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Departments of Internal Medicine (P.M.v.K., G.V., W.W.d.H., R.A.F., K.v.d.W., M.W., A.-J.v.d.L., S.W.J.L., L.J.H.) and Surgery (C.H.J.v.E), Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands; Department of Molecular Cell Biology (E.-J.M.S.), Research Institute for Growth and Development, University of Maastricht, NL-6200 MD Maastricht, The Netherlands; and Department of Immunology (E.C.), Berlex Bioscience Inc., Richmond, California 94006
Address all correspondence and requests for reprints to: Leo J. Hofland, Department of Internal Medicine, Erasmus Medical Center, Room Ee53ob, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: l.hofland{at}erasmusmc.nl.
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Abstract |
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Design: We evaluated the tumor-suppressive effects of type I interferons (IFN)-
2b and IFNß on the H295 and SW13 human ACC cell lines.
Results: As determined by quantitative RT-PCR analysis and immunocytochemistry, H295 and SW13 cells expressed the active type I IFN receptor (IFNAR) mRNA and protein (IFNAR-1 and IFNAR-2c subunits). Both IFN2b and IFNß1a significantly inhibited ACC cell growth in a dose-dependent manner, but the effect of IFNß1a (IC50 5 IU/ml, maximal inhibition 96% in H295; IC50 18 IU/ml, maximal inhibition 85% in SW13) was significantly more potent, compared with that of IFN2b (IC50 57 IU/ml, maximal inhibition 35% in H295; IC50 221 IU/ml, maximal inhibition 60% in SW13). Whereas in H295 cells both IFNs induced apoptosis and accumulation of the cells in S phase, the antitumor mechanism in SW13 cells involved cell cycle arrest only. Inhibitors of caspase-3, caspase-8, and caspase-9 counteracted the apoptosis-inducing effect by IFNß1a in H295 cells. In H295 cells, IFNß1a, but not IFN2b, also strongly suppressed the IGF-II mRNA expression, an important growth factor and hallmark in ACC.
Conclusions: IFNß1a is much more potent than IFN2b to suppress ACC cell proliferation in vitro by induction of apoptosis and cell cycle arrest. Further studies are required to evaluate the potency of IFNß1a to inhibit tumor growth in vivo.
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Introduction |
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In vitro and in vivo studies have shown the efficacy of type I interferons (IFNs) in the treatment of several tumors, alone or in combination with chemotherapy (7, 8). Type I IFNs, such as IFN, IFNß, and IFN
, interact with the same receptor complex [type I IFN receptor (IFNAR)], composed by two subunits, i.e. IFNAR-1 and IFNAR-2 (9, 10). The type I IFNs modulate tumor-suppressive activity through different mechanisms, including induction of cell cycle arrest and apoptosis (11). It has been described that type I IFNs are able to down-regulate the expression of IGF-II at the mRNA and protein level in some cancers (12, 13). Considering that IGF-II is highly expressed in more than 90% of the ACC and that this growth factor is involved in adrenal growth and tumorigenesis of ACC (14), type I IFNs may be of potential interest in the treatment of ACC.
To explore new possibilities of ACC treatment, we investigated the in vitro effects and mechanism of action of IFN2b and IFNß1a on the growth of two established human ACC cell lines, e.g. H295 and SW13.
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Materials and Methods |
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The human ACC cell line, NCI-H295R, was obtained from the American Type Culture Collection (Manassas, VA). The SW13 ACC cell line was obtained from ECACC (Salisbury, Wiltshire, UK) .
The cells were cultured in 75-cm2 culture flasks (Corning Costar, Amsterdam, The Netherlands) at 37 C in a humidified incubator containing 5% CO2. The culture medium consisted of a 1:1 mixture of DMEM and F12K medium, supplemented with 5% fetal calf serum, penicillin (1 x 105 U/liter), Fungizone (0.5 mg/liter), and L-glutamine (2 mmol/liter). Cells were harvested with trypsin (0.05%)-EDTA (0.53 mM) and resuspended in culture medium. Cell viability always exceeded 95%. Media and supplements were obtained from Gibco Bio-cult Europe (Invitrogen, Breda, The Netherlands).
Drugs and reagents
Human recombinant IFN2b (Roferon-A) was purchased from Roche (Almere, The Netherlands). Human recombinant IFNß1a was purchased from Serono Inc. (Rebif; Rockland, MA). Human recombinant IFNß1b was obtained from Schering (Mijdrecht, The Netherlands).
Caspase inhibitors Devd-cho, Lehd-cho, and Ietd-cho were purchased from Biosource (Brussels, Belgium).
Quantitative RT-PCR
For the detection of IFN receptors (IFNAR-1, IFNAR-2b, IFNAR-2c) and IGF-II, total RNA was isolated using a commercially available kit (high pure RNA isolation kit; Roche). cDNA synthesis and quantitative PCR using the TaqMan Gold nuclease assay was performed as described in detail previously (15). The primer and probe sequences were purchased from Biosource. The sequence of the primers IFNAR-1, IFNAR-2 total, IFNAR-2b, and IFNAR-2c as well as the concentrations of primers and probes used in the assay have been described by Vitale et al. (16). The sequences of the IGF-II primers were: IGF-II forward, 5'-CCAAGTCCGAGAGGGACGT-3'; IGF-II reverse, 5'-TTGGAAGAACTTGCCCACG-3'; and IGF-II probe, 5'-FAM-ACCGTGCTTCCGGACAACTTCCC-TAMRA-3'. Dilution curves were constructed for calculating the PCR efficiency for every primer set. PCR efficiencies were: IFNAR-1, 1.90 ± 0.02; IFNAR-2, 1.89 ± 0.03; IFNAR-2b, 1.68 ± 0.04; IFNAR-2c, 1.86 ± 0.06; and IGF-II, 2.01 ± 0.07. The estimated copy numbers were obtained according to the method described by Swillens et al. (17). The amount of mRNA was normalized to the total amount of RNA.
To exclude genomic DNA contamination in the RNA, the cDNA reactions were also performed without reverse transcriptase and amplified with each primer pair. To exclude contamination of the PCR mixtures, the reactions were also performed in the absence of cDNA template in parallel with cDNA samples. As a positive control for the PCR of type I IFN receptors and IGF-II, human DNA was amplified in parallel with the cDNA samples.
Immunocytochemistry
H295 and SW13 cells, cultured on coverslips (Invitrogen), were fixed with acetone (10 min at room temperature) and incubated for 30 min at room temperature with antibodies to human IFNAR-1 (rabbit polyclonal antibody; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and IFNAR-2c. Finally, a standard streptavidin-biotinylated alkaline phosphatase detection system (IL Immunologic, Duiven, The Netherlands) was used according to the manufacturer’s recommendations to visualize the bound antibodies.
The IFNAR-2c antibody (monoclonal antibody 27D11, provided by E.C., Berlex Biosciences) was generated against the purified IFNAR-2c ectodomain expressed in baculovirus and purified using a FLAG affinity column. This method was previously described for the generation of a monoclonal antibody against the IFNAR-1 ectodomain (18). The use and specificity of the IFNAR-2c antibody has been described previously (16, 19). Negative controls for the immunohistochemistry included omission of the primary antibody.
Cell proliferation assay
Cells were plated in 1 ml of medium in 24-well plates at a density of 105 cells/well for H295 and 5 x 103 cells/well for SW13. The plates were placed in a 37 C, 5% CO2 incubator. Two days later the cell culture medium was replaced with 1 ml/well medium containing the indicated concentrations of interferon in quadruplicate. After 3 and 6 d of treatment, the cells were harvested for DNA measurement. Plates for 6 d were refreshed after 3 d, and compounds were added again. Measurement of total DNA contents was performed as previously described (20).
Apoptosis assay
Apoptosis was evaluated by the analysis of the DNA fragmentation. After plating of 105 cells/well for H295 and 2 x 104 cells/well for SW13 on 24-well plates, cells were incubated at 37 C. After 2 d the cell culture medium was replaced with 1 ml/well medium containing various concentrations of drugs (IFN2b: 0–1000 IU/ml; IFNß1a: 0–100 IU/ml; IFNß1b: 0–100 IU/ml) in quadruplicate. After 1 d of incubation, apoptosis was assessed using a commercially available ELISA kit (Cell Death Detection ELISAPlus, Roche Diagnostic GmbH, Penzberg, Germany). The standard protocol supplied by the manufacturer was used. Apoptosis was expressed as percentage of control, untreated cells.
To evaluate the role of caspases involved in the induction of apoptosis after IFNß1a treatment, H295 cells (105 cells/well) were plated in 24-well plates and after 1 d of incubation; medium was changed by 1 ml of the medium containing 10 µM of different caspase inhibitors: Devd-cho (blocks caspase-3), Ietd-cho (blocks caspase-8), and Lehd-cho (blocks caspase-9). After 1 d of incubation, IFNß1a (5 IU/ml), in the absence or presence of the caspase inhibitors, was added, and the cells were incubated for an additional 24 h. Plates were then collected for detection of DNA fragmentation by ELISA, as described above.
Cell cycle analysis
Cells (2 x 105 for H295 and 5 x 105 for SW13) were plated on 12-well plates. After 1 d the medium was changed with fresh medium (control) or fresh medium plus IFN2b (500 and 1000 IU/ml) or IFNß1a (50 and 100 IU/ml). After 3 d of incubation (confluency of about 60–70%), cells were harvested by gentle trypsinization, washed with ice-cold phosphate-buffered calcium and magnesium-free saline (PBS) and collected by centrifugation. Cells were resuspended in 200 µl PBS and fixed in 70% ice-cold ethanol, followed by an overnight incubation at –20 C. After centrifugation, the cells were washed once with PBS and incubated for 30 min at 37 C in PBS containing 40 µg/ml propidium iodide (Sigma Aldrich, Zwijndrecht, The Netherlands) and 10 µg/ml of DNase-free RNase (Sigma Aldrich, Zwijndrecht, The Netherlands). For each tube, 20,000 cells were immediately measured on a FACScalibur flow cytometer (Becton Dickinson, Erembodegem, Belgium) and analyzed using CellQuest Pro Software.
Statistical analysis
All experiments were carried out at least three times and gave comparable results. For statistical analysis GraphPad Prism 3.0 (GraphPad Software, San Diego, CA) was used. The comparative statistical evaluation among groups was performed by ANOVA. When significant differences were found, a comparison between groups was made using the Newman-Keuls test. The unpaired Student t test was used to analyze the differences in concentration effect curves and effects in cell cycle modulation between different types of IFN preparations.
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Results |
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The effect of type I IFNs on cells is modulated by a common receptor. We generated specific primers and probes to determine the expression of IFNAR-1 and IFNAR-2 (total, short and long form) mRNA in H295 and SW13 cells by quantitative RT-PCR. In both cell lines, the presence of IFNAR-1, IFNAR-2 total, IFNAR-2b, and IFNAR-2c (Fig. 1
, upper panel) was detected. IFNAR-1 mRNA expression, was approximately three times higher than IFNAR-2 total in both cell lines. Among the IFNAR-2 subunits, IFNAR-2c was the form expressed at the relatively lowest level.
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). For IFNAR-1 as well as IFNAR-2c, staining was observed both in the cytoplasm and at the cell membrane (Fig. 1, middle panels). No staining was observed in the negative controls (Fig. 1, lower panels). Effects of two different types of IFNß on cell growth and apoptosis
We first evaluated the effect of two commercially available IFNß preparations on cell proliferation and proapoptotic activity in H295 cells, e.g. IFNß1a (Serono, amino acid sequence and glycosylation similar to native IFNß) and IFNß1b (Schering, one amino acid change and absence of glycosylation, compared with the native IFNß).
Inhibition of cell proliferation by IFNß1a was significantly stronger than with IFNß1b after 6 d of treatment (IC50 9.7 ± 2.7 vs. 87.0 ± 3.8 IU/ml, P < 0.01; maximal inhibitory effect on cell proliferation 88 ± 7 vs. 51 ± 10%, P < 0.01, respectively, for IFNß1a and IFNß1b). This is shown in Fig. 2A.
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). In subsequent experiments, we therefore decided to use IFNß1a.
Comparison between the effects of IFN2b and IFNß1a on cell proliferation
After 6 d of incubation, IFN2b and IFNß1a significantly suppressed the growth of H295 and SW13 cells in a dose-dependent manner (Fig. 3, upper panels). IFNß1a was significantly more potent than IFN2b, as determined by both the IC50 (H295: 5.4 ± 1.3 vs. 157.0 ± 2.3 IU/ml, respectively, P < 0.01; and SW13: 18.1 ± 1.3 vs. 221.1 ± 1.3 IU/ml, respectively, P < 0.01) and the maximal inhibition of cell proliferation after 6 d of treatment (H295: 96 ± 7 vs. 35 ± 2%, respectively, P < 0.01; and SW13: 85 ± 7 vs. 60 ± 3%, respectively, P < 0.01). After 6 d of incubation, IFNß1a induced in both cell lines a statistically significant inhibition of cell growth, already at very low concentrations (1 IU/ml, P < 0.01, vs. control). Moreover, the concentration required to obtain the maximal inhibition of proliferation was 100 IU/ml for IFNß1a and 10 times higher for IFN2b (1000 IU/ml). Comparable results were found by a [3H]thymidine incorporation assay (data not shown). There was no statistically significant difference between the IC50 values of 3 and 6 d of treatment (DNA control cells on d 3 and 6: 4330 ± 204 and 7257 ± 246 ng/well for H295, and 1685 ± 36 and 6870 ± 326 ng/well for SW13, respectively). H295 cells were slightly more sensitive to type I IFNs than SW13 cells in terms of IC50, whereas maximal inhibition of cell growth by IFN2b was highest in SW13 cells.
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In H295 cells, we did not observe any difference (IC50 or maximal inhibition) between the inhibitory effects of type I IFNs on forskolin-induced cortisol secretion on the one hand and on DNA content on the other hand. Cortisol production, normalized for DNA content, was not significantly changed by IFN treatment (data not shown). No detectable cortisol secretion by SW13 was found.
Induction of apoptosis by IFN2b and IFNß1a
After 1 d of treatment with IFNß1a and IFN2b, we measured the DNA fragmentation to investigate the induction of apoptosis (Fig. 3, lower panels). In H295, a potent dose-dependent induction of apoptosis was observed after IFNß1a treatment (EC50 5.0 ± 1.4 IU/ml). The maximal increase of DNA fragmentation induced by 100 IU/ml IFNß1a was about six times, compared with untreated control cells. IFNß1a stimulated apoptosis in H295 cells already at very low concentrations (1 IU/ml, P < 0.01). A less effective dose-dependent induction of apoptosis was observed after IFN2b treatment of H295 cells (EC50 249 ± 2 IU/ml). The maximal increase of DNA fragmentation by 1000 IU/ml IFN2b was about 2.5 times, compared with the control. Interestingly, DNA fragmentation was not observed in SW13 after treatment with IFN2b and IFNß1a. To study the role of caspases-3, -8, and -9 in the induction of apoptosis by IFNß1a in H295 cells, we evaluated whether specific caspase inhibitors were able to blunt IFNß-induced DNA fragmentation. As shown in Fig. 4, the stimulation of apoptosis by IFNß1a is completely blocked by Devd-cho, a specific inhibitor of caspase-3, whereas it is only partially counteracted by the use of specific inhibitors for caspase-8 (Ietd-cho) and caspase-9 (Lehd-cho).
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2b and IFNß1a on cell cycle progression
We also evaluated the effect of treatment with IFN2b (1000 and 500 IU/ml) and IFNß1a (100 and 10 IU/ml) on cell cycle distribution after 3 d of incubation (Table 1).
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2b 1000 IU/ml and IFNß1a 100 IU/ml: P < 0.001 vs. control in both cell lines; IFN2b 500 IU/ml and IFNß1a 50 IU/ml: P < 0.05 vs. control in H295 and P < 0.01 vs. control in SW13 cells), whereas there was no significant change in G1/G0 distribution (Table 1). The accumulation of cells in S phase after treatment with both IFNs was significantly higher in SW13 cells, compared with H295 cells. In addition, the proportion of cells in the G2/M phase decreased significantly, compared with control cells in both cell lines. These data suggest that ACC cells exhibit a prolonged stay in S phase and fail to transit into the G2/M phase after treatment with type I IFNs.
In H295 cells the cell cycle analysis revealed an increase in cells with subdiploid DNA content (sub-G0 phase) after treatment with IFNß1a (100 IU/ml: P < 0.001; 50 IU/ml: P < 0.05) or high-dose IFN2b (1000 IU/ml: P < 0.05), thereby confirming the induction of apoptosis by IFNß1a and IFN2b, as previously shown by the DNA fragmentation analyses.
Effects of IFN2b and IFNß1a treatment on IGF-II expression in ACC cells
The expression of IGF-II mRNA was detectable in H295 cells (20 ± 3 x 106 copies mRNA IGF-II per microgram RNA) but was undetectable in SW-13.
In H295 cells, the transcription of IGF-II gene is modulated by incubation with IFNß1a. We observed a potent and dose-dependent decrease in the number of copies of IGF-II mRNA after 3 d of treatment with IFNß1a (Fig. 5). In contrast, IFN2b (1000 IU/ml) was unable to modify the expression of IGF-II gene in H295 cells (data not shown). mRNA expression of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase was not affected by treatment with IFNß1a.
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Discussion |
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, -ß, -) are pleiotropic molecules that are able to modulate several processes, including immune response, cell differentiation, cell growth, and antitumor defense (11, 12). These cytokines activate a common receptor complex composed of two subunits: IFNAR-1 and IFNAR-2. IFNAR-1 is considered the signal subunit chain. There are three forms of IFNAR-2, which are differentially spliced products of the same gene. IFNAR-2a, the soluble form, acts as a regulator of the free IFNs (21, 22). IFNAR-2b, the short form, binds type I IFN but does not couple to signal transduction because it lacks the signal transduction tail (23); IFNAR-2c, the long form, constitutes together with the IFNAR-1 subunit the predominant active form of the type I receptor complex. In the present study, we evaluated tumor suppressive capability of type I IFNs (IFN2b, IFNß1a) in the human ACC cell lines H295 and SW13. First of all, the expression of mRNA and protein for the active subunits of type I IFN receptor (IFNAR-1 and IFNAR-2c) was demonstrated in H295 and SW13 cells by quantitative RT-PCR and immunocytochemistry, respectively. Both IFNs showed a dose-dependent inhibitory effect on cell proliferation. The antitumor activity of IFNß1a was significantly higher, compared with that of IFN2b in both cell lines. This appeared to be associated with an increased induction of apoptosis in H295 cells only and accumulation of cells in S phase in both cell lines. In addition, the maximal inhibition of SW13 cell growth by IFN2b appeared to be higher than that in H295 cells. This may be related to the absence of an effect of type I IFN on apoptosis and the more prominent role of cell cycle inhibition induced by type I IFNs in SW13 cells, compared with H295 cells, as was observed in the present study. The higher sensitivity of both ACC cell lines to IFNß1a may be explained by differences in the structure of IFN and IFNß. There is only 35% sequence identity between both IFNs (24, 25), and IFNß has a higher affinity for the type I IFN receptor, compared with IFN (26).
H295 cells were significantly more sensitive to IFNß1a treatment than SW13 cells. This could be a consequence of the IFN-mediated proapoptotic activity in H295 but not in SW13 cells, as shown by the increase in DNA fragmentation and increase in cells in subG0 phase after the treatment with IFNß1a or IFN2b. Because caspases play an important role in the induction of apoptosis, we evaluated the role of caspases in the induction of DNA fragmentation after IFNß1a treatment in H295 cells. In the classical model, caspases are divided into initiator caspases (such as caspase-8, -9) and executioner caspases (caspase-3, -6, -7), according to their function and sequence of activation. There are at least two major apoptotic pathways. The first involves the death receptor or extrinsic pathway that is initiated by TNF receptor family members that recruit adaptor and signaling molecules to assemble the death-inducing signaling complex. This complex leads to activation of caspase-8 and/or -10. An alternative mitochondrial pathway involves activation of caspase-9 on recruitment to the mitochondria by cytochrome c and apoptosis protease activation factor-1. More downstream, the initiator caspases lead to the activation of executioner caspase-3, -6, and -7, which in turn cleave specific proteins resulting in the DNA fragmentation (27, 28). In H295 cells the stimulation of DNA fragmentation by IFNß1a is completely blocked by a specific inhibitor of caspase-3, whereas it is only partially counteracted by the use of specific inhibitors for caspase-8 and -9. Therefore, IFNß1a seems to induce apoptosis through both the extrinsic and mitochondrial pathways in ACC.
At present, two recombinant IFNßs (IFNß1a and IFNß1b) are available as registered drugs. IFNß1a is produced in mammalian cells, with an amino acid sequence and glycosylation identical with that of natural IFNß. In contrast, IFNß1b is produced in Escherichia coli bacteria and is not glycosylated. Furthermore, IFNß1b has one amino acid sequence different from the native form of human IFNß. We observed in H295 cells that the effects of IFNß1a on cell proliferation inhibition and stimulation of DNA fragmentation were much more potent, compared with IFNß1b. This is in agreement with the observation that the absence of glycosylation in IFNß1b can reduce the biological activity (29). Because IFN is currently in clinical use for renal cell carcinoma and in trials for other malignancies, studies comparing the effects of IFNß and IFN in tumor cell models other than ACC will help determine whether the potent cytostatic and cytotoxic effects of IFNß are also present in other tumor models, rather than being restricted to selected tumor types.
IGF-II is considered to be an important growth factor in ACC. Therefore, we also evaluated the effects of IFNß1a on IGF-II mRNA expression. H295, but not SW13, cells expressed IGF-II. In H295 cells, IGF-II mRNA was inhibited in a dose-dependent manner by IFNß1a after 3 d of incubation. This effect was not observed after incubation with IFN2b. It is well known that in ACC an up-regulation of the IGF-II system represents a main pathway involved in the pathogenesis through inducing proliferation and inhibiting apoptosis (30). This could partially explain why only in H295 cells is IFNß1a able to induce apoptosis.
In conclusion, this is the first study showing that type I IFNs, particularly IFNß1a, are powerful inhibitors of proliferation of the H295 and SW13 human ACC cells. This effect is correlated with the induction of apoptosis and/or a cell cycle arrest in the S phase. Our findings support the clinical attractiveness to use IFNß in the treatment of ACC because of its ability to inhibit cell proliferation and stimulate apoptosis already at very low concentrations (1–10 IU/ml). In vivo, 12.3 IU/ml is the maximal IFNß serum concentration reported in healthy subjects after sc administration of this cytokine (31). In addition, recent studies report on the development of PEGylated IFNß with improved pharmacokinetic properties, compared with the unmodified protein (32). Finally, an important finding of the present study is that IFNß1a potently inhibits the expression of IGF-II at the transcriptional level. Further studies using primary ACC cultures as well as in vivo studies are required to evaluate these promising tumor suppressive effects, using IFNß1a not only as a single compound but also in combination with the currently used cytostatic drug mitotane.
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Footnotes |
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Disclosure statement: The authors have nothing to declare.
First Published Online August 15, 2006
Abbreviations: ACC, Adrenocortical carcinoma; IFN, interferon; IFNAR, type I IFN receptor.
Received March 21, 2006.
Accepted August 7, 2006.
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References |
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/ß-receptor interactions: a complex story unfolding. Curr Pharm Des 8:2131–2137[CrossRef][Medline]/ß signalling in immunity, oncogenesis and bone metabolism. Cancer Sci 94:405–411[CrossRef][Medline], ß, or 
using oligonucleotide arrays. Proc Natl Acad Sci USA 95:15623–15628-interferon on insulin-like growth factor-I, insulin-like growth factor-II and insulin-like growth factor binding protein-3 secretion by a human lung cancer cell line in vitro. J Endocrinol Invest 28:432–439[Medline]/ß receptor chain 1 in the structure and transmembrane signaling of the interferon /ß receptor complex. Proc Natl Acad Sci USA 91:9602–9606-2a determined by heteronuclear NMR spectroscopy in solution. J Mol Biol 274:661–675[CrossRef][Medline]
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, IFNß1b; 
, IFNß1a. Data are expressed as the percentage of control and represent the mean ± SEM. Control is set as 100% [mean DNA contents of control: 7660 ± 575 ng/well; mean absorbance (A405nm to A490nm) values of control: 0.095 ± 0.002].
, IFN
