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 Donepudi, M.
Right arrow Articles by Mokyr, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Donepudi, M.
Right arrow Articles by Mokyr, M. B.
The Journal of Immunology, 2001, 166: 6491-6499.
Copyright © 2001 by The American Association of Immunologists

Mechanism of Melphalan-Induced B7-1 Gene Expression in P815 Tumor Cells1

Manjula Donepudi2,*, Pradip Raychaudhuri*, Jeffrey A. Bluestone{dagger} and Margalit B. Mokyr3,*

* Department of Biochemistry and Molecular Biology, University of Illinois, Chicago, IL 60612; and {dagger} University of California, San Francisco Diabetes Center, Metabolic Research Unit/Hormone Research Institute, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that exposure of P815 tumor cells to melphalan (L-phenylalanine mustard; L-PAM) leads to up-regulation of B7-1 surface expression, and this L-PAM-induced up-regulation requires de novo RNA synthesis and is associated with accumulation of B7-1 mRNA. Here we show that the effect of L-PAM on B7-1 surface expression can be mimicked by exposing P815 tumor cells to oxidative stress but not to heat shock. Moreover, the antioxidant N-acetyl-L-cysteine prevented the L-PAM-induced accumulation of B7-1 mRNA in P815 tumor cells, suggesting that reactive oxygen species are involved in the transcriptional regulation of L-PAM-induced B7-1 gene expression. Although AP-1 and NF-{kappa}B are regarded as redox-sensitive transcription factors and the promoter/enhancer region of the B7-1 gene contains an AP-1 and an NF-{kappa}B binding site, exposure of P815 tumor cells to L-PAM led to rapid and transient activation only of NF-{kappa}B, but not AP-1, that bound specifically to a probe containing the respective binding site in the murine or human B7-1 gene. Moreover, exposure of P815 tumor cells to a cell-permeable peptide that selectively inhibits NF-{kappa}B activation by blocking the activation of the I{kappa}B-kinase complex was found to inhibit the L-PAM-induced B7-1 mRNA accumulation, indicating that NF-{kappa}B activation is essential for the L-PAM-induced B7-1 gene expression. Taken together, these results indicate that L-PAM leads to activation of B7-1 gene expression by activating NF-{kappa}B via a pathway that involves reactive oxygen species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability of anticancer drugs to facilitate the acquisition of antitumor immunity by tumor bearers has been recognized for quite some time (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Still, little is known about the role of the costimulatory molecules B7-1 and B7-2 in chemotherapy-induced potentiation of antitumor immunity in tumor bearers. Recently we have initiated studies to elucidate the importance of B7-1 and B7-2 expression for melphalan (L-phenylalanine mustard; L-PAM)4-induced acquisition of tumor-eradicating immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor with extensive metastases. Our studies revealed that the B7-1 and B7-2 molecules are important for the therapeutic outcome of low-dose L-PAM for MOPC-315 tumor bearers under conditions that depend on chemotherapy-induced acquisition of tumor-eradicating immunity by the tumor bearers (11). In addition, our studies illustrated that low-dose L-PAM leads to up-regulation of B7-1 and B7-2 surface expression on both tumor cells and host cells from MOPC-315 tumor bearers (11).

Numerous investigators observed that B7-2 expression was up-regulated earlier than B7-1 following stimulation of APCs with a variety of different stimuli including cytokines (e.g., GM-CSF), anti-CD40, or surface Ig ligation (12, 13, 14). Opposite expression patterns of these costimulatory molecules were observed following L-PAM therapy of MOPC-315 tumor bearers (15). Specifically, B7-1 was up-regulated at the tumor site within 24 h after L-PAM treatment (15), whereas B7-2 was up-regulated after 48 h (11), which is similar to the time of up-regulation of B7-2 expression on APC exposed to the other stimuli (12, 13, 14). In light of these observations, we considered the possibility that in contrast to B7-2, which is up-regulated indirectly by L-PAM (i.e., L-PAM induces TNF-{alpha} production (16) that in turn leads to up-regulation of B7-2 surface expression; Ref. 8, 17), B7-1 may be up-regulated by direct action of L-PAM on MOPC-315 tumor cells. To test this hypothesis, we determined initially whether in vitro exposure of MOPC-315 tumor cells from untreated mice to L-PAM results in rapid up-regulation of B7-1, but not B7-2, surface expression. Our studies revealed that within 24 h after in vitro exposure of MOPC-315 tumor cells to L-PAM, the tumor cells exhibited up-regulated B7-1 surface expression and unaltered B7-2 surface expression, which was high to start with (15). This effect of L-PAM was not restricted to MOPC-315 tumor cells, as preferential up-regulation of B7-1 surface expression was also observed within 24 h after in vitro exposure of P815 tumor cells to L-PAM (15). Subsequent studies into the mechanism through which L-PAM leads to up-regulation of B7-1 surface expression used P815 tumor cells because, unlike MOPC-315 tumor cells, P815 tumor cells do not constitutively express either the B7-1 or the B7-2 molecule on their surface (15). These studies revealed that up-regulation of B7-1 surface expression following in vitro exposure of tumor cells to L-PAM requires de novo RNA synthesis and is associated with rapid accumulation of B7-1 mRNA (15), indicating that the regulation of B7-1 expression is at the transcriptional level.

The observations that L-PAM activated B7-1 gene expression in tumor cells, which has important implications for an additional immune potentiating mechanism for L-PAM, prompted us to initiate studies to elucidate the mechanisms through which L-PAM leads to activation of B7-1 gene expression in tumor cells. Here we show that reactive oxygen species are involved in the transcriptional regulation of L-PAM-induced B7-1 gene expression. In addition, we illustrate that L-PAM leads to rapid and transient activation of the redox-sensitive transcription factor NF-{kappa}B, but not AP-1, which binds specifically to its cognate element in the enhancer/promoter region of the B7-1 gene. Moreover, NF-{kappa}B activation is shown to be essential for the L-PAM-induced B7-1 gene expression.


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

We have used primarily the P815 mastocytoma, which under normal conditions is negative for B7-1 and B7-2 surface expression (15, 18). P815 tumor cells were maintained in vitro, as previously described (15, 18), in low glucose DMEM supplemented with 10% FBS (Life Technologies, Grand Island, NY). In a few experiments we used the MOPC-315 plasmacytoma, which under normal conditions is negative for B7-1 surface expression but expresses relatively high levels of B7-2 (15). MOPC-315 tumor cells were maintained in vivo, as previously described (15, 19), in BALB/cAnNCrlBR mice 7–10 wk old, that were purchased from Charles River Breeding Laboratories (Wilmington, MA).

In vitro treatments of tumor cells

In the first set of experiments, P815 or MOPC-315 tumor cells were exposed in vitro to H2O2 for 15 min at a concentration ranging from 0.01 to 1.0 mM or exposed in vitro for 2 h to 42°C and subsequently cultured for 22–24 h at 37°C as previously described (15). In most experiments, P815 tumor cells were exposed in vitro to 15–30 nM L-PAM (Sigma, St. Louis, MO) for 30 min, unless otherwise stated, and then cultured for the indicated periods of time. In experiments evaluating the importance of reactive oxygen species for the L-PAM effect, P815 tumor cells were pretreated with the antioxidant N-acetyl-L-cysteine (NAC) for 1 h before their exposure to L-PAM as well as during the L-PAM treatment. A concentration of 25 mM NAC was chosen for these experiments based on reports by other investigators with other cell types and other treatment modalities (20, 21). Finally, in experiments assessing the importance of NF-{kappa}B activation for the L-PAM-induced B7-1 gene expression, we used a cell-permeable peptide of the following sequence DRQIKIWFQNRRMKWKKTALDWSWLQTE, which was shown to block the binding of the NF-{kappa}B essential modifier (NEMO) to the I{kappa}B-kinase (IKK) complex, thereby selectively inhibiting NF-{kappa}B activation (22). Specifically, P815 tumor cells were exposed to 250 µM of this peptide (which will be referred to as wild-type peptide) for 3 h before their exposure to L-PAM as well as during the L-PAM treatment. As control, P815 tumor cells were exposed for 3 h before the exposure to L-PAM as well as during the L-PAM treatment to 250 µM of a mutant peptide (DRQIKIWFQNRRMKWKKTALDASALQTE) in which W was substituted with A in the two underlined positions and, consequently, the peptide does not inhibit NEMO binding to the IKK complex (22).

Flow-cytometric analysis

B7-1 and B7-2 expression on P815 or MOPC-315 tumor cells was assessed, as previously described (15), through the use of PE-conjugated anti-B7-1 (16-10A1) and PE-conjugated anti-B7-2 (GL-1) mAb (PharMingen, San Diego, CA), respectively. As a control, we used the appropriate PE-conjugated isotype-matched normal IgG (NIgG; PharMingen). Flow-cytometric analysis of 10,000 viable cells was conducted on a Coulter EPICS Elite ESP (Coulter Electronics, Hialeah, FL). Each experiment was repeated at least three times, and the results of a representative experiment are provided in the form of a histogram.

RT-PCR

Total RNA was extracted from P815 tumor cells and subjected to reverse transcription followed by PCR with sense and antisense primers for B7-1, as previously described (15). {beta}-Actin (Stratagene, La Jolla, CA) served as a standard to normalize for the quantity of mRNA subjected to PCR in the various samples within the same experiment. PCR products were separated by electrophoresis on a 1% agarose gel containing ethidium bromide and visualized by UV light. The sizes of the PCR products were determined using a standard 100-bp DNA ladder (Life Technologies) and were found to be of the expected size. Each experiment was performed at least three times, and the results of a representative experiment are provided.

EMSA

Nuclear extracts were prepared according to the method of Osborn et al. (23), and 1 µg of nuclear proteins was incubated for 20 min at 25°C with 50 fM of a 32P-labeled double-stranded oligonucleotide probe. In experiments evaluating the activation of AP-1 or NF-{kappa}B that binds to the enhancer/promoter region of the murine B7-1 gene we used a probe corresponding to positions -124 to -103 that includes the consensus AP-1 binding site (5'-TCTAGTGTTAGTCACCCCACCC-3'), or a probe corresponding to positions -1455 to -1434 that includes the consensus NF-{kappa}B binding site (5'-GGAAAGGGGAAATTCCTGCCCC-3') (24). In experiments evaluating the activation of NF-{kappa}B that binds to the enhancer region of the human B7-1 gene we used a probe that corresponds to positions -2969 to -2945 that includes the consensus NF-{kappa}B binding site (5'-GGGAAAGGGGTTTTCCCAGCAGTCA-3') (25). Binding reactions contained 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 4% glycerol, 0.1% Nonidet P-40, and 0.1–0.250 µg poly(dI:dC). The free and protein-bound oligonucleotide probes were separated by electrophoresis on 5% polyacrylamide gel. Subsequently, the gels were dried and the bands were visualized by autoradiography. The specificity of binding was examined by competition with a 25-fold molar excess of the unlabeled specific oligonucleotide, and by competition with a 25-fold excess of an unlabeled mutant oligonucleotide in which the G in the third position from the 5' end of the NF-{kappa}B binding site was substituted with a C. Finally, each experiment was performed at least three times, and the results of a representative experiment are presented.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of exposure of tumor cells to H2O2 or heat shock on B7-1 and B7-2 surface expression

We have previously shown that in vitro exposure of MOPC-315 or P815 tumor cells to L-PAM, {gamma}-irradiation, or mitomycin C leads to selective up-regulation of B7-1 surface expression, and that this up-regulation of B7-1 expression occurs at the transcriptional level (15). Because reactive oxygen species were reported to be induced following exposure of cells to these anticancer modalities (26, 27, 28, 29), and to be important mediators of gene activation (20, 26, 28), experiments were undertaken to determine whether exposure of MOPC-315 or P815 tumor cells to H2O2 can also lead to selective up-regulation of B7-1 surface expression. Accordingly, MOPC-315 or P815 tumor cells were exposed in vitro for 15 min to H2O2 and then cultured for an additional 22–24 h before B7-1 or B7-2 surface expression was assessed. Because similar results were obtained with MOPC-315 and P815 tumor cells, we elected to present the data obtained with P815 tumor cells because untreated P815 tumor cells, unlike untreated MOPC-315 tumor cells, are negative not only for B7-1surface expression but also for B7-2 surface expression (15, 18). As seen in Fig. 1GoA, in vitro exposure of P815 tumor cells to H2O2 at a concentration ranging from 0.03 to 1.0 mM led to up-regulated B7-1 surface expression. The same preparation of P815 tumor cells remained negative for B7-2 surface expression even when exposed to 1.0 mM H2O2 (Fig. 1GoB). Thus, H2O2 mimics the effect of L-PAM, {gamma}-irradiation, and mitomycin C on B7-1 surface expression on tumor cells.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 1. Exposure of P815 cells to H2O2 leads to up-regulation of B7-1, but not B7-2, expression. P815 tumor cells were exposed in vitro for 15 min to H2O2 at a concentration ranging from 0.01 to 1.0 mM and subsequently cultured for an additional 22–24 h. At the end of the culture period, tumor cells treated with H2O2 as well as untreated tumor cells were stained with PE-conjugated NIgG (dotted line), or with PE-conjugated anti-B7-1 mAb (bold solid line) (A). P815 tumor cells that were exposed in vitro to 1.0 mM H2O2 as well as untreated tumor cells were also stained with PE-conjugated NIgG (dotted line) or with PE-conjugated anti-B7-2 mAb (bold solid line) (B).

 
Experiments were next conducted to determine whether exposure of P815 tumor cells to a different type of stress, heat shock, would also result in selective up-regulation of B7-1 surface expression. For this purpose P815 tumor cells were incubated for 2 h at 42°C and then transferred to 37°C for an additional 22–24 h, before B7-1 and B7-2 surface expression was assessed. As a reference point, the same batch of P815 tumor cells was exposed in vitro to L-PAM or H2O2. As seen in Fig. 2Go, although exposure of P815 tumor cells to L-PAM or H2O2 led to up-regulated B7-1 surface expression, heat shock treatment did not lead to elevated B7-1 surface expression. Thus, not all types of stress lead to elevated B7-1 surface expression on P815 tumor cells.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 2. Exposure of P815 cells to H2O2 or L-PAM, but not heat shock, leads to up-regulation of B7-1 surface expression. P815 cells were exposed in vitro to H2O2 (0.1 mM for 15 min), L-PAM (15 nM for 1 h), or heat shock (42°C for 2 h), and subsequently cultured for an additional 22–24 h. At the end of the culture period, tumor cells exposed to the different treatment modalities as well as untreated tumor cells were stained with PE-conjugated NIgG (dotted line) or with PE-conjugated anti-B7-1 mAb (bold solid line).

 
Effect of the antioxidant NAC on L-PAM-induced up-regulation of B7-1 gene expression in P815 tumor cells

Our observations that exposure of P815 tumor cells to H2O2 (but not to heat shock) leads to selective up-regulation of B7-1 surface expression, coupled with reports by other investigators that L-PAM can increase the levels of intracellular reactive oxygen species (30), prompted us to determine whether reactive oxygen species are involved in the L-PAM-induced up-regulation of B7-1 gene expression. Accordingly, we determined whether the antioxidant NAC would interfere with the ability of L-PAM to lead to up-regulation of B7-1 expression. For this purpose, we incubated P815 tumor cells with NAC for 1 h before their exposure to L-PAM as well as during the 1-h in vitro L-PAM treatment. Four hours after initiation of the L-PAM treatment, we evaluated the effect of NAC on B7-1 mRNA expression as an indication of the effect of NAC on B7-1 surface expression, because L-PAM-induced B7-1 surface expression on P815 tumor cells was previously shown to require de novo RNA synthesis and to be associated with accumulation of mRNA for B7-1 (15). In these studies, which used RT-PCR, we used RNA from B7-1-transfected P815 tumor cells (15, 31) as a positive control (Fig. 3Go, lane 1). As seen in Fig. 3Go, and in confirmation of our previous observations (15), untreated P815 tumor cells were negative for B7-1 mRNA expression (lane 2); however, a substantial level of B7-1 mRNA was evident by 4 h after initiation of the L-PAM treatment (lane 3). Treatment of P815 tumor cells with NAC prevented the L-PAM-induced accumulation of B7-1 mRNA (lane 4). These results indicate that reactive oxygen intermediates are involved in the transcriptional regulation of L-PAM-induced B7-1 gene expression.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3. The antioxidant NAC inhibits L-PAM-induced accumulation B7-1 mRNA. RNA derived from untreated P815 tumor cells (lane 2) or P815 tumor cells treated with L-PAM with (lane 4) or without (lane 3) NAC were subjected to RT-PCR with primers specific for B7-1 or {beta}-actin. RNA obtained from B7-1-transfected P815 tumor cells served as positive control (lane 1). The size of the PCR product for B7-1 was ~300 bp.

 
Assessment of the ability of L-PAM to activate AP-1 and/or NF-{kappa}B that bind specifically to the AP-1 or the NF-{kappa}B binding site in the promoter/enhancer of the murine B7-1 gene

Because 1) NF-{kappa}B and AP-1 are regarded as redox-sensitive transcription factors (26, 28, 32, 33, 34), and 2) the promoter/enhancer region of the B7-1 gene has an AP-1 and an NF-{kappa}B binding site (24, 35, 36), experiments were conducted to determine whether exposure of P815 tumor cells to L-PAM leads to activation of AP-1 and/or NF-{kappa}B that bind specifically to a probe containing the AP-1 or NF-{kappa}B binding site in the promoter/enhancer of the murine B7-1 gene. For this purpose, nuclear extracts were prepared from untreated P815 tumor cells or L-PAM-treated P815 tumor cells 30 or 60 min after initiation of the L-PAM treatment. The nuclear extracts were then evaluated for binding to a 32P-labeled double stranded oligonucleotide probe corresponding to 1) positions -124 to -103 or 2) positions -1455 to -1434 of the promoter/enhancer region of the murine B7-1 gene that includes the consensus AP-1 binding site (5'-TTAGTCA-3') or the consensus NF-{kappa}B binding site (5'-GGGAAATTCC-3'), respectively. As seen in Fig. 4Go, which provides the EMSA results with the probe containing the AP-1 binding site, although a protein-DNA complex was evident in the positive control lane with nuclear extract from HeLa cells (lane 1, panel B), a protein-DNA complex was not evident with nuclear extract obtained from untreated P815 tumor cells (lane 1, panel A) or with nuclear extract obtained from P815 tumor cells exposed in vitro to L-PAM 30 (lane 2, panel A) or 60 (lane 3, panel A) min earlier. The specificity of the protein-DNA complex with nuclear extract from HeLa cells is indicated by the fact that the complex was competed out with a 25-fold molar excess of the specific competitor (lane 2, panel B) but not with a 25-fold molar excess of an unrelated competitor (lane 3, panel B). These results illustrate that exposure of P815 tumor cells to L-PAM does not activate AP-1 that binds to the AP-1 binding site in the promoter of the murine B7-1 gene.



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 4. Nuclear extracts from L-PAM-treated P815 tumor cells do not exhibit increased binding to the AP-1 binding site in the promoter of the murine B7-1 gene. Nuclear extracts were obtained from untreated P815 tumor cells (lane 1) or L-PAM-treated P815 tumor cells 30 (lane 2) or 60 (lane 3) min after initiation of the L-PAM treatment (A). EMSA were performed using 1 µg nuclear protein and 50 fM of a 32P-labeled DNA probe containing the AP-1 binding site in the enhancer of the murine B7-1 gene. As a positive control, we performed EMSA with nuclear proteins from HeLa cells (lane 1, panel B). Specificity of the shifted protein-DNA complex was assessed by incubating nuclear proteins (1 µg) from HeLa cells with a 25-fold molar excess of unlabeled specific DNA probe (lane 2) or a 25-fold molar excess of unlabeled unrelated DNA probe (NF-{kappa}B, lane 3) (B).

 
The same nuclear extracts were simultaneously evaluated for binding to a probe containing the NF-{kappa}B binding site. As seen in Fig. 5GoA, two retarded bands of protein-DNA complexes were evident with nuclear extract from untreated P815 tumor cells (lane 1). However, both of these retarded bands of protein-DNA complexes were much more intense with nuclear extract obtained from P815 tumor cells exposed to L-PAM 30 min earlier (lane 2), but not with nuclear extract obtained from P815 tumor cells exposed to L-PAM 60 min earlier (lane 3). The specificity of the two protein-DNA complexes is indicated by the fact that the upper and lower complexes were competed out with a 25-fold molar excess of the specific competitor (Fig. 5GoB, lane 2) but not with a 25-fold molar excess of the mutant competitor (Fig. 5GoB, lane 3). Thus, exposure of P815 tumor cells to L-PAM leads to rapid and transient activation of NF-{kappa}B, but not AP-1, that binds specifically to the NF-{kappa}B binding site in the enhancer of the murine B7-1 gene.



View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 5. Nuclear extracts from L-PAM-treated P815 tumor cells exhibit increased binding to the NF-{kappa}B binding site in the enhancer of the murine B7-1 gene. Nuclear extracts were obtained from untreated P815 tumor cells (lane 1) or L-PAM-treated P815 tumor cells 30 (lane 2) or 60 (lane 3) min after initiation of the L-PAM treatment (A). EMSA were performed using 1 µg nuclear protein and 50 fM of a 32P-labeled DNA probe containing the NF-{kappa}B binding site in the enhancer of the murine B7-1 gene. Specificity of the shifted protein-DNA complexes was assessed by incubating nuclear proteins (1 µg) obtained from P815 cells that were exposed 30 min earlier to L-PAM (lane 1) with a 25-fold molar excess of unlabeled specific DNA probe (lane 2) or a 25-fold molar excess of unlabeled mutant DNA probe (lane 3) (B).

 
Assessment of the ability of L-PAM to activate NF-{kappa}B that binds specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene

In light of a recent report by Vereecque et al. (29) that exposure of primary cultures of human acute myeloid leukemic cells to {gamma}-irradiation induces up-regulation of B7-1 expression at both the protein and mRNA level, we conducted studies to determine whether nuclear extract from P815 tumor cells exposed in vitro to L-PAM can bind specifically to a probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene, which is not identical in sequence to NF-{kappa}B binding site in the enhancer of the murine B7-1 gene (24, 25). Accordingly, nuclear extracts obtained from untreated P815 tumor cells or L-PAM-treated P815 tumor cells 30 or 60 min after initiation of the L-PAM treatment were evaluated for their ability to bind to a 32P-labeled double stranded oligonucleotide probe corresponding to positions -2969 to -2945 in the enhancer of the human B7-1 gene that includes the consensus NF-{kappa}B binding site (5'-GGGGTTTTCC-3'). As seen in Fig. 6GoA, a single retarded band of protein-DNA complex was evident with nuclear extract from untreated P815 tumor cells (lane 1). The intensity of this band was greatly increased with nuclear extract from P815 tumor cells exposed 30 min earlier (lane 2), but not 60 min earlier (lane 3), to L-PAM. Two additional bands of protein-DNA complexes were also evident with nuclear extract from P815 tumor cells exposed 30 min earlier (lane 2), but not 60 min earlier, to L-PAM (lane 3). The specificity of the three protein-DNA complexes is indicated by the fact that addition of a 25-fold molar excess of unlabeled specific probe competed out all three bands (Fig. 6GoB, lane 2), whereas addition of a 25-fold molar excess of mutant probe did not compete out any of the bands (Fig. 6GoB, lane 3). Thus, exposure of P815 tumor cells to L-PAM leads to rapid and transient activation of NF-{kappa}B that binds specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 6. Nuclear extracts from L-PAM-treated P815 tumor cells exhibit increased binding to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene. Nuclear extracts were obtained from untreated P815 tumor cells (lane 1) or L-PAM-treated P815 tumor cells 30 (lane 2) or 60 (lane 3) min after initiation of the L-PAM treatment (A). EMSA were performed using 1 µg nuclear protein and 50 fM of a 32P-labeled DNA probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene. Specificity of the shifted protein-DNA complexes was assessed by incubating nuclear proteins (1 µg) obtained from P815 cells that were exposed 30 min earlier to L-PAM (lane 1) with a 25-fold molar excess of unlabeled specific DNA probe (lane 2) or a 25-fold molar excess of unlabeled mutant DNA probe (lane3) (B).

 
Identification of the members of the NF-{kappa}B family in the protein-DNA complexes formed with nuclear extract from L-PAM-treated P815 tumor cells and an oligonucleotide probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene

The NF-{kappa}B family of transcription factors consists of the homologous proteins p50, p52, p65, Rel B, and c-Rel, which dimerize in various combinations and activate or repress NF-{kappa}B-dependent gene transcription (37). To determine which members of the NF-{kappa}B family are present in the specific protein-DNA complexes formed with nuclear extract from P815 tumor cells exposed 30 min earlier to L-PAM, we exposed nuclear extract from L-PAM-treated P815 tumor cells to Abs directed against the various NF-{kappa}B family members before adding the 32P-labeled probe. As seen in Fig. 7Go, the fastest migrating third complex was competed with anti-p50 Ab (lane 2) and with anti-p65 Ab (lane 4); furthermore, in the presence of both anti-p50 and anti-p65 Ab, the third complex was competed away completely (lane 7), indicating that this complex contains heterodimers of the p50 and p65 subunits. In contrast, the second complex was competed away completely with Ab against p65 (lane 4), indicating that the second complex contains homodimers of p65. The composition of the less intense first complex is not clear, but it appears that it was diluted out with anti-p65 Ab (lane 4) and possibly anti-Rel B Ab (lane 5), suggesting that it may contain heterodimers of p65 and Rel B. Finally, Abs directed against p52 (lane 3) and c-Rel (lane 6) apparently did not compete or shift any of the complexes, suggesting that these subunits are not present in any of the complexes.



View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 7. Composition of the NF-{kappa}B complexes formed with nuclear extract of L-PAM-treated P815 tumor cells and an oligonucleotide probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene. Nuclear extract obtained from P815 tumor cells that were exposed 30 min earlier to L-PAM (lane 1) was incubated for 10 min with Abs against p50 (lane 2), p52 (lane 3), p65 (lane 4), Rel B (lane 5), c-Rel (lane 6), or p50 plus p65 (lane 7), and then incubated for 20 min with 32P-labeled probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene.

 
Effect of NAC on L-PAM-induced activation of NF-{kappa}B that binds specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene

Because the antioxidant NAC was found in the studies described above to prevent the L-PAM-induced accumulation of B7-1 mRNA in P815 tumor cells (Fig. 3Go), experiments were conducted to determine whether NAC will also inhibit the L-PAM-induced activation of NF-{kappa}B. For this purpose, P815 tumor cells were pretreated with NAC for 1 h before their exposure to L-PAM as well as during their 30-min exposure to L-PAM. Nuclear extract was prepared 30 min after initiation of the L-PAM treatment and incubated with a probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene. As seen in Fig. 8Go, the intensity of each of the three bands of protein-DNA complexes observed with nuclear extract from P815 tumor cells treated with L-PAM alone (lane 2) was substantially higher than the intensity of these complexes with nuclear extract from P815 tumor cells treated with L-PAM plus NAC (lane 3). Thus, NAC inhibits L-PAM-induced NF-{kappa}B activation, indicating that reactive oxygen species are involved in the NF-{kappa}B activation by L-PAM.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 8. The antioxidant NAC inhibits L-PAM-induced activation of NF-{kappa}B that binds specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene. Nuclear extracts were obtained from untreated P815 tumor cells (lane 1), or P815 tumor cells that were exposed to L-PAM 30 min earlier with (lane 3) or without (lane 2) NAC. EMSA were performed using 1 µg nuclear protein and 50 fM of a 32P-labeled DNA probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene.

 
Effect of a cell-permeable peptide that inhibits the binding of NEMO to the IKK complex on L-PAM-induced NF-{kappa}B activation and B7-1 gene expression

Experiments were undertaken to elucidate the importance of L-PAM-induced NF-{kappa}B activation for L-PAM-induced B7-1 gene expression. For this purpose, we took advantage of a recent report by May et al. (22) that a cell-permeable peptide that blocks the binding of NEMO to the IKK complex can selectively inhibit cytokine-induced NF-{kappa}B activation and NF-{kappa}B-dependent gene expression. Initially, we had to establish that this peptide (but not a mutant peptide that does not block NEMO binding to the IKK complex) inhibits L-PAM-induced NF-{kappa}B activation. For this purpose, P815 tumor cells were exposed to 250 µM of the wild-type or mutant peptide for 3 h before the L-PAM treatment as well as during the L-PAM treatment. Thirty minutes after initiation of the L-PAM treatment, nuclear extracts were prepared and incubated with a probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene. As seen in Fig. 9Go, the intensity of each of the three specific bands of the protein-DNA complexes observed with nuclear extract from P815 tumor cells treated with L-PAM alone (lane 2) or L-PAM plus the mutant peptide (lane 4) was substantially higher than the intensity of these complexes with nuclear extract from P815 tumor cells treated with L-PAM plus the wild-type peptide (lane 3). Thus, as expected based on the observations of May et al. (22) with cytokine-induced NF-{kappa}B activation, the wild-type peptide, which blocks NEMO binding to the IKK complex, also inhibits the L-PAM-induced NF-{kappa}B activation.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 9. A cell-permeable peptide that is known to block the binding of NEMO to the IKK complex inhibits L-PAM-induced activation of NF-{kappa}B that binds specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene. Nuclear extracts were obtained from untreated P815 tumor cells (lane 1) or P815 tumor cells that were exposed to L-PAM 30 min earlier with (lane 3) or without (lane 2) exposure to the cell-permeable peptide that blocks the binding of NEMO to the IKK complex. Concurrently, we obtained nuclear extract from P815 tumor cells exposed 30 min earlier to L-PAM with a mutant peptide that does not block the binding of NEMO to the IKK complex (lane 4). EMSA were performed using 1 µg nuclear protein and 50 fM of a 32P-labeled DNA probe containing the NF-{kappa}B binding site in the enhancer of the human B7-1 gene.

 
Experiments were next conducted to determine whether the peptide that was found to inhibit the L-PAM-induced NF-{kappa}B activation would also inhibit the L-PAM-induced B7-1 gene expression. For this purpose, P815 tumor cells were incubated with the wild-type or mutant peptide for 3 h before their exposure to L-PAM as well as during the 1 h in vitro L-PAM treatment. Four hours after initiation of the L-PAM treatment, we evaluated the effect of the wild-type and the mutant peptides on B7-1 mRNA expression. As seen in Fig. 10Go, and in confirmation of our previous observations (15), P815 tumor cells, which are negative for B7-1 mRNA expression (lane 1), expressed substantial levels of B7-1 mRNA 4 h after their exposure to L-PAM. Exposure of P815 tumor cells to the wild-type peptide, which inhibited the L-PAM-induced NF-{kappa}B activation, prevented the L-PAM-induced B7-1 mRNA accumulation (lane 3). In contrast, exposure of the P815 tumor cells to the mutant peptide, which did not inhibit the L-PAM-induced NF-{kappa}B activation, did not inhibit the L-PAM-induced B7-1 mRNA accumulation (lane 4). Thus, L-PAM-induced NF-{kappa}B activation is essential for L-PAM-induced B7-1 gene expression.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 10. Selective inhibition of NF-{kappa}B activation inhibits the L-PAM-induced accumulation of B7-1 mRNA in P815 tumor cells. RNA was obtained from untreated P815 tumor cells (lane 1) or P815 tumor cells that were treated with L-PAM with (lane 3) or without (lane 2) the cell-permeable peptide that blocks the binding of NEMO to the IKK complex (lane 3). Concurrently, RNA was obtained from P815 tumor cells that were treated with L-PAM with a mutant peptide that does not block the binding of NEMO to the IKK complex (lane 4). The RNA from the various groups was subsequently subjected to RT-PCR with primers specific for B7-1 or {beta}-actin. The size of the PCR product for B7-1 was ~300 bp.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown previously that in vitro exposure of MOPC-315 or P815 tumor cells to L-PAM leads to preferential up-regulation of B7-1 surface expression, without a concomitant increase in B7-2 surface expression (15). Moreover, the B7-1, which is induced on P815 tumor cells (which do not constitutively express either the B7-1 or B7-2) upon exposure to L-PAM was recently found to be functional. Specifically, we observed that anti-B7-1 mAb, but not anti-B7-2 mAb or isotype-matched Ig, inhibited the proliferation of splenic CD8+ T cells from 2C-transgenic mice bred onto a recombination-activating gene (RAG)-2-/- background (2C/RAG-/-) in response to stimulation with p2Ca (an octapeptide from {alpha}-ketoglutarate dehydrogenase)-pulsed L-PAM-pretreated P815 tumor cells (D. Sojka, S. O’Herrin, J. A. B., and M. B. M., unpublished observations). The preferential up-regulation of B7-1 surface expression may have important implications for the generation of an effective antitumor immune response in light of reports that in some situations costimulation via B7-1 and B7-2 leads to T cell differentiation along different pathways (38, 39, 40, 41, 42). Moreover, Gajewski (43) had shown in the P815 tumor system that B7-1-transfected tumor cells were superior to B7-2-transfected tumor cells in stimulating the generation of P815-specific CTLs. In the current studies we provide information regarding the mechanisms through which L-PAM initiates the selective up-regulation of B7-1 surface expression on P815 tumor cells.

We (15), as well as others (27, 29, 44), have recently shown that B7-1 surface expression can be selectively up-regulated on several different murine tumor cell lines, by exposing the tumor cells to a different anticancer modality, i.e., {gamma}-irradiation. Moreover, {gamma}-irradiation was reported to up-regulate B7-1 surface expression on primary cultures of human acute myeloid leukemic cell samples (29). Subsequent attempts to mimic the effect of {gamma}-irradiation on B7-1 surface expression with H2O2 revealed that while exposure of the murine AT3F hepatoma to {gamma}-irradiation resulted in a profound elevation in B7-1 mRNA and surface expression, exposure of these tumor cells for 60 min to H2O2 at a concentration of 2 mM, but not 1 mM, resulted in a slight elevation in B7-1 mRNA and surface expression (27). In contrast, exposure of the murine DA13b myeloid leukemic cell line for 60 min to 1 mM H2O2 (the only conditions reported) resulted in a substantial elevation in B7-1 surface expression, although not as profound as following exposure to 25 Gy {gamma}-irradiation (29). Neither one of these studies provided any data illustrating the effect of an antioxidant on {gamma}-irradiation-induced B7-1 surface expression. Here we extend the previous observations by demonstrating that H2O2 can also lead to selective up-regulation of B7-1 surface expression on P815 tumor cells. Moreover, elevation in B7-1 surface expression can be achieved by exposure of P815 tumor cells to H2O2 for only 15 min and at a much lower concentration than used in the previous studies (i.e., 0.03 vs 1.0 or 2.0 mM). Furthermore, we show the importance of reactive oxygen species for the L-PAM-induced up-regulation of B7-1 expression by demonstrating that pretreatment of P815 tumor cells with the antioxidant NAC prevented the L-PAM-induced B7-1 up-regulation.

We have recently observed that L-PAM administration to normal mice also resulted in rapid up-regulation of B7-1 gene expression in the spleen of such mice (V. Jovasevic, and M.B. Mokyr, unpublished observations). We have not as yet identified the splenic cell type(s) in which B7-1 gene expression was rapidly induced as a consequence of the L-PAM administration to normal mice. However, it is quite likely that APCs are among such cells given our observations that L-PAM leads to B7-1 gene expression via a pathway that involves reactive oxygen species and the observations of Rutault et al. (45) that reactive oxygen species function as "danger" signals to render dendritic cells more efficient in T cell activation by up-regulating the expression of costimulatory molecules and MHC class II molecules.

Several investigators have shown that different stimuli use reactive oxygen species as signaling messengers to activate transcription factors and induce gene expression (20, 26, 32, 33, 34, 46). For example, it was shown that activation of NF-{kappa}B by {gamma}-irradiation or phorbol ester can be inhibited by antioxidants (20, 26, 32, 33, 34, 46). Moreover, using cell lines overexpressing catalase, the H2O2-degrading enzyme, Schmidt et al. (47) have shown that among the reactive oxygen species, H2O2 acts as a messenger for NF-{kappa}B activation. In this regard it is interesting to note that Gorman et al. (30) have recently shown that exposure of HL60 cells to L-PAM leads to a rapid increase in intracellular peroxide level, but not superoxide level, and NAC, which was found in our studies to inhibit the L-PAM-induced B7-1 expression, has been shown by other investigators to directly reduce H2O2, ·OH, and HOCl, but not ·O2- (48, 49). Consistent with these observations, we show here that L-PAM leads to activation of NF-{kappa}B that binds specifically to the NF-{kappa}B binding site in the enhancer of the B7-1 gene, and this L-PAM-induced NF-{kappa}B activation is inhibited by pretreatment with NAC. Taken together these observations suggest that L-PAM leads to a rapid increase in the intracellular level of peroxide, which in turn leads to activation of NF-{kappa}B that binds selectively to the NF-{kappa}B binding site in the enhancer of the B7-1 gene.

Another redox-sensitive transcription factor is the AP-1. However, although in most studies AP-1 was shown to behave as an oxidative stress-responsive factor (34, 50), this was not the case in all studies. For example, in the studies by Meyer et al. (33) with HeLa cells AP-1 behaved like an anti-oxidative stress-responsive transcription factor, whereas NF-{kappa}B behaved like an oxidative stress-responsive transcription factor. Specifically, AP-1 was poorly activated following exposure of the HeLa cells to H2O2, and AP-1 activation by PMA was inhibited in the presence of H2O2. Moreover, antioxidants on their own activated AP-1. These observations led to the conclusion that the effect of oxidative stress on AP-1 activation may vary depending on the cell type studied and the modalities used, which in turn may result in a different pattern of transcription factor activation in different cell types exposed to the same modality or in the same cell line exposed to different modalities. Here we show that L-PAM leads to up-regulation of B7-1 expression via a mechanism that involves oxidative stress, and activation of NF-{kappa}B, but not AP-1, that binds specifically to its binding site in the promoter/enhancer of the B7-1 gene. It is not known at present whether the failure of L-PAM to activate AP-1 in P815 tumor cells is due to the cell type used (P815) and/or the modality used (L-PAM). However, AP-1 activation does not appear to be required for the L-PAM-induced B7-1 gene expression.

NF-{kappa}B in its active DNA-binding form is a heterogeneous collection of dimers (51). In fact, all five members of the NF-{kappa}B family can form homodimers or heterodimers, except Rel B, which can only form heterodimers (51). The different dimers show distinct preferences for DNA binding site sequences. Based on the existing model for the NF-{kappa}B binding site, which was developed based on crystal structure studies with a probe containing an NF-{kappa}B binding site and p50/p65 heterodimers, the sequences of the NF-{kappa}B binding sites are pseudosymmetric, i.e., a 5 bp sequence from the 5' end binds to p50, and a 4 bp sequence binds to p65 with a base pair spacer between them. The sequence of the first subsite that binds optimally to p50 can be 5'-GGGAA (52), as it is in the enhancer of the murine B7-1 gene (24) or 5'-GGGGT (51), as it is in the enhancer of the human B7-1 gene (25). In addition, the sequence of the second subsite that binds optimally to p65 (i.e., nucleotides 7–10 from the 5' end of the NF-{kappa}B binding site) is 5'-TTCC (52), which is identical to the second half of the NF-{kappa}B binding site in the enhancer of both mouse and human B7-1 gene (24, 25). Thus, it is not surprising that even though the NF-{kappa}B binding site in the enhancer of the murine and human B7-1 gene is not identical in sequence (i.e., 5'-GGGAAATTCC-3' and 5'-GGGGTTTTCC -3', respectively), the NF-{kappa}B that is activated following exposure of P815 tumor cells to L-PAM binds to the NF-{kappa}B binding site in the mouse as well as the human B7-1 gene.

Here we show that three different NF-{kappa}B dimers bound specifically to the NF-{kappa}B binding site in the enhancer of the B7-1 gene following exposure of P815 tumor cells to L-PAM. These included heterodimers of p50/p65, homodimers of p65, and conceivably heterodimers of p65/Rel B. Based on the above model and the sequence of the NF-{kappa}B binding site in the enhancer of the B7-1 gene, it is not surprising that one of the activated forms of NF-{kappa}B consists of heterodimers of p50/p65. In contrast, p65 homodimers have only one optimal subsite for binding to the NF-{kappa}B binding site in the enhancer of the B7-1 gene. However, as shown by Chen et al. (53), p65 homodimers can still bind to the NF-{kappa}B binding site by making base-specific contacts to the second subsite and only maintaining phosphate backbone contacts to the other (first) subsite of the NF-{kappa}B binding site (53).

Zhao et al. (25) have recently shown that nuclear extract derived from mature B cells (Raji cells) that constitutively express B7-1 bound specifically to an oligonucleotide probe containing the NF-{kappa}B binding site of the human B7-1 gene and formed two protein-DNA complexes. The faster migrating complex contained p50, c-Rel, and Rel B, whereas the slower moving complex contained heterodimers of p50/p65. In this regard it is interesting to note that in our studies L-PAM treatment of P815 tumor cells that activated B7-1 expression (on tumor cells that before the L-PAM treatment were negative for B7-1 expression) activated NF-{kappa}B that bound specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene and, as in the study by Zhao et al., one such activated NF-{kappa}B transcription factor was composed of heterodimers of p50/p65. However, in addition to the p50/p65 heterodimers, L-PAM treatment activated NF-{kappa}B transcription factors composed of homodimers of p65 and conceivably also heterodimers of p65/Rel B that bound specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene, which were not seen in the study by Zhao et al. (25). At present the function of the different dimers of the activated NF-{kappa}B that bind specifically to the NF-{kappa}B binding site in the enhancer of the B7-1 gene is not known. Consequently, it is not known what the significance is of the fact that in our studies, as in the studies by Zhao et al. (25), one of the activated transcription factors that bound specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene consisted of heterodimers of p50/p65, whereas the other activated transcription factors were different in the two studies. Finally, it is not known at present whether the differences seen in the composition of some of the activated NF-{kappa}B transcription factors that bound specifically to the NF-{kappa}B binding site in the enhancer of the human B7-1 gene in the two studies are the result of differences between the type of cell used (i.e., B7-1-expressing mature B cells vs mast cells) or the result of the "modalities" used to "induce" B7-1 expression (i.e., Raji cells that constitutively express B7-1 vs B7-1-negative P815 tumor cells that were induced to express B7-1 by exposure to L-PAM).

In the current studies we used a cell-permeable peptide that was previously shown to inhibit the binding of NEMO to the IKK complex (which consists of IKK{alpha} and IKK{beta}), thereby inhibiting IKK phosphorylation of the I{kappa}B{alpha} and I{kappa}B{beta}, which in turn inhibits the degradation of the I{kappa}B that is required for the translocation of NF-{kappa}B from the cytoplasm to the nucleus where it activates the specific cellular genes (22, 54). Our studies revealed that this peptide, which was previously shown to inhibit cytokine-induced NF-{kappa}B activation (22), also inhibits L-PAM-induced NF-{kappa}B activation. Moreover, our studies illustrated that this peptide inhibits L-PAM-induced B7-1 gene expression, indicating that NF-{kappa}B activation is essential for L-PAM-induced B7-1 gene expression.

It is interesting to point out at this stage that the NF-{kappa}B binding site in the enhancer of the IFN-{beta} gene, which is important for IFN-{beta} gene expression (55, 56), is identical in sequence to the sequence of the NF-{kappa}B binding site in the enhancer of the B7-1 gene (25, 53). Thus, although other transcription factors in addition to NF-{kappa}B are involved in the up-regulation of IFN-{beta} gene expression (55, 56), these observations prompted us to determine whether exposure of tumor cells to L-PAM in vitro also leads to up-regulation of IFN-{beta} gene expression. Our studies revealed that within 2 h after in vitro exposure of tumor cells to L-PAM there is a massive accumulation of IFN-{beta} mRNA, and this accumulation requires de novo RNA synthesis, indicating that L-PAM indeed leads to activation of IFN-{beta} gene expression (V. Jovasevic and M.B. Mokyr, manuscript in preparation). IFN-{beta} (not just oxidative stress) may in turn function as an inducible "danger" signal for the immune system (57).


   Footnotes
 
1 This work was supported by Research Grant R01 CA-76532 from the National Institutes of Health. Back

2 This work is in partial fulfillment of the requirements for the Doctor of Philosophy Degree. Back

3 Address correspondence and reprint requests to Dr. Margalit B. Mokyr, Department of Biochemistry and Molecular Biology (M/C 536), University of Illinois, 1819 West Polk Street, Chicago, IL 60612. E-mail address: mokyr{at}uic.edu Back

4 Abbreviations used in this paper: L-PAM, L-phenylalanine mustard; NAC, N-acetyl-L-cysteine; NIgG, normal IgG; NEMO, NF-{kappa}B essential modifier; IKK, I{kappa}B-kinase. Back

Received for publication December 6, 2000. Accepted for publication March 19, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Berd, D., M. J. Mastrangelo. 1988. Active immunotherapy of human melanoma exploiting the immunopotentiating effects of cyclophosphamide. Cancer Invest. 6:337.[Medline]
  2. Mokyr, M. B., E. Barker, L. M. Weiskirch, B. Y. Takesue, J. M. Pyle. 1989. Importance of Lyt 2+ T cells in the curative effectiveness of a low dose of melphalan for mice bearing a large MOPC-315 tumor. Cancer Res. 49:4597.[Abstract/Free Full Text]
  3. Awwad, M., R. J. North. 1989. Cyclophosphamide-induced immunologically mediated regression of a cyclophosphamide-resistant murine tumor: a consequence of eliminating precursor L3T4+ suppressor T-cells. Cancer Res. 49:1649.[Abstract/Free Full Text]
  4. Nagarkatti, M., D. M. Toney, P. S. Nagarkatti. 1989. Immunomodulation by various nitrosoureas and its effect on the survival of the murine host bearing a syngeneic tumor. Cancer Res. 49:6587.[Abstract/Free Full Text]
  5. North, R. J., M. Awwad. 1990. Elimination of cycling CD4+ suppressor T cells with an anti-mitotic drug releases non-cycling CD8+ T cells to cause regression of an advanced lymphoma. Immunology 71:90.[Medline]
  6. Yuan, L., Y. Kuramitsu, Y. Li, M. Kobayashi, M. Hosokawa. 1995. Restoration of interleukin-2 production in tumor-bearing rats through reducing tumor-derived transforming growth factor {beta} by treatment with bleomycin. Cancer Immunol. Immunother. 41:355.[Medline]
  7. Lattime, E. C., M. J. Mastrangelo, O. Bagasra, W. Li, D. Berd. 1995. Expression of cytokine mRNA in human melanoma tissues. Cancer Immunol. Immunother. 41:151.[Medline]
  8. Gorelik, L., Y. Bar-Dagan, M. B. Mokyr. 1996. Insight into the mechanism(s) through which TNF promotes the generation of T cell-mediated antitumor cytotoxicity by tumor bearer splenic cells. J. Immunol. 156:4298.[Abstract]
  9. Inagawa, H., T. Nishizawa, T. Honda, T. Nakamoto, K. Takagi, G. Soma. 1998. Mechanisms by which chemotherapeutic agents augment the antitumor effects of tumor necrosis factor: involvement of the pattern shift of cytokines from Th2 to Th1 in tumor lesions. Anticancer Res. 18:3957.[Medline]
  10. Bass, K. K., M. J. Mastrangelo. 1998. Immunopotentiation with low-dose cyclophosphamide in the active specific immunotherapy of cancer. Cancer Immunol. Immunother. 47:1.[Medline]
  11. Mokyr, M. B., T. V. Kalinichenko, L. Gorelik, J. A. Bluestone. 1998. Importance of the B7-2 molecule for low dose melphalan-induced acquisition of tumor-eradicating immunity by mice bearing a large MOPC-315 tumor. J. Immunol. 160:1866.[Abstract/Free Full Text]
  12. Lenschow, D. J., G. H. Su, L. A. Zuckerman, N. Nabavi, C. L. Jellis, G. S. Gray, J. Miller, J. A. Bluestone. 1993. Expression and functional significance of an additional ligand for CTLA-4. Proc. Natl. Acad. Sci. USA 90:11054.[Abstract/Free Full Text]
  13. Hathcock, K. S., G. Laszlo, C. Pucillo, P. Linsley, R. J. Hodes. 1994. Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function. J. Exp. Med. 180:631.[Abstract/Free Full Text]
  14. Lenschow, D. J., S. C. Ho, H. Sattar, L. Rhee, G. Gray, N. Nabavi, K. C. Herold, J. A. Bluestone. 1995. Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse. J. Exp. Med. 181:1145.[Abstract/Free Full Text]
  15. Sojka, D. K., M. Donepudi, J. A. Bluestone, M. B. Mokyr. 2000. Melphalan and other anticancer modalities up-regulate B7-1 gene expression in tumor cells. J. Immunol. 164:6230.[Abstract/Free Full Text]
  16. Gorelik, L., M. B. Mokyr. 1995. Low-dose-melphalan-induced up-regulation of type-1 cytokine expression in the s.c. tumor nodule of MOPC-315 tumor bearers and the role of interferon {gamma} in the therapeutic outcome. Cancer Immunol. Immunother. 41:363.[Medline]
  17. Kalinichenko, T. V., M. B. Mokyr. 1998. Limited importance of CD40/CD40L interaction in the B7-dependent generation of anti-MOPC-315 cytotoxic T lymphocyte activity by tumor bearer splenic cells stimulated in vitro in the presence of tumor necrosis factor. Cancer Immunol. Immunother. 46:293.[Medline]
  18. La Motte, R. N., M. A. Rubin, E. Barr, J. M. Leiden, J. A. Bluestone, M. B. Mokyr. 1996. Therapeutic effectiveness of the immunity elicited by P815 tumor cells engineered to express the B7-2 costimulatory molecule. Cancer Immunol. Immunother. 42:161.[Medline]
  19. Ben-Efraim, S., R. C. Bocian, M. B. Mokyr, S. Dray. 1983. Increase in the effectiveness of melphalan therapy with progression of MOPC-315 plasmacytoma tumor growth. Cancer Immunol. Immunother. 15:101.[Medline]
  20. Schreck, R., P. Rieber, P. A. Baeuerle. 1991. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-{kappa}B transcription factor and HIV-1. EMBO J. 10:2247.[Medline]
  21. Verhasselt, V., W. Vanden Berghe, N. Vanderheyde, F. Willems, G. Haegeman, M. Goldman. 1999. N-acetyl-L-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-{kappa}B inhibition. J. Immunol. 162:2569.[Abstract/Free Full Text]
  22. May, M. J., F. D’Acquisto, L. A. Madge, J. Glöckner, S. Pober, S. Ghosh. 2000. Selective inhibition of NF-{kappa}B activation by a peptide that blocks the interaction of NEMO with the IkB kinase complex. Science 289:1550.[Abstract/Free Full Text]
  23. Osborn, L., S. Kunkel, G. J. Nabel. 1989. Tumor necrosis factor {alpha} and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor {kappa}B. Proc. Natl. Acad. Sci. USA 86:2336.[Abstract/Free Full Text]
  24. Selvakumar, A., P. C. White, B. Dupont. 1993. Genomic organization of the mouse B-lymphocyte activation antigen B7. Immunogenetics 38:292.[Medline]
  25. Zhao, J., G. J. Freeman, G. S. Gray, L. M. Nadler, L. H. Glimcher. 1996. A cell type-specific enhancer in the human B7.1 gene regulated by NF-{kappa}B. J. Exp. Med. 183:777.[Abstract/Free Full Text]
  26. Baeuerle, P. A., T. Henkel. 1994. Function and activation of NF-{kappa}B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
  27. Morel, A., N. Fernandez, A. de La Coste, H. Haddada, M. Viguier, B. S. Polla, B. Antoine, A. Kahn. 1998. {gamma}-ray irradiation induces B7.1 costimulatory molecule neoexpression in various murine tumor cells. Cancer Immunol. Immunother. 46:277.[Medline]
  28. Pahl, H. L., P. A. Baeuerle. 1994. Oxygen and the control of gene expression. Bioessays 16:497.[Medline]
  29. Vereecque, R., G. Buffenoir, R. Gonzalez, N. Cambier, D. Hetuin, F. Bauters, P. Fenaux, B. Quesnel. 2000. {gamma}-ray irradiation induces B7.1 expression in myeloid leukaemic cells. Br. J. Haematol. 108:825.[Medline]
  30. Gorman, A., A. McGowan, T. G. Cotter. 1997. Role of peroxide and superoxide anion during tumour cell apoptosis. FEBS Lett. 404:27.[Medline]
  31. La Motte, R. N., A. H. Sharpe, J. A. Bluestone, M. B. Mokyr. 1999. Host B7-1 and B7-2 costimulatory molecules contribute to the eradication of B7-1-transfected P815 tumor cells via a CD8+ T cell-dependent mechanism. J. Immunol. 162:4817.[Abstract/Free Full Text]
  32. Meyer, M., H. L. Pahl, P. A. Baeuerle. 1994. Regulation of the transcription factors NF-{kappa}B and AP-1 by redox changes. Chem. Biol. Interact. 91:91.[Medline]
  33. Meyer, M., R. Schreck, P. A. Baeuerle. 1993. H2O2 and antioxidants have opposite effects on activation of NF-{kappa}B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 12:2005.[Medline]
  34. Pinkus, R., L. M. Weiner, V. Daniel. 1996. Role of oxidants and antioxidants in the induction of AP-1, NF-{kappa}B, and glutathione S-transferase gene expression. J. Biol. Chem. 271:13422.[Abstract/Free Full Text]
  35. Zhang, H., D. Haasch, K. B. Idler, G. F. Okasinski. 1996. Isolation and promoter mapping of the gene encoding murine co-stimulatory factor B7-1. Gene 183:1.[Medline]
  36. Hsing, Y., B. S. Hostager, G. A. Bishop. 1997. Characterization of CD40 signaling determinants regulating nuclear factor-{kappa}B activation in B lymphocytes. J. Immunol. 159:4898.[Abstract]
  37. Ghosh, S., M. J. May, E. B. Kopp. 1998. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
  38. Kuchroo, V. K., M. P. Das, J. A. Brown, A. M. Ranger, S. S. Zamvil, R. A. Sobel, H. L. Weiner, N. Nabavi, L. H. Glimcher. 1995. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell 80:707.[Medline]
  39. Freeman, G. J., V. A. Boussiotis, A. Anumanthan, G. M. Bernstein, X. Y. Ke, P. D. Rennert, G. S. Gray, J. G. Gribben, L. M. Nadler. 1995. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7- 2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 2:523.[Medline]
  40. Tsuyuki, S., J. Tsuyuki, K. Einsle, M. Kopf, A. J. Coyle. 1997. Costimulation through B7-2 (CD86) is required for the induction of a lung mucosal T helper cell 2 (TH2) immune response and altered airway responsiveness. J. Exp. Med. 185:1671.[Abstract/Free Full Text]
  41. Stremmel, C., E. A. Greenfield, E. Howard, G. J. Freeman, V. K. Kuchroo. 1999. B7-2 expressed on EL4 lymphoma suppresses antitumor immunity by an interleukin 4-dependent mechanism. J. Exp. Med. 189:919.[Abstract/Free Full Text]
  42. Girvin, A. M., M. C. Dal Canto, L. Rhee, B. Salomon, A. Sharpe, J. A. Bluestone, S. D. Miller. 2000. A critical role for B7/CD28 costimulation in experimental autoimmune encephalomyelitis: a comparative study using costimulatory molecule-deficient mice and monoclonal antibody blockade. J. Immunol. 164:136.[Abstract/Free Full Text]
  43. Gajewski, T. F.. 1996. B7-1 but not B7-2 efficiently costimulates CD8+ T lymphocytes in the P815 tumor system in vitro. J. Immunol. 156:465.[Abstract]
  44. Seo, A., F. Ishikawa, H. Nakano, H. Nakazaki, K. Kobayashi, T. Kakiuchi. 1999. Enhancement of B7-1 (CD80) expression on B-lymphoma cells by irradiation. Immunology 96:642.[Medline]
  45. Rutault, K., C. Alderman, B. M. Chain, D. R. Katz. 1999. Reactive oxygen species activate peripheral blood dendritic cells. Free Radic. Biol. Med. 26:232.[Medline]
  46. Pahl, H. L.. 1999. Activators and target genes of Rel/NF-{kappa}B transcription factors. Oncogene 18:6853.[Medline]
  47. Schmidt, K. N., E. B. Traenckner, B. Meier, P. A. Baeuerle. 1995. Induction of oxidative stress by okadaic acid is required for activation of transcription factor NF-{kappa}B. J. Biol. Chem. 270:27136.[Abstract/Free Full Text]
  48. Aruoma, O. I., B. Halliwell, B. M. Hoey, J. Butler. 1989. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic. Biol. Med. 6:593.[Medline]
  49. Cotgreave, I. A.. 1997. N-acetylcysteine: pharmacological considerations and experimental and clinical applications. Adv. Pharmacol. 38:205.
  50. Foletta, V. C., D. H. Segal, D. R. Cohen. 1998. Transcriptional regulation in the immune system: all roads lead to AP-1. J. Leukocyte Biol. 63:139.[Abstract]
  51. Gilmore, T. D.. 1999. The Rel/NF-{kappa}B signal transduction pathway: introduction. Oncogene 18:6842.[Medline]
  52. Chen, F. E., G. Ghosh. 1999. Regulation of DNA binding by Rel/NF-{kappa}B transcription factors: structural views. Oncogene 18:6845.[Medline]
  53. Chen, Y. Q., S. Ghosh, G. Ghosh. 1998. A novel DNA recognition mode by the NF-{kappa}B p65 homodimer. Nat. Struct. Biol. 5:67.[Medline]
  54. Li, X.-H., X. Fang, R. B. Gaynor. 2001. Role of IKK{gamma}/NEMO in assembly of the I{kappa}B kinase complex. J. Biol. Chem. 276:4494.[Abstract/Free Full Text]
  55. Doly, J., A. Civas, S. Navarro, G. Uze. 1998. Type I interferons: expression and signalization. Cell. Mol. Life Sci. 54:1109.[Medline]
  56. Kirchhoff, S., D. Wilhelm, P. Angel, H. Hauser. 1999. NF{kappa}B activation is required for interferon regulatory factor-1-mediated interferon {beta} induction. Eur. J. Biochem. 261:546.[Medline]
  57. Gallucci, S., P. Matzinger. 2001. Danger signals: SOS to the immune system. Curr. Opin. Immunol. 13:114.[Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. Sharma, A. A. George, B. N. Singh, N. C. Sahoo, and K. V. S. Rao
Regulation of Transcript Elongation through Cooperative and Ordered Recruitment of Cofactors
J. Biol. Chem., July 20, 2007; 282(29): 20887 - 20896.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. K. Rehman, S. Bertera, R. Bottino, A. N. Balamurugan, J. C. Mai, Z. Mi, M. Trucco, and P. D. Robbins
Protection of Islets by in Situ Peptide-mediated Transduction of the Ikappa B Kinase Inhibitor Nemo-binding Domain Peptide
J. Biol. Chem., March 7, 2003; 278(11): 9862 - 9868.
[Abstract] [Full Text] [PDF]

Home page
 CROBMHome page
S. T. Sonis
THE BIOLOGIC ROLE FOR NUCLEAR FACTOR-KAPPAB IN DISEASE AND ITS POTENTIAL INVOLVEMENT IN MUCOSAL INJURY ASSOCIATED WITH ANTI-NEOPLASTIC THERAPY
Critical Reviews in Oral Biology & Medicine, September 1, 2002; 13(5): 380 - 389.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Interv.Home page
F. D'Acquisto, M. J. May, and S. Ghosh
Inhibition of Nuclear Factor Kappa B (NF-B):: An Emerging Theme in Anti-Inflammatory Therapies
Mol. Interv., February 1, 2002; 2(1): 22 - 35.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
V. M. Jovasevic and M. B. Mokyr
Melphalan-Induced Expression of IFN-{beta} in MOPC-315 Tumor-Bearing Mice and Its Importance for the Up-Regulation of TNF-{alpha} Expression
J. Immunol., November 1, 2001; 167(9): 4895 - 4901.
[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 Donepudi, M.
Right arrow Articles by Mokyr, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Donepudi, M.
Right arrow Articles by Mokyr, M. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS