Mol. Hum. Reprod. Advance Access originally published online on February 8, 2006
Molecular Human Reproduction 2006 12(2):99-105; doi:10.1093/molehr/gah250
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IFN
pretreatment sensitizes human choriocarcinoma cells to etoposide-induced apoptosis
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
1 To whom correspondence should be addressed at: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. E-mail: pengjp{at}ioz.ac.cn
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Abstract |
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Choriocarcinoma is a malignant trophoblast-derived tumour, which can arise in any type of gestation. Cell proliferation assays showed that interferon
(IFN) alone significantly inhibited proliferation of choriocarcinoma JAR and JEG-3 cells. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assays and Hoechst staining indicated that IFN alone could not induce apoptosis of JAR and JEG-3 cells, but IFN could enhance the sensitivity of JAR cells to etoposide-induced apoptosis. RT–PCR and western blotting were performed to detect expression of apoptosis-related molecules IFNR, interferon regulatory factor-1 (IRF-1), p53 and pro-caspase 3. In JAR cells, etoposide increased expression of the proteins including IFNR, p53 and pro-caspase 3 as well as IRF-1 mRNA and IFN-pretreatment apparently promoted up-regulation of these molecules expression. In addition, the responses of IRF-1, p53 and pro-caspase 3 expression to IFN pretreatment were dose dependent. IRF-1 knock down assays demonstrated that IRF-1 directly mediated IFN pretreatment enhanced sensitivity of JAR cells to etoposide-induced apoptosis and that pro-caspase 3 was one of the target genes of IRF-1.
Key words:
apoptosis/IFN/IRF-1/P53/pro-caspase 3
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Introduction |
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Interferons (IFNs) are glycoproteins that exert antiproliferative effects on normal and malignant cell growth, as well as immunomodulatory and antiviral effects. In addition, several studies have reported that IFNs can induce or modulate apoptosis (Tamura et al., 1996
). IFNs are used in the therapy of human malignancies as a single agent as well as in combination with other chemotherapeutic agents. It has been reported that IFNß pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis (Chawla-Sarkar et al., 2002). Preliminary clinical studies with IFN
and the fluorinated pyrimidine, 5-fluorouracil, in patients with advanced colorectal carcinoma suggest that IFN may enhance the effects of the antimetabolite (Wadler and Schwartz, 1990). However, the effects of IFNs on cellular susceptibility to anticancer agents-induced apoptosis have not been elucidated.
The one type II species (IFN), mainly secreted by Th-1 lymphocytes and NK cells, is coded by a single gene on chromosome 12. IFN can directly inhibit human tumour cell growth and induce apoptosis (Ikeda et al., 2002). Extensive experiments in a range of animal cancer models suggest that endogenous IFN may be involved in immune surveillance of tumours via a combination of lymphocyte-mediated responses, direct actions on tumour cells and inhibition of tumour angiogenesis. Studies with interferon regulatory factor-1 (IRF-1) null mice have shown its prominent role in antiviral activity of IFN, cell cycle regulation and apoptosis (Chawla-Sarkar et al., 2003).
IFN promotes apoptosis, or increases susceptibility to apoptosis stimuli, in certain cell types (Ossina et al., 1997; Xu et al., 1998; Rathbun et al., 2000). It has been reported that in the U937 monoblastic leukaemia cell line, pretreatment with IFN enhanced sensitivity to apoptosis is triggered by antitumour agents (etoposide) (Tamura et al., 1996). In reproductive systems, it has been reported that IFN could induce apoptosis in ovarian carcinoma cells in vivo and in vitro (Wall et al., 2003). Prolongation of disease-free survival may emerge in using IFN as an adjuvant to high-dose chemotherapy for high-risk patients of ovarian cancer (Kim et al., 2002). However, little is known about the enhancing or inducing mechanism of IFN on tumour cell apoptosis (Park et al., 2002).
Choriocarcinoma is a malignant trophoblast-derived tumour, which can arise in any type of gestation. Resistance to chemotherapy is an important factor in failure in curing many patients with choriocarcinoma (Pandian et al., 2004).
In this study, results showed that IFN alone did not induce apoptosis of JAR and JEG-3 cells, but IFN enhanced apoptosis-induced by etoposide. In an attempt to understand the enhancing mechanism of IFN on tumour cell apoptosis induced by etoposide, we analysed the role of IFN with or without etoposide on apoptosis of both cell lines, and the mechanism of apoptosis was also discussed. This may provide a theoretic basis for choriocarcinoma therapy.
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Materials and methods |
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Cell culture and treatment of IFN
or etoposideThe human choriocarcinoma cell lines JAR and JEG-3 were generous gifts from Dr Yunshang Piao and Dr Yanling Wang (State Key Laboratory of Reproductive Biology, Institute of Zoology, CAS, Beijing). JAR and JEG-3 cells were maintained as a suspension in complete medium that contained RPMI 1640 and Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C under humidified 5% CO2 atmosphere. Human recombinant IFN
(Gibco) was reconstituted in sterile water to the concentration of 106 IU/ml and added into the medium to a final concentration of 50 IU/ml. Etoposide (BioVision, Mountain View, CA, USA) was prepared as a stock solution in dimethylsulphoxide (DMSO). The final concentration of DMSO in culture was 0.1%, which did not induce apoptosis. Before treatment, cells were seeded in culture medium, allowed to attach overnight and made quiescent by 24 h incubation in 0.5% serum medium. The following conditions were tested on JAR and JEG-3 cells: (i) 50 IU/ml IFN, (ii) 5 µM etoposide, (iii) IFN followed by etoposide. In the experiments with etoposide, cells were treated with 50 IU/ml IFN for 24 h, then one half of the cultures were treated with 5 µM etoposide dissolved in phosphate-buffered saline (PBS), and the other half were treated with PBS alone.
Cell proliferation assay
The cell proliferation assay was performed by using the Cell Titer 96 Aqueous One Solution cell proliferation assay (Promega, Madison, WI, USA). Cells were seeded in 96-well plates at low density (5000 cells per well) in culture medium, allowed to attach overnight and made quiescent by a 24 h incubation in 0.5% serum medium. Cells were treated with different concentrations of IFN for 24 h, and then some cells were treated with 5 µM etoposide dissolved in PBS, and others were treated with PBS alone. After 72 h, Cell Titer 96 Aqueous One Solution reagent was added to each well, and the plate was incubated for 4 h after which the absorbance was recorded at 490 nm with a 96-well plate reader.
Detection of apoptosis assay by TUNEL and Hoechst staining
In situ detection of apoptotic cells was performed on adherent cells cultured on chamber slides by using in situ Cell Death Detection Kit, Fluorescein (Roche, Mannheim, Germany). After treatment, JAR and JEG-3 cells undergoing cell death were identified. Briefly, air-dried cell samples were fixed with a freshly prepared fixation solution for 1 h at 15–25°C, and then incubated in permeabilization solution for 2 min on ice, and the TUNEL procedure was conducted according to the manufacturer’s instructions. For the correlation of TUNEL with nuclear morphology, cultures were counterstained with phosphatidylinositol (PI) (5 µg/ml) and coverslipped. To confirm the specificity of TUNEL, cultures were treated with 1 µg/ml DNase I (Sigma Chemical Co., St. Louis, MO, USA) at room temperature for 10 min to create positive controls. Terminal deoxynucleotidyl transferase (TdT) was omitted from the labelling reaction mixture in negative controls. Samples were viewed at excitation 488 nm /emission 512 nm by fluorescence microscopy (Leica Microsystems, Bensheim, Germany).
The fragmented nuclei were stained with Hoechst 33342 (Sigma Chemical Co.). Hoechst 33342 was diluted with PBS and added to the medium to the final concentration of 1 ng/ml. Cells were incubated for 15 min in an atmosphere of 5% CO2 at 37°C and studied with a fluorescence microscope (Leica Microsystems).
Total RNA isolation and RT–PCR
Total RNA was isolated from stimulated or non-stimulated JAR cells with TRIzol (Invitrogen, Carlsbad, CA, USA) by following the manufacturer’s instructions. All RT–PCR reagents including M-MLV reverse transcriptase and Taq DNA polymerase were purchased from Promega. RT reactions were performed by using 2 µg RNA. First-strand cDNA from 100 ng of total RNA was then used for PCR with oligonucleotide primers designed to amplify the target sequences given in Table I. PCR amplification was performed with 30 cycles of 30 s denaturation at 94°C, 1 min annealing at the temperature listed in Table I, and 1 min extension at 72°C. PCR products were detected by electrophoresis in 1.5% agarose gel (Promega). Minus RT was negative control. ß-Actin was used for ensuring equal total RNA used in RT–PCR. PCR signal intensities were analysed using Bio-Rad Quantity One software (Bio-Rad, Hercules, CA, USA). The results were normalized according to ß-actin. Three replicates were performed for all experiments.
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Western blotting
Whole cell lysates were prepared in 1x lysis buffer [50 mm Tris–HCl (pH 8), 1% Triton X-100, 10% glycerol, 1 mM EDTA, 250 mM NaCl, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 10 µg/ml pepstatin] for subsequent immunoblotting studies. The protein contents were determined by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Fifty micrograms of each protein sample were separated using a 12% polyacrylamide gel for 2 h and transferred onto Hybond nitrocellulose membrane (Amersham Life Science, Buckinghamshire, UK). After blocking for 3 h with 5% dried fat-free milk in Tris-buffered saline-Tween (TBST) 20 mM Tris–HCl, pH 7.6; 137 mM NaCl; 0.1% Tween-20, the membranes were incubated for 1 h with the primary antibody, a rabbit anti-IFNR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a rabbit anti- P53 (Santa Cruz Biotechnology) or a rabbit anti-pro-caspase 3 (Santa Cruz Biotechnology). The specific protein-antibody complex was detected by using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Santa Cruz Biotechnology) and an enhanced chemiluminescence (ECL) detection kit (Amersham Life Science).
For reprobing, the membranes were washed in stripping buffer (100 mM ß-mercaptoethanol, 20% sodium dodecyl sulphate (SDS), 62.5 mM Tris and pH 6.7) at 50°C for 30 min to strip off bound antibody after ECL detection. The membranes were reprobed with 1:300 polyclonal goat anti-actin (Santa Cruz Biotechnology), using the same procedure as described above. All experiments were repeated at least three times.
Synthesis of SiRNA
RNA from human JAR cells was isolated using TRIzol and transcribed into cDNA with M-MLV reverse transcriptase. Nucleotides 298–747 of the IRF-1 coding sequence were chosen as the target of RNAi. Primers containing T7 promoter and human IRF-1 sequence are summarized in Table I. A quantity of 100 nM of each primer was used in the PCR mixture. PCR conditions were 95°C for 5 min followed by 30 cycles of 94°C for 30 s, 1 min annealing at the temperature listed in Table 1, 72°C for 2 min and a final extension step at 72°C for 10 min. This product was then used to synthesize dsRNA followed by siRNA using the silencer siRNA cocktail kit (Ambion, Austin, TX, USA). The effectiveness of the RNAi experiments in our experimental conditions was confirmed using Ambion’s glyceraldehyde-3-phosphatedehydrogenase (GAPDH) positive control DNA template, according to the manufacturer’s instructions. A non-silencing siRNA against LH receptor was used as a negative control.
Transfectional analysis
One day before transfection, JAR cells were plated in 6-well plates so that they would be up to 30–50% confluence at the time of transfection. Hundred nM siRNA constructs were transfected into JAR cells by Lipofectamine 2000 (Invitrogen). The cells were incubated at 37°C in a CO2 incubator for 24 h. Cells were then treated with 50 IU/ml IFN for 24 h, and afterwards they were treated with 10 µM etoposide for additional 24 h. RT–PCR was undertaken to estimate the siRNA effect in reducing the level of IRF-1 expression.
FACS analysis
Cells to be examined for annexin V expression were washed with PBS and resuspended in 500 µl binding buffer (Annexin V-FITC Kit, Immunotech, Marseille, France), containing 1 µl annexin V-fluorescein isothiocyanate (FITC) stock and 5 µl 20 µg/ml PI to determine the phosphatidylserine (PS) exposure on the outer plasma membrane. After incubation for 10 min at room temperature in a light-protected area, the specimens were quantified by flow cytometry (FACS-vantage/Diva, BD Biosciences, San Jose, CA, USA), acquiring 10 000 events. All experiments were repeated at least three times.
Statistical analysis
Values were reported as the mean ± SEM from three independent samples. Statistical analysis was made by one-way analysis of variance (ANOVA), and when significant treatment effects were indicated, the Student-Newman-Keuls multirange test was employed to make pairwise comparisons of individual means.
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Results |
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Effect of IFN
on the proliferation of JAR and JEG-3 cellsWe examined the effects of varying concentrations of IFN
on proliferation of JAR and JEG-3 cells. Normal cells without IFN treatment were the control group. IFN could inhibit the proliferation of JAR cells (Figure 1). IFN 10, 25 and 50 IU/ml significantly decreased the number of JAR cells by 3.7% (P < 0.05), 5.6% (P < 0.01) and 10.5% (P < 0.01) compared to the control group, respectively. There was no significant difference in the number of JAR cells compared to the control group at the concentration of 5 IU/ml IFN. Low dose IFN could promote the proliferation of JEG-3 cells, whereas proliferation was inhibited by high-dose IFN. The number of JEG-3 cells was significantly increased by 6.5%(P < 0.01) and 4.0%(P < 0.05) compared to the control group at different concentrations of 5 and 10 IU/ml IFN, respectively, and decreased by 24.5% (P < 0.01) and 27.9% (P < 0.01) at 25 and 50 IU/ml IFN, respectively.
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Enhancement of susceptibility to etoposide-induced apoptosis by pretreatment with IFN
JAR and JEG-3 cells were exposed to IFN (50 IU/ml) for 24 h and then treated with 5 µM etoposide for an additional 72 h to induce apoptosis. Normal cells without IFN or etoposide treatment were the control group. As shown in Figure 2A, IFN alone significantly decreased the number of both cell types (P < 0.01 compared to control). Etoposide alone reduced the proliferation of JAR cells by approximately 21.5% and those of JEG-3 cells by 59.7%, whereas etoposide after pretreatment with IFN showed a reduction of 55.8% on JAR cells and 82.6% on JEG-3 cells, respectively. These results suggested that IFN could enhance the sensitivity of both JAR and JEG-3 cells to etoposide-induced reduction in cell proliferation.
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The cell proliferation assay (Figure 2B) showed that pretreatment of JAR cells with IFN significantly decreased the cell number compared to 5 µM etoposide without IFN pretreatment. IFN pretreatment at doses of 10, 25 and 50 IU/ml decreased JEG-3 cell number by 28.9% (P < 0.05), 17.6% (P < 0.01), 56.8% (P < 0.01), respectively compared to treatment with 5 µM etoposide alone. The results showed IFN promoted etoposide-induced reduction in JAR cell proliferation in a concentration-dependent manner. The number of JAR cells that were pretreated with 5 IU/ml IFN and subsequently with 5 µM etoposide did not differ from that treated with 5 µM etoposide alone. About 10, 25 and 50 IU/ml IFN pretreatment enhanced the reduction in JEG-3 cell number compared to 5 µM etoposide treatment alone.
To further assess the possible role of IFN in controlling apoptosis, cells were pretreated with or without IFN (50 IU/ml, 24 h) and then treated with the antitumour agent etoposide (5 µM, 72 h). TUNEL assay demonstrated that IFN-mediated enhancement of cell death was accompanied by an enhanced susceptibility to apoptosis in JAR (Figure 3) and JEG-3 cells (Figure 4). Cells without IFN treatment were both TUNEL negative (Figures 3C and 4C). TUNEL assay indicated that IFN alone could not induce apoptosis of JAR and JEG-3 cells (Figures 3D and 4D). Both cell types treated with 5 µM etoposide for 72 h were TUNEL positive (Figures 3E and 4E). Both cell types pretreated with IFN (50 IU/ml) for 24 h and further incubated with etoposide (5 M) for an additional 72 h (Figures 3F and 4F) were also TUNEL positive. It was apparent that samples pretreated with IFN showed much higher numbers of apoptotic cells than samples treated with etoposide alone. Cells stained after treatment with DNAse I were used as a positive control (Figures 3A and 4A), and those stained without terminal deoxynucleotide transferase were used as a negative control (Figures 3B and 4B).
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To confirm the above results, we examined nuclear morphological changes of JAR cells by determining nuclear condensation and fragmentation, another hallmark for apoptosis. Hoechst 33342 staining showed that a part of the cells displayed nuclear condensation at 72 h after 5 µM etoposide treatment. JAR cells without IFN or etoposide treatment are shown in Figure 5A. IFN pretreatment efficiently enhanced etoposide-induced apoptosis (Figure 5C and D) without having any significant effect on cell apoptosis when used alone (Figure 5B).
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Effect of IFN -pretreatment on apoptosis-related genes
To determine which protein is involved in the IFN promoting etoposide-induced apoptosis, we analysed expression of some apoptosis-related genes in JAR cells.
As it is known that IFNR is triggered by exogenous IFN, we wanted to study whether IFNR could mediate etoposide-induced apoptosis enhanced by IFN-pretreatment or not. Results indicated that etoposide increased IFNR expression, as did IFN pretreatment (Figure 6A).
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Because wild-type p53 protein was critical to initiate growth arrest and apoptosis, we investigated the change of p53 protein by using IFN to treat JAR cells that carried wild-type p53. As shown in Figure 6A, p53 was promoted by etoposide and further enhanced by IFN pretreatment, which suggested that the growth suppression in JAR cells was likely to be mediated by p53-dependent pathway.
Our results (Figure 6A) revealed that pro-caspase 3 protein was markedly increased with the treatment of etoposide and even more markedly increased by IFN pretreatment.
The results of western blot analysis showed that treatment of etoposide promoted the expression of IFNR, p53 and pro-caspase 3 proteins, all of which were enhanced by IFN-pretreatment. Furthermore, expression of p53 and pro-caspase 3 protein corresponding to IFN-pretreatment was dose-dependent (Figure 6B).
IRF-1 mediated IFN-enhanced apoptosis induced by etoposide
RT–PCR indicated that treatment of etoposide increased expression of IRF-1 mRNA and IFN-pretreatment apparently enhanced the up-regulation of IRF-1 mRNA expression (Figure 7IA). As shown in Figure 7IB, IFN alone significantly promoted the IRF-1 mRNA expression by 7.7% (P < 0.05 over the control). Etoposide increased IRF-1 mRNA expression by approximately 43.5% (P < 0.01), but even higher values were obtained when the cells were pretreated with IFN before etoposide stimulation.
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JAR cells were pretreated with different doses of IFN
for 24 h following etoposide treatment. Expression of IRF-1 mRNA was detected by RT–PCR at different concentrations of 25, 50 and 100 IU/ml, and it was steadily elevated with the increasing concentration of IFN (Figure 7IIA and B). The content of IRF-1 mRNA was significantly increased in cells pretreated with IFN compared to cells stimulated with 5 µM etoposide alone. It showed that expression of IRF-1 mRNA corresponding to IFN-pretreatment was dose dependent.
RNA interference assay was undertaken to detect the role of IRF-1 in IFN-enhanced apoptosis induced by etoposide. Figure 8IA showed that IRF-1-targeted siRNA was significantly effective in reducing IRF-1 mRNA. SiRNA cocktail constructs induced 41.3% knock-down of IRF-1 (Figure 8IB). The inhibition of expression of IRF-1 apparently weakened pro-caspase 3 expression (Figure 8II) and the apoptotic level (Figure 8III) promoted by IFN pretreatment. After pretreatment with 50 IU/ml IFN for 24 h and then by 10 µM etoposide for 24 h, JAR cells were transfected with siRNA. FACS assay showed that 50.4% of JAR cells became apoptotic (Figure 8IIIC), but in the untransfected cells only 17.2% of cells were annexin V/PI positive (Figure 8IIIB).
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Discussion |
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In our experiments, IFN
alone could inhibit proliferation of choriocarcinoma JAR and JEG-3 cells. TUNEL assay and Hoechst staining indicated that IFN alone could not induce apoptosis of both cell lines. These results showed that IFN could not affect proliferation of JAR cells by inducing apoptosis. Some papers reported that IFN could induce cell cycle arrest in certain cell types (Chawla-Sarkar et al., 2002). Cell death was not detected by trypan blue staining (figure not shown). So we speculated that IFN might affect proliferation of both cell lines by inducing cell cycle arrest.
Etoposide is one of the chemotherapeutic agents to treat gestational choriocarcinoma patients. Our results indicated that etoposide activated apoptosis induction in JAR and JEG-3 cells. Thus, we hypothesized that IFN might promote etoposide-induced apoptosis in both cell types. If so, IFN could be used in the therapy of human malignancies in combination with chemotherapeutic agents. To investigate the possibility, both cell types were pretreated with IFN for 24 h and further incubated with etoposide. In this study, IFN pretreatment could make choriocarcinoma cells sensitive to etoposide-induced apoptosis.
Novelli et al. reported that in human malignant T cells etoposide up-modulated interferon-gamma receptor (IFNR) expression. High IFNR expression is a marker for the T cell stress that precedes apoptosis (Novelli et al., 1997). We first estimated the expression of IFNR. Consistent with this, IFNR was up-regulated by etoposide in JAR cells. We also found that the up-regulation was further enhanced by IFN pretreatment.
Work performed in many laboratories over the past 10 years has demonstrated that the IFN receptor utilizes a specific signal transduction pathway termed the Jak–Stat pathway to regulate transcription of IFN-inducible genes leading to development of specific IFN-dependent cellular responses (Ikeda et al., 2002). However, downstream molecules of Jak-Stat pathway, which is thought to be involved in IFN-induced cell cycle arrest and apoptosis of cells, are still unknown. In this study, we detected some molecules that might participate in the process.
More recently, it has been recognized that activation of IRF-1 leads to the expression of genes in a number of different cellular processes, including antiviral response, regulation of the cell cycle and apoptosis (Pamment et al., 2002). Kano et al. (1999) reported that IRF-1 is an essential mediator in IFN-induced cell cycle arrest and apoptosis of primary cultured hepatocytes. It has been reported that IRF-1 plays a pivotal role in the IFN-mediated enhancement of Fas/CD95-mediated apoptosis (Tomita et al., 2003). Mice lacking the functional IRF-1 alleles grow without any spontaneous development of tumours. However, in view of the importance of multiple genetic events for the development of tumours in vivo and in vitro, it is conceivable that the IRF-1-deficient cells may manifest properties different from wild-type cells with respect to their susceptibility to transformation by ontogenesis (Donehower et al., 1992). In this study, we found that IFN pretreatment could up-regulate IRF-1 expression enhanced by etoposide. Furthermore, the effect of IFN on IRF-1 expression was dose dependent. IRF-1 knock down assay showed that IRF-1 directly mediated IFN promoting apoptosis sensitivity of JAR cells induced by etoposide.
Genetic evidence suggested that IRF-1 and p53 converged to suppress tumour development (Nozawa et al., 1999). When IRF-1 and p53 were co-expressed, their effect on reporter readout was synergistic rather than additive. Some studies reported that p53 is a downstream mediator of IRF-1 in IFN-induced cell cycle arrest of hepatocytes, although p53 is not involved in the cell death of hepatocytes (Kano et al., 1999). Our results suggested expression of p53 was promoted by etoposide, which could be further enhanced by IFN pretreatment. In addition, the enhancing relation was in a dose-dependent fashion. Growth suppression in JAR cells is probably mediated by p53-dependent pathway. It remains unclear whether p53 mediated apoptosis or cell growth arrest. Further investigations are warranted to clarify this phenomenon.
Several lines of evidence indicate that each caspase plays a different role in the apoptotic signalling pathway depending upon the cell type. Caspase 3 is one of the key molecules for inducing apoptosis in tumour cells. In our study, IFN pretreatment could up-modulate pro-caspase 3 (one of effector caspases) expression increased by etoposide in JAR cells. SiRNA of IRF-1 transfection inhibited the increase in pro-caspase 3 protein expression by IFN pretreatment. This suggested that pro-caspase 3 was one of the target genes of IRF-1 and a key molecule for inducing apoptosis in JAR cells.
P53 is a regulator of Bcl-2 and Bax gene expression in vitro and in vivo (Miyashita et al., 1994). In JAR cells, IFN pretreatment up-regulated p53 expression, and we asked whether p53 induced a decrease in Bcl-2 or an increase in Bax expression. We also detected other apoptosis-related gene expression including Fas/CD95. Immunoblot analysis showed that Bcl-2, Bax and Fas/CD95 protein in IFN-pretreatment JAR cells made no difference from those in non-pretreated cells (data not shown).
The various manipulations discussed above that modify the response of JAR cells to etoposide can be summarized by the following process. Etoposide promotes expression of IFNR, IRF-1, p53 and pro-caspase 3, and then promotes pro-caspase 3 expression leading to an increase of apoptosis levels. IFN-pretreatment promotes the susceptibility of JAR cells to etoposide-induced apoptosis. The promoting process might be summed up as follows: IFN pretreatment further up-regulated its receptor IFNR expression promoted by IFN, activated Jak-1, leading to phosphorylation and dimerization of STAT-1. IRF-1 gene has an IFN-activated site (GAS) that is strongly bound by STAT-1. Up-regulated IRF-1 further enhanced expression of pro-caspase 3. Then up-modulated pro-caspase 3 expression lead to an increase of apoptosis levels.
A survival benefit would be conferred to a cancer cell if it could selectively inhibit one or more apoptotic IFN-stimulated genes (ISGs) or acquire defects in IFN-signal transduction components (Chawla-Sarkar et al., 2003). One or all of these defects have been reported in a number of IFN-resistant cell lines, implicating defects in signal transduction as a probable cause of cellular resistance to IFNs. IFN alone cannot affect IRF-1 expression in JAR cells, which indicates JAR cells may have defects at the step in IFN signalling making themselves resistant to IFN-induced apoptosis. We speculate that etoposide activates IRF-1, and then resumes the apoptosis pathway induced by IFN.
Several chemotherapeutic drugs are routinely used in treating choriocarcinoma, but the clinical effectiveness is limited by the emergence of drug resistance in tumour cell population. Timely combination of chemotherapy drugs with IFN may thus provide a more effective way of inhibiting the progress of human malignant trophoblast cells through synergistic induction of their apoptosis (Kim et al., 2002).
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Acknowledgements |
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This work was funded by grants from the National Natural Science Foundation of China (No. 30370165) and supported by a grant from the Key Innovation Research Programs of the Chinese Academy of Sciences, No. KSCX2-SW-201.
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References |
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Chawla-Sarkar M, Leaman DW, Jacobs BS and Borden EC (2002) IFN-beta pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis. J Immunol 169,847–855.
Chawla-Sarkar M, Lindner DJ, Liu YF, Williams BR, Sen GC, Silverman RH and Borden EC (2003) Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis. Apoptosis 8,237–249.[CrossRef][Web of Science][Medline]
Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA Jr, Butel JS and Bradley A (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356,215–221.[CrossRef][Medline]
Ikeda H, Old LJ and Schreiber RD (2002) The roles of IFN gamma in protection against tumor development and cancer immunoediting. Cytokine Growth Factor Rev 13,95–109.[CrossRef][Web of Science][Medline]
Kano A, Haruyama T, Akaike T and Watanabe Y (1999) IRF-1 is an essential mediator in IFN-gamma-induced cell cycle arrest and apoptosis of primary cultured hepatocytes. Biochem Biophys Res Commun 257,672–677.[CrossRef][Web of Science][Medline]
Kim EJ, Lee JM, Namkoong SE, Um SJ and Park JS (2002) Interferon regulatory factor-1 mediates interferon-gamma-induced apoptosis in ovarian carcinoma cells. J Cell Biochem 85,369–380.[CrossRef][Web of Science][Medline]
Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, Hoffman B and Reed JC (1994) Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9,1799–1805.[Web of Science][Medline]
Novelli F, Allione A, Bernabei P, Rigamonti L and Forni G (1997) Antiblastic chemotherapy drugs up-modulate interferon-gamma receptor expression on human malignant T cells. Cancer Detect Prev 21,191–195.[Web of Science][Medline]
Nozawa H, Oda E, Nakao K, Ishihara M, Ueda S, Yokochi T, Ogasawara K, Nakatsuru Y, Shimizu S, Ohira Y et al. (1999) Loss of transcription factor IRF-1 affects tumor susceptibility in mice carrying the Ha-ras transgene or nullizygosity for p53. Genes Dev 13,1240–1245.
Ossina NK, Cannas A, Powers VC, Fitzpatrick PA, Knight JD, Gilbert JR, Shekhtman EM, Tomei LD, Umansky SR and Kiefer MC (1997) Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem 272,16351–16357.
Pamment J, Ramsay E, Kelleher M, Dornan D and Ball KL (2002) Regulation of the IRF-1 tumour modifier during the response to genotoxic stress involves an ATM-dependent signaling pathway. Oncogene 21,7776–7785.[CrossRef][Medline]
Pandian Z, Seckl MJ, Smith R and Lees DA (2004) Gestational choriocarcinoma: an unusual presentation with response to gemcitabine and surgery. BJOG 111,382–384.[Medline]
Park SY, Billiar TR and Seol DW (2002) IFN-gamma inhibition of TRAIL-induced IAP-2 upregulation, a possible mechanism of IFN-gamma-enhanced TRAIL-induced apoptosis. Biochem Biophys Res Commun 291,233–236.[CrossRef][Web of Science][Medline]
Rathbun RK, Christianson TA, Faulkner GR, Jones G, Keeble W, O’Dwyer M and Bagby GC (2000) Interferon-gamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members. Blood 96, 4204–4211.
Tamura T, Ueda S, Yoshida M, Matsuzaki M, Mohri H and Okubo T (1996) Interferon-gamma induces Ice gene expression and enhances cellular susceptibility to apoptosis in the U937 leukemia cell line. Biochem Biophys Res Commun 229,21–26.[CrossRef][Web of Science][Medline]
Tomita Y, Bilim V, Hara N, Kasahara T and Takahashi K (2003) Role of IRF-1 and caspase-7 in IFN-gamma enhancement of Fas-mediated apoptosis in ACHN renal cell carcinoma cells. Int J Cancer 104,400–408.[CrossRef][Medline]
Wadler S and Schwartz EL (1990) Antineoplastic activity of the combination of interferon and cytotoxic agents against experimental and human malignancies: a review. Cancer Res 50,3473–3486.
Wall L, Burke F, Barton C, Smyth J and Balkwill F (2003) IFN-gamma induces apoptosis in ovarian cancer cells in vivo and in vitro. Clin Cancer Res 9,2487–2496.
Xu X, Fu XY, Plate J and Chong AS (1998) IFN-gamma induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression. Cancer Res 58,2832–2837.
Submitted on October 12, 2005; revised on November 18, 2005; accepted on November 22, 2005
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|
 H.-F. Xia, J. Sun, Q.-H. Sun, Y. Yang, and J.-P. Peng Implantation-associated gene-1 (Iag-1): a novel gene involved in the early process of embryonic implantation in rat Hum. Reprod., July 1, 2008; 23(7): 1581 - 1593. [Abstract] [Full Text] [PDF]
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 J. C. Choi, R. Holtz, M. G. Petroff, N. Alfaidy, and S. P. Murphy Dampening of IFN-{gamma}-Inducible Gene Expression in Human Choriocarcinoma Cells Is Due to Phosphatase-Mediated Inhibition of the JAK/STAT-1 Pathway J. Immunol., February 1, 2007; 178(3): 1598 - 1607. [Abstract] [Full Text] [PDF]
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