The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aggarwal, S.
Right arrow Articles by Aggarwal, B. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aggarwal, S.
Right arrow Articles by Aggarwal, B. B.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
The Journal of Immunology, 2004, 173: 4368-4376.
Copyright © 2004 by The American Association of Immunologists

Melanoma Differentiation-Associated Gene-7/IL-24 Gene Enhances NF-{kappa}B Activation and Suppresses Apoptosis Induced by TNF

Sita Aggarwal*, Yasunari Takada*, Abner M. Mhashilkar{dagger}, Kerry Sieger{dagger}, Sunil Chada{dagger} and Bharat B. Aggarwal1,*

* Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, and {dagger} Introgen Therapeutics, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-{kappa}B or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-{kappa}B activation and on TNF-induced NF-{kappa}B activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-{kappa}B. However, TNF-induced NF-{kappa}B activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-{kappa}B-dependent reporter gene expression. Mda-7 transfection also potentiated NF-{kappa}B reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-{kappa}B reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-{kappa}B activation. TNF-induced expression of the NF-{kappa}B-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-{kappa}B by I{kappa}B{alpha} kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-{kappa}B, but potentiates TNF-induced NF-{kappa}B activation and NF-{kappa}B-regulated gene expression. Potentiation of NF-{kappa}B survival signaling by MDA-7 inhibits TNF-mediated apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The melanoma differentiation-associated gene-7 (mda-7)2 was first identified in 1995 by subtraction hybridization of cDNA libraries prepared from H0–1 melanoma cells treated with IFN-{beta} and mezerin (1). The expression of mda-7 mRNA was elevated in differentiation inducer-treated H0–1 cells. This gene encodes a novel protein of 206 aa, with a predicted size of 23.8 kDa. Mda-7 gene transfer suppresses the proliferation of human melanoma cells, and loss of MDA-7 protein correlates with increased invasion and metastasis in human melanoma (2, 3). Thus, the protein functions as a negative regulator of melanoma progression. Due to structural homology to other members of the IL-10 family of cytokines, chromosomal localization, and cytokine-like properties, MDA-7 has been redesignated IL-24 (4, 5, 6, 7, 8, 9). The soluble MDA-7 protein has been recently shown to signal through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2 (10).

Overexpression of MDA-7 has been shown to induce apoptosis in a wide variety of other cancer cells, besides melanoma, including lung, prostate, breast, cervical, and colorectal cancers; sarcoma; and glioblastoma (4, 11, 12, 13, 14, 15, 16). It has also been shown to suppress the growth of tumors in mouse xenografts and upon intratumoral injection in human tumors (12, 13). How MDA-7 induces apoptosis of various tumor cells is not fully understood, but Bax (17), dsRNA-dependent protein kinase (PKR) (18), and growth arrest and DNA damage family (19) have been reported to have roles. Gene delivery of mda-7 results in high levels of MDA-7 protein expression intracellularly in addition to secretion of a glycosylated from of the protein. The glycosylated secreted protein possesses potent antiantigenic properties that are mediated via the IL-22R in addition to its pro-Th1 cytokine activity (20, 21). We have recently determined that intracellular MDA-7 is responsible for cytotoxic activity in human lung cancer cells (22). Thus, the location of the MDA-7 protein can result in divergent effects.

Like most other apoptosis-inducing cytokines, including members of the TNF superfamily (23), MDA-7 has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF and IL-6 (21, 24). TNF can mediate either apoptotic or antiapoptotic effects (23). Although apoptosis is mediated through activation of caspases, the antiapoptotic effects are mediated through activation of the nuclear transcription factor NF-{kappa}B, which itself mediates the inflammatory effects of most cytokines. TNF-induced NF-{kappa}B-regulated genes, such as cyclooxygenase-2 (COX-2), cyclin D1, cellular inhibitors of apoptosis, and Bcl-xL, can mediate antiapoptosis. Because MDA-7 induces inflammatory cytokines, has been linked to differentiation and invasion of melanoma cells, and activates inflammatory and apoptotic signaling proteins, we postulated that MDA-7, alone or in combination with other cytokines, may activate NF-{kappa}B, and the latter can modulate apoptosis.

In this report we tested whether transient or stable transfection of MDA-7 alone or in combination with TNF can activate NF-{kappa}B or apoptosis. We found that MDA-7 alone did not activate NF-{kappa}B, but significantly enhanced TNF-induced NF-{kappa}B activation and NF-{kappa}B-regulated expression of cyclin D1 and COX-2. MDA-7 also suppressed TNF-induced apoptosis, and inhibition of NF-{kappa}B activation sensitized the cells to apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Rabbit polyclonal Ab against I{kappa}B{alpha} and an annexin V staining kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-TNF receptor (anti-TNFR) Abs were provided by Dr. M. Brockhaus (Hoffmann-La Roche, Basel, Switzerland). Goat anti-rabbit-HRP conjugate Ab was purchased from Bio-Rad (Hercules, CA), and goat anti-mouse-HRP conjugate Ab was obtained from BD Biosciences (Lexington, KY). Goat anti-rabbit Alexa 594 conjugate Ab and the Live/Dead assay kit were purchased from Molecular Probes (Eugene, OR). DMEM, FBS, and the calcium phosphate transfection kit were purchased from Invitrogen Life Technologies (Carlsbad, CA). Protein A/G-Sepharose beads were obtained from Pierce (Rockford, IL), [{gamma}-32P]ATP was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Anti-I{kappa}B{alpha} kinase-{alpha} (anti-IKK-{alpha}), anti-IKK-{beta} Abs, cell-permeable NF-{kappa}B essential modifier (NEMO; also called IKK-{gamma}) binding domain peptide (NBD; NH2-DRQIKIWFQNRRMKWKKTALDWSWLQTE-CONH2), and NBD control peptide (NBDC; NH2-DRQIKIWFQNRRMKWKK-CONH2) were gifts from Imgenex (San Diego, CA).

Cell culture

Human embryonic kidney 293 cells stably transfected with mda-7 were prepared as reported previously (21). These cells were cultured in DMEM containing 10% FBS.

Preparation of nuclear extracts and EMSA for NF-{kappa}B

Nuclear extracts were prepared as previously described (25). Briefly, 2 x 106 cells were washed with cold PBS and suspended in 0.4 ml of hypotonic lysis buffer containing protease inhibitors for 30 min. The cells were then lysed with 12.5 µl of 10% Nonidet P-40. The homogenate was centrifuged, and supernatant containing the cytoplasmic extracts was removed and stored frozen at –80°C. The nuclear pellet was resuspended in 25 µl of ice-cold nuclear extraction buffer. After 30 min of intermittent mixing, the extract was centrifuged, and supernatants containing nuclear extracts were secured. The protein content was measured by the Bradford method. If the samples were not used immediately, they were stored at –80°C. Nuclear extracts (8 µg) prepared from TNF-treated or untreated cells were incubated with 32P end-labeled, 45-mer, double-stranded NF-{kappa}B oligonucleotide from HIV-1 long terminal repeat (5'-TTGTTACAAGGGACTTTCCGCT GGGGACTTTCCAG GGAGGCGTGG-3'; underlined sequence indicates NF-{kappa}B binding site) for 15 min at 37°C, and the DNA-protein complex was resolved in a 6.6% native polyacrylamide gel. The radioactive bands from the dried gels were visualized and quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.

Western blot analysis

Thirty to 50 µg of cytoplasmic protein extracts, prepared as previously described (26), were resolved on 10% SDS-PAGE. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and probed with Abs against COX-2, cyclinD1, TNFR1, or TNFR2 (1/3000 dilution) for 1 h. Thereafter, the blot was washed, exposed to HRP-conjugated secondary Abs for 1 h, and finally detected by chemiluminescence (ECL; Amersham Biosciences, Arlington Heights, IL).

Immunocytochemistry for NF-{kappa}B p65 localization

293 cells and 293-mda-7 cells were plated 1 day previously on a glass chamber slide for adherence and treated with and without TNF. Removed chambers and slides were air-dried for 1 h at room temperature and fixed with cold acetone. After a brief washing in PBS, slides were blocked with 5% normal goat serum for 1 h and then incubated with rabbit polyclonal anti-human p65 Ab (dilution, 1/100). After overnight incubation, the slides were washed and then incubated with goat anti-rabbit Alexa 594-conjugated Ab (1/100) for 1 h and counterstained for nuclei with Hoechst 33342 (50 ng/ml) for 5 min. Stained slides were mounted with mounting medium (Sigma-Aldrich, St. Louis, MO) and analyzed under an epifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan). Pictures were captured using Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and MetaMorph version 4.6.5 software (Universal Imaging, Downingtown, PA).

NF-{kappa}B-dependent reporter gene expression

The effects of MDA-7 transfection on TNF, TNFR-associated death domain (TRADD), TNFR-associated factor 2 (TRAF2), NF-{kappa}B-inducing kinase (NIK), I{kappa}B{alpha} kinase (IKK), and p65 (trans-activation subunit of NF-{kappa}B)-induced NF-{kappa}B-dependent reporter gene expression were measured as described previously (27, 28). Briefly, 293 and 293-mda-7 cells (0.5 million/well) were plated in six-well plates, left overnight for adherence, and transfected with various plasmids the next day by the calcium phosphate method.

Flow cytometric analysis of TNFR expression

For analysis of TNFR expression, cells were harvested by gentle scraping, centrifuged, and resuspended in Dulbecco’s PBS containing 1% FBS and 0.1% sodium azide. The cells were preincubated with 10% goat serum for 20 min and washed, then monoclonal mouse IgG anti-TNFR Abs were added; utr-9 and utr-1 Abs are specific for TNFR1/p60 and TNFR2/p80, respectively. After 1-h incubation at 4°C, the cells were washed and incubated for an additional 1 h with a FITC-conjugated goat anti-mouse IgG mAb. The cells were analyzed using a FACSCalibur flow cytometer and CellQuest acquisition and analysis programs (BD Biosciences, Mountain View, CA).

Apoptosis assay

Apart from subcellular localization, we also analyzed the effect of TNF, NBDC, and NBD on 293 and 293-mda-7 cells on cell killing using the live-dead (green-red) assay. IKK is composed of IKK-{alpha}, IKK-{beta}, and IKK-{gamma} (also called NEMO). The N-terminal {alpha}-helical region of NEMO has been shown to interact with the C-terminal segment of IKK-{alpha} and IKK-{beta}. A small peptide from the C terminus of IKK-{alpha} and IKK-{beta} NEMO has been shown to block this interaction. To make it cell-permeable, the NBD peptide was conjugated to a small sequence from the antennapedia homeodomain. This peptide has been shown to specifically suppress NF-{kappa}B activation. The peptide without the antennapedia homeodomain was used as a control. Cells were grown in chamber slides overnight for adherence, then treated with TNF, NBD, NBDC, or the indicated combinations. After 24- or 48-h incubation, the cells were stained with the live-dead assay reagents for 30 min at room temperature according to the manufacturer’s protocol. Cells were then examined under the fluorescence microscope and counted for live/dead (green/red) ratio.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The goal of this study was to determine whether MDA-7, alone or in combination with cytokines such as TNF, can modulate the activation of NF-{kappa}B or apoptosis. As target cells, we used human embryonic kidney cell line 293. These cells were stably transfected with the mda-7 gene and previously characterized (20, 21, 22). Stable expression of MDA-7 in these cells was verified by Western blot analysis (Fig. 1A). These cells express intracellularly a doublet of 30 and 23 kDa and secrete a 40-kDa glycosylated protein as previously reported (21).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 1. A, Expression of MDA-7 protein in stably transfected human embryonic kidney 293 cells. Whole cell extracts were prepared from 293 and stably transfected 293-mda-7 cells, and Western blot analysis was performed using anti-MDA-7 Ab. B, Effect of MDA-7 on TNF-induced NF-{kappa}B activation. 293 and 293-mda-7 cells (2 x 106/ml) were incubated at 37°C with the indicated concentrations of TNF for 30 min, nuclear extracts were prepared, and then assayed for NF-{kappa}B activity by EMSA as described in Materials and Methods. C, Effect of MDA-7 on the time course of TNF-induced NF-{kappa}B activation. 293 and 293-mda-7 cells (2 x 106/ml) were incubated at 37°C with 100 pM TNF for the indicated times, nuclear extracts were prepared, and then assayed for NF-{kappa}B activation by EMSA. D, Supershift analysis and specificity of NF-{kappa}B complex. Nuclear extracts were prepared from untreated or 100 pM TNF-treated cells (2 x 106/ml), incubated for 15 min with the indicated Abs and cold NF-{kappa}B probe, and then assayed for NF-{kappa}B activation by EMSA. E, Effect of MDA-7 on TNF-induced nuclear translocation of p65 NF-{kappa}B subunit. 293 and 293-mda-7 cells were plated in chamber slides, then treated with 100 pM TNF for 30 min. Then the slides were analyzed for the distribution of p65 NF-{kappa}B subunit by immunocytochemistry as described in Materials and Methods (magnification, x200).

 
MDA-7 alone does not activate NF-{kappa}B, but potentiates TNF-induced NF-{kappa}B activation

Nontransfected and stably mda-7-transfected cells were stimulated with increasing concentrations of TNF as indicated, prepared nuclear extracts, and assayed for NF-{kappa}B by EMSA. As shown in Fig. 1B, MDA-7 expression alone did not activate NF-{kappa}B. However, TNF activated NF-{kappa}B by 3-fold in nontransfected cells, but by 11-fold in the mda-7-transfected cells. In nontransfected cells, NF-{kappa}B activation was maximal (4.5-fold) at 60 min after TNF exposure, but in mda-7-transfected cells it was maximal (11-fold) at 30 min (Fig. 1C).

Various combinations of Rel/NF-{kappa}B proteins constitute an active NF-{kappa}B heterodimer that binds to a specific sequence in DNA (29, 30). To show that the retarded band visualized by EMSA in TNF-treated cells was indeed NF-{kappa}B, we incubated nuclear extracts from TNF-treated cells with Abs to either the p50 (NF-{kappa}B1) or the p65 (RelA) subunit of NF-{kappa}B. Both shifted the band to a higher molecular mass (Fig. 1D), thus suggesting that the TNF-activated NF-{kappa}B complex consisted of p50 and p65 subunits. Neither preimmune serum nor the irrelevant Ab anti-cyclin D1 had any effect. Excess unlabeled NF-{kappa}B oligo (100-fold), but not the mutated oligo, caused the band to completely disappear.

To demonstrate that MDA-7 enhances the TNF-induced translocation of p65 subunit of NF-{kappa}B to the nucleus, we subjected TNF-treated and untreated mda-7-transfected 293 cells to immunocytochemistry. Fig. 1E clearly demonstrates that MDA-7 enhanced translocation of the p65 subunit of NF-{kappa}B to the nucleus.

MDA-7 potentiates TNF-induced NF-{kappa}B-dependent reporter gene expression

DNA binding alone does not always correlate with NF-{kappa}B-dependent gene transcription, suggesting that additional regulatory steps are required (31). To determine NF-{kappa}B-dependent reporter gene expression, we transiently transfected 293 and 293-mda7 cells with the NF-{kappa}B secretory alkaline phosphatase (SEAP) reporter construct, and then stimulated them with the indicated concentrations of TNF. MDA-7 alone had no effect on the NF-{kappa}B reporter activity, but 1 nM TNF activated NF-{kappa}B reporter activity by only 4-fold in the 293 cells (Fig. 2A) and by >15-fold in mda-7-transfected cells (Fig. 2A). The potentiation of NF-{kappa}B-mediated gene expression by TNF was dose dependent. These results indicate that enhanced DNA binding of NF-{kappa}B correlated with enhanced NF-{kappa}B-dependent reporter gene expression.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. A, Effect of MDA-7 on TNF-induced NF-{kappa}B-dependent reporter gene expression. 293, 293-neo (empty vector-transfected neomycin resistant clone), and 293-mda-7 cells were transiently transfected with NF-{kappa}B containing plasmid linked to the SEAP gene. After 24 h of transfection, cells were exposed to the indicated concentrations of TNF for next 24 h. Supernatants were collected and assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the nontransfected controls; bars indicate the SE. B, Effect of MDA-7 on NF-{kappa}B-dependent reporter gene expression induced by TNF-signaling proteins. 293 and 293-mda-7 cells were transiently transfected with the indicated plasmids along with an NF-{kappa}B-containing plasmid linked to the SEAP reporter gene. Where indicated, cells were exposed to 1 nM TNF or plasmid for TNF-signaling protein. After 24 h, supernatants were collected and assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the nontransfected controls; bars indicate the SD. C, Effect of transient transfected MDA-7 on TNF-induced NF-{kappa}B-dependent reporter gene expression. 293 cells were transiently transfected with mda-7 plasmid along with the NF-{kappa}B-containing plasmid linked to the SEAP reporter gene. After 24 h, cells were exposed to TNF for next 24 h. Supernatants were collected and assayed for SEAP activity as described in Materials and Methods. Results are plotted as fold activity over the nontransfected controls; bars indicate the SD.

 
TNF-induced NF-{kappa}B activation is mediated through sequential interaction of the TNFR with TRADD, TRAF2, NIK, and IKK-{beta}, resulting in phosphorylation of I{kappa}B{alpha} (23, 30). To delineate the site of action of MDA-7 in the TNF-signaling pathway leading to NF-{kappa}B activation, cells were transfected with TRADD-, TRAF2-, NIK-, IKK-, and p65-expressing plasmids along with NF-{kappa}B reporter plasmid, and then monitored NF-{kappa}B-dependent SEAP expression in 293 and 293-mda-7 cells. MDA-7 significantly enhanced the NF-{kappa}B reporter activity induced by TRADD and TRAF2, but not that induced by NIK, IKK, and p65 plasmids (Fig. 2B). These results indicate that MDA-7 enhances the TNF-induced binding of NF-{kappa}B protein to the DNA, and this potentiation is lost downstream of TRAF2.

We also examined whether transient transfection of cells with mda-7 plasmid potentiates TNF-induced NF-{kappa}B-reporter activity. As shown in Fig. 2C, MDA-7 enhanced TNF-induced NF-{kappa}B-dependent gene expression.

Secretion of MDA-7 is not required for the potentiation of TNF-induced NF-{kappa}B activation

To determine whether MDA-7 secretion is required for MDA-7 activity, we transfected 293 cells with MDA-7 lacking the secretion signal (Fig. 3A). Fluorescence microscopy revealed that MDA-7 without the secretion sequence was located in the cytoplasm, whereas full-length protein showed a typical punctate staining in the cell (data not shown). Neither full-length MDA-7 with signal sequence nor cytoplasmic MDA-7 without the signal sequence alone induced NF-{kappa}B reporter activity. However, both the cytoplasmic and full-length MDA-7 enhanced TNF-induced NF-{kappa}B reporter activity (Fig. 3B). There was no significant difference in the activity between the effects of the two forms of MDA-7. These results indicate that MDA-7 need not be secreted to potentiate TNF-induced NF-{kappa}B activation.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 3. Secretion of MDA-7 is not needed for TNF-induced NF-{kappa}B activation. A, The architecture of secreted and cytoplasmic MDA-7 in the present studies. B, Effect of cytoplasmic and full-length MDA-7 constructs on TNF-induced NF-{kappa}B dependent reporter gene expression. 293 cells were transiently transfected with full-length (FL) and signal sequence-truncated (Cyto) mda-7 plasmids. Thereafter, cells were cotransfected with NF-{kappa}B SEAP reporter plasmids. Where indicated, cells were exposed to 1 nM TNF. After 24 h, supernatants were collected and assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity over the nontransfected controls; bars indicate the SD.

 
MDA-7 enhances the TNF-induced expression of NF-{kappa}B-regulated COX-2 and cyclin D1

Whether MDA-7 modulates the expression of NF-{kappa}B-dependent gene products COX-2 and cyclin D1 (32, 33, 34) induced by TNF was also investigated. Treatment with 0.1 nM TNF induced COX-2 and cyclin D1 expression in 293 cells (Fig. 4), and mda-7 transfection enhanced this induction. Interestingly, MDA-7 alone induced a small amount of COX-2 and cyclin D1 expression. These results clearly demonstrate that MDA-7 potentiates the TNF-induced expression of both cyclin D1 and COX-2.



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 4. MDA-7 potentiates TNF-induced expression of COX-2 and cyclin D1 proteins. 293 and 293-mda-7 cells (2 x 106/ml) were treated with 100 pM TNF for the indicated times, whole cell extracts were prepared, and Western blot analysis was performed using specific Abs.

 
MDA-7 suppresses the TNF-induced apoptosis of cells

MDA-7 activates apoptosis in a variety of cells (7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19), whereas NF-{kappa}B activation can suppress it (35, 36, 37). Thus, how MDA-7 affects TNF-induced apoptosis was also investigated. As shown in Fig. 5, TNF induced apoptosis in 293 cells, and MDA-7 suppressed this induction (from 57 to 4%). TNF-induced apoptosis was time dependent, as indicated by an increase from 24 to 48 h.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 5. A, MDA-7 inhibits TNF-induced apoptosis. 293 and 293-mda-7 cells were grown in chamber slides in either the presence or the absence of 1 nM TNF. After 24 or 48 h, cells were assayed for apoptosis by the live-dead method as described in Materials and Methods. Cells were then examined under a fluorescence microscope and counted for live/dead (green/red) ratio. Results are expressed as the percentage of cell killing over that in the respective controls; bars indicate the SD in B.

 
NBD suppresses TNF-induced NF-{kappa}B activation and enhances apoptosis

The results shown above indicate that cells that express higher level of NF-{kappa}B (MDA-7-expressing) are more resistant to TNF-induced apoptosis. Whether NF-{kappa}B activation is linked to apoptosis was investigated.

To establish that TNF-induced NF-{kappa}B activation is linked to apoptosis, we used the NBD and NBDC. Treatment of cells with NBDC had no effect, whereas NBD suppressed TNF-induced NF-{kappa}B activation (data not shown). Results in Fig. 6 indicate that treatment of 293 cells with NBD enhanced TNF-induced apoptosis from 46 to 80% in 293 cells (Fig. 6A) and from 10 to 56% in mda-7-expressing 293 cells (Fig. 6B). Control NBD peptide had no significant effect. These results indicate that suppression of NF-{kappa}B activation does indeed enhance TNF-induced apoptosis.



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 6. NBD enhances TNF-induced apoptosis. 293 and 293-mda-7 cells were grown in chamber slides in the presence or the absence of 25 µM NBD and NBDC for 48 h as described in Materials and Methods, followed by treatment with 1 nM TNF for 24 h. Cells were then examined under a fluorescence microscope and counted for live/dead (green/red) ratio. Results are expressed as the percentage of cell killing over that in the respective controls; bars indicate the SD in B.

 
MDA-7 up-regulates TNFR expression

Whether increased TNF-induced NF-{kappa}B activation by MDA-7 is mediated through the up-regulation of TNFR was investigated. Western blot analysis of cell extracts prepared from the control and mda-7-transfected cells indicate that MDA-7 up-regulated both the p60 (TNFR-1) and p80 (TNFR-2) forms of TNFR proteins (Fig. 7A). Quantitation of the results indicated that the up-regulation was small, but significant (Fig. 7B). FACS analysis confirmed the findings (Fig. 7C).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 7. A, Effect of MDA-7 on cell surface expression of TNFR protein. Cytoplasmic extracts were prepared from 293 and 293-mda-7 cells (2 x 106/ml) and assayed for the expression level of TNFRs by Western blot analysis as described in Materials and Methods. Results are expressed as receptor expression normalized with {beta}-actin as arbitrary units over the respective controls; bars indicate the SD in B. C, Effect of MDA-7 on the cell surface expression of TNFRs by flow cytometric analysis. One million cells were harvested and Ab-labeled for analysis of the expression of TNFR1/p60 (dotted lines) and TNFR2/p80 (broken lines). Cells were incubated first with mouse anti-TNFR mAbs, followed by FITC-conjugated goat anti-mouse mAb. The negative control (shaded) consisted of unstained cells. Cells labeled with second Ab alone are shown as solid lines.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
mda-7 is a melanoma differentiation associated gene whose product has been renamed IL-24. This cytokine has been shown to suppress the growth of tumor cells, induce inflammatory cytokines, and up-regulate the immune system. In the present report we demonstrate that MDA-7 expression alone does not activate NF-{kappa}B, but, instead, enhances TNF-induced NF-{kappa}B activation, as indicated by DNA binding, p65 nuclear translocation, and NF-{kappa}B-dependent reporter assay. MDA-7 also enhanced NF-{kappa}B reporter activation induced by TRADD and TRAF2. The secretion of MDA-7 was not required for the potentiation of TNF-induced NF-{kappa}B activation. TNF-induced expression of the NF-{kappa}B-regulated gene products cyclin D1 and COX-2 were significantly up-regulated by the expression of MDA-7. Furthermore, mda-7 transfection abolished TNF-induced apoptosis.

Our results clearly show that stable expression of MDA-7 can neither activate DNA binding of NF-{kappa}B nor induce NF-{kappa}B-dependent reporter gene expression. The up-regulation of TNF, IL-1, and IL-6 by MDA-7 in PBMC reported previously (21) thus must be NF-{kappa}B independent. Cell type-dependent activation of NF-{kappa}B has been reported previously (38). Thus, it is possible that MDA-7 can activate NF-{kappa}B in PBMC, but not in the 293 cells we used in current studies.

How MDA-7 up-regulates TNF-induced NF-{kappa}B activation is not clear. TNF-induced NF-{kappa}B activation requires the sequential recruitment of TNFR, TRADD, TRAF2, NIK, and IKK. MDA-7 enhanced the NF-{kappa}B reporter activity induced by TRADD and TRAF2, but had no significant effect on NIK-, IKK-, or p65-induced NF-{kappa}B activation. Thus, MDA-7 must act at a step downstream from TRAF2. MDA-7 has been shown to activate PKR (18). PKR has been shown to activate NF-{kappa}B by phosphorylating I{kappa}B{alpha} (39). More recently, PKR was shown to stimulate NF-{kappa}B regardless of its kinase function by interacting with the IKK (40, 41). PKR has also been implicated in the synergistic activation of NF-{kappa}B by TNF and IFN-{gamma} in preneuronal cells (42). Like MDA-7, IFN-{gamma} alone does not activate NF-{kappa}B (43). Thus, it is possible that the potentiation of TNF-induced NF-{kappa}B activation by MDA-7 is mediated through PKR.

Structurally, MDA-7 is a novel member of the IL-10 superfamily (8). However, it differs from IL-10 in that the latter inhibits NF-{kappa}B activation (44), whereas we show in this study that MDA-7 synergizes with other agents for NF-{kappa}B activation. Similarly, IL-10 inhibits TNF, IL-6, and IL-1 production (44), but MDA-7 induces it (21). MDA-7 also differs from IL-10 in that expression of MDA-7 is lost during melanoma development (1, 2, 3, 8), whereas the IL-10 level rises (2).

Our results indicate that the secretion of MDA-7 is not needed for it to synergize with TNF for NF-{kappa}B activation. MDA-7 has been shown to interact with two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2 (10). It is possible that these receptors are functioning in an intracrine manner as described for fibroblast growth factor and for platelet-derived growth factor (45). Our results also show that MDA-7 induces small, but significant, levels of TNFRs. It is unlikely that induction of TNFRs is responsible for enhanced NF-{kappa}B activation induced by MDA-7. Previously, we have shown that although TNFRs are induced by IFN-{gamma} (46), the induction of TNFRs is not normally rate-limiting in TNF signaling (47). Moreover, mda-7 expression in 293 cells does not induce TNF expression (data not shown).

Whether the effects of MDA-7 as described in this study are receptor mediated is not yet known. Because 293 cells do not express MDA-7 receptors, the effects of MDA-7 described in this study appear to be receptor independent. We have recently shown that intracellular MDA-7 expression can activate the unfolded protein response stress pathway (22) and the PKR-like kinase. Both PKR-like kinase and unfolded protein response have been shown to regulate NF-{kappa}B activation via phosphorylation of eukaryotic initiation factor-2{alpha}.

We found that MDA-7 enhanced the TNF-induced expression of cyclin D1 and COX-2; both of these gene products are known to be regulated by NF-{kappa}B (32, 33, 34). Cyclin D1, which is overexpressed in a wide variety of tumors, is a cell cycle protein required for transition of cells from G1 to S phase (33). In our study this novel activity of MDA-7 was seen only when it was combined with TNF. The latter has been shown to induce the proliferation of various tumor cells (47, 48, 49, 50). Whether MDA-7 can also induce the proliferation of cells is not clear. COX-2 is another gene product that is induced synergistically by MDA-7 together with TNF. COX-2 expression has also been linked with cell proliferation (51).

Our results indicate that TNF induces apoptosis and that the expression of mda-7 suppresses TNF-induced apoptosis. Because activation of NF-{kappa}B has been linked with suppression of apoptosis (35, 36, 37), it is possible that MDA-7 suppresses apoptosis through activation of NF-{kappa}B. The antiapoptotic effects of MDA-7 may also be mediated through the induction of cyclin D1 and COX-2 expression. Our results are in agreement with a previous report that MDA-7 can activate STAT3 (52), and STAT3 activation can also lead to expression of cyclin D1 (53) and suppression of apoptosis (54). Although MDA-7 is known to induce apoptosis in some tumor cells (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), it appears to suppress TNF-{alpha}-mediated apoptosis in 293 cells, as described in this study. Why MDA-7 alone induces apoptosis in some tumor cells, but not in 293 cells, is not clear. The balance between proapoptotic and antiapoptotic proteins plays a critical role in determining cell fate. Overexpression of the antiapoptotic protein E1B in 293 cells (9) may prevent MDA-7-mediated apoptosis in these cells. This is similar to other cytokines, such as TNF (23). The suppression of TNF-induced apoptosis by MDA-7 demonstrates a cross-talk between the two cytokines. Interestingly, both T cells and macrophages secrete TNF and also produce MDA-7; their relative expression levels may regulate cell fate. Overall, the studies we describe unveil several novel activities of MDA-7 and indicate that this novel cytokine can synergize with TNF for NF-{kappa}B activation and for NF-{kappa}B-mediated gene expression and can suppress the apoptotic effects of TNF.


   Footnotes
 
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 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Box 143, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail address: aggarwal{at}mdanderson.org Back

2 Abbreviations used in this paper: mda, melanoma differentiation-associated gene; COX, cyclooxygenase; IKK, I{kappa}B{alpha} kinase; NBD, NEMO binding domain peptide; NBDC, NEMO binding domain control peptide; NEMO, NF-{kappa}B essential modifier; NIK, NF-{kappa}B-inducing kinase; PKR, dsRNA-dependent protein kinase; SEAP, secretory alkaline phosphatase; TRADD, TNFR-associated death domain; TRAF, TNFR-associated factor. Back

Received for publication May 5, 2004. Accepted for publication August 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jiang, H., J. J. Lin, Z. Z. Su, N. I. Goldstein, P. B. Fisher. 1995. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 11:2477.[Medline]
  2. Ellerhorst, J. A., V. G. Prieto, S. Ekmekcioglu, L. Broemeling, S. Yekell, S. Chada, E. A. Grimm. 2002. Loss of MDA-7 expression with progression of melanoma. J. Clin. Oncol. 20:1069.[Abstract/Free Full Text]
  3. Ekmekcioglu, S., J. Ellerhorst, A. M. Mhashilkar, A. A. Sahin, C. M. Read, V. G. Prieto, S. Chada, E. A. Grimm. 2001. Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas. Int. J. Cancer 94:54.[Medline]
  4. Huang, E. Y., M. T. Madireddi, R. V. Gopalkrishnan, M. Leszczyniecka, Z. Su, I. V. Lebedeva, D. Kang, H. Jiang, J. J. Lin, D. Alexandre, et al 2001. Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties. Oncogene 20:7051.[Medline]
  5. Chaiken, I. M., W. V. Williams. 1996. Identifying structure-function relationships in four-helix bundle cytokines: towards de novo mimetics design. Trends Biotechnol. 14:369.[Medline]
  6. Kotenko, S. V., C. D. Krause, L. S. Izotova, B. P. Pollack, W. Wu, S. Pestka. 1997. Identification and functional characterization of a second chain of the interleukin-10 receptor complex. EMBO J. 16:5894.[Medline]
  7. Sauane, M., R. V. Gopalkrishnan, D. Sarkar, Z. Z. Su, I. V. Lebedeva, P. Dent, S. Pestka, P. B. Fisher. 2003. MDA-7/IL-24: novel cancer growth suppressing and apoptosis inducing cytokine. Cytokine Growth Factor Rev. 14:35.[Medline]
  8. Chada, S., R. B. Sutton, S. Ekmekcioglu, J. Ellerhorst, J. B. Mumm, W. W. Leitner, H. Y. Yang, A. A. Sahin, K. K. Hunt, K. L. Fuson, et al 2004. MDA-7/IL-24 is a unique cytokine-tumor suppressor in the IL-10 family. Int. Immunopharmacol. 4:649.[Medline]
  9. Jiang, H., Z. Z. Su, J. J. Lin, N. I. Goldstein, C. S. Young, P. B. Fisher. 1996. The melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc. Natl. Acad. Sci. USA 93:9160.[Abstract/Free Full Text]
  10. Wang, M., Z. Tan, R. Zhang, S. V. Kotenko, P. Liang. 2002. Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2. J. Biol. Chem. 277:7341.[Abstract/Free Full Text]
  11. Su, Z. Z., M. T. Madireddi, J. J. Lin, C. S. Young, S. Kitada, J. C. Reed, N. I. Goldstein, P. B. Fisher. 1998. The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc. Natl. Acad. Sci. USA 95:14400.[Abstract/Free Full Text]
  12. Lebedeva, I. V., Z. Z. Su, Y. Chang, S. Kitada, J. C. Reed, P. B. Fisher. 2002. The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells. Oncogene 21:708.[Medline]
  13. Saeki, T., A. Mhashilkar, X. Swanson, X. H. Zou-Yang, K. Sieger, S. Kawabe, C. D. Branch, L. Zumstein, R. E. Meyn, J. A. Roth, et al 2002. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene 21:4558.[Medline]
  14. Su, Z., I. V. Lebedeva, R. V. Gopalkrishnan, N. I. Goldstein, C. A. Stein, J. C. Reed, P. Dent, P. B. Fisher. 2001. A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells. Proc. Natl. Acad. Sci. USA 98:10332.[Abstract/Free Full Text]
  15. Saeki, T., A. Mhashilkar, S. Chada, C. Branch, J. A. Roth, R. Ramesh. 2000. Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther. 7:2051.[Medline]
  16. Su, Z. Z., I. V. Lebedeva, D. Sarkar, R. V. Gopalkrishnan, M. Sauane, C. Sigmon, A. Yacoub, K. Valerie, P. Dent, P. B. Fisher. 2003. Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in a p53-independent manner. Oncogene 22:1164.[Medline]
  17. Cao, X. X., I. Mohuiddin, S. Chada, A. M. Mhashilkar, M. K. Ozvaran, D. J. McConkey, S. D. Miller, J. C. Daniel, W. R. Smythe. 2002. Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Mol. Med. 8:869.[Medline]
  18. Pataer, A., S. A. Vorburger, G. N. Barber, S. Chada, A. M. Mhashilkar, H. Zou-Yang, A. L. Stewart, S. Balachandran, J. A. Roth, K. K. Hunt, et al 2002. Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR). Cancer Res. 62:2239.[Abstract/Free Full Text]
  19. Sarkar, D., Z. Z. Su, I. V. Lebedeva, M. Sauane, R. V. Gopalkrishnan, K. Valerie, P. Dent, P. B. Fisher. 2002. Mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc. Natl. Acad. Sci. USA 99:10054.[Abstract/Free Full Text]
  20. Ramesh, R., A. M. Mhashilkar, F. Tanaka, Y. Saito, C. D. Branch, K. Sieger, J. B. Mumm, A. L. Stewart, A. Boquoi, L. Dumoutier, et al 2003. Melanoma differentiation-associated gene 7/interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor. Cancer Res. 63:5105.[Abstract/Free Full Text]
  21. Caudell, E. G., J. B. Mumm, N. Poindexter, S. Ekmekcioglu, A. M. Mhashilkar, X. H. Yang, M. W. Retter, P. Hill, S. Chada, E. A. Grimm. 2002. The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24. J. Immunol. 168:6041.[Abstract/Free Full Text]
  22. Sieger, K. A., A. M. Mhashilkar, A. Stewart, R. B. Sutton, R. W. Strube, S. Y. Chen, A. Pataer, S. G. Swisher, E. A. Grimm, R. Ramesh, et al 2004. The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells. Mol. Ther. 9:355.[Medline]
  23. Aggarwal, B. B.. 2003. Signalling pathways of the TNF superfamily: a double-edged sword. Nat. Rev. Immunol. 3:745.[Medline]
  24. Liao, Y. C., W. G. Liang, F. W. Chen, J. H. Hsu, J. J. Yang, M. S. Chang. 2002. IL-19 induces production of IL-6 and TNF-{alpha} and results in cell apoptosis through TNF-{alpha}. J. Immunol. 169:4288.[Abstract/Free Full Text]
  25. Chaturvedi, M. M., A. Mukhopadhyay, B. B. Aggarwal. 2000. Assay for redox-sensitive transcription factor. Methods Enzymol. 319:585.[Medline]
  26. Manna, S. K., A. Mukhopadhyay, B. B. Aggarwal. 2000. Leflunomide suppresses TNF-induced cellular responses: effects on NF-{kappa}B, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis. J. Immunol. 165:5962.[Abstract/Free Full Text]
  27. Manna, S. K., A. Mukhopadhyay, B. B. Aggarwal. 2000. IFN-{alpha} suppresses activation of nuclear transcription factors NF-{kappa}B and activator protein 1 and potentiates TNF-induced apoptosis. J. Immunol. 165:4927.[Abstract/Free Full Text]
  28. Darnay, B. G., J. Ni, P. A. Moore, B. B. Aggarwal. 1999. Activation of NF-{kappa}B by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-{kappa}B-inducing kinase: identification of a novel TRAF6 interaction motif. J. Biol. Chem. 274:7724.[Abstract/Free Full Text]
  29. Garg, A., B. B. Aggarwal. 2002. Nuclear transcription factor-{kappa}B as a target for cancer drug development. Leukemia 16:1053.[Medline]
  30. Karin, M., Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu. Rev. Immunol. 18:621.[Medline]
  31. Nasuhara, Y., I. M. Adcock, M. Catley, P. J. Barnes, R. Newton. 1999. Differential I{kappa}B kinase activation and I{kappa}B{alpha} degradation by interleukin-1{beta} and tumor necrosis factor-{alpha} in human U937 monocytic cells: evidence for additional regulatory steps in {kappa}B-dependent transcription. J. Biol. Chem. 274:19965.[Abstract/Free Full Text]
  32. Guttridge, D. C., C. Albanese, J. Y. Reuther, R. G. Pestell, A. S. Baldwin, Jr. 1999. NF-{kappa}B controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol. Cell. Biol. 19:5785.[Abstract/Free Full Text]
  33. Hinz, M., D. Krappmann, A. Eichten, A. Heder, C. Scheidereit, M. Strauss. 1999. NF-{kappa}B function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol. Cell. Biol. 19:2690.[Abstract/Free Full Text]
  34. Yamamoto, K., T. Arakawa, N. Ueda, S. Yamamoto. 1995. Transcriptional roles of nuclear factor {kappa}B and nuclear factor-interleukin-6 in the tumor necrosis factor {alpha}-dependent induction of cyclooxygenase-2 in MC3T3–E1 cells. J. Biol. Chem. 270:31315.[Abstract/Free Full Text]
  35. Van Antwerp, D. J., S. J. Martin, T. Kafri, D. R. Green, I. M. Verma. 1996. Suppression of TNF-{alpha}-induced apoptosis by NF-{kappa}B. Science 274:787.[Abstract/Free Full Text]
  36. Beg, A. A., D. Baltimore. 1996. An essential role for NF-{kappa}B in preventing TNF-{alpha}-induced cell death. Science 274:782.[Abstract/Free Full Text]
  37. Wang, C. Y., M. W. Mayo, A. S. Baldwin, Jr. 1996. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-{kappa}B. Science 274:784.[Abstract/Free Full Text]
  38. Bonizzi, G., J. Piette, M. P. Merville, V. Bours. 1997. Distinct signal transduction pathways mediate nuclear factor-{kappa}B induction by IL-1{beta} in epithelial and lymphoid cells. J. Immunol. 159:5264.[Abstract]
  39. Kumar, A., J. Haque, J. Lacoste, J. Hiscott, B. R. Williams. 1994. Double-stranded RNA-dependent protein kinase activates transcription factor NF-{kappa}B by phosphorylating I{kappa}B. Proc. Natl. Acad. Sci. USA 91:6288.[Abstract/Free Full Text]
  40. Bonnet, M. C., R. Weil, E. Dam, A. G. Hovanessian, E. F. Meurs. 2000. PKR stimulates NF-{kappa}B irrespective of its kinase function by interacting with the I{kappa}B kinase complex. Mol. Cell Biol. 20:4532.[Abstract/Free Full Text]
  41. Ishii, T., H. Kwon, J. Hiscott, G. Mosialos, A. E. Koromilas. 2001. Activation of the I{kappa}B{alpha} kinase (IKK) complex by double-stranded RNA-binding defective and catalytic inactive mutants of the interferon-inducible protein kinase PKR. Oncogene 20:1900.[Medline]
  42. Cheshire, J. L., B. R. Williams, A. S. Baldwin, Jr. 1999. Involvement of double-stranded RNA-activated protein kinase in the synergistic activation of nuclear factor-{kappa}B by tumor necrosis factor-{alpha} and {gamma}-interferon in preneuronal cells. J. Biol. Chem. 274:4801.[Abstract/Free Full Text]
  43. Chaturvedi, M. M., M. Higuchi, B. B. Aggarwal. 1994. Effect of tumor necrosis factors, interferons, interleukins, and growth factors on the activation of NF-{kappa}B: evidence for lack of correlation with cell proliferation. Lymphokine Cytokine Res. 13:309.[Medline]
  44. Wang, P., P. Wu, M. I. Siegel, R. W. Egan, M. M. Billah. 1995. Interleukin (IL)-10 inhibits nuclear factor {kappa}B (NF{kappa}B) activation in human monocytes: IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J. Biol. Chem. 270:9558.[Abstract/Free Full Text]
  45. Jouanneau, J., J. Gavrilovic, D. Caruelle, M. Jaye, G. Moens, J. P. Caruelle, J. P. Thiery. 1991. Secreted or nonsecreted forms of acidic fibroblast growth factor produced by transfected epithelial cells influence cell morphology, motility, and invasive potential. Proc. Natl. Acad. Sci. USA 88:2893.[Abstract/Free Full Text]
  46. Aggarwal, B. B., T. E. Eessalu, P. E. Hass. 1985. Characterization of receptors for human tumour necrosis factor and their regulation by {gamma}-interferon. Nature 318:665.[Medline]
  47. Aggarwal, B. B., T. E. Eessalu. 1987. Induction of receptors for tumor necrosis factor-{alpha} by interferons is not a major mechanism for their synergistic cytotoxic response. J. Biol. Chem. 262:10000.[Abstract/Free Full Text]
  48. Sugarman, B. J., B. B. Aggarwal, P. E. Hass, I. S. Figari, M. A. Palladino, Jr, H. M. Shepard. 1985. Recombinant human tumor necrosis factor-{alpha}: effects on proliferation of normal and transformed cells in vitro. Science 230:943.[Abstract/Free Full Text]
  49. Giri, D. K., B. B. Aggarwal. 1998. Constitutive activation of NF-{kappa}B causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells: autocrine role of tumor necrosis factor and reactive oxygen intermediates. J. Biol. Chem. 273:14008.[Abstract/Free Full Text]
  50. Aggarwal, B. B., L. Schwarz, M. E. Hogan, R. F. Rando. 1996. Triple helix-forming oligodeoxyribonucleotides targeted to the human tumor necrosis factor (TNF) gene inhibit TNF production and block the TNF-dependent growth of human glioblastoma tumor cells. Cancer Res. 56:5156.[Abstract/Free Full Text]
  51. Frungieri, M. B., S. Weidinger, V. Meineke, F. M. Kohn, A. Mayerhofer. 2002. Proliferative action of mast-cell tryptase is mediated by PAR2, COX2, prostaglandins, and PPAR{gamma}: possible relevance to human fibrotic disorders. Proc. Natl. Acad. Sci. USA 99:15072.[Abstract/Free Full Text]
  52. Dumoutier, L., C. Leemans, D. Lejeune, S. V. Kotenko, J. C. Renauld. 2001. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J. Immunol. 167:3545.[Abstract/Free Full Text]
  53. Masuda, M., M. Suzui, R. Yasumatu, T. Nakashima, Y. Kuratomi, K. Azuma, K. Tomita, S. Komiyama, I. B. Weinstein. 2002. Constitutive activation of signal transducers and activators of transcription 3 correlates with cyclin D1 overexpression and may provide a novel prognostic marker in head and neck squamous cell carcinoma. Cancer Res. 62:3351.[Abstract/Free Full Text]
  54. Greten, F. R., C. K. Weber, T. F. Greten, G. Schneider, M. Wagner, G. Adler, R. M. Schmid. 2002. Stat3 and NF-{kappa}B activation prevents apoptosis in pancreatic carcinogenesis. Gastroenterology 123:2052.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
T. Matsumiya, S. M. Prescott, and D. M. Stafforini
IFN-{epsilon} Mediates TNF-{alpha}-Induced STAT1 Phosphorylation and Induction of Retinoic Acid-Inducible Gene-I in Human Cervical Cancer Cells
J. Immunol., October 1, 2007; 179(7): 4542 - 4549.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
Y. Oida, B. Gopalan, R. Miyahara, C. D. Branch, P. Chiao, S. Chada, and R. Ramesh
Inhibition of nuclear factor-{kappa}B augments antitumor activity of adenovirus-mediated melanoma differentiation-associated gene-7 against lung cancer cells via mitogen-activated protein kinase kinase kinase 1 activation
Mol. Cancer Ther., April 1, 2007; 6(4): 1440 - 1449.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
S. Shishodia, G. Sethi, M. Konopleva, M. Andreeff, and B. B. Aggarwal
A Synthetic Triterpenoid, CDDO-Me, Inhibits I{kappa}B{alpha} Kinase and Enhances Apoptosis Induced by TNF and Chemotherapeutic Agents through Down-Regulation of Expression of Nuclear Factor {kappa}B-Regulated Gene Products in Human Leukemic Cells
Clin. Cancer Res., March 15, 2006; 12(6): 1828 - 1838.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
S. Aggarwal, S.-W. Kim, K. Cheon, F. H. Tabassam, J.-H. Yoon, and J. S. Koo
Nonclassical Action of Retinoic Acid on the Activation of the cAMP Response Element-binding Protein in Normal Human Bronchial Epithelial Cells
Mol. Biol. Cell, February 1, 2006; 17(2): 566 - 575.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. Floryk and E. Huberman
Differentiation of Androgen-Independent Prostate Cancer PC-3 Cells Is Associated with Increased Nuclear Factor-{kappa}B Activity
Cancer Res., December 15, 2005; 65(24): 11588 - 11596.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aggarwal, S.
Right arrow Articles by Aggarwal, B. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aggarwal, S.
Right arrow Articles by Aggarwal, B. B.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS