL2 luciferase reporter (37). The human MnSOD expression vector, pBKSOD, was constructed by inserting human MnSOD cDNA into the multiclonal site of a mammalian cell expression vector pBKCMV (Stratagene, La Jolla, Calif.). The pBKSOD vector was successfully expressed in MCF-7 cells by transient transfection. The pHßApr-1 MnSOD vector driven by human ß-actin promoter was a kind gift from Dr. St. Clair at the University of Kentucky, and was used to establish the stable MnSOD-expressing MCF-7 cell line (21).

Cells
A MnSOD overexpressing cell line used for these experiments has been described (21). Wild-type MCF-7 cells and the vector control lines were cultured in the IMEM containing sodium pyruvate (1 mM), bovine insulin (10 mg/ ml), and 10% heat-inactivated fetal bovine serum as described before. The stable human MnSOD transfected cell line was grown in the same IMEM medium with 20% fetal bovine serum and 10 mM sodium pyruvate. All cell lines were maintained at approximately the same passage number when used to measure transcription factors.

Transfection and luciferase reporter assay
For AP-1, NF-{kappa}B, or SP-1 luciferase reporter transfection, MCF-7, MCF + MTX, or MCF + SOD cell lines were cultured in 12-well tissue culture dishes for 24 h and transfected with 2–4 µg AP-1 or NF-{kappa}B luciferase reporter and 0.75 µg of ß-galactosidase vector using LipofectAMINE. For the study of transient overexpression, 0.45 ml reduced serum medium OPTI (Life Technologies) containing 4 µg luciferase reporter, 0.75 µg ß-galactosidase vector, various amounts of pBKSOD plasmids, and 2 µl of LipofectAMINE was used for transfection. An equal amount of cDNA per well was obtained by adding the blank pBKCMV vector. Transfection was stopped by adding 0.5 ml complete medium 36 h after transfection. To measure the luciferase activity, 100 µl of whole cell lysate was mixed with an equal volume of the luciferase assay reagent (Promega) and luciferase activity was measured by a luminometer (Monolight 2010, Analytical Luminescence Laboratory, San Diego, Calif.) for 10 s. The luciferase activity was normalized by ß-galactosidase activity measured with 15–30 µl of the same cell lysate.

Electrophoretic mobility shift assay (EMSA)
An AP-1 binding sequence from the human collagenase promoter region, 5'-AGCATGAGTCAGACACCTCTGGC-3' [human (h)-collagenase AP-1, position from -73 to -54], NF-{kappa}B binding sequence 5'-TACAAGGGACTTTCCGCTGG-GGACTTTCCAG-3' from HIV-LTR (position from -110 to -80), and SP-1 binding sequence 5'-ATT CGA TCG GGG CGG GGC GAG C-3' (Promega) were synthesized and labeled with 32P-ATP using the end-labeling method (Life Technologies). For competition controls, the AP-1 (5'-CGCTTGATGAGTCAGCCGGAA-3') and NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3') gel shift oligonucleotides were also used in some binding reactions (Promega). Three micrograms of nuclear protein was added to the DNA binding buffer with 5 x 104 cpm 32P-labeled oligonucleotide probe, 1.5 µg poly dI.dC, and 3 µg bovine serum albumin. The reaction mixture was incubated on ice for 10 min, followed by incubation at room temperature for 20 min. The DNA–protein complexes were resolved in a 6% nondenaturing acrylamide gel and separated by electrophoresis at room temperature for 1.5 to 2 h. The gel was dried and exposed to Kodak X-OMAT film at -70°C overnight. The radioactivity of gel shift bands was measured for quantitation.

Western blot
Western blot was performed using 10–15 µg protein and rabbit antiserum to human kidney MnSOD or human placental CuZnSOD, as characterized earlier (34, 38), and alkaline phosphatase-linked second antibody. Staining was carried in alkaline phosphatase reaction solution (Pierce, Rockford, Ill.) with 5-bromo-4-chloro-3-indolyl phosphate and NBT for 1–5 min. Alternatively, the protein blot was washed with phosphate-buffered saline, incubated with horseradish-peroxidase-linked antibody, and visualized with the ECL kit (Amersham, Arlington Heights, Ill.). For quantitation, the Western blots were subjected to densitometry scanning analysis.

SOD activity gels
Cells of 100 mm tissue culture dishes were washed in 10 ml of phosphate-buffered saline and harvested by scraping with a sterile rubber policeman and resuspended in phosphate-buffered solution. The cell suspension was sonicated on ice and SOD activity was visualized in SOD activity gels (39). Briefly, protein samples were loaded onto 12.5% polyacrylamide gel with 5% stacking gel after preelectrophoresis for 1 h. After electrophoresis at 4°C for 2 h, the gel was stained in solution containing sodium cyanide to visualize only the MnSOD bands.

Oligonucleotide primers
The following oligonucleotide primers were used for this study—primers for human MnSOD cDNA: 21-mer, 5'-GGCATCAGCCGGTAGCACCAG-3', located at the 5' noncoding region and 23-mer, 5'-CTGCAGTACTCTATACC-ACTACA-3', located at the 3' noncoding region (21, 40); primers for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Gene Bank M17851): 5'-ATCCCATCACCATCTTCCAG-3' (20-mer, sense, located at 248–268 base); 5'-GCCATCACGCCACAGTTTCC-3' (20-mer, antisense, located 610–630). For amplification of AP-1 and NF-{kappa}B target gene, human collagenase I, stromelysin I, interleukin 1 (IL-1), and IL-6 expression were detected using the following primers—collagenase I (41): 5'-ATT GGA GCA GCA AGA GGC TGG GA-3' [sense] and 5'-TTC CAG GTA TTT CTG GAC TAA GT; stromelysin I (41): GCA TAG AGA CAA CAT AGA GCT-3' [sense] and 5'-TTC TAG ATA TTT CTG AAC AAG-3'; IL-1 (42): GTC TCT GAA TCA GAA ATC CTT CTA TC-3' [sense] and 5'-CAT GTC AAA TTT CAC TGC TTC ATC C-3' [antisense]; IL-6 (43): GTA GCC GCC CCA CAC AGA CAG CC-3' [sense] and GCC ATC TTT GGA AGG TTC AGG-3' [antisense].

RNA preparation and RT-PCR
Expression of exogenous and endogenous MnSOD in MCF-7 cells was determined by the reverse-transcriptase polymerase chain reaction (RT-PCR) method as described earlier (40). Two micrograms of RNA purified from different MCF-7 cell lines and treated by TPA was used for reverse transcription. PCR was performed with primers for MnSOD or different target genes of AP-1 and NF-{kappa}B under optimized running conditions. PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. For radioactive quantitation, dCTP was replaced by 1.25 µCi 32P-dCTP in each reaction and product radioactivity was measured by a radioactivity detector at 0 ~ 30 cycles.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transrepression of AP-1 and NF-{kappa}B in stable MnSOD transfectants
Subunits of AP-1 and NF-{kappa}B cross talk (12), and both factors may be responsible for signaling tumor promoter induced transformation (14, 15, 36). We have demonstrated that overexpression of MnSOD in the human breast cancer MCF-7 cell line caused phenotypic changes in cell culture and diminished tumor growth in nude mice (21). Thus, it is logical to ask whether MnSOD-induced tumor suppression is related to an alteration in these two transcription factors. In the present study it was found that transcriptional activity of AP-1 and NF-{kappa}B, but not control transcription factor SP-1 ( Fig. 1A–C), was inhibited in the MCF-7 cell line overexpressing MnSOD, which showed a 5.7-fold increase of MnSOD activity compared to the control cell lines (21). Transactivation of AP-1 and NF-{kappa}B determined by luciferase reporters was 52% and 45%, respectively, compared to the wild-type MCF-7 cells ( Fig. 1A, B). DNA binding activity of AP-1 and NF-{kappa}B but not SP-1 was also reduced in the MnSOD stable transfectants ( Fig. 1A–C, lower panel).



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  		Figure 1. Inhibition of AP-1 and NF-{kappa}B activity in MnSOD stable transfectants. Twenty thousand cells of wild-type (MCF-7), vector control (Vector), or MnSOD transfected (MnSOD) cell lines were cotransfected with collagenase AP-1-luciferase (A), NF-{kappa}B-luciferase (B), or SP-1-luciferase (C) and ß-galactosidase reporters. Luciferase activity was measured using 100 µl whole cell lysate and 100 µl luciferase substrate. The value of luciferase activity was normalized by ß-galactosidase activity and the activity of transcription factors was standardized by the value of wild-type MCF-7 cells. Data are presented as mean ±SEM of three experiments. DNA binding ability of (lower panel) was conducted with 3 µg nuclear protein prepared from MCF-7 (MCF-7), vector control (Vector), or MnSOD transfected (MnSOD) cell lines with 5 x 104 cpm 32P-labeled collagenase AP-1 oligonucleotides. Binding specificity was measured by competition with 100-fold excess of nonradioactive oligonucleotides (lane C).

Expression of MnSOD in MCF-7 cells cotransfected with MnSOD cDNA and AP-1 or NF-{kappa}B luciferase reporter
To analyze the relationship between transcription factor activity and MnSOD expression, the optimal MnSOD expression after transfection with different amounts of cDNA in wild-type MCF-7 cells was determined. Figure 2 shows that both immunoreactive and functional MnSOD was proportional to the amount of cDNA transfected in MCF-7 cells detected by Western blotting and MnSOD activity gel ( Fig. 2A, B). The highest protein level was obtained by transfection with 4 µg MnSOD DNA, with a 8.9-fold increase according to densitometry measurement in Western blot. The increased MnSOD immunoreactive protein level was correlated with the increases in MnSOD activity demonstrated in a MnSOD activity gel ( Fig. 2B). To verify MnSOD cDNA expression in cotransfection, MnSOD was detected in cells after cotransfection of MnSOD cDNA with AP-1 or NF-{kappa}B reporter ( Fig. 2C). The transfected MnSOD expression was detected after cotransfection with luciferase reporter and MnSOD cDNA. As an internal control, the CuZnSOD immunoreactive protein did not change with or without expression of MnSOD, as measured by band densitometry ( Fig. 2C). The ratio of MnSOD to CuZnSOD was calculated and showed a maximum of fourfold increase in MnSOD protein level for either AP-1 or NF-{kappa}B reporters (0.82 when cotransfected with AP-1 and 0.89 with NF-{kappa}B reporter) compared to the ratio in cells transfected with reporter and control vector only ( Fig. 2C, lanes 1 and 2).




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  		Figure 2. MnSOD Expression in MCF-7 cells by transient transfection. A) MnSOD immunoreactive protein level. MCF-7 cells (1x105 cells) were transfected with different amounts of pBKSOD plasmid by the LipofectAMINE method. An equal amount of DNA per dish was achieved by adding pBKCMV vector DNA. Protein samples were prepared 36 h after transfection, and 10 µg protein from each dish was used for Western blotting. Lanes 1–7 contained cells transfected with 0 to 8 µg pBKSOD plasmid. Lane 8 contained partially purified human kidney MnSOD protein (30 ng) as a positive control. The quantitation was calculated by using the densitometry values. B) Increase of MnSOD activity detected by MnSOD activity gel. Two hundred micrograms of total cell protein from duplicate dishes of experiment A was separated by electrophoresis in native 12.5% polyacrylamide gel for 2–3 h at 4°C. After electrophoresis, the gel was stained in NBT solution containing sodium cyanide to visualize only the MnSOD. Lanes 1–3 contained cells transfected with 1, 2, 4 µg MnSOD cDNA; lane 4 contained cells transfected with 4 µg of pBKCMV plasmid as control. C) Increase of MnSOD but not CuZnSOD in MCF-7 cells after cotransfection with MnSOD cDNA and AP-1 or NF-{kappa}B luciferase reporter. To determine MnSOD expression in cotransfection with luciferase reporter, MCF-7 cells were cotransfected with 4 µg AP-1 or NF-{kappa}B luciferase reporters with 0–4 µg pBKSOD plasmid. A protein sample was prepared 36 h after transfection and Western blotting was performed using MnSOD and CuZnSOD antibodies. The radio of MnSOD to CuZnSOD was calculated by using the densitometry value.

Effect of transient MnSOD expression on AP-1 and NF-{kappa}B
Under the cotransfection conditions described above, luciferase reporter activity was measured to determine the effect of MnSOD expression on AP-1 and NF-{kappa}B transcriptional activity. Figure 3A shows that inhibition of AP-1 paralleled MnSOD expression and that the transcriptional activity of another transcription factor, SP-1, was not affected. There was no significant AP-1 inhibition observed when cells were cotransfected with 0.5 or 1.0 µg MnSOD cDNA, amounts that did not induce detectable increased MnSOD protein and activity ( Fig. 2). This result was in agreement with the AP-1 transrepression observed in the MnSOD stable transfectants ( Fig. 1A). Unexpectedly, transient expression of MnSOD produced the opposite effect on NF-{kappa}B activity. Figure 3A shows that NF-{kappa}B transactivation paralleled the amount of MnSOD DNA transfection. Cotransfection of NF-{kappa}B reporter with 1 µg MnSOD DNA, which showed little increase in MnSOD/CuZnSOD ratio ( Fig. 2C, lane 3) but caused significant NF-{kappa}B transactivation (2.5-fold) and cotransfection with 2 µg MnSOD DNA, induced a 3.6-fold NF-{kappa}B transactivation ( Fig. 3A). Since hydrogen peroxide has been reported to increase NF-{kappa}B transactivation (44, 45), it was speculated that the observed difference in NF-{kappa}B activity could be due to the levels of hydrogen peroxide. It was also found that the stable cell line overexpressing MnSOD must be cultured in 10 mM sodium pyruvate in order for the cells to grow (21). All the other cells were grown in 1 mM sodium pyruvate. Pyruvate has been shown to be a strong hydrogen peroxide scavenger; it reacts stoichiometrically with H2O2, and this reaction protects cells from peroxide toxicity (46). Therefore, we performed transient transfection in the presence of various concentration of pyruvate. As shown in Fig. 3B, if the transfection was done in 10 mM pyruvate, NF-{kappa}B was not induced and was in fact inhibited at the highest concentration of MnSOD cDNA, whereas pyruvate did not induce an effect on SP-1 and MnSOD induced AP-1 transrepression ( Fig. 3B). Thus, these studies show that if cells are grown in high concentrations of pyruvate (and hydrogen peroxide is therefore removed), then NF-{kappa}B transactivation is inhibited in both stable and transient experiments. Conversely, if the cells are grown in low concentrations of pyruvate, then NF-{kappa}B is greatly activated by MnSOD overexpression.



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  		Figure 3. Activation of NF-{kappa}B by transient expression of MnSOD and sodium pyruvate inhibited MnSOD-induced NF-{kappa}B activity. MCF-7 cells were cotransfected with 4 µg AP-1, NF-{kappa}B or SP-1 luciferase reporter, 0.75 µg of ß-galactosidase reporter, and the indicated amounts of pBKSOD cDNA. Cells were cultured for 12 h in medium with 1 mM sodium pyruvate and then with 1 mM (A) or 10 mM (B) sodium pyruvate for 24 h. Luciferase activity was measured with 100 µl cell lysate and normalized to the value of control cells without MnSOD transfection. Each set of data represents four experiments (mean ±SEM).

Down-regulation of AP-1 and NF-{kappa}B responsive genes in MnSOD stable transfectants
To determine the target genes in MnSOD-induced tumor repression, genes related to cell growth and regulated by AP-1 or NF-{kappa}B were analyzed. Expression of collagenase I and stromelysin I (responsive to AP-1) and of IL-1 and IL-6 (responsive to NF-{kappa}B) was determined in MnSOD stable transfectants. Figure 4 shows that the mRNA level of collagenase I was reduced and that of stromelysin I was absent in the MnSOD stable cell line compared to the control MCF-7 cells. Since these two matrix metalloproteinase genes are related to cell proliferation and tumor metastasis (47, 48), down-regulation of this group of genes may reflect an important antitumor function of MnSOD. IL-1 and IL-6 responsive to NF-{kappa}B transactivation after different stimulation were also absent in the MnSOD stable transfectants, whereas both were clearly detected in the wild-type and vector control MCF-7 cells ( Fig. 4). These results indicate that multigenic regulation responsive to either AP-1 or NF-{kappa}B or both is responsible for the cell phenotypic change after MnSOD overexpression.



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  		Figure 4. AP-1 and NF-{kappa}B responsive genes were down-regulated in MnSOD transfectants. Total RNA was prepared from MCF-7 (lane 2), vector control (lane 3), and MnSOD stable transfectants (lane 4); RT-PCR was performed with the indicated primers of AP-1 responsive genes collagenase I, stromelysin I, NF-{kappa}B responsive genes IL-1 and IL-6, and GAPDH as described in Materials and Methods. Lane 1 is a control containing the same reaction shown in lane 2 without reverse transcription.

Induction of endogenous MnSOD in wild-type MCF-7 cells by TPA
TPA inhibits proliferation (49, 50) and induces differentiation in MCF-7 cells in a dose-dependent pattern (28, 29). Significant morphological changes in MCF-7 cells were induced by TPA in a dose range of 15—150 nM (data not shown). To determine that TPA-induced MCF-7 cell differentiation is related to an induction of the MnSOD expression, we analyzed the endogenous MnSOD expression in MCF-7 cells treated by varied concentrations of TPA. The results show a dose-dependent pattern of MnSOD induction by TPA ( Fig. 5A, B). TPA at 100 nM produced a 4.5-fold MnSOD protein level similar to that in the MnSOD stable transfectants ( Fig. 5A, lane 2). These results indicate that up-regulation of the endogenous MnSOD gene, which is not mutated in MCF-7 cells (21, 40; unpublished observations), may contribute to the inhibitory effect on cell growth induced by TPA.



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  		Figure 5. TPA up-regulated endogenous MnSOD expression. A) The endogenous MnSOD transcripts were measured by RT-PCR in MCF-7 cells after TPA treatment using human MnSOD and GAPDH cDNA primers. B) Quantitation by radioactivity detector for labeled endogenous MnSOD transcripts as shown in panel A at 0 ~ 30 cycles. Data were normalized and presented as a percentage of the value of cells with 100 nM TPA at 30 cycles.

AP-1 and NF-{kappa}B transrepression induced by TPA
To determine that TPA induced endogenous MnSOD expression may also affect transcription factors, AP-1 and NF-{kappa}B transcriptional activity was measured in MCF-7 cells after TPA treatment (100 nM for 18 h). Transrepression of both AP-1 and NF-{kappa}B activity (60%) but not SP-1 was observed, with a sixfold increase in MnSOD protein level after TPA treatment ( Fig. 6A, B). In agreement with transrepression, DNA binding ability of AP-1 and NF-{kappa}B, but not SP-1, was reduced ( Fig. 6C). These results indicate that AP-1 and NF-{kappa}B transrepression is induced by TPA, which may be a downstream event from TPA-induced MnSOD expression and account for signaling cell differentiation. It was also observed that TPA further decreased AP-1 and NF-{kappa}B activity in the MnSOD-overexpressing cell line (data not shown), indicating that a synergistic effect of MnSOD and TPA in modulation of transcription factor activity may exist.



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  		Figure 6. TPA increased endogenous MnSOD protein and inhibited AP-1 and NF-{kappa}B activity. A) MCF-7 cells (1x105) were transfected with AP-1, NF-{kappa}B, or SP-1 reporters, and luciferase activity was measured after treatment with TPA for 18 h. B) Western blotting of MnSOD and CuZnSOD was performed with or without TPA treatment as described in panel A. C) Gel shifting with 5 x 104 cpm 32P-labeled AP-1, NF-{kappa}B, or SP-1 oligonucleotides and 3 µg nuclear protein from MCF-7 cells with or without TPA treatment. Lane C contained the same reaction as in lane 1 competed with 100% access of nonradioactive oligonucleotides.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The signaling pathway underlying MnSOD-induced tumor suppression needs to be elucidated. The present study demonstrated that both AP-1 and NF-{kappa}B activity is inhibited and genes responsive to AP-1 and NF-{kappa}B were down-regulated in the stable MnSOD overexpressing cell line. Modulation of NF-{kappa}B activity was likely related to the hydrogen peroxide concentration produced in MnSOD transfected cells. TPA functions as an inducer of differentiation in MCF-7 cells, up-regulates MnSOD expression, and induces AP-1 and NF-{kappa}B transrepression. These results indicate an important function of MnSOD in signaling cell phenotypic changes in human cancer cells.

Signal transduction via protein kinase C (PKC) is known to influence gene transcription rates by modulation of the activity of a variety of transcription factors, including AP-1 and NF-{kappa}B (51). We chose these two transcription factors in our present study because 1) both AP-1 and NF-{kappa}B are regulated via phosphorylation/dephosphorylation processes (52), which are activated by PKC activators such as TPA, and antioxidants suppress the induced NF-{kappa}B transactivation (53); 2) subunits of AP-1 and NF-{kappa}B cross talk and induce mutual transactivation (12); 3) AP-1 (35, 54) or NF-{kappa}B (55, 56), or both (13–15), are indicated in signaling neoplastic transformation; 4) MnSOD is involved in metabolism of ROS (superoxide to hydrogen peroxide), and both superoxide and hydrogen peroxide affect AP-1 and NF-{kappa}B activity; 5) both AP-1 and NF-{kappa}B binding sequences are found in the human MnSOD gene promoter (57–59). The present results indicate that long-term overexpression of human MnSOD inhibits both AP-1 and NF-{kappa}B activity, which may account for the suppression of the malignant phenotype. One might argue that the changes seen in these experiments were not large (around 50% inhibition); however, the changes are of the same order of magnitude of those seen in reduction of in vitro cell growth [50% (21, 22)]. This result indicates that inhibition at this level induced by expressing MnSOD is efficient in suppressing tumor malignant phenotype, and higher inhibition on both transcription factors may be lethal to cells that can be screened out during cell cloning. Supporting this result, data also indicate a down-regulation of AP-1 and NF-{kappa}B target genes in the MnSOD stable transfectants. Transcription of collagenase I and stromelysin I was low or absent in MnSOD stable cell line compared to the control MCF-7 cells. Since these matrix metalloproteinase genes are related not only to tumor metastasis but also tumor proliferation (47, 48), down-regulation of this group of genes in addition to others responsive to NF-{kappa}B may reflect an important antitumor function of MnSOD. These results indicate that regulation of multigene levels responsive to either AP-1 or NF-{kappa}B, or both, is responsible for the cell phenotypic changes after MnSOD overexpression.

Transiently expressing MnSOD in the present study induced an opposite response of AP-1 and NF-{kappa}B, which is in agreement with the reported result that AP-1 and NF-{kappa}B respond differently to ROS (H2O2) -induced oxidant stress and to the antioxidants PDTC (pyrrolidine dithiocarbamate) and NAC (N-acety-L-cysteine) (59). PDTC and NAC enhanced TPA-induced AP-1 activity but were ineffective in activating NF-{kappa}B binding activity. On the other hand, H2O2 strongly induces the NF-{kappa}B transactivation response to TPA, but the antioxidants PDTC and NAC suppress TPA-induced NF-{kappa}B transactivation (60). Many reported differences in the responsiveness of AP-1 and NF-{kappa}B may have been due to differences in the levels of superoxide and/or hydrogen peroxide scavengers in the tissue culture medium; in the present study, when hydrogen peroxide was removed by high levels of pyruvate, similar inhibitions of AP-1 and NF-{kappa}B transrepression were observed. The H2O2-induced NF-{kappa}B transactivation is likely specific since it occurs at low concentrations of H2O2 treatment and other DNA binding protein is not likely to be affected (11). The decreased NF-{kappa}B activity induced in the stable MnSOD transfectants is probably due to the higher concentration of pyruvate (10 mM) in cell culture medium needed for the growth of the MnSOD transfectants (21). Transfer of the wild-type MCF-7 cells to high concentrations of pyruvate (10 mM) and culture for up to 4 days did not induce an obvious increase of MnSOD protein level as measured by Western blotting (data not shown). This indicates that decreased AP-1 and NF-{kappa}B activity in the transient MnSOD transfectants is not due to an induction of MnSOD by pyruvate. However, pyruvate reacts with H2O2, producing the less toxic carbon dioxide and water (46, 61) and thus down-regulating the H2O2 level necessary for signaling for high NF-{kappa}B activity. This is supported by the data from transiently MnSOD expression, which induced NF-{kappa}B transactivation; pyruvate inhibited this activation. These results are also in agreement with the observation that NF-{kappa}B transactivation was inhibited in catalase and glutathione peroxidase transfectants, but induced by an increase in H2O2 levels and in CuZnSOD transfectants (24–26). It is not clear whether up-regulation of H2O2 or down-regulation of O2·- (superoxide radical) after MnSOD transfection is the key step for the change of signaling for AP-1 and/or NF-{kappa}B. However, our present data suggest that down-regulation of O2·- after MnSOD transfection is related to AP-1 transrepression whereas up-regulation of H2O2 is related to NF-{kappa}B transactivation.

While this manuscript was in preparation, two papers have appeared that support our observations. Kiningham and St. Clair (62) have shown that overexpression of MnSOD selectively modulates the activity of Jun-associated transcription factors in fibrosarcoma cells. MnSOD was overexpressed in a mouse fibrosarcoma cell line. EMSA indicated an inverse correlation between MnSOD activity and DNA binding ability of AP-1 and CREB. No change was seen in the DNA binding of NF-{kappa}B or p53. AP-1 activity measured with an AP-1-dependent Luc reporter was reduced by as much as 20-fold in a high MnSOD-overexpressing clone. Thus, the paper shows that transcription factor activity can be modulated by overexpression of MnSOD; it disagrees with the present work in that no changes in NF-{kappa}B were observed. This disagreement suggests a cell type specificity regarding which transcription factors are modulated. Another paper recently demonstrated that thioredoxin peroxidase also plays a regulatory role in NF-{kappa}B activation (63). Like glutathione peroxidase, thioredoxin peroxidase (TPX) removes hydrogen peroxide from cells. Overexpression of TPX led to inhibition of NF-{kappa}B DNA binding activity. Overexpression of TPX also inhibited tumor necrosis factor- and TPA-dependent intracellular activation of NF-{kappa}B (both DNA binding and transactivation). It was hypothesized that overexpression of TPX affects the phosphorylation of I{kappa}B-{alpha}, thus activating the p65 and p50 complex. Specificity was shown by the demonstration that SP-1, SRE, and HTLV-1 were not affected by TPX overexpression.

The effects of TPA are thought to be mediated by modulation of PKC activity (64), which is the upstream event for cell signaling of transcription activation regulated by transcription factors such as AP-1 and NF-{kappa}B. Although TPA acts as a tumor promoter in several in vivo and in vitro transformation models, in human breast cancer cells TPA inhibits proliferation and induces cell differentiation (28, 29). TPA was included in the present experiment to compare transcription factor activity in TPA-induced cell differentiation and MnSOD-induced phenotypic changes. Up-regulation of the endogenous MnSOD gene and both AP-1 and NF-{kappa}B transrepression were observed in the wild-type MCF-7 cells after TPA treatment. These results indicate that although TPA induced endogenous MnSOD expression, the underlying signaling pathway of NF-{kappa}B transrepression may be different from that observed in MnSOD transfection. A report (12) has indicated that the subunits of AP-1 and NF-{kappa}B cross talk and may lead to a mutual activation. As a result, NF-{kappa}B inhibition induced by TPA or by long-term expression of MnSOD could result at least in part from a reduced signaling cross from AP-1. Direct evidence of protein–protein interaction between AP-1 and NF-{kappa}B subunits such as cJun or cFos with p65 could be informative. We are currently working on the possible interactions between these two transcription factors and their roles in cell proliferation and neoplastic transformation. A recent report has also indicated that TPA-induced MCF-7 growth inhibition is related to Erk 2 MAP kinase activation and junB induction (65), which has shown low activity in DNA binding (66) and is a negative regulator in AP-1 transactivation (67). In disagreement with the present results, TPA induced TRE-related transactivation as detected by a synthetic 5xTRE reporter, whereas AP-1 transrepression was observed in the present experiment when using a reporter with one AP-1 binding site from the collagenase promoter region. It has also been indicated that JunB can transactivate only through multiple but not single TRE (67). Thus, the difference in AP-1 activity may come from the dominant AP-1 components, especially those that are JunB induced, and the characteristics of the reporter constructs used.

In conclusion, this study indicates a role for MnSOD in modulation of transcription factors AP-1 and NF-{kappa}B, which caused a down-regulation of the target genes responsible for the malignant tumor phenotypes. Thus, inhibition of AP-1 and NF-{kappa}B by reconstitution of MnSOD expression in tumor cells may provide a potential to study signal pathways related to transcription factor activity and tumor phenotype.


   ACKNOWLEDGMENTS
 
This work was supported by a National Cancer Institute grant (CA 41267) to Dr. L. W. Oberley at the University of Iowa. The authors would like to thank Dr. St. Clair of the University of Kentucky for a kind gift of the pHßApr-1MnSOD vector used to establish the stable MnSOD-expressing MCF-7 cell line. By acceptance of this article, the publisher or recipient acknowledges the right of the U. S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.


   FOOTNOTES
 
2 Present address: SAIC, NCI-FCRDC, Frederick, MD 21702, USA.

1 Correspondence: Gene Regulation Section, Laboratory of Biochemical Physiology, National Cancer Institute, Frederick, MD 21702–1201, USA. E-mail: lij{at}mail.ncifcrf.gov.

3 Abbreviations: CAT, catalase; CuZnSOD, copper- and zinc-containing superoxide dismutase; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPX, glutathione peroxidase; H2O2, hydrogen peroxide; interleukin, IL; IMEM, improved minimum Eagle's medium; MnSOD, manganese-containing superoxide dismutase; NAC, N-acety-L-cysteine; NBT, nitroblue tetrazolium; O2·-, superoxide radical; PDTC, pyrrolidine dithiocarbamate; PKC, protein kinase C; ROS, reactive oxygen species; RT-PCR, reverse-transcriptase polymerase chain reaction; TPA, 12-O- tetradecanoylphorbol-13-acetate; TRE, TPA response element; TPX, thioredoxin peroxidase.

Received for publication June 4, 1998. Revision received July 24, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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