Thioredoxin (Trx) is a small redox-active protein that provides reducing equivalents for key cysteine residues of proteins through thiol-disulfide exchange, such as the transcription factor nuclear factor-κB (NF-κB). NF-κB activation has been associated previously with cell growth and the inhibition of apoptosis. We have shown in earlier studies that overexpression of Trx in MCF-7 cells increases anchorage-independent growth. In this study, the activation of NF-κB was examined as a mechanism through which Trx overexpression might promote anchorage-independent growth. Constitutive NF-κB activity is elevated 4–7-fold in Trx-overexpressing cells. NF-κB activity was inhibited in these cells by expressing a dominant-negative mutant of the IκBα protein (IκBαM). Expression of IκBαM in Trx-overexpressing cells dramatically reduced the Trx-associated increase in NF-κB activity but did not affect anchorage-dependent or -independent growth. The results suggest that increased growth in MCF-7 cells overexpressing Trx is not mediated by increased activation of the transcription factor, NF-κB. Additionally, activator protein-1 (AP-1), another transcription factor associated with growth, was increased up to 10-fold in Trx-overexpressing cells. Thus, AP-1 activation might contribute to the growth-promoting effect of Trx.

NF-κB3 is a dimeric transcription factor complex that binds specifically to 10-bp κB sites on DNA (1, 2). NF-κB is comprised of two classes of DNA-binding proteins; class I (p50 and p52) does not have transactivation domains and is derived from cytoplasmic precursors that have to be processed to release the DNA-binding proteins; and class II [cRel, RelA (p65), and RelB], which contain transactivation domains. Homodimers of p50 act as inhibitors of NF-κB-mediated transactivation, whereas homodimers composed of p65 and c-Rel transactivate NF-κB. However, the most active NF-κB transactivating complexes are heterodimers comprised of class I and class II proteins. The term NF-κB generally refers to the heterodimer composed of p50 and RelA subunits (1). NF-κB activity is regulated in a number of ways. IκB proteins (IκBα, IκBβ, IκBγ, IκBε, Bcl-3, and IκBR) bind to both class I and II NF-κB proteins, masking a nuclear localization sequence, thus, sequestering the NF-κB complex in the cytoplasm. Phosphorylation of IκBα by a specific serine kinase, IKKα, leads to polyubiquitination of IκBα, targeting it for proteolytic degradation (3). This unmasks the nuclear localization sequence of NF-κB, allowing it to move to the nucleus and initiate transactivation. The redox state of NF-κB is also important for its activity. The Cys62 residue of the p50 subunit of NF-κB is redox-regulated and critical for its binding to DNA. Deletion of the Cys62 residue of p50 decreases both the specificity and affinity of DNA binding (4).

NF-κB plays a role in tumor development. v-Rel, a truncated and mutated form of avian c-Rel, is constitutively located in the nucleus of chicken fibroblasts and is highly oncogenic in birds (5). A naturally occurring mutant of p65 has been reported to transform rat embryo fibroblasts (6). Furthermore, the Tax protein of the leukemogenic virus HTLV-1 is an activator of NF-κB, and Tax-induced tumors in mice can be suppressed by antisense NF-κB constructs (7). NF-κB has been associated with inhibition of apoptosis, which could contribute to increased tumor growth. Treatment of fibroblasts and macrophages from RelA knockout mice (8) or treatment of different cell types with a dominant-negative inhibitor of IκBα (IκBαM; Ref. 9) greatly increases sensitivity of the cells to apoptosis induced by TNF-α and other agents.

Trxs are low molecular weight (Mr 10,000–12,000) redox proteins found in both prokaryotic and eukaryotic cells (10). The Cys residues at the conserved -Cys32-Gly-Pro-Cys35-Lys active site of human Trx undergo reversible oxidation-reduction, catalyzed by the NADPH-dependent flavoprotein, Trx reductase (11). Trx acts extracellularly as a growth promoter (12). Intracellularly, Trx can scavenge reactive oxygen species through the enzyme, Trx peroxidase (13). Trx also reduces key Cys residues on transcription factors including the glucocorticoid receptor (4, 14), NF-κB (15), and, indirectly through Ref-1/HAPE, activator protein-1 (AP-1, Fos/Jun heterodimer; Ref. 16).

Trx is found at high levels in a number of human primary tumors (17, 18). Many human primary lung and colon cancers exhibit increased expression of Trx mRNA compared with normal corresponding tissue from the same subjects (19, 20). Experimental evidence suggests that Trx can both stimulate cancer cell growth and inhibit apoptosis. We have shown that MCF-7 breast cancer cell lines that stably overexpress Trx exhibit increased clonogenic growth in soft agar and form tumors more rapidly in scid mice than empty vector transfected control cells (21). These cells do not overexpress Trx reductase. Additionally, MCF-7 cells expressing a redox-inactive form of Trx grow poorly in soft agar and fail to form tumors in scid mice. In mouse WEHI7.2 lymphoid cells, stable transfection and expression of human Trx inhibits apoptosis induced by a variety of agents including dexamethasone, etoposide, and thapsigargin (22).

The reported association between Trx and NF-κB prompted us to examine NF-κB activation in MCF-7 cells overexpressing Trx. We found that NF-κB is constitutively elevated in the Trx-overexpressing cells. Inhibition of NF-κB activation by stable expression of a transdominant-negative of IκBα (IκBαM; Ref. 9) dramatically reduced constitutive NF-κB activity. However, this resulted in only a small reduction in anchorage-dependent growth and no effect on anchorage-independent growth. Thus, the increased growth observed in Trx-overexpressing MCF-7 cells does not appear to be mediated by an increase in constitutive NF-κB activity.

Establishment of Cell Lines

Establishment of MCF-7 human breast cancer cell lines overexpressing Trx used in these studies have been described previously (21). The cell lines were maintained in DMEM containing 10% FBS under 6% CO2 at 37°C unless otherwise specified. An MCF-7 cell line overexpressing Trx (Trx9) and an empty vector (pDC304neo) transfected cell line (Neo3) were used to create double transfected cell lines. The transdominant-negative IκBαM cDNA (9) was obtained from Dr. Douglas Green (La Jolla, CA) and in conjunction with the empty vector (pCMXhygro) was used to produce stably transfected MCF-7 Trx9 and Neo3 cell lines. Briefly, the Neo3 and Trx9 MCF-7 cells were grown to 50% confluence in 100-mm culture plates. The medium was then aspirated and replaced with DMEM supplemented with 1% FBS. Ten μg of each plasmid DNA were mixed with the cationic lipid, N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate (Boehringer Mannheim, Indianapolis, IN), and mixed with the cells according to the manufacturer’s protocol. Clones were selected by limiting dilution in 400 units/ml hygromycin over 3–4 weeks and designated with subsequent numbering (e.g., Neo3/Hygro1). All studies were conducted on clonal cell lines. Expression of the IκBαM protein was confirmed in each clonal outgrowth by Western blot and incorporation of the empty vector was determined by PCR.

Western Blot

Whole-cell pellets (5 × 106 cells) were washed twice in PBS and then resuspended in 50 μl PBS and lysed by the addition of 50 μl of 60 mm Tris-base (pH 6.8), 2% SDS, 5.8 mm β-mercaptoethanol, and 20% glycerol. Lysates were boiled for 10 min and centrifuged at 12,800 × g for 5 min, and protein was quantified using the Coomassie protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein (25 μg) were separated by SDS-PAGE (5% stacker and 10% resolving) and electroblotted to polyvinylidene difluoride membranes. Blots were stained with Coomassie blue to confirm transfer and equal loading and then blocked in Tris-buffered saline with 0.05% Tween 20 (TBS-T) and 5% milk for 1 h at 22°C. The blots were incubated in fresh blocking solution and probed for 4 h with a 1:1000 dilution of anti-IκBα primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were washed three times for 5 min in TBS-T and then incubated with a 1:3000 dilution of peroxidase-conjugated secondary antibody (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) in TBS-T for 1 h at 22°C. Blots were again washed three times for 5 min in TBS-T and then developed by enhanced chemiluminescence (Pierce).

Transactivation Assays

NF-κB transactivation was measured using a double κB repeat attached to a CAT reporter in a pCMV transfection vector (pNFCAT) or the 197-bp TaqI/HindIII fragment of the HIV-1 long terminal repeat in the pGL2 luciferase vector (Promega Corp., Madison, WI; Ref. 23). AP-1 activation was measured using the collagenase promoter in the pGL2 luciferase vector (23). Each cell line was seeded in triplicate into 35-mm plates and allowed to attach overnight. The LipoTAXI transfection reagent (Stratagene, La Jolla, CA) was mixed with pNFCAT (3.7 μg/plate) or pGL2-collagenase (3.7 μg/plate) and pCMVβgal (1.7 μg/plate) in DMEM with 2% FBS and added to the cells for 24 h. Subsequently, the medium was replaced with DMEM alone, 5%, or 10% FBS for an additional 24 h before measuring CAT using a specific ELISA kit and protocol (Boehringer Mannheim). Luciferase activity was measured using 100 μg of cell protein with a luminometer, and transfection efficiency from both experiments was corrected by normalizing to β-galactosidase activity in the same cell lysates. Alternatively, pGL2-HIV (3 μg/plate) and pCMVβgal (1.7 μg/plate) were used to measure NF-κB activity. Cells were transfected using the LipoTAXI transfection reagent for 6 h in serum-free DMEM, at which time the FBS concentration was increased to 1 or 2.5% for an additional 18 h. Subsequently, the medium was replaced with fresh DMEM supplemented with 1 or 2.5% FBS for an additional 24 h before measuring luciferase activity. Cells were lysed in reporter lysis buffer according to the protocol provided by Promega. Luciferase activity was measured as described above.

Proliferation and Colony Formation

Anchorage-dependent Growth.

Each cell line was grown as a monolayer in DMEM supplemented with 1 or 2.5% FBS. Cells were plated in triplicate at 4 × 104 cells/well in a 12-well plate and allowed to grow for 72 h. Each well was trypsinized, and the cells were resuspended in a total volume of 1 ml and counted using a hemacytometer.

Anchorage-independent Growth.

Cell lines were seeded in triplicate in 0.3% agar in DMEM supplemented with 1 or 2.5% FBS at 500 cells/ml (1 ml/well) and incubated 7–10 days. Five hundred μl of INT stain [1 mg/ml 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride hydrate; Aldrich, Milwaukee, WI] was then added to each well and incubated over night. Colonies containing more than ∼50 cells were counted manually with a dissecting scope.

Anchorage-independent Growth in Low Serum.

Anchorage-independent growth was assessed by colony formation in 1, 2.5, and 5% serum. After 10 days of growth, colonies formed by the Trx-overexpressing MCF-7 cells (Trx9, Trx12, and Trx20) were more numerous and larger in size compared with wild-type and control MCF-7 (Neo3) cells (Fig. 1,A). These three Trx clones overexpressed Trx message by 0.4-, 0.5-, and 0.8-fold, respectively (21). When quantitated and normalized to the number of control colonies, the Trx-overexpressing cells formed from 1.6- to 2.2-fold more colonies (Fig. 1 B).

NF-κB Transactivation: Trx- and Redox-inactive, Trx-expressing MCF-7 Cells.

Constitutive levels of NF-κB activity were increased 4–7-fold in the Trx-overexpressing clones (Trx9 and Trx12) compared with the empty vector cells (Neo3; Fig. 2). There was no difference between wild-type MCF-7 cells and the Neo3 cells. These observations lead us to examine a potential relationship between increased NF-κB activity and increased anchorage-independent growth in the Trx-overexpressing MCF-7 cells.

IκBαM Expression.

NF-κB activation was inhibited by expressing a mutant protein (IκBαM) that sequesters NF-κB in the cytosol (9). IκBαM and its cognate empty vector (pCMXhygro) were stably transfected into MCF-7 cells containing either the pDC304neo empty vector (Neo3) or pDC304-Trx (Trx9). Incorporation of the empty vector was confirmed through PCR by amplifying a fragment of the hygromycin resistance gene from total cellular DNA (not shown). Expression of IκBαM was analyzed by Western blot (Fig. 3). The IκBαM protein migrates slightly faster than native IκBα due to the deletion of the COOH-terminal PEST sequence. The relative abundance of the IκBαM protein was nearly equivalent to that of native IκBα, as has been observed in other cell lines (9, 24).

NF-κB Transactivation: Trx9/IκBαM-expressing MCF-7 Cells.

Stable transfection and expression of IκBαM caused a 2–3.6-fold inhibition of constitutive NF-κB activity in the Neo3 cells and a 3.8–6.7-fold inhibition in the Trx9 cells at serum concentrations of 0, 5, and 10% (Fig. 4). Constitutive NF-κB activity in the dual empty-vector control cells was equivalent to wild-type MCF-7 cells. There was a small increase in activity in the Trx9/Hygro4 cells as the serum concentration in the medium increased from 0 to 5% and in the Trx9/IκBαM8 cells as the serum concentration in the medium increased from 0 to 10%. However, serum did not appreciably affect NF-κB activity in MCF-7 cells that were not transfected with Trx. Thus, serum increased NF-κB activity in Trx-transfected cells, but expression of IκBαM inhibited NF-κB activity in both control and Trx-overexpressing MCF-7 cells.

Anchorage-dependent and -independent Growth in Low Serum.

Expression of IκBαM inhibited both anchorage-dependent and -independent growth in the Neo3 cells expressing IκBαM (Neo3/IκBαM8) compared with the dual empty vector control cells (Neo3/Hygro1) and wild-type MCF-7 cells (Fig. 5). Monolayer growth was inhibited by 45 and 23% at 1 and 2.5% FBS, respectively, and colony formation was reduced by 49 and 32%. However, the increases in anchorage-independent growth conferred by Trx overexpression were not reduced by expression of IκBαM compared with the cells containing the pCMX empty vector and overexpressing Trx (Trx9/Hygro4). There was a slight but statistically significant reduction in anchorage-dependent growth in two of the three Trx-overexpressing clones examined, but growth was still significantly increased compared with the empty vector control cells. Thus, expression of IκBαM in MCF-7 cells inhibits both anchorage-dependent and -independent growth at low serum concentrations, whereas Trx overexpression appears to overcome this inhibition.

AP-1 Transactivation: Trx9/IκBαM-expressing MCF-7 Cells.

We also measured constitutive levels of AP-1 activation in IκBαM-expressing cell lines (Neo3/IκBαM8 and Trx9/IκBαM8), their cognate empty vector controls (Neo3/Hygro1 and Trx9/Hygro4), and wild-type MCF-7 cells. AP-1 activity was increased 8–10-fold in the Trx-overexpressing cells (Neo3/Hygro1 versus Trx9/Hygro4; Fig. 6). There was also an increase in AP-1 activity in the Neo3/IκBαM8-expressing cells compared with the dual empty vector controls (Neo3/Hygro1) that was greater at higher serum concentrations. Thus, expression of IκBαM does not seem to effect constitutive AP-1 activation in control cells. However, in low serum, expression of IκBαM reduced the increase in constitutive AP-1 activation conferred by the overexpression of Trx.

Activation of NF-κB is an early response to cytokine stimulation in lymphoid and other cells and may be at least partly responsible for increased proliferation in response to these agents (25, 26). Activation of NF-κB has also been reported to block apoptosis caused by TNF-α (8, 9). Thus, NF-κB activation can directly stimulate cell proliferation and indirectly increase growth by inhibiting spontaneous apoptosis. We chose MCF-7 breast cancer cells because apoptosis does not seem to be a major factor in their growth kinetics, possibly because they lack detectable levels of caspase 3 (27, 28). This makes them an ideal cell line to study the growth effects of Trx uncomplicated by effects on apoptosis. Indeed, we did not observe any appreciable cell death in our experiments (<5%), as indicated by cells detaching from the tissue culture plates.

Transient transfection of cells with Trx has been reported to both increase (4) and to decrease (29) NF-κB activity. One reason for these discrepancies may be the use of various reporter constructs containing different NF-κB promoter elements (30). The subcellular localization of Trx in different cell lines might also account for differential NF-κB activation. Although predominantly cytoplasmic, Trx has been found in the nucleus of some cells (31). For example, we have observed Trx in both the cytoplasm and nucleus in WEHI7.2 cells (22) and in MCF-7 breast cancer cells (21), whereas in HT-29 colon cells, Trx appears to be localized exclusively in the cytoplasm.4 The Cys62 residue of the p50 subunit of NF-κB has to be reduced to bind DNA (4), and Trx has been reported to bind to a peptide fragment of NF-κB containing the Cys62 residue (15). Thus, an increase in Trx in the nucleus might lead to an increase in constitutive NF-κB activity. Trx also has antioxidant properties mediated by Trx peroxidases (32), which could also explain an inhibition of oxidant-induced NF-κB activation by Trx in the cytoplasm (33).

In our experiments, an inhibition of constitutive NF-κB activity in wild-type cells and cells containing the pDC304neo empty vector and IκBαM was associated with up to a 50% decrease in monolayer growth and colony formation compared with cells containing both empty vector constructs. Thus, constitutive NF-κB activity seems to play a significant role in the rate of MCF-7 cell growth. The effects on growth resulting from inhibition of NF-κB in Trx-overexpressing cells was also studied. Monolayer growth was only slightly reduced in cells expressing IκBαM compared with the control cell line, which suggests that increased NF-κB activity is at most only partly responsible for the growth advantage conferred by overexpression of Trx. Thus, it appears that although constitutive NF-κB activity is increased by overexpression of Trx in MCF-7 cells, it is probably not responsible for the increased growth kinetics in Trx-overexpressing cells. It is important to note, however, that expression of IκBαM did not inhibit NF-κB activity in the Trx-overexpressing cells to the level of the control cells. Thus, we cannot discount the possibility that a threshold level of NF-κB activity exists and, that above this level, the growth rate is increased.

If NF-κB activity is not involved, there must be other mechanisms through which an increase in Trx augments growth. One possibility is the direct activation of other transcription factors regulated by Trx, e.g., AP-1. Activation of AP-1 is associated with increased proliferation in many cell lines (34, 35). We observed a 7–8-fold increase in the constitutive level of AP-1 activity in cells overexpressing Trx. Surprisingly, AP-1 activity was decreased by IκBαM in the Trx-overexpressing cells; however, AP-1 activity was increased in these cells with an increase in the serum concentration. Cross-talk between AP-1 and NF-κB has been reported, and inhibition of AP-1 activity can result in an inhibition of NF-κB activity (23). Conversely, in the Trx-overexpressing cells, inhibition of NF-κB appears capable of inhibiting AP-1 activity. Serum appears to partly overcome the inhibition of AP-1 and NF-κB activation in the Trx-transfected cells, which may be due to Trx augmenting growth factors in the serum, although the actual factors mediating this effect are unknown (12, 20).

In conclusion, the overexpression of Trx in MCF-7 cells increases the constitutive activation of both NF-κB and AP-1. The inhibition of NF-κB, however, did not reverse the growth advantage conferred by Trx at low serum. This growth advantage may be mediated through increased AP-1 activity or via an altogether different mechanism.

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

Supported by NIH Grants F32 CA76774 (to A. J. F.), CA48725 (to A. G. and G. P.), and CA77204 (to A. G. and G. P.).

            
3

The abbreviations used are: NF-κB, nuclear factor-κB; Trx, thioredoxin; AP-1, activator protein 1; IκBαM, inhibitor κ Bα mutant; FBS, fetal bovine serum; CAT, chloramphenicol acetyltransferase; β-gal, β-galactosidase.

      
4

Unpublished observation.

Fig. 1.

Effects of Trx overexpression on anchorage-independent growth in low serum. A, wild-type MCF-7 breast cancer cells (panel 1) and clones stably transfected with empty vector (Neo3; panel 2) or Trx (Trx9, Trx12, and Trx20; panels 3–5, respectively) were grown as colonies in soft agar in DMEM containing 5% FBS for 10 days. Colonies were stained with INT and photographed. B, colonies growing in 1, 2.5, or 5% FBS and >60 μm in size were enumerated after 10 days. The colony number is represented as the percentage of growth of the control cells (Neo3/Hygro1). Values are the means of quadruplicate determinations; bars, SE. Two additional experiments yielded similar results. wt, wild type.

Fig. 1.

Effects of Trx overexpression on anchorage-independent growth in low serum. A, wild-type MCF-7 breast cancer cells (panel 1) and clones stably transfected with empty vector (Neo3; panel 2) or Trx (Trx9, Trx12, and Trx20; panels 3–5, respectively) were grown as colonies in soft agar in DMEM containing 5% FBS for 10 days. Colonies were stained with INT and photographed. B, colonies growing in 1, 2.5, or 5% FBS and >60 μm in size were enumerated after 10 days. The colony number is represented as the percentage of growth of the control cells (Neo3/Hygro1). Values are the means of quadruplicate determinations; bars, SE. Two additional experiments yielded similar results. wt, wild type.

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Fig. 2.

NF-κB activity in wild-type (wt) MCF-7 cells and MCF-7 cells stably transfected with the empty vector (Neo3) or Trx (Trx9 and Trx12). Relative NF-κB activity was measured using a NF-κB/Luciferase reporter construct as described in “Materials and Methods.” Luciferase activity was measured with a luminometer and is expressed as the fold induction of activity found in the empty vector control cells (Neo3). Values have had background activity subtracted and then corrected for transfection efficiency with pCMVβ-gal. Values are the means of triplicate determinations; bars, SE. Two additional experiments yielded similar results.

Fig. 2.

NF-κB activity in wild-type (wt) MCF-7 cells and MCF-7 cells stably transfected with the empty vector (Neo3) or Trx (Trx9 and Trx12). Relative NF-κB activity was measured using a NF-κB/Luciferase reporter construct as described in “Materials and Methods.” Luciferase activity was measured with a luminometer and is expressed as the fold induction of activity found in the empty vector control cells (Neo3). Values have had background activity subtracted and then corrected for transfection efficiency with pCMVβ-gal. Values are the means of triplicate determinations; bars, SE. Two additional experiments yielded similar results.

Close modal
Fig. 3.

Western blot showing expression of IκBαM in MCF-7 cell lines. Equal amounts of protein (25 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride. The blot was probed and developed as described in “Materials and Methods.” The double-transfected cell lines used in this and subsequent experiments are as follows: Lane 1, wild type; Lane 2, Neo3/Hygro1; Lane 3, Neo3/IκBαM8; Lane 4, Trx9/Hygro4; Lane 5, Trx9/IκBαM1; Lane 6, Trx9/IκBαM4; and Lane 7, Trx9/IκBαM8. Right, native IκBα and IκBαM proteins. Left, migration of prestained molecular weight markers. Three additional experiments yielded similar results.

Fig. 3.

Western blot showing expression of IκBαM in MCF-7 cell lines. Equal amounts of protein (25 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride. The blot was probed and developed as described in “Materials and Methods.” The double-transfected cell lines used in this and subsequent experiments are as follows: Lane 1, wild type; Lane 2, Neo3/Hygro1; Lane 3, Neo3/IκBαM8; Lane 4, Trx9/Hygro4; Lane 5, Trx9/IκBαM1; Lane 6, Trx9/IκBαM4; and Lane 7, Trx9/IκBαM8. Right, native IκBα and IκBαM proteins. Left, migration of prestained molecular weight markers. Three additional experiments yielded similar results.

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Fig. 4.

NF-κB activity in wild-type, control, and IκBαM-expressing cells measured with a CAT reporter construct. Cells were transfected with the reporter construct for 24 h in DMEM with 2% FBS, then treated for 24 h with DMEM alone, 5% FBS, or 10% FBS. The cell lines used are as follows: Lane 1, wild type; Lane 2, Neo3/Hygro1; Lane 3, Neo3/IκBαM8; Lane 4, Trx9/Hygro4; Lane 5, Trx9/IκBαM1; Lane 6, Trx9/IκBαM4; and Lane 7, Trx9/IκBαM8, and values have been normalized to the control cells (Neo3/Hygro1). The amount of CAT protein was measured by ELISA at 405 nm and represents the relative amount of NF-κB activity. Values are the means of three determinations (bars, SE) and are corrected for transfection efficiency with pCMVβ-gal as described in “Materials and Methods.” Two additional experiments yielded similar results.

Fig. 4.

NF-κB activity in wild-type, control, and IκBαM-expressing cells measured with a CAT reporter construct. Cells were transfected with the reporter construct for 24 h in DMEM with 2% FBS, then treated for 24 h with DMEM alone, 5% FBS, or 10% FBS. The cell lines used are as follows: Lane 1, wild type; Lane 2, Neo3/Hygro1; Lane 3, Neo3/IκBαM8; Lane 4, Trx9/Hygro4; Lane 5, Trx9/IκBαM1; Lane 6, Trx9/IκBαM4; and Lane 7, Trx9/IκBαM8, and values have been normalized to the control cells (Neo3/Hygro1). The amount of CAT protein was measured by ELISA at 405 nm and represents the relative amount of NF-κB activity. Values are the means of three determinations (bars, SE) and are corrected for transfection efficiency with pCMVβ-gal as described in “Materials and Methods.” Two additional experiments yielded similar results.

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Fig. 5.

Effects of IκBαM expression on cell growth. A, each double-transfected cell line and wild-type cells were grown as a monolayer in DMEM supplemented with 1 and 2.5% FBS. Cells were seeded in triplicate at 4 × 104/wells in a 12-well plate, allowed to grow for 72 h, and then trypsinized and counted. The cell number is represented as the percentage of growth of the control cells containing both empty vectors (Neo3/Hygro1). Each cell line used is listed on the axis. B, each cell line was grown in 0.3% agar in DMEM supplemented with 1 and 2.5% FBS. Cells were seeded at 500 cells/well in a 24-well plate and allowed to grow for 10 days. Colonies were stained overnight, and those containing >50 cells were counted. The colony number is represented as the percentage of growth of the control cells (Neo3/Hygro1). Values represent the means of three determinations; bars, SE (Neo3/Hygro1 versus Neo3/IκBαM8: ∗, P < 0.001; Trx9/Hygro4 versus Trx9/IκBαM1 and Trx9/IκBαM8: ∗∗, P < 0.01, paired t test).

Fig. 5.

Effects of IκBαM expression on cell growth. A, each double-transfected cell line and wild-type cells were grown as a monolayer in DMEM supplemented with 1 and 2.5% FBS. Cells were seeded in triplicate at 4 × 104/wells in a 12-well plate, allowed to grow for 72 h, and then trypsinized and counted. The cell number is represented as the percentage of growth of the control cells containing both empty vectors (Neo3/Hygro1). Each cell line used is listed on the axis. B, each cell line was grown in 0.3% agar in DMEM supplemented with 1 and 2.5% FBS. Cells were seeded at 500 cells/well in a 24-well plate and allowed to grow for 10 days. Colonies were stained overnight, and those containing >50 cells were counted. The colony number is represented as the percentage of growth of the control cells (Neo3/Hygro1). Values represent the means of three determinations; bars, SE (Neo3/Hygro1 versus Neo3/IκBαM8: ∗, P < 0.001; Trx9/Hygro4 versus Trx9/IκBαM1 and Trx9/IκBαM8: ∗∗, P < 0.01, paired t test).

Close modal
Fig. 6.

AP-1 activity in wild-type, control, and IκBαM-expressing cells measured with a luciferase reporter construct. Cells were transfected with the reporter construct for 24 h in DMEM with 2% FBS and then treated for 24 h with DMEM alone, 5% FBS, or 10% FBS. The cell lines used are listed, and values are represented as the fold induction of control cells (Neo3/Hygro1). Values have had background activity subtracted and then corrected for transfection efficiency with pCMVβ-gal. Values are the means of triplicate determinations; bars, SE. An additional experiment yielded similar results. (Trx9/Hygro4 versus Trx9/IκBαM8: ∗, P < 0.001; ∗∗, P < 0.01, paired t test).

Fig. 6.

AP-1 activity in wild-type, control, and IκBαM-expressing cells measured with a luciferase reporter construct. Cells were transfected with the reporter construct for 24 h in DMEM with 2% FBS and then treated for 24 h with DMEM alone, 5% FBS, or 10% FBS. The cell lines used are listed, and values are represented as the fold induction of control cells (Neo3/Hygro1). Values have had background activity subtracted and then corrected for transfection efficiency with pCMVβ-gal. Values are the means of triplicate determinations; bars, SE. An additional experiment yielded similar results. (Trx9/Hygro4 versus Trx9/IκBαM8: ∗, P < 0.001; ∗∗, P < 0.01, paired t test).

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