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The Journal of Immunology, 2003, 170: 6329-6337.
Copyright © 2003 by The American Association of Immunologists

Coordinate Regulation of IFN Consensus Sequence-Binding Protein and Caspase-1 in the Sensitization of Human Colon Carcinoma Cells to Fas-Mediated Apoptosis by IFN-{gamma}

Kebin Liu and Scott I. Abrams1

Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} is thought to be essential for the regulation of antitumor reactions. However, the degree of responsiveness of malignant cells to IFN-{gamma} may have a profound influence on the overall efficacy of an antitumor response. In this study, we examined the molecular basis by which IFN-{gamma} differentially sensitized human primary and metastatic colon carcinoma cells to Fas-mediated apoptosis. To that end, we analyzed IFN-{gamma}-induced gene expression at the genome scale, followed by an analysis of the expression and function of specific genes associated with IFN-{gamma}- and Fas-mediated signaling. We found that although both cell populations exhibited a similar gene expression profile at the genome scale in response to IFN-{gamma}, the expression intensities of the IFN-{gamma}-regulated genes were much greater in the primary tumor. Noteworthily, two genes, one involved in IFN-{gamma}-mediated signaling, IFN consensus sequence-binding protein (ICSBP), and one involved in Fas-mediated signaling, caspase-1, were clearly shown to be differentially induced between the two cell lines. In the primary tumor cells, the expression of ICSBP and caspase-1 was strongly induced in response to IFN-{gamma}, whereas they were weakly to nondetectable in the metastatic tumor cells. Functional studies demonstrated that both caspase-1 and ICSBP were involved in Fas-mediated apoptosis following IFN-{gamma} sensitization, but proceeded via two distinct pathways. This study also reports for the first time the expression of ICSBP in a nonhemopoietic tumor exhibiting proapoptotic properties. Overall, in a human colon carcinoma cell model, we identified important functional contributions of two IFN-{gamma}-regulated genes, ICSBP and caspase-1, in the mechanism of Fas-mediated death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} is a pleiotropic proinflammatory cytokine integral for the regulation of type 1 cellular immune reactions, including those relevant for antitumor activity (1, 2). In that setting, IFN-{gamma} may be important not only at the effector cell level, but also at the target cell level, where it may alter tumor-associated biologic properties, such as immunogenicity, antigenicity, and even responsiveness to apoptotic stimuli (3, 4, 5, 6, 7). Consequently, from an immunotherapy standpoint, the degree or extent of responsiveness of malignant cells to IFN-{gamma} may have a profound influence on the overall efficacy of an antitumor lymphocyte-mediated response.

IFN-{gamma}, secreted primarily by activated T cells and NK cells after antigenic encounter, has been shown to be involved in the regulation of apoptotic processes, including sensitization of target cells to Fas-mediated death (8, 9). Although the molecular mechanisms underlying the regulation of Fas-mediated apoptosis by IFN-{gamma} remain to be fully understood, it has been reported that this might occur in a number of different cell types through induction of Fas expression (8, 10). Because Fas is an important receptor-mediated signaling pathway for triggering apoptotic death by both innate and adaptive elements of the immune system (11, 12, 13), disengagement of such a cell death mechanism in neoplastic cells might confer a selective survival advantage for tumor escape from IFN-{gamma}-mediated effector mechanisms.

In human colon tumorigenesis, immunohistochemical evidence suggests that diminished Fas expression is a common occurrence of an advancing neoplastic phenotype (14, 15, 16, 17). Furthermore, in functional studies, it has been shown that IFN-{gamma} can sensitize human primary colon carcinoma cells to Fas-mediated apoptosis involving enhanced Fas expression (18, 19). However, in contrast to what was observed with the primary tumor, IFN-{gamma} failed to sensitize its metastatic counterpart to Fas-mediated death (5). This model system consisted of two naturally occurring cell lines, termed SW480 and SW620, which have been previously characterized as primary and metastatic colon adenocarcinoma cell lines, respectively, established from the same patient (20). The SW620 cell line was derived as a lymph node metastasis identified 6 mo later during disease relapse. In fact, both cell lines were isolated from the patient without any prior chemotherapy. To better understand the molecular basis for the differential sensitization of these primary and metastatic colon carcinoma cells to Fas-mediated apoptosis by IFN-{gamma}, we analyzed IFN-{gamma}-induced gene expression at the genome scale, followed by an analysis of the expression and function of specific genes associated with IFN-{gamma}- and Fas-mediated signaling. We identified an overall diminished responsiveness of the metastatic population in the induction of IFN-{gamma}-regulated gene expression and, in particular, in two key genes: one involved in IFN-{gamma}-mediated signaling and the other in Fas-mediated signaling. These findings support the hypothesis that an altered or reduced responsiveness of certain neoplastic populations to IFN-{gamma}-regulated gene expression may contribute to a more apoptotic-resistant phenotype.


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

The HLA-A2+, SW480 (CCL-228), and SW620 (CCL-227) colon adenocarcinoma cell lines (20) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). As described in the ATCC product sheets, the SW480 and SW620 cell lines were acquired by the ATCC after their establishment from the patient and have been cryopreserved since then. According to the ATCC data, the SW480 and SW620 cell lines were received at passages 91 and 79, and cryopreserved at passages 99 and 86, respectively. Experiments described in this study were conducted for up to an additional 25–30 passages in vitro, without any evidence of phenotypic or functional changes (in terms of Fas expression). Transgenic mice with spontaneously arising primary and metastatic mammary carcinoma (21), now backcrossed in a C57BL/6 (H-2b) background, were kindly provided by S. Gendler (Mayo Clinic, Scottsdale, AZ) via J. Schlom (National Institutes of Health, Bethesda, MD). This transgenic mouse model was originally produced by expression of the polyomavirus middle T Ag via germline introduction of the middle T oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer (21). These mice were reported to develop multifocal mammary adenocarcinoma palpable by 5 wk of age. By 3 mo of age, a significant proportion of these tumor-bearing mice developed metastatic lesions in the lung (21). In our colony, the primary tumor was resected from a progressively growing mammary lesion, while the metastatic tumor from the same mouse (>120 days of age) was established from lung digests. Briefly, the appropriate tissues were removed, digested for 4–6 h at room temperature with an enzyme mixture containing hyaluronidase (0.1 mg/ml), collagenase (1 mg/ml), and DNase I (30 U/ml) (all enzymes obtained from Sigma-Aldrich, St. Louis, MO), then washed and maintained in culture. Experiments with these mouse mammary tumor cell lines were conducted within 10–15 passages of their establishment. All tumor cell lines were verified to be of nonhemopoietic origin based, in part, on the absence of detectable cell surface expression of CD45, as determined by flow cytometry. All tumor cell lines were mycoplasma negative, as determined by PCR analysis using the Mycoplasma Detection Kit from the ATCC.

Measurement of apoptotic cell death

Tumor cells were either untreated or pretreated overnight (18–24 h) with human rIFN-{gamma} (sp. act. 2.4 x 107 U/mg; 250 U/ml) (Biogen Research, Cambridge, MA). Apoptotic cell death was measured by the TUNEL assay (22) or propidium iodide (PI)2 staining. For the TUNEL assay, untreated or IFN-{gamma}-pretreated tumor cells were incubated at 37°C for 18–24 h in the absence or presence of an apoptosis-inducing anti-Fas mAb, clone CH-11 (Immunotech, Westbrook, ME) (23) or an isotype-matched (IgM) control Ab (MOPC-104E; ICN Biomedicals, Aurora, OH). An apoptosis detection kit (R&D Systems, Minneapolis, MN) was then used to quantitate the percentage of TUNEL+ cells. Briefly, cells were fixed with a 3.7% formaldehyde solution at room temperature for 10 min, washed in saline, and permeabilized in Cytonin reagent for 30 min at room temperature. Cells were then washed and incubated with TdT and biotinylated nucleotides for 1 h at 37°C, followed by incubation with streptavidin conjugated to a fluorescent tag (FITC) for 10 min at room temperature. Cells were then washed again and analyzed immediately by flow cytometry. For PI measurement, tumor cells were incubated with a PI/RNase solution (R&D Systems) for 10 min at room temperature, and analyzed immediately by flow cytometry. The rationale for the selection of the IFN-{gamma} dose used in this study (250 U/ml) was based on earlier experiments that demonstrated that this concentration was optimal for the sensitization of the SW480 primary tumor cell line to Fas-mediated death (5). Furthermore, doses as high as 1000 U/ml had no effect on sensitizing the SW620 metastatic cell line to Fas-mediated death (5).

Cell surface marker analysis

Untreated or IFN-{gamma}-pretreated tumor cells (250 U/ml for 18–24 h, as previously described in Ref.5) were incubated with an anti-Fas mAb (clone DX-2; BD PharMingen, San Diego, CA), or an isotype-matched control, followed by washing and incubation with an affinity-purified, FITC-conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Cells were then analyzed by flow cytometry.

RT-PCR analysis and DNA sequencing

Total RNA was isolated from cells, as previously described (24, 25), and used for the first strand cDNA synthesis using the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA). The cDNA was then used as templates for PCR amplification of the indicated transcripts. The following parameters were used: 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C for 30 cycles for amplification of all cDNA except mouse IFN consensus sequence-binding protein (ICSBP) and caspase-1, which were amplified for 25 cycles. The PCR primers were listed in Table I. To sequence the human Fas coding sequence, RT-PCR was performed essentially as described above, except that the annealing temperature used was 55°C to amplify the full-length cDNA. The PCR primers were as follows: forward, 5'-ATGCTGGGCATCTGGACCCTC-3'; reverse, 5'-CACTCTAGACCAAGCTTTGG-3'. The PCR-amplified Fas cDNA fragment was then cloned into plasmid pCR4 Blunt-TOPO (Invitrogen), according to the manufacturer’s instructions. The plasmid was then used to transform TOP10 cells (Invitrogen), amplified, and then purified. DNA sequencing was conducted using the standard fluorescent dye terminator method.


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  		Table I. PCR primer sequences used in this study

 
cDNA microarrays, probes, hybridization, and analysis of data

Total RNA and mRNA were isolated from cells, as previously described (24, 25), and used for cDNA probe preparation. In general, mRNA (0.8 µg) was used for each cDNA probe preparation. cDNA probes were synthesized using the FairPlay microarray labeling kit (Stratagene, La Jolla, CA). The cDNA probes made from mRNA isolated from SW620 cells were then labeled with Cy3 monofunctional reactive dye (Amersham Biosciences, Piscataway, NJ), and cDNA probes made from mRNA isolated from SW480 cells were labeled with Cy5 monofunctional dye (Amersham Biosciences). The appropriate Cy3- and Cy5-labeled probes were combined, along with 10 µg Cot-1 DNA (Invitrogen), 4 µg yeast tRNA, and 10 µg poly(dA) in a final volume of 15 µl, and incubated at 98°C for 1 min. The denatured probes were mixed with 15 µl 2x hybridization buffer (50% formamide, 10x SSC, and 0.1% SDS). The hybridization solution and cDNA probe mixtures were added to the processed National Cancer Institute (NCI) human cDNA microarray slides (NCI, Advanced Technology Center, Gaithersburg, MD), which were then placed in hybridization chambers and incubated at 43°C for 16 h. The cDNA microarray chips used in these experiments consisted of 9128 nonredundant cDNA clones. The slides were washed for 5 min in 2x SSC and 0.1% SDS, for 5 min in 1x SSC, for 5 min in 0.2x SSC, for 5 min in 0.05x SSC, and then spun dried. Fluorescence images were captured using a Genepix 4000 (Axon Instruments, Union City, CA). Both image and signal intensity data were loaded onto a database supported by the Center for Information Technology of the National Institutes of Health. Cy3:Cy5 intensity ratios from each gene or cDNA clone were calculated and subsequently normalized to ratios of overall signal intensity from the corresponding channel in each hybridization. The normalized data were then extracted from the database as text files, and analyzed using computer software JMP (SAS Institute, Cary, NC) to compare the gene expression profiles quantitatively. For clustering analysis, Cluster and TreeView programs (26) were used to analyze the gene expression patterns in a one-dimensional hierarchical clustering to generate gene dendrograms based on the pairwise calculation of the Pearson coefficient of normalized fluorescence ratios as measurements of similarity and linkage clustering. The clustered data were loaded into TreeView and displayed by the graded color scheme.

Caspase inhibition assay

Tumor cells were pretreated with IFN-{gamma}, and then recultured in the absence or presence of peptide-based caspase inhibitors with specificity for caspase-1, Z-YVAD-FMK, or Z-LEVD-FMK (ICN Biomedicals) at a final concentration of 20 µM, as directed by the manufacturer. A negative control peptide, Z-FA-FMK (ICN Biomedicals), was included in a parallel set of cultures. The cells were preincubated with the various peptides at 37°C for 30 min, followed by the addition of CH-11 or MOPC-104E as an isotype control Ab (1 µg/ml). The cultures were then incubated at 37°C for 24 h, collected, stained with PI, and analyzed by flow cytometry for the percentages of dead cells.

Stable transfection of SW480 and SW620 cells with human ICSBP

SW480 and SW620 cells were transfected with the mammalian expression plasmid pcDNA3.1 (Invitrogen) containing the human ICSBP gene, kindly provided by B. Levi (Technion, Haifa, Israel) via E. Eklund (Northwestern University, Chicago, IL). Groups of tumor cells were also transfected with the vector control plasmid, pcDNA3.1, lacking the human ICSBP gene. Transfections were performed using LipofectAMINE 2000 reagent (Invitrogen), according to the manufacturer’s instructions. The transfected cells were propagated and maintained in culture medium containing Geneticin (0.75 mg/ml) (Invitrogen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential expression of Fas by SW480 and SW620 cells

SW480 and SW620 cells were analyzed for Fas expression in the absence or presence of IFN-{gamma} treatment at both the RNA and protein levels. In untreated tumor cells, the Fas transcript level was higher in SW480 cells as compared with SW620 cells (Fig. 1A). After IFN-{gamma} treatment, the Fas transcript levels increased markedly in both cell lines as early as 4 h after cytokine exposure; however, the magnitude of the increase was again greater in SW480 cells as compared with SW620 cells (Fig. 1A). Similarly, cell surface expression of Fas was also higher on SW480 cells as compared with SW620 cells, before or after treatment with IFN-{gamma} for 24 h (Fig. 1B). Based on their mean fluorescence intensity values (Fig. 1C), the relative number of Fas molecules on the cell surface correlated well with the Fas transcript levels in both cell lines before and after IFN-{gamma} treatment.



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  		FIGURE 1. Differential Fas expression and sensitivity to apoptotic death induced by anti-Fas mAb in SW480 and SW620 cells. A, RT-PCR analysis of Fas transcript levels in SW480 and SW620 cells. Tumor cells were incubated in the absence (indicated by 0) or presence of IFN-{gamma} for 4 or 24 h. Total RNA was then isolated, which was then used for cDNA synthesis and PCR amplification using Fas-specific and {beta}-actin-specific primers. {beta}-actin was used as normalization standard. B, Cell surface expression of Fas in SW480 (a) and SW620 (b) cells, as measured by flow cytometry. The histograms of untreated cells are represented by the solid thin lines, and the histograms of IFN-{gamma}-treated cells are indicated by solid thick lines. The isotype control staining for the IFN-{gamma}-treated cells is indicated by a gray-filled area. C, The relative Fas expression levels were quantified by measuring the mean fluorescence intensity values of tumor cells stained with anti-Fas mAb, as shown in B. Data represent the mean ± SEM of three separate experiments. D, Apoptotic death in SW480 (a and b) and SW620 (c and d) cells, as measured by the TUNEL assay. Tumor cells were incubated in the absence (a and c) or presence (b and d) of IFN-{gamma} overnight, and then treated with either CH-11 (solid line) or an isotype-matched control Ab (gray-filled area).

 
The sensitivity of these two cell lines to Fas-mediated death was then determined in response to CH-11, and the extent of apoptotic death was analyzed by TUNEL staining (Fig. 1D). In the absence of IFN-{gamma} treatment, SW480 cells showed a weak response toward CH-11 (Fig. 1Da). However, the percentage of apoptotic SW480 cells induced by CH-11 was substantially increased after IFN-{gamma} treatment (Fig. 1Db). As a specificity control, an isotype-matched control Ab failed to trigger detectable apoptotic death. In contrast to what was observed with SW480 cells, pretreatment of SW620 cells with IFN-{gamma} had little, if any, effect on their subsequent sensitivity to CH-11 (Fig. 1D, c and d), despite the fact that Fas expression in SW620 cells was substantially up-regulated by IFN-{gamma} (Fig. 1, A and B). Because SW620 cells up-regulated Fas expression at both the RNA and protein levels, but did not respond functionally to Fas engagement, we considered the possibility that these cells might express a nonfunctional or mutated Fas receptor. To examine that possibility, we cloned the full-length Fas cDNA from SW620 cells. However, DNA sequencing analysis found no mutations in the entire Fas coding region (data not shown).

Genome scale analysis of IFN-{gamma}-induced alterations in gene expression of SW480 and SW620 cells

To begin to understand the molecular basis for the differential sensitization of SW480 and SW620 cells to Fas-mediated apoptosis by IFN-{gamma}, we analyzed IFN-{gamma}-regulated gene expression. We first examined the effects of IFN-{gamma} on gene expression at the genome scale in SW480 and SW620 cells at different time points after cytokine exposure. SW480 and SW620 cells were pretreated with IFN-{gamma} for either 4 or 24 h, and then were collected for gene expression profiling by cDNA microarray analysis. The cDNA microarray chips used in these experiments consisted of 9128 nonredundant cDNA clones. Three independent experiments under each experimental condition were conducted. We set up an arbitrary cutoff of log2 = 0.3 (1.24-fold) to select genes (cDNA clones) whose expression levels were increased (larger than 0.3) or decreased (less than -0.3). To match the selection criteria, the ratios in all three replicated experiments must have been larger than 0.3 or less than -0.3. This arbitrary cutoff was chosen based on the notion that a lower cutoff number would generate or qualify a larger number of genes for the selection and comparison. We further reasoned that the analysis of data from a larger pool of genes would better represent the patterns of gene expression at the genome scale. By this selection criteria, we collectively identified 2275 genes (cDNA clones) whose expression levels were increased, and 1081 genes (cDNA clones) whose expression levels were decreased in at least one time point and in SW480 cells, SW620 cells, or both populations. However, there were 97 genes (cDNA clones) that overlapped between both categories of up- and down-regulated genes. Therefore, a total of 3259 discrete genes (cDNA clones) was identified whose expression levels were altered by IFN-{gamma}. The large number of genes affected by this standard (cutoff), consequently, provided an extensive set of data points for the generation of more comprehensive and unique molecular signatures for comparative purposes after IFN-{gamma} treatment.

Next, we used Cluster array data clustering and Treeview visualization programs (26) to analyze the expression profiles of these 3259 genes (cDNA clones). Log2-concerted expression data from these 3259 genes (cDNA clones) measured across three independent hybridization reactions were subjected to one-dimensional hierarchical clustering to generate gene dendrograms based on the pairwise calculation of the Pearson correlation coefficient of ratios as measures of similarity and complete linkage clustering. The reordered table from Cluster was imported into the Treeview program and displayed by a graded color scheme (Fig. 2A). In general, these 3259 genes (cDNA clones) displayed three distinct expression patterns (Fig. 2A): those that were down-regulated at both time points in both cell lines (group III); those that were up-regulated at both time points in both cell lines (group II); and those that represented a unique expression pattern between the two cell lines (group I). The vast majority of the 3259 genes (cDNA clones) (i.e., groups II and III) exhibited a similar qualitative expression pattern between the two cell lines after IFN-{gamma} treatment over a 24-h period (Fig. 2A). However, at a quantitative level, it was clear that the increase or decrease in mRNA levels of genes up- and down-regulated was greater in SW480 cells as compared with SW620 cells at both time points after IFN-{gamma} treatment (Fig. 2A). The average log ratios of the up-regulated genes were 0.3 ± 0.07 and 0.41 ± 0.04 at 4 and 24 h, respectively, in SW480 cells, and 0.13 ± 0.04 and 0.14 ± 0.02 for 4 and 24 h, respectively, in SW620 cells (Fig. 2B). The average log ratios of the down-regulated genes were -0.41 ± 0.02 and -0.27 ± 0.04 at 4 and 24 h, respectively, in SW480 cells, and -0.21 ± 0.03 and -0.19 ± 0.02 at 4 and 24 h, respectively, in SW620 cells (Fig. 2B). It is important to point out, however, that the data shown in Fig. 2B are reported as the mean ± SD for a given cell line at a given time point and, therefore, may include genes overlapping between time points or between cell lines. However, the actual numbers of discrete genes up-regulated or down-regulated were 2275 and 984, respectively.



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  		FIGURE 2. Comparison of genome scale gene expression profiles of SW480 and SW620 after IFN-{gamma} treatment. A, Diagram of complete-linkage hierarchical clustering and array clustering of SW480 and SW620 cells based on the expression profiles of 3259 genes (cDNA clones) whose expression levels were either increased or decreased by IFN-{gamma} treatment. SW480 or SW620 cells were first treated with IFN-{gamma} for 4 or 24 h, as shown, and then prepared for cDNA microarray analysis, as described in Materials and Methods. Untreated tumor cells were also prepared and used as the reference for hybridization with the corresponding IFN-{gamma}-treated preparation. A total of three independent hybridization reactions was conducted, and the data from these three replicated experiments are labeled as 1, 2, or 3 (shown to the right). Each colored square represents a single cDNA clone based on the ratio of expression between IFN-{gamma}-treated cells and the corresponding untreated cells. Essentially, three distinct gene expression patterns were observed, and designated as groups I, II, or III. The scale of intensity ratio is shown, with red indicating an increase and green indicating a decrease in gene expression. B, Results summarizing the intensity ratios of genes up-regulated (above zero) or down-regulated (below zero) in SW480 (filled bars) or SW620 (gray bars) cells after treatment with IFN-{gamma} for 4 or 24 h. For the genes up-regulated in SW480 cells at 4 and 24 h, n = 964 and 1717, respectively. For the genes up-regulated in SW620 cells at 4 and 24 h, n = 293 and 65, respectively. For the genes down-regulated in SW480 cells at 4 and 24 h, n = 673 and 360, respectively. For the genes down-regulated in SW620 cells at 4 and 24 h, n = 396 and 198, respectively. Data are reported as the mean ± SD for a given cell line at a given time point. Given the large number of genes compared, the p values for the comparisons between SW480 and SW620 cells at both time points were highly significant and closely approached zero.

 
Differential expression patterns of specific IFN-{gamma}-regulated genes in SW480 and SW620 cells

We next focused our efforts on changes in the expression levels of two groups of genes: the IFN regulatory factors (IRFs) and genes involved in the Fas-signaling pathway, including the caspases. Among the 10 known IRFs, IRF1 and ICSBP were both significantly up-regulated in SW480 cells, whereas only IRF1 was up-regulated in SW620 cells after IFN-{gamma} treatment, as determined by cDNA microarray analysis. RT-PCR analysis confirmed that IRF1 was up-regulated in both cell lines, and that ICSBP was up-regulated only in SW480 cells (Fig. 3). In parallel experiments conducted at the same time, IRF2, IRF3, IRF6, and IFN-stimulated gene factor 3 {gamma} were expressed in both cell lines, but their expression levels were not changed by IFN-{gamma} treatment, whereas IRF4, IRF5, IRF7, and vIRF were not detected in either cell line (data not shown). Importantly, both the basal and IFN-{gamma}-inducible levels of STAT1 expression were comparable between both cell lines, suggesting that IFN-{gamma}-mediated signaling immediately downstream from the receptor was functionally intact.



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  		FIGURE 3. IFN-{gamma} induction of caspase-1, STAT1, IRF1, and ICSBP in SW480 and SW620 cells. Tumor cells were incubated in the absence or presence of IFN-{gamma}, as in Fig. 1, and harvested 4 or 24 h later to isolate total RNA, which were then used for cDNA synthesis and PCR amplification using gene-specific primers. {beta}-actin was used as normalization standard. These results are representative of three separate experiments.

 
The Fas-mediated signaling pathway has been extensively studied, and a large number of genes involved in that pathway have now been identified (16, 27). Collectively, from our cDNA microarray and RT-PCR studies, we did not identify any of the following known genes in the Fas-signaling pathway whose expression level was demonstrably changed by IFN-{gamma}: Fas-associated death domain protein, cellular inhibitor of apoptosis protein 1, cellular inhibitor of apoptosis protein 2, X-linked inhibitor of apoptosis protein, survivin, Fas-associated phosphatase 1, cellular FLICE inhibitory protein, long form, cellular FLICE inhibitory protein, short form, receptor interacting protein, Bcl-XL, Bcl-Xs, Bcl-2, Bax, second mitochondria-derived activator of caspases, and I-{kappa}B{alpha} (data not shown). cDNA microarray analysis, however, revealed that caspases-1 and -7 were up-regulated by IFN-{gamma} in SW480 cells and caspase-7 in SW620 cells. RT-PCR analysis also revealed that the caspase-1 transcript in SW480 cells was detectable after 4 h and strongly expressed after 24 h of IFN-{gamma} treatment (Fig. 3). In contrast, the caspase-1 transcript was not detectable in SW620 cells 4 h after IFN-{gamma} treatment, and only marginally detectable 24 h after IFN-{gamma} treatment (Fig. 3). Multiple bands of caspase-7 were detected by RT-PCR in both SW480 and SW620 cells, indicating the likely existence of multiple variants (data not shown). In parallel experiments conducted at the same time, we found that caspases-2, -3, -4, -6, -8, and -9 were expressed in both cell lines, but their expression levels were apparently unchanged by IFN-{gamma} treatment, whereas caspases-5 and -10 were not detected in either cell line (data not shown).

Differential expression patterns of ICSBP and caspase-1 in a matched pair of primary and metastatic mouse mammary carcinoma cell lines

We next sought to extend the association or link between ICSBP and/or caspase-1 expression in a mouse tumor model of primary and metastatic disease. We have established a matched pair of primary and metastatic tumor cell lines from a transgenic mouse colony with spontaneously arising mammary carcinoma (21), which was thought to more closely resemble the natural process of tumor progression. In this setting, the metastases form within the lungs of those mice with advanced primary tumor burden. Using this model, we examined the expression of ICSBP and caspase-1, as well as other transcripts in both cell lines, with and without IFN-{gamma} treatment (Fig. 4). As with the human colon carcinoma studies (Fig. 3), we found a very similar pattern of expression of ICSBP and caspase-1. In the primary tumor line, termed D4387B, both transcripts were expressed at a low level and further up-regulated after IFN-{gamma} treatment, whereas both transcripts were not detectable in the metastatic counterpart, termed D4387L, even after IFN-{gamma} treatment. In contrast, both cell lines expressed STAT1 and IRF1, suggesting that signaling through the IFN-{gamma} receptor was intact.



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  		FIGURE 4. Induction of ICSBP, caspase-1, IRF1, and STAT1 in primary and metastatic mouse mammary carcinoma cells. A matched pair of primary (D4387B) and metastatic (D4387L) mouse mammary tumor cell lines was incubated in the absence (-) or presence (+) of mouse rIFN-{gamma}, in a manner similar to that of Figs. 1 and 3, and harvested 24 h later to isolate total RNA, which were then used for cDNA synthesis and PCR amplification using gene-specific primers. Mouse {beta}-actin was used as normalization standard. These results are representative of three separate experiments.

 
IFN-{gamma} sensitized SW480 cells to Fas-mediated apoptosis through the induction of caspase-1

The differential induction of caspase-1 in SW480 and SW620 cells by IFN-{gamma} indicated that caspase-1 might have mechanistically contributed, at least in part, to the differential sensitivity of these cell lines to Fas-mediated apoptosis. To address that possibility, we first examined the functional role of caspase-1 in Fas-mediated apoptosis in IFN-{gamma}-treated SW480 cells. IFN-{gamma}-treated SW480 cells were incubated with CH-11 in the absence or presence of two peptide-based caspase-1-specific inhibitors (tested separately) or a negative control peptide (Fig. 5). In the absence of any inhibitor, CH-11 induced >45% cell death (Fig. 5, Aa and B). In the presence of the two different caspase-1-specific inhibitors, CH-11-induced cell death was substantially reduced, whereas the negative control peptide had no inhibitory effect (Fig. 5, Ac and d and B). These data suggested that IFN-{gamma} sensitized SW480 cells to Fas-mediated apoptosis involving a caspase-1-dependent pathway.



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  		FIGURE 5. Inhibition of apoptotic cell death by peptide-based caspase-1-specific inhibitors. SW480 cells were pretreated with IFN-{gamma}, as in Fig. 1, and then incubated with CH-11 (1 µg/ml) (solid thick line) in the absence (a) or presence of caspase-1 inhibitors, Z-YVAD-FMK (c) or Z-LEVD-FMK (d), for 24 h. Z-FA-FMK was used as a negative control peptide (b). Cell death was measured by PI staining. Cells incubated with the isotype control for CH-11 in the absence of any inhibitor are shown by a gray-filled histogram. B, Percentage of cell death (mean ± SEM) of three separate experiments.

 
Functional role of ICSBP in Fas-mediated apoptosis of SW480 and SW620 cells

ICSBP is a transcription factor with dual functionality in that it can either activate or repress downstream target genes involved in apoptotic processes, and has been reported to be expressed in hemopoietic cells (28, 29). As with caspase-1, ICSBP was selectively induced in SW480 cells after IFN-{gamma} treatment, and remained undetectable in SW620 cells. We hypothesized that the defect in ICSBP induction by IFN-{gamma} might have led to the failure of caspase-1 induction and, consequently, the inability to initiate Fas-induced apoptosis in SW620 cells. If that were the case, then the introduction of ICSBP should render SW620 cells, at least to some extent, responsive to Fas-mediated apoptosis. To that end, ICSBP was ectopically expressed in SW620 cells by transfection with a mammalian expression vector containing the human ICSBP coding sequence. Similarly, SW480 cells were transfected with the same expression vector. The prediction in this study was that it may further potentiate Fas-mediated lysis of SW480 cells.

As shown earlier, SW480 cells, but not SW620 cells, expressed ICSBP mRNA after IFN-{gamma} treatment, as determined by both cDNA microarray and RT-PCR analyses (Fig. 3). After transfection with the plasmid encoding the human ICSBP gene, both tumor cell lines expressed the ICSBP transcript level without any IFN-{gamma} treatment (Fig. 6, lanes 5 and 11). After exposure to IFN-{gamma}, the expression of ICSBP in the SW480-ICSBP transfectants increased even further, which was most likely due to the accumulation of both ectopic and IFN-{gamma}-inducible endogenous sources (Fig. 6, lane 6). In contrast, the constitutive or basal expression level of ICSBP in the SW620-ICSBP transfectants remained unchanged after IFN-{gamma} treatment, consistent with the notion that ectopic introduction of ICSBP was the only detectable source in SW620 cells (Fig. 6, lanes 11 and 12). However, ectopic expression of ICSBP in SW620 cells, as well as SW480 cells, did not activate caspase-1 expression (Fig. 6, lanes 11 and 5, respectively). On the contrary, ectopic expression of ICSBP repressed the transcription of caspase-1 in both IFN-{gamma}-treated SW480 cells (Fig. 6, lane 6 vs 2 or 4) and SW620 cells (Fig. 6, lane 12 vs 8 or 10). SW480 or SW620 cells transfected with the empty vector as a negative control showed patterns of ICSBP and caspase-1 mRNA expression (before or after IFN-{gamma} treatment) similar to that of the nontransfected tumor cell lines (Fig. 6, lanes 1–4 and 7–10).



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  		FIGURE 6. ICSBP and caspase-1 expression in SW480 and SW620 cells ectopically transfected with ICSBP. Untransfected, vector-transfected, or ICSBP-transfected SW480 and SW620 cells were incubated in the absence (-) or presence (+) of IFN-{gamma} for 24 h. The mRNA levels of ICSBP and caspase-1 were then analyzed by RT-PCR using gene-specific primers. {beta}-actin was used as normalization standard. These results are representative of three separate experiments, except for the vector control groups, which are representative of two separate experiments.

 
We next examined the effects of ectopic expression of ICSBP on Fas-mediated apoptosis in both SW480 and SW620 cells. Ectopic expression of ISCBP did not efficiently sensitize SW480 or SW620 cells to Fas-mediated apoptosis without prior IFN-{gamma} treatment (Fig. 7, A, a1–f1, and B). However, ectopic expression of ICSBP resulted in ~20% cell death of IFN-{gamma}-treated SW620 cells in response to CH-11 (Fig. 7, A, d2 and e2 vs f2, and B), and dramatically enhanced death in IFN-{gamma}-treated SW480 cells (Fig. 7, A, a2 and b2 vs c2, and B). SW480 or SW620 cells transfected with the empty vector as a negative control showed patterns of sensitivity to Fas-mediated apoptosis (before or after IFN-{gamma} treatment) similar to that of the nontransfected tumor cell lines (Fig. 7, A and B).



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  		FIGURE 7. Enhancement of Fas-mediated apoptosis by ectopic expression of ICSBP in a caspase-1-independent manner. A, Fas-mediated cell death, as determined by PI staining. Untransfected (a and d), vector-transfected (b and e), or ICSBP-transfected (c and f) SW480 (a–c) and SW620 (d–f) cells were incubated in the absence (1) or presence (2) of IFN-{gamma} for 24 h, followed by treatment with CH-11 (solid line) or an isotype-matched control Ab (gray-filled area) for an additional 20 h. Cells were then harvested, stained with PI, and analyzed by flow cytometry. B, The percentage of cell death, as shown in A, was quantified as the mean ± SEM of three independent experiments. C, Effect of caspase-1 inhibition on Fas-mediated death of ICSBP transfectants. Untransfected (a and c) and ICSBP-transfected (b and d) SW480 (a and b) and SW620 (c and d) cells were incubated in the presence of IFN-{gamma} for 24 h. The IFN-{gamma}-pretreated tumor cells were then incubated in the absence (1) or presence of a negative control peptide, Z-FA-FMK (2), or caspase-1-specific inhibitor, Z-LEVD-FMK (3), for 30 min, before treatment with CH-11 (solid line) or an isotype-matched Ab (gray-filled area) for another 20 h. Cells were then harvested, stained with PI, and analyzed by flow cytometry. D, The percentage of cell death, as shown in C, was quantified as the mean ± SEM of three independent experiments.

 
Despite the precise levels of expression of ICSBP and caspase-1, Fas-mediated death of both cell lines was observed only under conditions in which both ICSBP and caspase-1 were coexpressed or detectable. Because ectopic expression of ICSBP repressed caspase-1 transcription, it was unlikely that the enhancement of apoptosis in IFN-{gamma}-treated SW480 cells by forced ICSBP expression was exclusively through a caspase-1 pathway. However, this did not preclude the possibility that overexpression of ICSBP affected caspase-1 activation at the protein level. To determine whether the enhanced sensitivity of IFN-{gamma}-treated tumor cells to Fas-mediated apoptosis by ectopic ICSBP expression was linked to caspase-1, we assessed the effects of a caspase-1-specific inhibitor on CH-11-mediated death of these ICSBP transfectants. As expected, the caspase-1-specific inhibitor strongly blocked Fas-mediated apoptosis of IFN-{gamma}-treated SW480 tumor cells (Fig. 7, Ca1–a3 and D). In contrast, this same caspase-1 inhibitor only partially blocked Fas-mediated death of the ICSBP-transfected, IFN-{gamma}-treated SW480 cells (Fig. 7, Cb1–b3 and D), strongly implicating a role for a caspase-1-independent mechanism. Therefore, the extent of contribution of a caspase-1-dependent or -independent pathway to this death response correlated with the balance or level of expression of ICSBP and caspase-1. Finally, this same caspase-1 inhibitor blocked Fas-mediated death of IFN-{gamma}-treated ICSBP-transfected SW620 cells (Fig. 7, Cd1–d3 and D), consistent with the low level of caspase-1 expression by these transfectants (Fig. 6). However, because the overall magnitude of Fas-mediated death was much lower with SW620 cells as compared with SW480 cells, it was more difficult to unmask or discriminate a caspase-1-independent component under these conditions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IFN-{gamma} is a key proinflammatory cytokine produced by type 1-directed immune reactions (1). IFN-{gamma} mediates numerous biologic activities, including those that are essential for antitumor activity in therapeutic paradigms, as well as the control of both spontaneous and chemically induced tumor formation in animal models (3, 4, 6). Some of these antineoplastic effects have been attributed to the direct modulation of antigenic and apoptotic properties of tumor cells, consequently rendering them more susceptible to immune recognition and attack. Thus, if IFN-{gamma} is crucial for the regulation of tumor growth or antitumor immune responses, then cancerous cells that develop an altered or diminished responsiveness to IFN-{gamma}-regulated gene expression may exhibit a unique biologic advantage that favors tumor escape and metastatic development. For example, tumor cells that intrinsically develop resistance to IFN-{gamma}-regulated, Fas-mediated apoptosis may evade FasL-dependent antitumor interactions.

In human colon carcinoma, Fas expression has been reported to be down-regulated during tumor formation and progression from benign to malignant disease (14). SW480 and SW620 cells are a pair of human primary and metastatic colon carcinoma cell lines, respectively, derived from the same patient (20). Both cell lines displayed a differential pattern of sensitivity to Fas-mediated apoptosis, in that the primary tumor, but not the metastatic tumor, harbored an IFN-{gamma}-inducible Fas-responsive phenotype. The inability of IFN-{gamma} to render SW620 cells Fas sensitive most likely reflected a postreceptor signaling blockade, because IFN-{gamma} treatment still efficiently up-regulated the level of cell surface Fas expression at both the RNA and protein levels (Fig. 1). Furthermore, DNA sequence analysis of the coding region of the Fas gene, including the death domain, in SW620 cells revealed it to be wild type. Therefore, although the degree of Fas induction by IFN-{gamma} was somewhat lower in SW620 cells as compared with SW480 cells (Fig. 1), the intensity of cell surface Fas expression alone did not seem solely to dictate a Fas-resistant phenotype. These observations implied that the expression of other genetic aberrations in the Fas-signaling pathway downstream of receptor engagement was most likely additionally responsible for the Fas-resistant phenotype of the metastatic cell line. Therefore, we next compared gene expression patterns of SW480 and SW620 cells after IFN-{gamma} treatment at the genome scale (Fig. 2). Although global gene expression profiles were generally similar between both cell lines after IFN-{gamma} treatment, the intensity of genes up- or down-regulated by IFN-{gamma} was much greater in the primary tumor. In fact, such quantitative differences in the gene expression intensities were observed across a large gamut of genes, suggesting that the differential responses of the primary and metastatic cell lines to Fas-mediated apoptosis, as well as other IFN-{gamma}-associated activities, were not limited to an altered expression pattern of just a few genes, but rather the induction of a larger spectrum of IFN-{gamma}-regulated genes.

Binding of IFN-{gamma} to its cell surface receptor leads to the activation of transcription factors known as STATs (30). Activated STATs form dimers and translocate to the nucleus, where they bind to well-defined DNA sequences and activate a family of secondary transcription factors, termed the IRFs (31, 32). In this study, we observed that STAT1 was significantly up-regulated in both cell lines following IFN-{gamma} treatment (Fig. 3). Because STAT1 was up-regulated comparably in both cell lines, it was unlikely that SW620 cells were defective in the IFN-{gamma} receptor. However, ICSBP levels were significantly lower in SW620 cells as compared with SW480 cells (Fig. 3). This finding suggested that biochemical or functional differences observed between this primary-metastatic pair in terms of their response toward IFN-{gamma} might reside, at least in part, at the level of this transcription factor, which functions downstream of STAT1. How ICSBP becomes less responsive to IFN-{gamma} induction remains unclear, and warrants further study.

In addition to the identification of a key member of the IFN-{gamma}-signaling pathway, we identified a component of the Fas-signaling pathway that was also clearly differentially expressed between both cell lines. Caspases are known as essential mediators of apoptosis induced by a number of proapoptotic stimuli, including those that directly activate the Fas pathway (16, 27). In this study, we detected a significant up-regulation of caspase-1 mRNA in SW480 cells as early as 4 h after IFN-{gamma} treatment (Fig. 3). In contrast, in SW620 cells, caspase-1 mRNA was not observed after 4 h, and only weakly detectable after 24 h of exposure to IFN-{gamma}. Moreover, based on inhibitor studies, we demonstrated a functional role for caspase-1 in Fas-mediated apoptosis of IFN-{gamma}-treated SW480 cells (Figs. 5–7). These findings suggested that the failure of caspase-1 induction by IFN-{gamma}, as with ICSBP induction, may have contributed, at least in part, to the resistance of the SW620 cells to Fas-mediated apoptosis. Furthermore, we extended the link between IFN-{gamma}-regulated gene expression of ICSBP and caspase-1 and malignant phenotype using a matched pair of primary and metastatic tumor cell lines derived from a transgenic mouse model with spontaneously arising mammary carcinoma (Fig. 4). Although future studies are warranted to explore this model in detail, these data, at least at this time, lend additional support to the potential physiologic relevance of the connection between IFN-{gamma}-regulated expression of ICSBP and/or caspase-1 and metastatic phenotype.

As mentioned above, ICSBP has been reported to be expressed in cells of the immune system (33, 34) and harbors multiple functions (33, 35). It can associate with IRF1 and act as a transcriptional repressor (28, 36, 37). The observation that peptide-based inhibitors with specificity for caspase-1 blocked Fas-mediated death in IFN-{gamma}-treated SW480 cells (Figs. 5 and 7) indicated that IFN-{gamma} sensitized SW480 cells to Fas-mediated apoptosis through the induction of caspase-1. However, the ability of caspase-1 inhibitors to only partially block Fas-mediated apoptosis in IFN-{gamma}-treated, ICSBP-transfected SW480 cells (Fig. 7) indicated that ectopic expression of ICSBP also initiated a caspase-1-independent apoptotic death pathway, the exact nature of which requires further investigation. Therefore, the extent of contribution of a caspase-1-dependent or -independent pathway to this death response correlated with the level of expression of ICSBP and caspase-1 seemingly in a coordinate manner (Fig. 6). Although both untransfected and ICSBP-transfected, IFN-{gamma}-treated SW480 cells expressed both ICSBP and caspase-1 mRNA, the ratio or balance of these two transcripts seemed to correlate or determine which pathway dominated. In the case of SW620 cells, because the overall extent of Fas-mediated death was much lower with IFN-{gamma}-treated ICSBP-transfected SW620 cells (Fig. 7), it was more difficult to unmask a caspase-1-independent component under those conditions.

Taken collectively, we propose that IFN-{gamma} treatment of SW480 cells induces IRF1, which, in turn, induces the transcriptional activation of caspase-1 (38, 39), followed by a caspase-1-dependent mechanism of apoptosis ensuing Fas ligation (40, 41). However, under conditions of ectopic expression of ICSBP, the enhanced production of ICSBP can result in binding to IRF1, which in turn can repress caspase-1 transcription (28, 42). Although the precise nature of the specific interactions in this model requires further study, ectopic expression of ICSBP was able to further potentiate Fas-mediated apoptosis of IFN-{gamma}-treated SW480 cells and partially in IFN-{gamma}-treated SW620 cells (Fig. 7). What also remains to be understood in detail is the status of caspase-1 and ICSBP expression in normal colonocytes. Because normal colonocytes have been reported to be Fas sensitive (14, 16), it is reasonable to postulate that caspase-1 and/or ICSBP are adequately expressed in normal colonocytes, or their expression levels can be elevated to an even higher degree in response to IFN-{gamma}, as compared with primary and metastatic lesions from the same patients. Thus, the machinery for these two apoptotic pathways identified in this study might naturally and operationally exist in normal colonocytes, whereas the primary tumor and, to a lesser extent, the metastatic tumor might have already begun to develop defects in the induction or expression of caspase-1 and/or ICSBP.

Although we were indeed able to partially restore Fas sensitivity to IFN-{gamma}-treated SW620 cells by ICSBP transfection, it resulted only in ~20% apoptotic cell death. This low, but detectable level of susceptibility to Fas-mediated apoptosis may reflect, at least in part, the low level of expression of ectopically expressed ICSBP (Fig. 6). Taken together, we propose that the molecular basis for the inability of IFN-{gamma} to sensitize the metastatic tumor to Fas-mediated apoptosis occurred at multiple levels. We showed both at the genome scale gene expression level and at the level of individual genes that down-regulation of Fas was one, but unlikely the sole factor that contributed to the failure of the metastatic cell line to display sensitivity to Fas-mediated apoptosis. Rather, resistance was most likely the consequence of deficiences in the responsiveness of a wide range of IFN-{gamma}-regulated genes, including, but not limited to, caspase-1 and ICSBP. Moreover, these data described for the first time the expression of ICSBP in nonhemopoietic tumors (Figs. 3 and 4). Previously, defective or diminished ICSBP induction or expression has been linked to the development of malignancies of hemopoietic origin (33, 43). In a broader sense, the overall diminished or altered responsiveness of certain neoplastic populations to IFN-{gamma}-regulated gene expression may have implications for enhanced tumor survival and escape.


   Acknowledgments
 
We thank Drs. Levi and Eklund for providing the human ICSBP gene; Drs. S. Gendler and J. Schlom for the transgenic mice; E. McDuffie for technical assistance; and D. Weingarten for editorial assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Scott I. Abrams, Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 5B46, 10 Center Drive, Bethesda, MD 20892-1402. E-mail address: sa47z{at}nih.gov Back

2 Abbreviations used in this paper: PI, propidium iodide; ICSBP, IFN consensus sequence-binding protein; IRF, IFN regulatory factor. Back

Received for publication December 20, 2002. Accepted for publication April 4, 2003.


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D. Yang, N. ud Din, D. D. Browning, S. I. Abrams, and K. Liu
Targeting Lymphotoxin {beta} Receptor with Tumor-Specific T Lymphocytes for Tumor Regression
Clin. Cancer Res., September 1, 2007; 13(17): 5202 - 5210.
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D. Yang, M. Thangaraju, K. Greeneltch, D. D. Browning, P. V. Schoenlein, T. Tamura, K. Ozato, V. Ganapathy, S. I. Abrams, and K. Liu
Repression of IFN Regulatory Factor 8 by DNA Methylation Is a Molecular Determinant of Apoptotic Resistance and Metastatic Phenotype in Metastatic Tumor Cells
Cancer Res., April 1, 2007; 67(7): 3301 - 3309.
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K. Liu, S. A. Caldwell, K. M. Greeneltch, D. Yang, and S. I. Abrams
CTL Adoptive Immunotherapy Concurrently Mediates Tumor Regression and Tumor Escape
J. Immunol., March 15, 2006; 176(6): 3374 - 3382.
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 Cancer Res.Home page
K. Liu, S. A. Caldwell, and S. I. Abrams
Immune Selection and Emergence of Aggressive Tumor Variants as Negative Consequences of Fas-Mediated Cytotoxicity and Altered IFN-{gamma}-Regulated Gene Expression
Cancer Res., May 15, 2005; 65(10): 4376 - 4388.
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K. Liu, S. A. Caldwell, and S. I. Abrams
Cooperative Disengagement of Fas and Intercellular Adhesion Molecule-1 Function in Neoplastic Cells Confers Enhanced Colonization Efficiency
Cancer Res., February 1, 2005; 65(3): 1045 - 1054.
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K. Liu, E. McDuffie, and S. I. Abrams
Exposure of Human Primary Colon Carcinoma Cells to Anti-Fas Interactions Influences the Emergence of Pre-existing Fas-Resistant Metastatic Subpopulations
J. Immunol., October 15, 2003; 171(8): 4164 - 4174.
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