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The Journal of Immunology, 2000, 164: 6230-6236.
Copyright © 2000 by The American Association of Immunologists

Melphalan and Other Anticancer Modalities Up-Regulate B7-1 Gene Expression in Tumor Cells1

Dorothy K. Sojka*, Manjula Donepudi*, Jeffrey A. Bluestone2,{dagger} and Margalit B. Mokyr2,3,*

* Department of Biochemistry and Molecular Biology, University of Illinois, Chicago, IL 60612; and {dagger} The Ben May Institute for Cancer Research, Committee on Immunology, and Department of Pathology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we show that administration of low-dose melphalan (L-PAM, L-phenylalanine mustard) to mice bearing a large MOPC-315 plasmacytoma led to a rapid up-regulation of B7-1 (CD80), but not B7-2 (CD86), expression on the surface of MOPC-315 tumor cells. This L-PAM-induced preferential up-regulation of B7-1 surface expression was due, at least in part, to a direct effect of L-PAM on the tumor cells, as in vitro exposure of MOPC-315 tumor cells to L-PAM led to the preferential up-regulation of B7-1 surface expression. Moreover, in vitro exposure of MOPC-315 tumor cells to two other anticancer modalities, {gamma}-irradiation and mitomycin C, resulted in the preferential up-regulation of B7-1 surface expression. This effect was not restricted to MOPC-315 tumor cells, as preferential up-regulation of B7-1 surface expression was observed also following in vitro exposure of the P815 mastocytoma (that is negative for both B7-1 and B7-2 surface expression) to any of the three anticancer modalities. The up-regulation of B7-1 surface expression following in vitro exposure of tumor cells to L-PAM, {gamma}-irradiation, or mitomycin C required de novo protein and RNA synthesis, and was associated with the accumulation of mRNA for B7-1 within 4–8 h, indicating that the regulation of B7-1 expression is at the RNA transcriptional level. These results have important implications for an additional immune-potentiating mechanism of these anticancer modalities in clinical setting.


   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, 11). In fact, several anticancer drugs (e.g., cyclophosphamide (1, 4, 5, 6, 8, 10, 11), L-PAM4 (2), 1,3-bis(2-chloroethyl)-1-nitrosourea (7), vinblastine (9), and bleomycin (3)) have been shown to enhance the acquisition of T cell-mediated antitumor responses in a variety of animal tumor models (1, 2, 3, 6, 7, 8, 9) and in patients with advanced melanoma (4, 10, 11) or advanced renal carcinoma (5). Studies into the mechanisms through which the anticancer drugs enhance the acquisition of T cell-dependent tumor-eradicating immunity in tumor bearers revealed that the chemotherapy leads to a shift in the cytokine profile from anti-inflammatory cytokines (such as TGF-ß and IL-10) with inhibitory activity for CTL generation toward proinflammatory cytokines (e.g., TNF-{alpha}, IFN-{gamma}, and GM-CSF) that favor the development of antitumor cell-mediated immunity (12, 13, 14, 15, 16, 17, 18).

Very few studies have attempted to date to elucidate the role of the costimulatory molecules B7-1 and B7-2 in the chemotherapy-induced potentiation of the development of antitumor immunity in tumor bearers. We have recently initiated studies to elucidate the importance of B7-1 and B7-2 expression for L-PAM-induced acquisition of T cell-dependent tumor-eradicating immunity in hitherto immunosuppressed mice bearing a large MOPC-315 tumor (19). Specifically, we determined the effect of anti-B7-1 or anti-B7-2 mAb on the curative effectiveness of low-dose L-PAM for MOPC-315 tumor bearers under conditions in which the therapeutic outcome of the chemotherapy depends on the contribution of T cell-dependent immunity for tumor eradication (2, 20, 21). Our studies revealed that B7-1 and B7-2 are important for the curative effectiveness of low-dose L-PAM for MOPC-315 tumor bearers. At present it is not known whether the B7-1 and the B7-2 molecules that are important for the curative outcome of low-dose L-PAM for mice bearing a large MOPC-315 tumor are expressed on host APCs and/or on tumor cells. However, L-PAM-induced up-regulation of B7-1 and/or B7-2 expression on tumor cells and/or host APCs would be expected to promote the acquisition of tumor-eradicating immunity (22, 23, 24, 25, 26, 27).

In this study, we show that MOPC-315 tumor cells that are negative for B7-1 surface expression are induced in vivo to express substantial levels of B7-1 within 24 h after low-dose L-PAM therapy of mice bearing a large tumor. This L-PAM- induced up-regulation of B7-1 surface expression is due, at least in part, to a direct effect of the anticancer drug on the tumor cells. The ability to up-regulate B7-1 surface expression on tumor cells is not limited to the anticancer drug L-PAM or to the tumor line MOPC-315, as 1) two other anticancer modalities, {gamma}-irradiation and mitomycin C, also up-regulated B7-1 surface expression on MOPC-315 tumor cells, and 2) any of these three anticancer modalities up-regulated B7-1 surface expression on the P815 mastocytoma. The up-regulation of B7-1 surface expression following exposure of tumor cells to any of these three anticancer modalities required de novo protein and RNA synthesis, and was associated with a rapid accumulation of mRNA for B7-1. Taken together, the current studies suggest that one of the mechanisms through which anticancer modalities exert their immunopotentiating effect in tumor bearers involves the up-regulation of B7-1 surface expression by up-regulating B7-1 gene expression.


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

The MOPC-315 plasmacytoma was maintained in vivo, as previously described (2), in BALB/cAnNCrlBR mice 7–10 wk old, which were purchased from Charles Rivers Breeding Laboratories (Wilmington, MA). Routinely, 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 and leads to the appearance of palpable tumors in 4–5 days. The tumor nodules were excised on day 10–12 after tumor inoculation, when they reached 18–22 mm in diameter. Single cell suspensions were prepared by mechanical disruption between glass slides, and the cells were then exposed to the anticancer modalities. The P815 mastocytoma was maintained in vitro, as previously described (28), in low glucose DMEM supplemented with 10% FBS (Life Technologies, Grand Island, NY).

L-PAM therapy

A fresh stock solution of L-PAM (Sigma, St. Louis, MO) was prepared as previously described (2). A dose of 2 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 be curative for most BALB/c mice bearing a large MOPC-315 tumor, but only in cooperation with host CD8+ T cell-dependent antitumor immunity that emerges after the chemotherapy (20, 21). Significant regression of the s.c. tumor nodules is evident within 4–5 days after the L-PAM administration, with complete regression of the s.c. tumor nodules within 8–10 days after the chemotherapy (2, 20, 21). In the current experiments, s.c. tumor nodules were excised on day 1 after the chemotherapy, and single cell suspensions were prepared by mechanical disruption between glass slides. The single cell suspensions were examined for B7-1 and B7-2 surface expression by flow cytofluorometry.

In vitro exposure of MOPC-315 or P815 tumor cells to anticancer modalities

MOPC-315 tumor cells or P815 tumor cells were exposed in vitro to one of the following three anticancer modalities: L-PAM, {gamma}-irradiation, or mitomycin C. Unless otherwise stated, the L-PAM treatment consisted of exposing the tumor cells for 1 h to 15 nM L-PAM, as previously described (29). The {gamma}-irradiation treatment consisted of exposing the tumor cells to 40 GY from a 137Cs source (The J. L. Shepherd and Associates model 143-68 irradiator), and the mitomycin C treatment (Sigma) consisted of exposing the tumor cells to 50 µg/ml mitomycin C for 30 min. After the completion of the treatments with the anticancer modalities, the tumor cells were washed and subsequently cultured in vitro at a concentration of 0.5–0.75 x 106 cells/ml for 24 h, unless otherwise stated. The culture medium for the P815 cells consisted of low glucose DMEM supplemented with 10% FBS, while the culture medium for the MOPC-315 cells consisted of DMEM supplemented with 10% FBS, 0.1 mM nonessential amino acids (Life Technologies), and 2 x 10-5 2 ME (Sigma).

Flow-cytometric analysis

Assessment of B7-1 and B7-2 expression on MOPC-315 and P815 tumor cells was done with the aid 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 Ig (PharMingen). In experiments assessing B7-1 and B7-2 expression on MOPC-315 tumor cells, we gated on a population of cells that based on light scatter properties consisted almost exclusively of MOPC-315 tumor cells with contaminating B220+ cells and MAC-1+ cells constituting less than 5%. 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.

Reverse transcriptase PCR

Total RNA was extracted, as previously described (17), and subjected to reverse transcription and PCR with sense and antisense primers for B7-1 (5'-CTGTCCAAGTCAGTGAAAGAT for the sense primer, and 5'-GGACAACTTTACTAAAGCCA for the antisense) or B7-2, (5'-TATTTCAATGGGACTGCATAT for the sense, and 5'-CGATCACTGACAGTTCTGTTA for the antisense), which were synthesized by Integrated DNA Technologies (Coralville, IA). The cycling conditions for B7-1 and B7-2 consisted of 5 min at 94°C, 25 cycles of 15 s at 94°C, 15 s at 52°C, 30 s at 72°C, followed by a 7-min extension at 72°C. ß-Actin (Stratagene, La Jolla, CA) served as a standard to normalize for the quantity of mRNA subjected to RT-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 PCR products were determined relative to a standard 100-bp DNA ladder (Life Technologies) and were found to be of the expected size.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo administration of low-dose L-PAM to mice bearing a large MOPC-315 tumor leads to rapid up-regulation of B7-1, but not B7-2, expression on the surface of MOPC-315 tumor cells

We have previously shown that B7-1 and B7-2 contribute to the curative effectiveness of low-dose L-PAM for mice bearing a large MOPC-315 tumor under conditions that depend on the acquisition of CD8+ T cell-dependent tumor-eradicating immunity (19). In this study, we show that MOPC-315 tumor cells derived from the s.c. tumor nodule of untreated mice bearing a large (~20-mm) MOPC-315 tumor are negative for B7-1 expression, but express high levels of B7-2 on their surface (Fig. 1Go). However, within 24 h after low-dose chemotherapy of mice bearing a large MOPC-315 tumor, MOPC-315 tumor cells were found to up-regulate in vivo B7-1 surface expression with no change in the level of B7-2 surface expression, which was high to start with (Fig. 1Go). Thus, administration of low-dose L-PAM to mice bearing a large MOPC-315 tumor leads to rapid up-regulation of in vivo B7-1, but not B7-2, expression on the surface of MOPC-315 tumor cells.



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  		FIGURE 1. Low-dose L-PAM administered i.p. to mice bearing a large s.c. MOPC-315 tumor leads to rapid up-regulation of in vivo B7-1, but not B7-2, surface expression on the tumor cells. MOPC-315 tumor cells from untreated mice (Untreated Control) or from mice injected 1 day earlier with 2 mg/kg L-PAM (L-PAM) were stained with PE-conjugated NIgG (dotted line) and PE-conjugated anti-B7-1 (bold solid line) or PE-conjugated anti-B7-2 (bold solid line) mAb.

 
In vitro exposure of MOPC-315 tumor cells to L-PAM leads to up-regulation of B7-1, but not B7-2, surface expression

Experiments were conducted to determine whether the up-regulation of in vivo B7-1 expression on MOPC-315 tumor cells that was observed following low-dose L-PAM therapy of MOPC-315 tumor bearers was due, at least in part, to a direct effect of L-PAM on MOPC-315 tumor cells. Accordingly, we assessed the effect of exposing MOPC-315 tumor cells in vitro to L-PAM on B7-1 and B7-2 surface expression. In these studies, we employed a concentration of 15 nM L-PAM in light of our previous observations that in vitro exposure of MOPC-315 tumor cells to 15 nM L-PAM enhances the stimulatory capacity of the tumor cells for the in vitro generation of anti-MOPC-315 CTL activity (29). Specifically, MOPC-315 tumor cells were exposed in vitro for 1 h to 15 nM L-PAM and then cultured for 24 h before assessing the effect of the L-PAM treatment on B7-1 and B7-2 surface expression. As seen in Fig. 2Go, L-PAM-treated MOPC-315 tumor cells exhibited in vitro up-regulated B7-1 surface expression and unaltered B7-2 surface expression. The concentration of L-PAM used (i.e., 15 nM) was found in subsequent dose-response studies to be optimal for the up-regulation of B7-1 surface expression on MOPC-315 tumor cells (data not shown). Thus, L-PAM can act directly on MOPC-315 tumor cells, leading to up-regulation of B7-1, but not B7-2, surface expression.



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  		FIGURE 2. In vitro exposure of MOPC-315 tumor cells to L-PAM leads to up-regulation of B7-1, but not B7-2, surface expression. MOPC-315 tumor cells were exposed in vitro for 1 h to 15 nM L-PAM and subsequently cultured for an additional 24 h. At the end of the culture period, L-PAM-treated tumor cells (L-PAM) as well as untreated tumor cells (Untreated Controls) were stained with PE-conjugated NIgG (dotted line), or with PE-conjugated anti-B7-1 (bold solid line) or PE-conjugated anti-B7-2 (bold solid line) mAb.

 
In vitro exposure of MOPC-315 tumor cells to {gamma}-irradiation or mitomycin C also leads to up-regulation of B7-1, but not B7-2, surface expression

Experiments were conducted to determine whether in vitro exposure of MOPC-315 tumor cells to two other anticancer modalities also results in preferential up-regulation of B7-1 surface expression. Specifically, MOPC-315 tumor cells were exposed in vitro to {gamma}-irradiation or to mitomycin C, and 24 h later the tumor cells were examined for B7-1 and B7-2 surface expression. As seen in Fig. 3Go, in vitro exposure of MOPC-315 tumor cells to {gamma}-irradiation (A) or mitomycin C (B), like in vitro exposure of MOPC-315 tumor cells to L-PAM (Fig. 2Go), resulted in up-regulation of B7-1 surface expression. In addition, in vitro exposure of MOPC-315 tumor cells to {gamma}-irradiation or mitomycin C, like in vitro exposure of MOPC-315 tumor cells to L-PAM (Fig. 2Go), did not result in up-regulation of B7-2 surface expression. Thus, in vitro exposure of MOPC-315 tumor cells to {gamma}-irradiation and mitomycin C, like in vitro exposure of MOPC-315 tumor cells to L-PAM, leads to rapid up-regulation of B7-1, but not B7-2, surface expression.



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  		FIGURE 3. In vitro exposure of MOPC-315 tumor cells to {gamma}-irradiation or mitomycin C leads to up-regulation of B7-1, but not B7-2, surface expression. MOPC-315 tumor cells were exposed in vitro to {gamma}-irradiation (40 Gy) (A) or mitomycin C (50 µg/ml for 30 min) (B), and subsequently cultured for an additional 24 h. At the end of the culture period, tumor cells that were exposed to {gamma}-irradiation or to mitomycin C as well as untreated tumor cells were stained with PE-conjugated NIgG (dotted line), or with PE-conjugated anti-B7-1 (bold solid line) or PE-conjugated anti-B7-2 (bold solid line) mAb.

 
Preferential up-regulation of B7-1 expression on the surface of P815 tumor cells exposed in vitro to L-PAM, {gamma}-irradiation, or mitomycin C

In an attempt to determine whether our observations that L-PAM, {gamma}-irradiation, and mitomycin C can up-regulate B7-1 surface expression on MOPC-315 tumor cells can be extended to other tumors, we assessed the effect of exposing the P815 mastocytoma to these three anticancer modalities on the level of B7-1 and B7-2 surface expression. The P815 mastocytoma was chosen for these studies since, unlike the MOPC-315 plasmacytoma, it normally does not express either the B7-1 or the B7-2 molecule on its surface (28). Specifically, we examined the expression of B7-1 and B7-2 on the surface of P815 tumor cells that were exposed in vitro 24 h earlier to one of the above three anticancer modalities. As seen in Fig. 4Go, untreated P815 tumor cells were negative for B7-1 and B7-2 surface expression. However, within 24 h after exposure to either L-PAM (A), {gamma}-irradiation (B), or mitomycin C (C), P815 tumor cells expressed substantial levels of B7-1 on their surface. In contrast, P815 tumor cells exposed 24 h earlier to either one of the three anticancer modalities remained negative for B7-2 expression. Thus, B7-1 surface expression is up-regulated on the surface of two distinct tumor cell lines in response to L-PAM, {gamma}-irradiation, and mitomycin C. Furthermore, B7-2 expression is unaltered even under conditions wherein the tumor cells are negative for B7-2 expression before exposure to the anticancer modalities.



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  		FIGURE 4. In vitro exposure of P815 tumor cells to L-PAM, {gamma}-irradiation, or mitomycin C leads to up-regulation of B7-1, but not B7-2, surface expression. P815 tumor cells were exposed in vitro to L-PAM (15 nM for 1 h) (A), {gamma}-irradiation (40 Gy) (B), or mitomycin C (50 µg/ml for 30 min) (C), and subsequently cultured for an additional 24 h. At the end of the culture period, tumor cells that were exposed to the anticancer modalities as well as untreated tumor cells were stained with PE-conjugated NIgG (dotted line), or with PE-conjugated anti-B7-1 (bold solid line) or PE-conjugated anti-B7-2 (bold solid line) mAb.

 
In light of reports that the B7-2 is up-regulated earlier than the B7-1 after stimulation of APCs with other modalities (e.g., cytokines or anti-CD40 mAb) (30, 31, 32), we next considered the possibility that there is a temporal difference in the surface expression of B7-1 and B7-2 following in vitro exposure of P815 tumor cells to the above anticancer modalities. To test this possibility, P815 tumor cells were exposed in vitro to 15 nM L-PAM and subsequently cultured for 3, 6, 12, 18, and 24 h before examining the tumor cells for B7-2 surface expression. As seen in Fig. 5GoA, P815 tumor cells exposed to L-PAM remained negative for B7-2 surface expression at all time points after exposure to the anticancer drug. In contrast, L-PAM-treated P815 tumor cells expressed substantial levels of B7-1 on their surface as early as 12 h after the exposure to L-PAM, and the level of B7-1 expressed on the surface of the P815 tumor cells increased progressively thereafter until 24 h after the L-PAM exposure (Fig. 5GoB). No further increase in the level of B7-1 surface expression, or any B7-2 surface expression, was noted when P815 tumor cells were examined 48 h after the L-PAM treatment (data not shown).



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  		FIGURE 5. P815 tumor cells exposed in vitro to L-PAM remained negative for B7-2 surface expression at 3, 6, 12, 18, and 24 h after the L-PAM exposure, but became positive for B7-1 surface expression by 12 h after the L-PAM exposure. P815 tumor cells were exposed in vitro to L-PAM and subsequently cultured for an additional 3, 6, 12, 18, or 24 h. At the end of the culture period, tumor cells that were exposed to L-PAM as well as untreated tumor cells were stained with PE-conjugated NIgG (dotted line), or with PE-conjugated anti-B7-1 (bold solid line in B) or PE-conjugated anti-B7-2 (bold solid line in A) mAb.

 
Protein synthesis is required for the up-regulation of B7-1 surface expression following in vitro exposure of P815 tumor cells to L-PAM, {gamma}-irradiation, or mitomycin C

Experiments were conducted to determine whether protein synthesis is required for the up-regulation of B7-1 surface expression on P815 tumor cells exposed to L-PAM, {gamma}-irradiation, or mitomycin C. This was done by assessing the effect of cycloheximide, a known inhibitor of protein synthesis, on the level of B7-1 surface expression on P815 tumor cells exposed in vitro to the anticancer modalities. As seen in Fig. 6Go, cycloheximide led to a drastic decrease in the level of B7-1 expressed on the surface of P815 tumor cells exposed in vitro to L-PAM (A), {gamma}-irradiation (B), or mitomycin C (C) when measured 24 h after treatment with the anticancer modalities. Thus, protein synthesis is required to fully realize the potentiating effect of L-PAM, {gamma}-irradiation, or mitomycin C for B7-1 surface expression on P815 tumor cells.



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  		FIGURE 6. Cycloheximide-mediated inhibition of the up-regulation of B7-1 surface expression on P815 tumor cells exposed in vitro to L-PAM, {gamma}-irradiation, or mitomycin C. P815 tumor cells were exposed in vitro to L-PAM (A), {gamma}-irradiation (B), or mitomycin C (C) in the presence or absence of cycloheximide (CHX, 10 µg/ml) during the exposure to the anticancer modalities as well as during the subsequent 24-h culture period. P815 tumor cells exposed 24 h earlier to the anticancer modalities as well as untreated P815 tumor cells were stained with PE-conjugated NIgG (dotted line), or with PE-conjugated anti-B7-1 mAb (bold solid line).

 
RNA synthesis is required for the up-regulation of B7-1 surface expression following in vitro exposure of P815 tumor cells to L-PAM, {gamma}-irradiation, or mitomycin C

Initially, experiments were conducted to determine whether L-PAM, {gamma}-irradiation, or mitomycin C leads to accumulation of mRNA for the B7-1 in P815 tumor cells. In these studies, which utilized RT-PCR, we used RNA from the B7-1-transfected P815 tumor cells that we have previously described (28) as a positive control, and illustrated that they contain high levels of mRNA for B7-1 (Fig. 7Go, row I, lane 1). RT-PCR with RNA from parental P815 tumor cells revealed no mRNA for B7-1 (Fig. 7Go, row I, lane 2). Substantial levels of mRNA for B7-1 were evident, however, at 4 and 8 h after treatment of the parental P815 tumor cells with L-PAM (Fig. 7GoA, row I, lanes 3 and 4), or with {gamma}-irradiation (Fig. 7GoB, row I, lanes 3 and 4). mRNA for B7-1 was also evident in P815 tumor cells after treatment with mitomycin C, however, only at 8 h after the treatment (Fig. 7GoC, row I, lanes 3 and 4). RT-PCR for B7-2 expression with RNA from our B7-2-transfected P815 tumor cells (28) as a positive control (Fig. 7Go, row II, lane 1) revealed that parental P815 tumor cells were negative for mRNA for B7-2 (Fig. 7Go, row II, lane 2) and remained negative for mRNA for B7-2 also at 4 and 8 h after treatment of the parental P815 tumor cells with L-PAM (Fig. 7GoA, row II, lanes 3 and 4), {gamma}-irradiation (Fig. 7GoB, row II, lanes 3 and 4), or mitomycin C (Fig. 8GoC, row II, lanes 3 and 4). Thus, treatment of P815 tumor cells with L-PAM, {gamma}-irradiation, or mitomycin C leads to the accumulation of mRNA for B7-1, but not mRNA for B7-2.



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  		FIGURE 7. Accumulation of mRNA for B7-1, but not for B7-2, following treatment of P815 tumor cells with L-PAM, {gamma}-irradiation, or mitomycin C. RNA derived from untreated P815 tumor cells (lane 2) or tumor cells exposed to L-PAM (A), {gamma}-irradiation (B), or mitomycin C (C) 4 h (lane 3), or 8 h (lane 4) earlier was subjected to RT-PCR with primers specific for B7-1 (row I) or B7-2 (row II). RNA obtained from B7-1-transfected P815 tumor cells (lane 1, row I) and RNA obtained from B7-2-transfected P815 tumor cells (lane 1, row II) served as positive controls. ß-actin served as a standard to normalize for the quantity of mRNA subjected to RT-PCR in the various samples. The size of the PCR products for B7-1 and B7-2 was ~300 bp.

 


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  		FIGURE 8. Actinomycin D-mediated inhibition of the up-regulation of B7-1 surface expression on P815 tumor cells exposed in vitro to L-PAM, {gamma}-irradiation, or mitomycin C. P815 tumor cells were exposed in vitro to L-PAM (A), {gamma}-irradiation (B), or mitomycin C (C) in the presence or absence of actinomycin D (Actino D, 1 µg/ml) during the exposure to the anticancer modalities as well as during the subsequent 24-h culture period. P815 tumor cells exposed 24 h earlier to the anticancer modalities as well as untreated P815 tumor cells were stained with PE-conjugated NIgG (dotted line), or with PE-conjugated anti-B7-1 mAb (bold solid line).

 
We next determined whether RNA synthesis is required for the up-regulation of B7-1 surface expression on P815 tumor cells exposed to L-PAM, {gamma}-irradiation, or mitomycin C. This was done by assessing the effect of actinomycin D, a known inhibitor of RNA synthesis, on the level of B7-1 surface expression on P815 tumor cells exposed in vitro to the anticancer modalities. As seen in Fig. 8Go, actinomycin D prevented completely the up-regulation of B7-1 expression on the surface of P815 tumor cells exposed to L-PAM (A), {gamma}-irradiation (B), or mitomycin C (C). Thus, de novo synthesis of mRNA for B7-1 is essential for the L-PAM-, {gamma}-irradiation-, and mitomycin C-induced up-regulation of B7-1 surface expression on P815 tumor cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that MOPC-315 tumor cells are negative for B7-1 surface expression. However, within 24 h after treatment of mice bearing a large MOPC-315 tumor with low-dose L-PAM, MOPC-315 tumor cells derived from the s.c. tumor nodule of such mice express in vivo substantial levels of B7-1 on their surface. At present it is not known whether B7-1 expression on the surface of MOPC-315 tumor cells from low-dose L-PAM-treated MOPC-315 tumor bearers is important for the therapeutic outcome of low-dose L-PAM for MOPC-315 tumor bearers. However, such a possibility seems likely for the following reasons. First, the B7-1 molecule was previously shown, with the aid of anti-B7-1 mAb (that masks B7-1 expression on tumor cells and on host APCs), to contribute to the therapeutic outcome of low-dose L-PAM therapy for MOPC-315 tumor bearers (19) under conditions that require the acquisition of T cell-dependent antitumor immunity by the hitherto immunosuppressed tumor bearers (2, 20, 21). Second, transfection of the B7-1 gene into tumor cells was shown to enhance the ability of the tumor cells to elicit tumor-eradicating immunity (22, 23, 24, 28, 33).

In principal, two different mechanisms could account for the up-regulation of in vivo B7-1 expression on the surface of MOPC-315 tumor cells from low-dose L-PAM-treated MOPC-315 tumor bearers. Accordingly, the L-PAM may act directly on the tumor cells leading to up-regulation of B7-1 surface expression and/or the L-PAM may act on host cells, which in turn would bring about the up-regulation of B7-1 surface expression on the MOPC-315 tumor cells. In this study, we show that the up-regulation of B7-1 surface expression on MOPC-315 tumor cells from low-dose L-PAM-treated MOPC-315 tumor bearers is due, at least in part, to a direct effect of the anticancer drug on tumor cells, as in vitro exposure of MOPC-315 tumor cells to L-PAM resulted in up-regulation of B7-1 surface expression within 24 h after the treatment.

In vitro exposure of MOPC-315 tumor cells to two other anticancer modalities, {gamma}-irradiation and mitomycin C, was also found to up-regulate B7-1 surface expression. To our knowledge, this is the first demonstration that exposure of cells to L-PAM or mitomycin C leads to the up-regulation of B7 surface expression. Information is available, however, regarding the ability of {gamma}-irradiation to up-regulate B7-1 surface expression. Specifically, Seo et al. (34) have shown that in vitro exposure of a different B cell tumor (the A20.2J lymphoma) to {gamma}-irradiation resulted in the up-regulation of B7-1 surface expression. In addition, they have shown that the B7-1 that is expressed on the {gamma}-irradiated A20.2J cells was important for the enhanced efficiency of the {gamma}-irradiated A20.2J cells in stimulating an OVA-specific Th1 clone to produce IL-2 in response to OVA. Our current studies extend the observations of Seo et al. with regard to the ability of {gamma}-irradiation to up-regulate B7-1 surface expression by demonstrating that {gamma}-irradiation can up-regulate B7-1 expression not only on a B cell tumor, but also on a non-B cell tumor, the P815 mastocytoma. Moreover, our studies provide information regarding the molecular basis for the {gamma}-irradiation-induced up-regulation of B7-1 surface expression by illustrating that the {gamma}-irradiation-induced up-regulation of B7-1 surface expression requires de novo protein and mRNA synthesis.

The current studies demonstrate that L-PAM, {gamma}-irradiation, and mitomycin C lead to preferential up-regulation of B7-1 without concomitant increases in B7-2 mRNA or protein expression. Although in the MOPC-315 tumor system the lack of an effect on B7-2 expression could be attributed to the relatively high constitutive level of B7-2 expression on the tumor cells, P815 tumor cells are B7-2 negative and the anticancer modalities still did not cause increased expression of this costimulatory molecule. 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 (35, 36, 37, 38, 39). Moreover, Gajewski (40) 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 CTL response.

The regulation of B7-1 expression appears to be at the RNA transcriptional level because the RNA synthesis inhibitor actinomycin D completely prevented the up-regulation of B7-1 surface expression on P815 tumor cells exposed to L-PAM, {gamma}-irradiation, or mitomycin C. Consistent with this finding is our observation that accumulation of mRNA for B7-1 was evident in P815 tumor cells within 4–8 h after in vitro exposure to the anticancer modalities. At present we do not know which transcription factors are activated in P815 tumor cells following exposure to the anticancer modalities and are critical for the up-regulation of the B7-1 gene expression. However, NF-{kappa}B and AP-1 are likely candidates because: 1) {gamma}-irradiation (41, 42, 43) as well as mitomycin C (44, 45) were shown to activate NF-{kappa}B and AP-1 in other cell lines, and 2) the promoter/enhancer region of the B7-1 gene contains an NF-{kappa}B and an AP-1 response element (46, 47, 48, 49). Studies are currently underway to assess the importance of NF-{kappa}B and/or AP-1 for the up-regulation of B7-1 gene expression by the anticancer modalities.

B7-1 is not the only gene whose expression is up-regulated as a consequence of exposure to anticancer modalities. In fact, {gamma}-irradiation, which is the best-studied anticancer modality in this regard, was shown to up-regulate the expression of genes encoding cytokines such as TNF-{alpha}, IL-1ß, IL-6, and TGF-ß (42, 50, 51, 52), as well as the expression of surface molecules such as ICAM-1 (51) and E-selectin (53). In addition, the up-regulated expression of at least some of these genes (e.g., TNF-{alpha} and IL-6) was shown to result from elevated mRNA synthesis, leading to the conclusion that at least part of the {gamma}-irradiation-induced cellular responses are mediated by up-regulation of gene expression (42). In fact, subsequent studies revealed that transcriptional modulation is a critical control point in the up-regulation of cellular processes as a consequence of {gamma}-irradiation (41, 42, 43).

Costimulation of T cells through CD28/B7 interaction was shown to play an important role in eliciting tumor-eradicating immunity, and inadequate costimulation has been suggested to contribute to tumor progression in immunocompetent hosts (22, 23, 24). Therefore, in an attempt to elicit a more powerful antitumor immunity, several groups of investigators have introduced the B7-1 gene into tumor cells (that express on their surface tumor-associated Ags in the context of MHC) (22, 23, 24). Indeed, numerous studies utilizing a variety of tumor types have shown that B7-1 transfectants can trigger the development of sufficient tumor-eradicating immunity to lead to their rejection and provide immunoprotection against a challenge with unmodified (B7-negative) parental tumor cells (22, 23, 24, 28, 33, 38). The ability to rapidly induce B7-1 surface expression on tumor cells by exposing the tumor cells to L-PAM, mitomycin C, or {gamma}-irradiation offers a fast and efficient method to endow these cells with the expression of a surface molecule that is important for the elicitation of tumor-eradicating immunity, without the labors and risks associated with transfecting or infecting (e.g., with a recombinant replication-defective adenovirus) the tumor cells with the B7-1 gene.


   Footnotes
 
1 This work was supported by Research Grant R01 CA-76532 from the National Institutes of Health. M.D.’s work is in partial fulfillment of the requirements for the Doctor of Philosophy Degree. Back

2 J.A.B. and M.B.M. are co-senior authors. 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. Back

4 Abbreviations used in this paper: L-PAM, L-phenylalanine mustard; NIgG, normal IgG. Back

Received for publication January 4, 2000. Accepted for publication April 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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