IL-18 E42A mutant is resistant to the inhibitory effects of HPV-16 E6 and E7 oncogenes on the IL-18-mediated immune response

Cancer Letters 229 (2005) 261–270 www.elsevier.com/locate/canlet

IL-18 E42A mutant is resistant to the inhibitory effects of HPV-16 E6 and E7 oncogenes on the IL-18-mediated immune response Kyung-Ae Leea, Kyung-Joo Choa, Soo-Hyun Kimb, Jung-Hyun Shima, Jong-Seok Lima,c, Dae-Ho Choc, Min-Sung Songb, Charles A. Dinarellob, Do-Young Yoona,* a

Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology, Yuseong, P. O. Box 115, Daejeon 305-600, South Korea b Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, CO80262 c Department of Biological Science, Sookmyung Women’s University, Hyochangwongil 52, Yongsan-ku, Seoul 140-742, South Korea Received 23 March 2005; accepted 14 June 2005

Summary Our previous studies showed that the down-modulation of IL-18-induced immune response caused by oncoproteins E6 and E7 as one of the mechanisms underlying immune escape in HPV-induced cervical cancer cells. E42 residue of IL-18 also appears to be critical in the activity of IL-18. Single point mutation E42 in IL-18 show promise in the study of IL-18 binding motifs for HPV oncoproteins. We attempted to ascertain whether site-specific IL-18 mutant E42A would modulate the inhibitory effects of IL-18-induced immune responses via the HPV 16 E6 and E7 oncoproteins. Compared to wild type IL-18, E42A-induced IFN-g production was not inhibited by HPV 16 E6 and E7. In vitro and in vivo binding assays have also revealed that E6 and E7 do not result in the inhibition of the binding of E42A to its IL-18 receptor alpha chain. There were no effects on the E42A-induced phosphorylations of p38 and JNK observed in the presence of E6 or E7. The degradation of IkB by E42A was not affected by E6 or E7 in NK0 cells. Moreover, E42A-induced NF-kB activation was also not inhibited by these oncoproteins. These results suggest that E42A is a stronger activator than wild type IL-18, and is not susceptible to inhibition by the HPV oncoproteins E6 and E7. Thus, it is suggested that E42A could be used in immunotherapy for patients with cervical cancer. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: IL-18; E42A mutant; E6; E7

1. Introduction

* Corresponding author. Tel.: C82 42 860 4218; fax: C82 42 860 4593. E-mail address: [email protected] (D.-Y. Yoon).

Cervical cancer is one of the leading causes of female death from cancer worldwide. Human papillomaviruses (HPVs) are recognized as the primary cause of cervical cancer. Among them,

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.06.042

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specific types of HPV (16, 18 and several others) have been identified as causative agents in at least 90% of cervical cancers, and have also been implicated in more than 50% of other anogenital cancers [1]. HPVs are characterized by circular, double-stranded DNA genomes, approximately 8 kb in size, and encode eight genes of which E5, E6, and E7 exhibit transformative properties. Viral oncoproteins E6 and E7 are selectively retained and expressed in carcinoma cells infected with HPV type 16 and cooperated in the immortalization and transformation of primary keratinocytes. E6 and E7 oncoproteins interact and interfere with the functions of the tumor suppressor proteins p53 and retinoblastoma protein (pRb), respectively [2–4]. Cervical carcinoma or keratinocyte cell lines, immortalized by HPV 16 E6 and E7, also alter the expression of several immunoregulatory cytokines [5–7]. We have previously reported that the downmodulation of IL-18-induced immune response by E6 and E7 is one of the mechanisms underlying immune escape in HPV-induced cervical cancer cells [8,9]. Both E6 and E7 oncoproteins down-regulated IL-18 expression in the HaCaT keratinocytes and C33A cervical carcinoma cells, and also bound to IL-18Ra. Furthermore, these oncoproteins, inhibited IL-18 binding to its receptor in vitro and in vivo, and inhibited the IL-18-induced production of interferong (IFN-g) [8,9]. IL-18 is a pro-inflammatory cytokine [10] as well as an immunostimulatory cytokine, which plays an important role in the host defense mechanism against infection and cancer. Upon binding to IL-18 receptors, IL-18 represents diverse biological functions, such as the stimulation of NK cells’ lytic activity and T cell proliferation as well as the induction of IFN-g, granulocyte/macrophage colony stimulating factor (GM-CSF) by activated T cells, and Th1 cell responses [11]. These biological activities of IL-18 are normally mediated via the activation of MAP kinases, following several transcription factors, such as nuclear factor-kappa B (NF-kB), signal transducers, activators of transcription 3 (STAT3), and activator protein-1 (AP-1). Thus, the biological functions of IL-18 are initiated via cell surface receptor binding, followed by a kinase cascade which includes kinases, such as myeloid differentiation factor 88 (MyD88), IL-1 receptor activated

kinase (IRAK), and TNF receptor-associated factor-6 (TRAF-6) [12–16]. IL-18, coupled with IL-12, plays an important role in both anti-tumor immunity and protective effects against infection, including viral infection. IL-18-binding protein (IL-18BP) is a constitutively expressed natural antagonist of IL-18. When IL-18BP binds to IL-18, it neutralizes the biological activities of IL-18 and competes with cell surface receptors [17,18]. Using computer models of human IL-18, two charged residues, Glu-42 (E42) and Lys-89 (K89), which interact with oppositely charged amino acid residues of IL-18BP, have been identified [18,19]. Based on the molecular modeling, Glu-42 was mutated to Ala (E42A) because point mutations are useful for understanding protein–protein interactions [19,20]. Compared with wild type IL-18, the E42A mutant exhibited an increase in biological activity. Glu-42 is a critical amino acid residue in the structure and function of IL-18, and is important for binding to cell surface receptors for signal transduction. A single point mutation of E42 in the mature IL-18 is anticipated to be useful in studies of the inhibitory effects of HPV oncoproteins on IL-18. In the present study, we attempted to determine whether the sitespecific IL-18 mutant E42A would modulate the inhibitory effects of IL-18-induced immune responses by HPV 16 E6 and E7.

2. Materials and methods 2.1. Antibodies and reagents The followings were purchased: RPMI 1640 culture medium, mouse monoclonal anti-actin, and alkaline phosphatase conjugated goat anti-mouse IgG (Sigma Chemical Co., St. Louis, MO); human IL-2, monoclonal anti-human IL-18, and monoclonal antihuman IL-18Ra (R&D system, Minneapolis, MN); IL-15 (Peprotech, Rocky Hill, NJ); polyclonal rabbit anti-IkBa, antibodies against C-19 of HPV 16 E6 protein and mouse monoclonal anti-HPV 16 E7 IgG2a, mouse monoclonal anti-NF-kB p65, rabbit polyclonal anti-JNK and polyclonal anti-p38 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-phospho-JNK and polyclonal anti-phospho-p38 (Cell Signaling Technology, Beverly, MA);

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peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, RA); recombinant human IL-12p70 (IL-12), and Opt EIA human IFN-g ELISA kit (PharMingen, San Diego, CA); Talon affinity resins (Clonetech Laboratories, Palo Alto, CA); glutathione–Sepharose agarose (Amersham Pharmacia, Little Chalfont, UK); Ni2C– NTA agarose (Qiagen, West Sussex, UK); Immunobilon-P membrane (Millipore, Bedford, MA). Monoclonal anti-human IL-18 Ab clone 8 (IgG2a) was made and characterized as previously described [8,9]. IL-18BPa was provided from Serono Pharmaceutical Research Institute (Geneva, Switzerland). 2.2. Cells NK0 cells, a subclone of the original NK92 cell line [21], were maintained in RPMI supplement with 10% FBS, 50 pg/ml IL-2 and 200 pg/ml IL-15 at 37 8C in a humidified incubator with 5% CO2 as described [19]. 2.3. Protein expression and purification Plasmids of wild type IL-18 and mutant E42A were prepared and used. IL-18 wild type and E42A were inserted into pPROEXeHTA, transformed into the competent DH5 (Invitrogen, Carlsbad, CA) as described [19], and purified by using Talon column. E6 and E7 were inserted into pET28a (Novagen, Madison, USA) and expressed in Escherichia coli BL21 (DE3), respectively, and purified with Ni2C– NTA as previously described [22,23]. In order to use for the nonspecific binding experiment, plasmid expressing recombinant OVA were prepared. In brief, total RNA was isolated from EG7-OVA (H-2bOVA-transfectant of EL-4; ATCC) by acidic phenol– guanidinium thiocyanate–chloroform extraction. OVA insert was amplified by RT-PCR, using primer sets comprising 5 0 -GCC TTC AGC AGC TTG AGA GT-3 0 (sense) and 5 0 -GAA TGG ATG GTC AGC CCT AA-3 0 (antisense). PCR product was double digested by restriction enzymes, BamH I and Xho I. An OVA fragment was cloned into the pET28a(C) to construct 6x his tagged OVA. Transformation of pET28a/OVA into E. coli BL21 (DE3) pLys S (Pharmacia) was performed to express 6x his-OVA. Plasmids of hisPPAR-g2-LBD was prepared and used. His tagged

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PPAR-g2-LBD was inserted into pET28a, expressed in E. coli BL21 (DE3) and purified with Ni2C–NTA as previously described [24]. 2.4. IFN-g assay To investigate the effect of oncoproteins on IL-18induced IFN-g production, NK0 cells in RPMI 1640 medium were seeded into 96 well plates in 200 ml at 5!105 cells/ml and treated with E6 or E7 in the presence of 0.5 ng/ml IL-12 and inducing agents, such as human IL-18 wild type or mutant E42A. After 20 h at 37 8C in a humidified incubator with 5% CO2, the culture supernatants were collected for IFN-g measurements as previously described [9]. 2.5. In vitro binding assay GST-IL-18Ra was bound to glutathione–Sepharose, and the purified mature IL-18 was used for in vitro binding. The purified IL-18 wild type and E42A were bound to IL-18Ra. The competitive bindings of IL-18 and E6 and E7 oncoproteins to IL-18Ra were performed as previously described [9]. The proteins bound to IL-18Ra were detected by Western blot analyses. In brief, binding assays were performed by combining GST-IL-18Ra immobilized on glutathione–Sepharose with 20 mg purified mature IL-18 proteins in a binding buffer (PBS containing 0.5% Triton X-100). A constant amount of IL-18 and bead-bound IL-18Ra was incubated with an increasing amount of oncoproteins ranging from 10 to 100 mg in a binding buffer at 4 8C for 1 h. Then the unbound proteins were washed, and Western blot analyses were performed using specific Abs. 2.6. Radioiodination of human IL-18 Each 20 mg of mature human IL-18 and E42A mutant in Tris iodination buffer (25 mM Tris–HCl, pH 7.5, 0.4 M NaCl) were radiolabeled with 2 mCi of Na125I and IODO–GENw pre-coated iodination tube (PIERCE Biotechnology, Inc., Rockford, IL) according to the manufacturer’s recommendations. The reaction was stopped with an excess of tyrosine. Unbound 125I was removed by chromatography on Sephadex G-25 equilibrated in Tris/NaCl/EDTA buffer containing 25 mM Tris–HCl, pH 7.5, 0.4 M NaCl, 5 mM EDTA

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and 0.05% Sodium azide. Each fraction was counted, and the peak fractions were pooled as previously described [19]. The pooled radiolabeled 125I-IL-18 or 125 I-E42A containing w3.5!106 cpm/mg was used for competition binding assays. Human NK0 cells (1!106, 0.2 ml) were suspended in a binding solution (cell culture medium containing 2% FBS and 0.02% sodium azide) containing increasing concentrations of 125 I-IL-18 in order to determine the optimum binding of 125 I-IL-18 onto the cell surface receptor. Eppendorf tubes were mixed for 1 h at 4 8C. The mixtures were then washed two times at 4 8C with the binding solution, and cell bound 125I-IL-18 was measured using gamma counter. 2.7. In vivo binding assay 125

I-IL-18 or 125I-E42A (2.14!106 cpm/320 ng) was incubated with NK0 cells (1!106, 0.2 ml) in the absence or presence of 100-fold molar excess of E6 or E7 oncoproteins, and respective unlabeled IL-18 or E42A for 1 h at 4 8C. The cells were then washed two times at 4 8C with the binding solution, and bound 125 I-IL-18 or 125I-E42A was measured using a gamma counter as previously described [19]. 2.8. Immunoblotting NK0 cells (5!106 cells) were washed and incubated in RPMI at 37 8C for 2 h. The cells were incubated in RPMI containing 20 ng/ml of IL-18 or E42A in presence or absence of E6 or E7 oncoproteins for 10 min at 37 8C, and washed twice with PBS. Cytosolic proteins were prepared by lysing cells in a hypotonic buffer containing 10 mM HEPES, 1.5 mM MgCl2 containing 1 mg/ml aprotinin, 1 mg/ml leupeptin and 1 mM PMSF, pH 8.5 on ice for 15 min at 4 8C. The nuclei were pelleted by centrifugation at 3000!g for 5 min. Nuclear lysis was performed in a hypertonic buffer containing 30 mM HEPES, 1.5 mM MgCl2, 450 mM KCl, 0.3 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1 mM PMSF, 1 mg/ml of aprotinin and 1 mg/ml leupeptin. The phosphorylated proteins were prepared by lysing cells in a lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EGTA, 25 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 mg/ml of aprotinin and 1 mg/ml leupeptin, pH 7.2)

on ice for 15 min at 4 8C. The protein concentration of the cell lysate was determined by Bradford method. Fifty microgram of protein per lane was electrophoresed in 10% SDS-PAGE, transferred to PVDF membrane (Millipore, Bedford, MA) and incubated with anti-IkBa, anti-p38, anti-JNK, anti-phospho-p38 and anti-phospho-JNK, or anti-NF-kB overnight at 4 8C, followed by 1 h incubation with respective secondary antibodies conjugated with horseradish peroxidase and development by ECL (Amersham Pharmacia, Little Chalfont, UK). 2.9. Electrophoretic mobility shift assay NK cells were lysed with a hypotonic buffer (10 mM HEPES and 1.5 mM MgCl2, pH 7.5), and the nuclei were pelleted by centrifugation at 3000!g for 5 min. Nuclear lysis was performed in a hypertonic buffer. After lysis, the samples were centrifuged at 13, 000!g for 10 min, and supernatant was retained for use in the DNA binding assay. An NF-kB binding site (5 0 -AGT TGA GGG GAC TTT CCC AGG C-3 0 ) was used as probes (Promega, Madison, WI). Two doublestranded deoxyoligonucleotides were end-labeled using T4 kinase and [g-32P] ATP. Then the labeled nucleotides were purified by gel filtration. Nuclear extracts (5–10 mg) were incubated with poly (dI–dC) and 32P-labeled DNA probe in a binding buffer (100 mM KCl, 30 mM HEPES, 1.5 mM MgCl2, 0.3 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1 mM PMSF, 1 mg/ml of aprotinin, and 1 mg/ml of leupeptin) at room temperature for 10 min in a total volume of 20 ml. DNA binding activity was separated from the free probe using 5% polyacrylamide gel in 0.5!TBE buffer (44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA). After electrophoresis, the gel was dried on 3 mm paper and subjected to hautoradiography. 2.10. Statistical analysis Data are presented as meanGSD of at least three independent experiments performed in triplicate. Differences between means were assessed by oneway analysis of variance. Data was analyzed for statistical significance using ANOVA. The minimum level of significance was set at P!0.05.

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3. Results 3.1. The effects of E6 and E7 on IFN-g production by wild type IL-18 or E42A mutant As our previous reports revealed [9], IL-18induced IFN-g production was inhibited by both the E6 and E7 oncoproteins of HPV 16 (Fig. 1A). In addition, the site-specific IL-18 mutant E42A resulted in about a 200% increase in the production of IFN-g in the NK0 cell line (Fig. 1), as previously reported [19,20]. Therefore, we attempted to determine whether the IL-18 mutant E42A would effect the modulation of the inhibitory effects of E6 and E7 oncoproteins on IL-18-induced IFN-g production. The E6 or E7 did not affect E42A-induced IFN-g production in NK0 cells (Fig. 1B). IL-18 wild type or E42A-induced IFN-g production was inhibited by IL18 binding protein (IL-18BP), neutralizing anti-hIL18Ra, or neutralizing anti-hIL-18 (Fig. 1). 3.2. The effects of E6 or E7 on the binding of wild type IL-18 or E42A to IL-18R in vitro and in vivo

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either E6 or E7 was specifically binding to IL-18R. The in vivo binding assay was also performed by incubating 125I-IL-18 or 125I-E42A with NK0 cells, in the absence or presence of a 100-fold molar excess of E6 and E7, and unlabeled IL-18 or E42A. The binding of wild type IL-18 to its receptor IL-18R was inhibited by excess unlabeled wild type IL-18, or by E6 or E7 oncoprotein, while

IL-18 WT

A IL-18BP (200) anti-IL-18 (200) E7 (200) IL-18 + E7 (20) IL-12 E6 (200) E6 (20) Cont IL-18BP (200 ng/ml) anti-IL-18 (200 ng/ml) E7 (200 ng/ml) E7 (20 ng/ml) E6 (200 ng/ml) E6 (20 ng/ml) IL-18 (20 ng/ml) IL-12 (0.5 ng/ml) Cont

* * * * * *

2

E6 and E7 oncoproteins inhibited the binding of IL-18 to its receptor IL-18Ra in both in vitro and in vivo binding assays, as previously described [9]. In order to confirm whether site-specific IL-18 mutant E42A would be prevented from binding to its receptor IL-18Ra by E6 or E7, in vitro binding assays were performed with recombinant proteins, wild type IL-18 or mutant E42A, and oncoproteins E6 or E7. Wild type IL-18 or mutant E42Aas well as E6 or E7 bound to IL-18Ra in a dose-dependent manner. As the doses of E6 and E7 proteins were increased, the binding of wild type IL-18 to its receptor IL-18Ra was increasingly inhibited, while E6 and E7 appeared to exert no significant effects on the binding of E42A to IL-18Ra (Fig. 2B, C). In order to ascertain whether tagged recombinant proteins would affect the binding of IL-18 or E42A to IL-18Ra, in vitro binding assays were also performed with recombinant tagged or similarly sized control proteins, such as his-ovalbumin (OVA) or his PPAR-g2-LBD (Fig. 2A). The recombinant tagged control proteins appeared to have no effects on the binding between IL-18 or E42A and IL-18Ra, supporting the notion that

3

B

4

5 6 7 8 9 10 IFN- γ (ng/ml)

11

E42A *

IL-18BP (200) anti-IL-18 (200) E7 (200) E42A + E7 (20) IL-12 E6 (200) E6 (20) Cont IL-18BP (200 ng/ml) anti-IL-18 (200 ng/ml) E7 (200 ng/ml) E7 (20 ng/ml) E6 (200 ng/ml) E6 (20 ng/ml) IL-18 (20 ng/ml) IL-12 (0.5 ng/ml) Cont

*

2

4

6

8 10 12 14 16 18 20 IFN- γ (ng/ml)

Fig. 1. The effects of HPV oncoprotein E6 or E7 on wild type IL-18 or IL-18 mutant E42A-induced IFN-g production. IL-18 (A) or E42A (B) (20 ng/ml), IL-18BP, neutralizing anti-IL-18 antibody and oncoproteins (20, 200 ng/ml) were coincubated at room temperature for 30 min, and administered to NK0 cells (1!105) coincubated with IL-12 (0.5 ng/ml). After 20 h, IFN-g was measured, as is described in Section 2. The bar represents the meansGSD of five independent experiments. *significantly different from control (P!0.05).

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3.4. The effects of E6 or E7 on IkB degradation by wild type IL-18 or E42A In order to investigate the effects of E6 or E7 on IL-18 signaling through its binding to IL-18R, we measured IkB degradation induced by wild type IL18 or E42A in NK0 cells. IkB was completely degraded by both wild type IL-18 and E42A, as determined by immunoblot. The degradation of IkB by wild type IL-18 was blocked by the neutralization of anti-IL-18Ra antibody and was ameliorated by the presence of E6 or E7 in NK0 cells, while no IkB was observed when the E42A mutant was treated in the presence of E6 or E7 (Fig. 5A). Thus, supporting the notion that E42A-mediated IkB degradation was not inhibited by these oncoproteins. IL-18-induced NF-kB p65 expression was also inhibited by E6 or E7, whereas E42A mutant-induced p65 was not affected by E6 or E7, as determined by immunoblot (Fig. 5B). 3.5. The effects of E6 or E7 on wild type IL-18 or E42A induced the transcriptional activities of NF-kB Fig. 2. The effects of E6 and E7 on the binding of wild type IL-18 or E42A to IL-18Ra in vitro. Competitions of OVA, PPARg (A), E6 (B) or E7 (C) proteins with IL-18 protein for glutathione–Sepharose bead-bound IL-18Ra were analyzed, and the proteins bound to IL18R were detected by Western blot analyses. Binding assays were described in Section 2.

E6 and E7 appeared to have no effect on the binding of E42A to the cell surface receptor. IL-18 binding protein (IL-18BP) inhibited IL-18 binding to the cell surface receptor, while OVA also had no effect on E42A’s binding to the cell surface IL-18 receptor (Fig. 3). 3.3. The effects of E6 or E7 on MAP kinase phosphorylation by wild type IL-18 or E42A We then investigated the effects of E6 or E7 on IL18-induced MAP kinase phosphorylation. JNK and p38 phosphorylations induced by wild type IL18 that were inhibited by E6 or E7 in the NK0 cells, neither E6 nor E7 appeared to have any effect on the E42A-induced phosphorylation of JNK and p38 (Fig. 4).

In order to investigate the inhibitory effects of E6 or E7 on IL-18 wild type or E42A-induced NF-kB activation, we examined IL-18 induced NF-kB

E42A +

OVA E7 E6 IL-18BP

*

IL-18 Ctrl

*

OVA IL-18 +

E7 E6

* *

IL-18BP

*

IL-18 Ctrl 0

* 20

40

60

80

100

120

IL-18 binding (%)

Fig. 3. The effects of E6 and E7 on the binding of wild type IL-18 or E42A to IL-18Ra in vivo. Competition for cell surface receptors of E6 or E7 proteins with type IL-18 or E42A were analyzed, and bound 125I-IL-18 was detected by a gamma counter, as described in Section 2.

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267

Fig. 4. The effects of E6 or E7 oncoproteins on MAP kinase phosphorylation induced by wild type IL-18 or mutant E42A. After treatment of the NK0 cell with IL-18 or E42A (20 ng/ml) in the presence or absence of oncoproteins or IL-18 neutralizing agent, cytosolic proteins were electrophoresed and transferred to PVDF membrane. Phosphorylated JNK and p38 were detected using specific antibodies, as described in Section 2.1.

activation in the presence of E6 or E7, using an EMSA. HPV 16 E6 and E7 interfered with IL-18 wild type-induced NF-kB activation in NK0 cells. However, treatment with E6 or E7 in the presence of mutant E42A had no effect on NF-kB activity (Fig. 6).

4. Discussion Human papillomavirus (HPV) 16 is the most common virus that cause human cervical carcinoma. Viral oncoproteins E6 and E7 interact and interfere with the functions of tumor suppressor proteins p53

Fig. 5. The effects of E6 or E7 oncoproteins on IkB degradation and NF-kB expression induced by wild type IL-18 or mutant E42A. IL-18 or E42A (20 ng/ml), neutralizing anti-IL-18Ra or anti-IL-18 antibody, and E6 or E7 (2 mg/ml) were coincubated, and applied into NK0 cells (5! 106) for 15 min. Cells were solubilized, cytosolic and nuclear proteins were extracted, subjected to SDS-PAGE, and immunoblotted as described in Section 2.

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Fig. 6. The effects of E6 or E7 oncoproteins on NF-kB activation by wild type IL-18 or mutant E42A. IL-18 or E42A (20 ng/ml), antiIL-18 or IL-18BP (200 ng/ml) and oncoproteins (200 ng/ml) were preincubated, and treated into NK0 cells (5!106 coincubated with IL-12 (1 ng/ml) for 2 h. Nuclear proteins were extracted and incubated with radiolabeled oligonucleotides, which contained a binding site for NF-kB. The resulting complexes were analyzed by EMSA, as described in Section 2.

and retinoblastoma protein (pRb), respectively [1–4]. However, E6 or E7 also functions by targeting other molecules independent of tumor suppressor proteins [25–28]. We previously reported that the downmodulation of IL-18-induced immune response by E6 and E7 constitutes a common mechanism underlying immune escape in HPV-induced cervical cancer cells [9]. Using point mutations of IL-18, we attempted to determine whether site-specific IL-18 mutants would modulate the inhibitory effects of IL18-induced immune responses by HPV 16 E6 and E7. In the present study, point mutations of IL-18 evidenced significant structural changes, which influenced the activity of IL-18 with regard to HPV 16 E6 and E7 oncoproteins. The functionality of the IL-18 mutant E42A was assessed by the induction of cytokines such as IFN-g, competition for receptor binding in in vitro pull-down assay, in vivo binding assay, induction of IkB degradation, MAP kinase phosphorylation, and activations of transcriptional factors, such as NF-kB, using EMSA. The E42A mutant enhanced IFN-g production by 2-fold

compared to wild type IL-18 in the NK0 cell line, and neither E6 nor E7 inhibited E42A-induced IFN-g production (Fig. 1). In fact, E6 and E7 exerted no inhibitory effects on the E42A-induced signaling transduction via the IL-18 receptor. In contrast, the wild type IL-18 induced signalings such as IkB degradation, MAPK phosphorylation, and activations of transcriptional factors which were blocked or inhibited by E6 or E7 oncoproteins (Figs. 4–6). These results suggested that the E42A mutant is too strong to be inhibited by oncoproteins. According to the results obtained with the IL-18 mutant E42, we expected that E42 would be an important amino acid residue in IL18 signaling, competing with E6 and E7. In our study, E42A and IL-18 were both shown to bind directly to IL-18Ra, which was demonstrated both in the in vitro binding assay (Fig. 2) and in the in vivo experiment using intact NK0 cells with 125I-labeled IL-18 or 125IE42A (Fig. 3). E6 and E7 oncoproteins inhibited the binding of IL-18 to its receptor IL-18R, while E6 and E7 appeared to exert no significant effects on the binding of the E42A mutant to IL-18Ra and to the cell surface receptor (Fig. 3). The binding effect of E42A demonstrated that E6 and E7 had no effects on the binding between mutant E42A and IL-18R. Based on the E42A effects on receptor binding and IFN-g signaling, we further investigated the manner in which E42A influenced the activation of several transcriptional factors associated with IL-18 signaling. IL-18 induces the activation of several transcription factors, such as NF-kB and AP-1, and also caused IkB degradation. IL-18-dependent IkB degradation in NK0 cells resulted in the NF-kB release for nuclear translocation. HPV 16 oncoproteins E6 or E7 blocked the complete IL-18-induced degradation of IkB in NK0 cells, while the oncoproteins had no effect on E42A-induced IkB degradation (Fig. 5A). EMSA also demonstrated that HPV 16 E6 and E7 interfered with IL-18 wild type-induced NF-kB activation in the NK0 cell line. However, E6 and E7 failed to affect E42Ainduced NF-kB activation (Fig. 6). These data show that E6 and E7 affect wild type IL-18-induced activation of transcriptional factors, but are insufficient to influence E42A mutant-induced signaling. The cross-linking of 125I-IL-18 to its receptor on NK cells revealed that the E42 mutant competitively displaced 125I-IL-18 from the receptor [19,20]. These results suggest that E42A is a critical amino acid

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residue of IL-18, which binds quite tightly to its surface receptors, functioning as a strong activator of IFN-g production, which is resistant to the inhibitory effects of the E6 or E7 oncoproteins. If wild type IL-18 is used in cervical cancer immunotherapies, the functions of wild type IL-18 would remain susceptible to the inhibitory influences of E6 and E7. However, when the E42A mutant is expressed on cervical cancer cells, biological activities will be enhanced without interfering with HPV 16 oncoproteins E6 and E7, and thus could exert a more pronounced anti-tumor effect. Therefore, mutant E42A may be used in immunotherapy for HPVinfected cells.

[7]

[8]

[9]

[10]

Acknowledgements [11]

This work was supported by a grant from KRIBB Research Initiative Program and a grant (PF032100100) from Plant Diversity Research Center of 21st Century Frontier Research Program funded by Ministry of Science and Technology and by grant No. RTI04-03-07 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE). The authors thank Mr Noh, Young-Woock for cloning his-OVA expression vector.

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