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The Journal of Immunology, 2001, 167: 4895-4901.
Copyright © 2001 by The American Association of Immunologists

Melphalan-Induced Expression of IFN-{beta} in MOPC-315 Tumor-Bearing Mice and Its Importance for the Up-Regulation of TNF-{alpha} Expression1

Vladimir M. Jovasevic and Margalit B. Mokyr2

Department of Biochemistry and Molecular Biology, University of Illinois, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that administration of a low-dose of melphalan (L-phenylalanine mustard; L-PAM) to mice bearing a large s.c. MOPC-315 tumor leads to up-regulation of TNF-{alpha} expression, which is first evident at the mRNA level at 24 h after the chemotherapy. In this study, we show accumulation of IFN-{beta} mRNA in the spleen and tumor nodule of such mice as early as 1 h after the chemotherapy followed by elevated production of IFN-{beta} protein. IFN-{beta} protein in turn was found to be important for the L-PAM-induced up-regulation of TNF-{alpha} expression, as neutralization of IFN-{beta} inhibited the L-PAM-induced up-regulation of TNF-{alpha} mRNA expression in MOPC-315 tumor cells. In addition, L-PAM failed to up-regulate TNF-{alpha} expression in spleen cells from mice in which signaling by IFN-{beta} is deficient. Studies into the mechanism through which L-PAM leads to rapid accumulation of IFN-{beta} mRNA revealed that it requires de novo RNA synthesis, indicating that the regulation is at the transcriptional level. However, it did not require de novo protein synthesis, indicating that activation of pre-existing transcription factors is sufficient for IFN-{beta} gene expression. The L-PAM-induced accumulation of IFN-{beta} mRNA was mimicked with H2O2 and was prevented with the antioxidant N-acetyl-L-cysteine, indicating that reactive oxygen species are involved in the transcriptional regulation of L-PAM-induced IFN-{beta} gene expression. Thus, the IFN-{beta} gene is an early response gene that is activated in response to L-PAM via a pathway that involves reactive oxygen species, and IFN-{beta} in turn plays an important role in L-PAM-induced TNF-{alpha} up-regulation.


   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 some time (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In fact, several anticancer drugs (e.g., cyclophosphamide (CY)3 (1, 4, 8, 9, 10), L-PAM (2, 3), N,N'-bis(2-chloroethyl)-N-nitrosourea (6), vinblastine (5), and bleomycin (7)) have been shown to enhance the acquisition of T cell-mediated antitumor immunity in a variety of animal tumor systems (1, 2, 3, 4, 5, 6, 7) and in patients with advanced melanoma (8, 10) or advanced renal carcinoma (9). Studies into the mechanisms through which the anticancer drugs enhance the acquisition of T cell-mediated antitumor immunity in tumor bearers revealed that the chemotherapy leads to a shift in the cytokine profile from anti-inflammatory cytokines (e.g., TGF-{beta}, IL-10, and/or IL-4) with inhibitory activity for the generation of cell-mediated immunity toward proinflammatory cytokines (e.g., TNF-{alpha}, IFN-{gamma}, and/or GM-CSF) that favor the development of cell-mediated immunity (7, 11, 12, 13, 14, 15, 16, 17, 18). In these studies, however, the up-regulation of expression of the proinflammatory cytokines and/or the down-regulation of the anti-inflammatory cytokines at the mRNA level was first noted at least 24 h after the chemotherapy (7, 11, 12, 13, 14, 15, 18). None of these studies attempted to identify earlier events after the chemotherapy that may have contributed to the chemotherapy-induced shift in the cytokine profile at the tumor site.

The current studies were undertaken to identify an earlier event after the chemotherapy that may be involved in chemotherapy-induced shift in the cytokine profile at the tumor site. The MOPC-315 tumor system and the anticancer drug L-PAM were selected for these studies in light of our previous observations that 1) elevated expression of TNF-{alpha} mRNA is first evident in the tumor nodule of mice bearing a large MOPC-315 tumor at 24 h after the administration of low-dose L-PAM (18) and 2) TNF-{alpha} is critical for the L-PAM-induced acquisition of CD8+ T cell-mediated tumor-eradicating immunity by the hitherto immunosuppressed MOPC-315 tumor bearers (18). Specifically, we examined the possibility that L-PAM leads to rapid activation of a type I IFN gene expression, and the type I IFN in turn plays an important role in L-PAM-induced up-regulation of TNF-{alpha} expression. A type I IFN seemed to be a likely candidate for this purpose for the following reasons. First, Schiavoni et al. (19) have recently reported elevated expression of type I IFNs in the spleen of normal mice at 6 h after CY administration (the earliest time point studied), which was associated with subsequent elevation in biological activity of type I IFNs in the sera of the CY-treated mice. Second, type I IFNs were reported to favor the development of a type 1 cytokine response in nontumor systems (20, 21, 22, 23) and to prime mice for elevated production of TNF-{alpha} in response to LPS (24). As a representative of the type I IFNs, we decided to focus our attention on IFN-{beta} and not IFN-{alpha} because there is a single IFN-{beta}, whereas there are multiple subtypes of IFN-{alpha} (25). In addition, L-PAM was recently found to activate NF-{kappa}B (26) and the promoter/enhancer region of the IFN-{beta} gene (but not IFN-{alpha} genes) has an NF-{kappa}B binding site that is one of the binding sites that is important for IFN-{beta} gene activation (27, 28). Finally, IFN-{beta} was recently shown to be activated rapidly after viral infection without the need for de novo protein synthesis, while the induction of several members of the IFN-{alpha} gene family (e.g., IFN-{alpha} 2, 5, 6, and 8) occurred several hours later and required protein synthesis (29). Furthermore, exogenous IFN-{beta} induced IFN-{alpha} production in virally infected cells in which protein synthesis was inhibited, indicating not only that IFN-{beta} is an early response gene but also that IFN-{beta} can stimulate IFN-{alpha} production by signaling via its receptor (29).

Here, we show that the IFN-{beta} gene is an early response gene that is activated rapidly after L-PAM therapy in both the spleen and the tumor nodule of MOPC-315 tumor bearers via a pathway that involves reactive oxygen species. In addition, we illustrate that IFN-{beta} plays an important role in L-PAM-induced up-regulation of TNF-{alpha} expression, suggesting that L-PAM may initiate its immunopotentiating effect in MOPC-315 tumor bearers by rapidly activating the IFN-{beta} gene.


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

Female BALB/cAnNCrlBR mice 7–10 wk old were purchased from Charles Rivers Breeding Laboratories (Wilmington, MA). Mice deficient in IFN-{alpha}{beta} receptor (IFN-{alpha}{beta} R-/-) on 129 Ev/Sv background as well as wild-type 129 Ev/Sv mice were kindly provided by Drs. H. Virgin and R. Schreiber (Washington University, St. Louis, MO) (30). These mice were kept under germfree conditions in our Barrier Animal Facility until initiation of the experiments.

Tumors

The MOPC-315 plasmacytoma was maintained in vivo, as previously described (2), in BALB/cAnNCrlBR. Briefly, mice were inoculated s.c. with 1 x 106 viable tumor cells, a dose that is at least 300-fold greater than the minimal lethal tumor dose. Tumor nodules were excised on days 10–12 after tumor inoculation, when they reached 18–22 mm in diameter, and single-cell suspensions were prepared by mechanical disruption between glass slides. For the sake of simplicity such cells will be referred to as tumor cells, although a few host cells are also present in the tumor nodules (3).

L-PAM therapy

A fresh stock solution of L-PAM (Sigma, St. Louis, MO) was prepared, as previously described (2), just before use. A dose of 2.0 mg/kg body weight (low dose) was administered i.p. to mice bearing a large (~20 mm) tumor that resulted from the s.c. inoculation of 1 x 106 MOPC-315 tumor cells 10–12 days earlier. This dose of drug was previously shown to lead to the acquisition of CD8+ T cell-dependent antitumor immunity via a mechanism that involves L-PAM-induced TNF production, and the antitumor immunity in turn was shown to eradicate a large tumor burden not eradicated by the direct antitumor effect of L-PAM (2, 3, 18). In the current experiments, spleens and tumor nodules were excised within 24 h after the L-PAM administration.

In vitro exposure to L-PAM or H2O2

Cells derived from normal mice or tumor-bearing mice were exposed in vitro for 1 h to 15 nM L-PAM, as previously described (31, 32). After completion of the L-PAM treatment, the cells were washed and cultured at a concentration of 0.5–0.75 x 106 cells/ml in DMEM supplemented with 10% FBS and 0.1 mM nonessential amino acids (Life Technologies, Grand Island, NY). In experiments assessing the importance of RNA synthesis, protein synthesis, or reactive oxygen species for L-PAM-induced accumulation of IFN-{beta} mRNA, MOPC-315 tumor cells were treated with actinomycin D (1 µg/ml) (32), cycloheximide (10 µg/ml) (32), or N-acetyl-L-cysteine (NAC; 25 mM) (33), respectively, for 1 h before their exposure to L-PAM, as well as during the L-PAM treatment. In experiments assessing the effect of anti-IFN-{beta} mAb on L-PAM-induced up-regulation of TNF-{alpha} mRNA expression, at the completion of the in vitro L-PAM treatment, the MOPC-315 tumor cells were cultured for 23 h in the presence or absence of rat anti-mouse IFN-{beta} mAb (U.S. Biological, Swampscott, MS) at a concentration that can neutralize 100 U IFN-{beta}/ml. Finally, in experiments assessing the ability of H2O2 to up-regulate IFN-{beta} mRNA expression, MOPC-315 tumor cells were treated for 15 min with H2O2 (at a concentration of 0.1–1.0 mM), at the end of which the cells were washed and cultured under the same conditions as described above for the L-PAM-treated cells.

RT-PCR

Total RNA was extracted, as previously described (14, 26), and subjected to RT-PCR with primers specific for IFN-{beta} (5'-CCATCCAAGAGATGCTCCAG-3' for the sense primer and 5'-GTGGAGAGCAGTTGAGGACA-3' for the antisense) (19). The cycling conditions consisted of 5 min at 94°C and 30 cycles of 40 s at 94°C, 40 s at 62°C, and 1 min at 72°C, and a final 10-min extension at 72°C (19). {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. In some experiments, we conducted PCR with primers specific for TNF-{alpha} (5'-GTTCTATGGCCCAGACCCTCATCACA-3' for the sense and 5'-TCCCAGGTATATGGGTTCATACC-3' for the antisense) according to the protocol we have previously described (14). 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 50- or 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.

Western blot analysis

Tumor cells derived from the s.c. tumor nodules of untreated mice or mice treated 18 h earlier with low-dose L-PAM were cultured for 6 h in RPMI 1640 medium. Culture supernatants (containing 20 µg of total protein) were mixed with sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 100 mM DTT, and 0.01% bromphenol blue), boiled, and loaded onto 15% SDS-PAGE. Concurrently, 5, 10, and 30 ng of recombinant murine IFN-{beta} (Calbiochem-Novabiochem, San Diego, CA) were also mixed with the sample buffer, boiled, and loaded onto the gel. The proteins were separated and electroblotted to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) and the membrane was blocked with 5% nonfat dried milk in TBS containing 0.1% Tween 20. Subsequently, the membranes were probed with rat anti-mouse IFN-{beta} mAb (U.S. Biological) in TBS containing 0.1% Tween 20, incubated with a secondary Ab (goat anti-rat IgG (H + L); Caltag Laboratories, San Francisco, CA), and visualized by an ECL Western blotting detection system (Pierce, Rockford, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of in vivo administration of low-dose L-PAM to mice bearing a large s.c. MOPC-315 tumor on IFN-{beta} mRNA expression in their spleen and tumor nodule

Experiments were conducted to determine whether administration of low-dose L-PAM to MOPC tumor bearers would result in rapid up-regulation of IFN-{beta} mRNA expression in their spleen and/or s.c. tumor nodule. For this purpose, mice bearing a large (~20 mm in diameter) s.c. MOPC-315 tumor were given an i.p. injection of 2.0 mg/kg L-PAM and the level of IFN-{beta} mRNA expression in their spleen and tumor nodule was determined 1, 2, 4, and 6 h after the chemotherapy relative to the level of IFN-{beta} mRNA expression in the spleen and tumor nodule of untreated MOPC-315 tumor bearers. As seen in Fig. 1Go, elevated expression of IFN-{beta} mRNA was evident in the spleen as well as tumor nodule of MOPC-315 tumor bearers at all time points examined, although the elevation in IFN-{beta} mRNA expression was more profound at 6 h after the L-PAM administration than at the earlier time points. Thus, low-dose L-PAM leads to rapid accumulation of IFN-{beta} mRNA in the spleen and tumor nodule of MOPC-315 tumor bearers.



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  		FIGURE 1. Rapid accumulation of IFN-{beta} mRNA in the spleen and tumor nodule of mice bearing a large MOPC-315 tumor following low-dose L-PAM therapy. Mice bearing a large (~20 mm) s.c. MOPC-315 tumor were given an i.p. injection of 2.0 mg/kg L-PAM and 1 (lane 2), 2 (lane 3), 4 (lane 4), and 6 h (lane 5) later their spleens and tumor nodules were excised. RNA extracted from the spleens (A) or the s.c. tumor nodules (B) of the L-PAM-treated mice as well as RNA extracted from the spleen or tumor nodule of untreated mice (lane 1) was subjected to RT-PCR with primers specific for IFN-{beta} or {beta}-actin. The size of the PCR product for IFN-{beta} was ~150 bp.

 
Effect of in vitro exposure of MOPC-315 tumor cells to L-PAM on IFN-{beta} mRNA expression

Experiments were performed to determine whether the L-PAM-induced elevation in the expression of IFN-{beta} mRNA in the tumor nodule of MOPC-315 tumor bearers is the result of drug-induced accumulation of IFN-{beta} mRNA in cells that were present in the tumor nodule before the chemotherapy or the result of chemotherapy-induced migration of cells with elevated IFN-{beta} mRNA expression into the tumor nodule. In these experiments, cells from the s.c. tumor nodule of MOPC-315 tumor bearers were treated in vitro with L-PAM under conditions that were previously shown to enhance the stimulatory capacity of the tumor cells for the in vitro generation of anti-MOPC-315 CTL activity (31), and that consisted of 1-h exposure to 15 nM L-PAM. The earliest time point after initiation of the in vitro L-PAM treatment that cells were examined for IFN-{beta} mRNA expression was 2 h, because in addition to the 1-h L-PAM treatment the cells were washed three times before the extraction of their RNA. As seen in Fig. 2GoA, elevated expression of IFN-{beta} mRNA was evident in cells derived from the s.c. tumor nodule of MOPC-315 tumor bearers at 2 and 4 h after initiation of the in vitro L-PAM treatment. Thus, L-PAM can rapidly induce accumulation of IFN-{beta} mRNA in cells present in the tumor nodule of MOPC-315 tumor bearers.



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  		FIGURE 2. In vitro exposure to L-PAM of cells freshly obtained from the s.c. tumor nodule of MOPC-315 tumor bearers or cells derived from an established in vitro line of MOPC-315 tumor cells leads to rapid accumulation of IFN-{beta} mRNA. Freshly obtained cells from the s.c. tumor nodule of mice bearing a large MOPC-315 tumor (A) or MOPC-315 tumor cells from an established in vitro line (B) were exposed in vitro for 1 h to 15 nM L-PAM. RNA extracted from the L-PAM-treated cells at 2 (lane 2) or 4 h (lane 3) after initiation of the L-PAM treatment as well as RNA extracted from untreated cells (lane 1) was subjected to RT-PCR with primers specific for IFN-{beta} or {beta}-actin.

 
We would like to stress at this stage that for the sake of simplicity we will refer to cells freshly obtained from the s.c. tumor nodule of untreated MOPC-315 tumor bearers as MOPC-315 tumor cells, even though the tumor nodules contain a few host cells (2, 3). It should be noted, however, that in vitro exposure to L-PAM was found to lead to rapid accumulation of IFN-{beta} mRNA also in an established in vitro line of MOPC-315 tumor cells (which is devoid of host cells; Fig. 2GoB). Thus, MOPC-315 tumor cells represent one of the cell types that displays up-regulated IFN-{beta} mRNA expression following exposure of cells from MOPC-315 tumor nodules to L-PAM.

Effect of low-dose L-PAM administration to MOPC-315 tumor bearers on IFN-{beta} secretion by their tumor cells

Experiments were conducted to determine whether the L-PAM-induced elevation in IFN-{beta} expression at the mRNA level is also manifested in L-PAM-induced elevation in IFN-{beta} expression at the protein level. For this purpose, MOPC-315 tumor cells from untreated or low-dose L-PAM-treated mice were cultured in vitro for 6 h and the presence of IFN-{beta} protein in the supernatant of the cultured tumor cells was determined by Western blot analysis. As reference point, we subjected concurrently 5, 10, and 30 ng of rIFN-{beta} to Western blot analysis. As seen in Fig. 3Go, although no IFN-{beta} protein was detected in supernatant of cultured tumor cells from untreated mice, IFN-{beta} protein was clearly evident in supernatant of cultured tumor cells from low-dose L-PAM-treated mice. Thus, L-PAM treatment leads to up-regulated IFN-{beta} expression not only at the mRNA level but also at the protein level.



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  		FIGURE 3. L-PAM up-regulates IFN-{beta} production at the protein level. Tumor cells derived from the s.c. tumor nodule of untreated MOPC-315 tumor-bearing mice (lane 1) or MOPC-315 tumor-bearing mice treated 18 h earlier with low-dose L-PAM (lane 2) were placed in culture for 6 h. The presence of IFN-{beta} in the supernatants of these cultures was evaluated by Western blot analysis. As reference point, we provide the results with 5 (lane 3), 10 (lane 4), and 30 ng of rIFN-{beta} (lane 5) subjected simultaneously to Western blot analysis.

 
Assessment of the importance of IFN-{beta} for L-PAM-induced up-regulation of TNF-{alpha} expression in MOPC-315 tumor cells

Experiments were conducted to determine whether IFN-{beta} is important for L-PAM-induced up-regulation of TNF-{alpha} mRNA expression. For this purpose, we assessed the effect of IFN-{beta} neutralization, through the use of anti-IFN-{beta} mAb, on L-PAM-induced accumulation of TNF-{alpha} mRNA. We chose to assess TNF-{alpha} mRNA expression at 24 h after initiation of the L-PAM treatment since this was the earliest time point at which L-PAM therapy led to up-regulation of TNF-{alpha} mRNA expression at the tumor site (14), and since this was the earliest time after in vitro exposure of MOPC-315 tumor cells to L-PAM that elevated expression of TNF-{alpha} mRNA was seen (data not shown). In the current experiments, MOPC-315 tumor cells were treated in vitro with L-PAM for 1 h and subsequently cultured for an additional 23 h in the presence or absence of rat anti-mouse IFN-{beta} mAb, at the end of which TNF-{alpha} mRNA expression was determined. As seen in Fig. 4Go, anti-IFN-{beta} mAb led to a decrease in TNF-{alpha} mRNA expression in L-PAM-treated MOPC-315 tumor cells, indicating that IFN-{beta} plays an important role in the L-PAM-induced up-regulation of TNF-{alpha} mRNA expression.



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  		FIGURE 4. IFN-{beta} is important for L-PAM-induced up-regulation of TNF-{alpha} mRNA expression in MOPC-315 tumor cells. RNA was extracted from MOPC-315 tumor cells that were exposed in vitro to L-PAM for 1 h and then cultured for an additional 23 h in the presence (lane 3) or absence (lane 2) of anti-IFN-{beta} mAb. RNA extracted from these cultures as well as RNA extracted from untreated MOPC-315 tumor cells that were cultured in vitro for 23 h (lane 1) was subjected to RT-PCR with primers specific for TNF-{alpha} or {beta}-actin. The size of the PCR product for TNF-{alpha} was ~310 bp.

 
Assessment of the importance of IFN-{beta} for L-PAM-induced up-regulation of TNF-{alpha} expression in spleen cells

Since low-dose L-PAM leads to rapid accumulation of IFN-{beta} mRNA, not only in the tumor nodule of MOPC-315 tumor bearers but also in their spleens, we considered the possibility that L-PAM therapy which leads to subsequent elevation in TNF-{alpha} mRNA expression in tumor nodules of these mice would also do so in their spleens. Accordingly, we determined the effect of low-dose L-PAM administration to MOPC-315 tumor bearers on TNF-{alpha} mRNA expression in their spleens at 6, 12, and 24 h after the chemotherapy. As seen in Fig. 5GoA, whereas no accumulation of TNF-{alpha} mRNA was evident in the spleens of low-dose L-PAM-treated MOPC-315 tumor bearers at 6 h after the chemotherapy, a very slight, if any, accumulation was evident at 12 h after the chemotherapy with much more profound accumulation at 24 h after the chemotherapy. Thus, in spleens of MOPC-315 tumor-bearing mice, like in their tumor nodules, the L-PAM-induced up-regulated IFN-{beta} mRNA expression is followed by up-regulated TNF-{alpha} mRNA expression.



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  		FIGURE 5. L-PAM up-regulates TNF-{alpha} mRNA expression in spleen cells from MOPC-315 tumor-bearing BALB/c mice as well as in spleen cells from normal 129 Ev/Sv mice. RNA extracted from spleen cells from untreated MOPC-315 tumor-bearing BALB/c mice (lane 1) or MOPC-315 tumor-bearing BALB/c mice treated 6 (lane 2), 12 (lane 3), or 24 h (lane 4) earlier with low-dose L-PAM was subjected to RT-PCR with primers specific for TNF-{alpha} or {beta}-actin (A). In addition, RNA extracted from spleen cells from normal129 Ev/Sv mice that were exposed in vitro to L-PAM for 1 h and then cultured for an additional 23 h (lane 2), or RNA extracted from untreated spleen cells from 129 Ev/Sv mice that were cultured in vitro for 23 h (lane 1) was also subjected to RT-PCR with primers specific for TNF-{alpha} or {beta}-actin (B).

 
Next, since L-PAM was found to lead to rapid up-regulation of IFN-{beta} mRNA expression, not only in the tumor nodule and the spleen of MOPC-315 tumor bearers but also in the spleen of normal mice (data not shown), we considered the possibility that L-PAM may also lead to up-regulation of TNF-{alpha} mRNA expression in normal spleen cells. Our rationale for testing this possibility was that if exposure of normal spleen cells to L-PAM leads indeed to elevated expression of TNF-{alpha} mRNA, we should be able to use mice in which signaling via IFN-{beta} is deficient (i.e., IFN-{alpha}{beta}R-/-) to confirm the importance of IFN-{beta} for L-PAM-induced up-regulation of TNF-{alpha} mRNA expression. Since the mice in which signaling via IFN-{beta} is deficient are on the 129 Ev/Sv background, we determined whether in vitro exposure of spleen cells from wild-type 129 Ev/Sv mice to L-PAM leads to elevated expression of TNF-{alpha} mRNA at 24 h after initiation of the L-PAM treatment. As seen in Fig. 5GoB, in vitro exposure of spleen cells from normal 129 Ev/Sv mice to L-PAM resulted in elevated expression of TNF-{alpha} mRNA at 24 h after initiation of the L-PAM treatment.

Finally, experiments were conducted to determine whether in vitro exposure to L-PAM of spleen cells from normal IFN-{alpha}{beta} R-/- mice would also result in up-regulated expression of TNF-{alpha} mRNA at 24 h after initiation of the L-PAM treatment. As seen in Fig. 6GoA, L-PAM failed to up-regulate TNF-{alpha} mRNA expression in spleen cells from mice in which signaling by IFN-{beta} is deficient, even though the L-PAM treatment was associated with elevated expression of IFN-{beta} mRNA in spleen cells from the IFN-{alpha}{beta} R-/- mice (Fig. 6GoB). Thus, signaling through the IFN-{alpha}{beta} R is important for L-PAM-induced up-regulation of TNF-{alpha} expression.



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  		FIGURE 6. L-PAM up-regulates IFN-{beta} mRNA expression, but fails to up-regulate TNF-{alpha} mRNA expression in spleen cells from mice deficient in the IFN-{alpha}{beta} R (IFN-{alpha}{beta} R-/-). Spleen cells from IFN-{alpha}{beta} R-/- on 129 Ev/Sv background were exposed in vitro to L-PAM for 1 h and subsequently cultured for 23 h (lane 2). RNA extracted from these cultures as well as RNA extracted from untreated spleen cells that were cultured in vitro for 23 h (lane 1) was subjected to RT-PCR with primers specific for TNF-{alpha} (A) or IFN-{beta} (B) as well as with primers specific for {beta}-actin.

 
Effect of the RNA synthesis inhibitor actinomycin D on L-PAM-induced accumulation of IFN-{beta} mRNA

Experiments were next conducted to gain some insights into the mechanism through which L-PAM leads to up-regulated expression of IFN-{beta} mRNA. Initially we determined whether RNA synthesis is required for the L-PAM-induced rapid accumulation of IFN-{beta} mRNA. This was done by assessing the effect of actinomycin D, a known inhibitor of RNA synthesis, on the L-PAM-induced accumulation of IFN-{beta} mRNA. As seen in Fig. 7GoA, pretreatment of MOPC-315 tumor cells with actinomycin D before their treatment with L-PAM, as well as during the L-PAM treatment, prevented completely the L-PAM-induced accumulation of IFN-{beta} mRNA. Thus, de novo RNA synthesis is required for the L-PAM-induced rapid accumulation of IFN-{beta} mRNA, indicating that the regulation is at the transcriptional level.



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  		FIGURE 7. The RNA synthesis inhibitor actinomycin D, but not the protein synthesis inhibitor cycloheximide, inhibits the L-PAM-induced accumulation of IFN-{beta} mRNA. MOPC-315 tumor cells were exposed in vitro to L-PAM with (lane 3) or without (lane 2) 1 µg/ml actinomycin D (A) or 10 µg/ml cycloheximide (B). RNA extracted from these cells 2 h after initiation of the in vitro L-PAM treatment as well as RNA extracted from untreated MOPC-315 tumor cells (lane 1) was subjected to RT-PCR with primers specific for IFN-{beta} or {beta}-actin.

 
Effect of the protein synthesis inhibitor cycloheximide on L-PAM-induced accumulation of IFN-{beta} mRNA

Experiments were conducted to determine whether protein synthesis is required for the L-PAM-induced up-regulation of IFN-{beta} mRNA expression. Accordingly, we determined the effect of cycloheximide, a known inhibitor of protein synthesis, on the L-PAM-induced accumulation of IFN-{beta} mRNA. As seen in Fig. 7GoB, pretreatment of MOPC-315 tumor cells with cycloheximide before their treatment with L-PAM, as well as during the L-PAM treatment, did not inhibit the L-PAM-induced up-regulation of IFN-{beta} mRNA expression. In fact, MOPC-315 tumor cells treated with L-PAM plus cycloheximide expressed at 2 h after initiation of the L-PAM treatment a higher level of IFN-{beta} mRNA than did MOPC-315 tumor cells treated with L-PAM alone. Thus, L-PAM-induced IFN-{beta} gene activation does not require de novo protein synthesis.

Effect of exposure of MOPC-315 tumor cells to H2O2 on IFN-{beta} mRNA expression

In light of reports that L-PAM can increase the level of intracellular reactive oxygen species (34), coupled with reports that H2O2 induces rapid phosphorylation of RAX (a cellular activator of dsRNA-dependent protein kinase (PKR)) (35), as well as reports that PKR can activate IFN-{beta} gene expression (36), experiments were conducted to determine whether exposure of MOPC-315 tumor cells to H2O2 would also result in up-regulation of IFN-{beta} gene expression. For this purpose, MOPC-315 tumor cells were exposed in vitro for 15 min to H2O2 at a concentration ranging from 0.1 to 1.0 mM, and the level of IFN-{beta} mRNA expression was determined 2 h after initiation of the H2O2 treatment relative to the level of IFN-{beta} mRNA expression by untreated tumor cells. As seen in Fig. 8Go, in vitro exposure of MOPC-315 tumor cells to 0.3 or 1.0 mM, and to a lesser extent to 0.1 mM, H2O2, resulted in accumulation of IFN-{beta} mRNA. Thus, H2O2 mimics the effect of L-PAM on IFN-{beta} mRNA accumulation.



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  		FIGURE 8. In vitro exposure of MOPC-315 tumor cells to H2O2 leads to rapid accumulation of IFN-{beta} mRNA. MOPC-315 tumor cells were exposed in vitro for 15 min to 0.1 (lane 2), 0.3 (lane 3), or 1.0 mM H2O2 (lane 4) . RNA extracted from the H2O2-treated MOPC-315 tumor cells 2 h after initiation of the H2O2 treatment as well as RNA extracted from untreated MOPC-315 tumor cells (lane 1) was subjected to RT-PCR with primers specific for IFN-{beta} or {beta}-actin.

 
Effect of the antioxidant NAC on L-PAM-induced accumulation of IFN-{beta} mRNA in MOPC-315 tumor cells

The above observations that H2O2 mimics the effect of L-PAM on IFN-{beta} mRNA accumulation, coupled with reports by other investigators that L-PAM can increase the level of intracellular reactive oxygen species (34), prompted us to determine whether the antioxidant NAC can inhibit the L-PAM-induced accumulation of IFN-{beta} mRNA in MOPC-315 tumor cells. As seen in Fig. 9Go, treatment of MOPC-315 tumor cells with NAC 1 h before the L-PAM treatment, as well as during the L-PAM treatment, prevented the L-PAM-induced accumulation of IFN-{beta} mRNA. These results indicate that reactive oxygen species are involved in L-PAM-induced accumulation of IFN-{beta} mRNA.



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  		FIGURE 9. The antioxidant NAC inhibits the L-PAM-induced accumulation of IFN-{beta} mRNA. MOPC-315 tumor cells were exposed in vitro to L-PAM with (lane 3) or without (lane 2) 25 mM NAC. RNA extracted from these cells 2 h after initiation of the in vitro L-PAM treatment as well as RNA extracted from untreated MOPC-315 tumor cells (lane 1) was subjected to RT-PCR with primers specific for IFN-{beta} or {beta}-actin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous studies have shown that anticancer drugs can lead to a shift in the cytokine profile at the tumor site from anti-inflammatory cytokines toward proinflammatory cytokines (7, 11, 12, 13, 14, 15, 16, 17, 18). In these studies, the up-regulation of proinflammatory cytokine production (e.g., TNF-{alpha} and/or IFN-{gamma}) at the mRNA level was first evident 24 h or longer after the chemotherapy. However, earlier events after the chemotherapy that may have contributed to the chemotherapy-induced up-regulation of proinflammatory cytokine production have not been studied. In this study, we show that the widely used anticancer drug L-PAM leads to rapid accumulation of IFN-{beta} mRNA by activating IFN-{beta} gene expression via a mechanism that does not require de novo protein synthesis and involves reactive oxygen species. IFN-{beta} in turn plays an important role in L-PAM-induced subsequent up-regulation of TNF-{alpha} mRNA expression. In light of our previous observations demonstrating the importance of TNF-{alpha} production for the immunopotentiating activity of L-PAM for mice bearing large MOPC-315 tumors (18, 37), the current observations suggest that IFN-{beta} is important for the realization of the immunopotentiating effects of L-PAM in the MOPC-315 tumor system.

Schiavoni et al. (19) have recently shown that another widely used anticancer drug, CY, administered to normal mice leads, by 6 h after its administration, to elevated expression of IFN-{beta} mRNA in the spleens of these mice, which is followed by elevated production of a biologically active cytokine. Although the study by Schiavoni et al. (19) did not determine whether IFN-{beta} mRNA expression is elevated sooner than 6 h after CY administration, the time at which accumulation of IFN-{beta} mRNA was evident was sooner than the earliest time point after CY that up-regulation of expression of mRNA for TNF-{alpha} and/or IFN-{gamma} was seen by other investigators in tumor-bearing mice (7, 12) or in patients with malignancies (11). Thus, the timing of IFN-{beta} up-regulation in the study by Schiavoni et al. (19) is consistent with a potential role for IFN-{beta} in chemotherapy-induced up-regulation of TNF-{alpha} and/or IFN-{gamma} mRNA expression. Here, we extend the observations of Schiavoni et al. (19) by demonstrating that a different anticancer drug, L-PAM, can lead to up-regulation of IFN-{beta} mRNA expression in tumor-bearing mice, and it can do so within 1 h after the administration of the drug. In addition, we extend the observations of Schiavoni et al. (19) by demonstrating that IFN-{beta} is actually important for chemotherapy-induced up-regulation of TNF-{alpha} mRNA expression. Finally, we extend the observations of Schiavoni et al. (19) by providing some information on the molecular mechanism for L-PAM-induced rapid accumulation of IFN-{beta} mRNA.

We show here that in addition to up-regulating IFN-{beta} mRNA expression in the spleen of MOPC-315 tumor bearers, low-dose L-PAM therapy also leads to rapid up-regulation of IFN-{beta} mRNA expression in the s.c. tumor nodule of the mice. Moreover, we demonstrate that in vitro exposure of cells derived from the s.c. tumor nodule of mice bearing a large MOPC-315 tumor to L-PAM also results in rapid up-regulation of IFN-{beta} mRNA expression. These observations indicate that the up-regulation of IFN-{beta} mRNA expression at the tumor site in low-dose L-PAM-treated MOPC-315 tumor bearers is not necessarily the result of L-PAM-induced migration of host cells with up-regulated IFN-{beta} mRNA expression into the tumor nodule. At present we do not know in which cell types the L-PAM actually induced rapid accumulation of IFN-{beta} mRNA. However, the fact that in vitro exposure of an established in vitro line of MOPC-315 tumor cells to L-PAM also resulted in rapid elevation in IFN-{beta} mRNA expression indicates that tumor cells, not only host cells, are one such cell type. Regardless of the cell types that display up-regulated IFN-{beta} mRNA expression as a consequence of exposure to L-PAM, IFN-{beta} production plays an important role in L-PAM-induced subsequent up-regulation of TNF-{alpha} mRNA expression.

We show here that de novo RNA synthesis is required for L-PAM-induced accumulation of IFN-{beta} mRNA, indicating that the regulation is at the transcriptional level. In addition, we show that protein synthesis is not required for the L-PAM-induced de novo synthesis of IFN-{beta} mRNA, indicating that activation of latent transcription factors is sufficient for the L-PAM-induced activation of IFN-{beta} gene expression. This is not surprising, given the fact that the transcription factors required for IFN-{beta} gene activation were found to be NF-{kappa}B, c-jun/ATF2 (AP-1), and IFN regulatory factor, and each of these transcription factors is activated following phosphorylation events (38). The signaling pathway(s) that leads to L-PAM-induced IFN-{beta} gene activation is not known at present. However, it involves reactive oxygen species since the antioxidant NAC was found to block completely the ability of L-PAM to activate IFN-{beta} gene expression. Thus, it is possible that RAX is activated by oxidative stress (35), which in turn activates the dsRNA-dependent PKR pathway which leads to IFN-{beta} gene expression (36).

In the current study, we focused our attention on the role of IFN-{beta} in L-PAM-induced up-regulation of TNF-{alpha} expression as a prototype of type 1 cytokine that is up-regulated at the mRNA level 24 h or longer after the chemotherapy (14). TNF-{alpha} mRNA expression was chosen for our studies in light of our previous observations illustrating that TNF production is critical for low-dose L-PAM-induced: 1) acquisition of CTL activity by CD8+ T cells from hitherto immunosuppressed mice bearing large MOPC-315 tumors, and 2) cure of MOPC-315 tumor bearers under conditions that require the L-PAM-induced acquisition of CD8+ T cell-dependent tumor-eradicating immunity (14, 18, 37). Given the importance of TNF production for low-dose L-PAM-induced acquisition of tumor-eradicating immunity in the MOPC-315 tumor system, our current observations demonstrating that IFN-{beta} plays an important role in L-PAM-induced up-regulation of TNF-{alpha} mRNA expression suggest that IFN-{beta} plays an important role in low-dose L-PAM-induced acquisition of tumor-eradicating immunity by MOPC-315 tumor bearers as well as in the curative effectiveness of low-dose L-PAM for MOPC-315 tumor bearers.

At a first glance, it may seem surprising that L-PAM did not lead to rapid activation of TNF-{alpha} gene expression in the MOPC-315 tumor system, particularly since ionizing irradiation was reported to lead to rapid activation of TNF-{alpha} gene expression in HL-60, certain human sarcoma, and peripheral human T cells (39, 40, 41). Moreover, the enhancer of the TNF-{alpha} gene, like the enhancer of the IFN-{beta} gene, contains an NF-{kappa}B response element, which was shown in some studies to be important for TNF-{alpha} gene activation (42), and L-PAM, like ionizing irradiation, activates NF-{kappa}B (26). However, other transcription factors, which are not required for IFN-{beta} gene activation, were shown to be required for TNF-{alpha} gene activation (e.g., Erg-1 and Sp1) (43), and these transcription factors may not be activated rapidly following exposure of MOPC-315 tumor cells or normal mouse spleen cells to L-PAM. In fact, even ionizing radiation, which was shown to rapidly induce TNF-{alpha} gene expression in some human cells, failed to do so in others under the same experimental conditions (39). Moreover, different stimuli were recently shown to utilize different transcription factors to activate TNF-{alpha} gene expression (43). For example, while NFAT binding to the enhancer of TNF-{alpha} was shown to be required for TNF-{alpha} gene activation by calcineurin-dependent stimuli, it was found to be of no functional relevance in LPS stimulation of TNF-{alpha} gene expression (43). Regardless of the exact reason(s) why IFN-{beta} and not TNF-{alpha} is an early response gene that is induced rapidly by L-PAM in MOPC-315 tumor cells and in murine spleen cells, IFN-{beta} is important for the L-PAM-induced up-regulation of TNF-{alpha} mRNA expression.

In summary, our studies provide the first demonstration that a widely used anticancer drug leads to up-regulation of TNF-{alpha} mRNA expression, at least in part, by rapidly inducing the activation of the IFN-{beta} gene. Hence, our studies have identified an early event that occurs as a consequence of chemotherapy with L-PAM and conceivably other anticancer drugs and is important for the initiation of the immunopotentiating activity of the chemotherapy in tumor bearers (14, 18, 37).


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

2 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 60612-7334. E-mail address: mokyr{at}uic.edu Back

3 Abbreviations used in this paper used: CY, cyclophosphamide; L-PAM, L-phenylalanine mustard; NAC, N-acetyl-L-cysteine; PKR, dsRNA-dependent protein kinase. Back

Received for publication June 8, 2001. Accepted for publication August 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hengst, J. C. D., M. B. Mokyr, S. Dray. 1981. Cooperation between cyclophosphamide tumoricidal activity and host antitumor immunity in the cure of mice bearing large MOPC-315 tumors. Cancer Res. 41:2163.[Abstract/Free Full Text]
  2. Mokyr, M. B., E. Barker, L. 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. Takesue, B. Y., J. M. Pyle, M. B. Mokyr. 1990. Importance of tumor-specific cytotoxic CD8+ T-cells in eradication of a large subcutaneous MOPC-315 tumor following low-dose melphalan therapy. Cancer Res. 50:7641.[Abstract/Free Full Text]
  4. 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]
  5. North, R. J., M. Awwad. 1990. Elimination of cycling CD4+ suppressor T-cells with anti-mitotic drug releases non-cycling CD8+ T-cells to cause regression of an advanced lymphoma. Immunology 71:90.[Medline]
  6. 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]
  7. Yuan, L., Y. Kuramitsu, Y. Li, M. Kobayashi, M. Hosokawa. 1995. Restoration of interleukin-2 production in tumor-bearing rats through reduction of tumor-derived transforming growth factor {beta} by treatment with bleomycin. Cancer Immunol. Immunother. 41:355.[Medline]
  8. Berd, D., M. J. Mastrangelo. 1988. Active immunotherapy of human melanoma exploiting the immunopotentiating effects of cyclophosphamide. Cancer Invest. 6:337.[Medline]
  9. Sahasrabudhe, D. M., J. B. deKernion, J. E. Pontes, D. M. Ryan, R. W. O’Donnell, D. M. Marquis, G. S. Mudholkar, C. S. McCune. 1986. Specific immunotherapy with suppressor function inhibition for metastatic renal cell carcinoma. J. Biol. Response Modif. 5:581.[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. Lattime, E. C., M. J. Mastrangelo, O. Bagasra, W. Li, D. Berd. 1995. Expression of cytokine mRNA in human melanoma tissue. Cancer Immunol. Immunother. 41:151.[Medline]
  12. Inagawa, H., T. Nishicawa, T. Honda, T. Nakamoto, K. Takagi, G.-I. 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]
  13. Gorelik, L., A. Prokhorova, M. B. Mokyr. 1994. Low-dose melphalan-induced shift in the production of a TH2-type cytokine to a TH1-type cytokine in mice bearing a large MOPC-315 tumor. Cancer Immunol. Immunother. 39:117.[Medline]
  14. 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]
  15. Mokyr, M. B., T. V. Kalinichenko, L. Gorelik. 1997. Potentiation of antitumor CTL response by GM-CSF involves a B7-dependent mechanism. Cell. Immunol. 178:152.[Medline]
  16. Weiskirch, L., Y. Bar-Dagan, M. B. Mokyr. 1994. Transforming growth factor-{beta}-mediated down-regulation of antitumor cytotoxicity of spleen cells from MOPC-315 tumor-bearing mice engaged in tumor eradication following low-dose melphalan therapy. Cancer Immunol. Immunother. 38:215.[Medline]
  17. Matar, P., V. R. Rozados, A. D. Gonzalez, D. G. Dlugovitzky, R. D. Bonfil, O. G. Scharovsky. 2000. Mechanism of antimetastatic immunopotentiation by low-dose cyclophosphamide. Eur. J. Cancer 36:1060.
  18. Gorelik, L., M. Rubin, A. Prokhorova, M. B. Mokyr. 1995. Importance of TNF production for the curative effectiveness of low-dose melphalan therapy for mice bearing a large MOPC-315 tumor. J. Immunol. 154:3941.[Abstract]
  19. Schiavoni, G., F. Mattei, T. Di Puchio, S. M. Santini, L. Bracci, F. Belardelli, E. Proietti. 2000. Cyclophosphamide induces type I interferon and augments the number of CD44high T lymphocytes in mice: implications for strategies of chemoimmunotherapy of cancer. Blood 95:2024.[Abstract/Free Full Text]
  20. Belardelli, F.. 1995. Role of interferons and other cytokines in the regulation of immune responses. APMIS 103:161.[Medline]
  21. Cousens, L. P., R. Peterson, S. Hsu, A. Dorner, J. D. Altman, R. Ahmed, C. A. Biron. 1999. Two roads diverged: interferon {alpha} 47 {beta}- and interleukin 12-mediated pathways in promoting T cell interferon {gamma} responses during viral infection. J. Exp. Med. 189:1315.[Abstract/Free Full Text]
  22. Santini, S. M., C. Lapenta, M. Logozzi, S. Parlato, M. Spada, T. Di Pucchio, F. Belardelli. 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J. Exp. Med. 191:1777.[Abstract/Free Full Text]
  23. Nguyen, K. B., L. P. Cousens, L. A. Doughty, G. C. Pien, J. E. Durbin, C. A. Biron. 2000. Interferon {alpha} 47 {beta}-mediated inhibition and promotion of interferon {gamma}: STAT1 resolves a paradox. Nat. Immunol. 1:70.[Medline]
  24. Doughty, L. A., K. B. Nguyen, J. E. Durbin, C. A. Biron. 2001. A role for IFN-{alpha}{beta} in virus infection-induced sensitization to endotoxin. J. Immunol. 166:2658.[Abstract/Free Full Text]
  25. Biron, C. A., K. B. Nguyen, G. C. Pien, L. P. Cousens, T. P. Salazar-Mather. 1999. Natural killer cells in antiviral defense: function and regulation of innate cytokines. Annu. Rev. Immunol. 17:189.[Medline]
  26. Donepudi, M., P. Raychaudhuri, J. A. Bluestone, M. B. Mokyr. 2001. Mechanism of melphalan-induced B7-1 gene expression in P815 tumor cells. J. Immunol. 166:6491.[Abstract/Free Full Text]
  27. 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]
  28. Vilcek, J., G. Sen. 1996. IFNs and other cytokines. B. N. Fields, and D. M. Knipe, and P. M. Howley, eds. Virology 375.-399. Lippincott-Raven, Philadelphia.
  29. Mariè, I., J. E. Durbin, D. E. Levy. 1998. Differential viral induction of distinct interferon-{alpha} genes by positive feedback through interferon regulatory factor-7. EMBO J. 17:6660.[Medline]
  30. Leib, D. A., M. A. Machalek, B. R. G. Williams, R. H. Silverman, H. W. Virgin. 2000. Specific phenotypic restoration of an attenuated virus by knockout of host resistance gene. Proc. Natl. Acad. Sci. USA 97:6097.[Abstract/Free Full Text]
  31. Bocian, R. C., S. Dray, S. Ben-Efraim, M. B. Mokyr. 1985. Melphalan-induced enhancement of tumor cell immunostimulatory capacity as a mechanism for the appearance of potent antitumor immunity in the spleen of mice bearing a large metastatic MOPC-315 tumor. Cancer Immunol. Immunother. 20:61.[Medline]
  32. 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]
  33. 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]
  34. Ito, T., M. Yang, W. S. May. 1999. RAX, a cellular activator for double-stranded RNA-dependent protein kinase during stress signaling. J. Biol. Chem. 274:15427.[Abstract/Free Full Text]
  35. Young, Y.-L., L. F. L. Reis, J. Pavlovic, A. Aguzzi, R. Schäfer, A. Kumar, B. R. G. Williams, M. Aguet, C. Weissmann. 1995. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J. 14:6095.[Medline]
  36. Gorman, A., A. McGowan, T. G. Cotter. 1997. Role of peroxide and superoxide anion during tumour cell apoptosis. FEBS Lett. 404:27.[Medline]
  37. Gorelik, L., Y. Bar-Dagan, M. B. Mokyr. 1996. Insight into the mechanism(s) through which tumor necrosis factor promotes the generation of T cell-mediated antitumor cytotoxicity by tumor bearer splenic cells. J. Immunol. 156:4298.[Abstract]
  38. Smith, E. J., I. Mariè, A. Prakash, A. Garcia-Sastre, D. E. Levy. 2001. IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or I{kappa}B kinase but is blocked by vaccinia virus E3L protein. J. Biol. Chem. 276:8951.[Abstract/Free Full Text]
  39. Hallahan, D. E., D. R. Spriggs, D. W. Kufe, R. R. Weichselbaum. 1989. Increased tumor necrosis factor {alpha} mRNA after cellular exposure to ionizing radiation. Proc. Natl. Acad. Sci. USA 86:10104.[Abstract/Free Full Text]
  40. Hallahan, D. E., V. Virudachalam, M. L. Sherman, E. Huberman, D. W. Kufe, R. R. Weichselbaum. 1991. Tumor necrosis factor gene expression is mediated by protein kinase C following activation by ionizing radiation. Cancer Res. 51:4565.[Abstract/Free Full Text]
  41. Weill, D., F. Gay, M. G. Tovey, S. Chouaib. 1996. Induction of tumor necrosis factor {alpha} expression in human T lymphocytes following {gamma} irradiation. J. Interferon Cytokine Res. 16:395.[Medline]
  42. Young, S.-H., J. Ye, D. G. Frazer, X. Shi, V. Castranova. 2001. Molecular mechanism of TNF-{alpha} production in 1–3-{beta}-glucan (zymosan) activated macrophages. J. Biol. Chem. 276:20781.[Abstract/Free Full Text]
  43. Tsai, E. Y., J. V. Falvo, A. V. Tsytsykova, A. K. Barczak, A. M. Reimold, L. H. Glimcher, M. J. Fenton, D. C. Gordon, I. F. Dunn, A. E. A. Goldfeld. 2000. Lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo. Mol. Cell. Biol. 16:6084.



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