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• W2074443441 abstract "Interferon-γ (IFNγ) has an antiproliferative effect on a variety of tumor cells. However, many tumor cells resist treatment with IFNs. Here, we show that IFNγ fails to inhibit the growth of some types of oral squamous cell carcinoma (OSCC) cells that possess a fully functional IFNγ/STAT1 (signal transducer and activator of transcription-1) signaling pathway. IFNγ inhibited the growth of the HSC-2, HSC-3, and HSC-4 OSCC cell lines. However, Ca9–22 cells were resistant to IFNγ despite having intact STAT1-dependent signaling, such as normal tyrosine phosphorylation, DNA binding activity, and transcriptional activity of STAT1. The growth inhibition of HSC-2 cells resulted from S-phase arrest of the cell cycle. IFNγ inhibited cyclin A2 (CcnA2)-associated kinase activity, which correlated with the IFNγ-mediated down-regulation of CcnA2 and Cdk2 expression at both the transcriptional and post-transcriptional level in HSC-2 cells but not in Ca9–22 cells. RNAi-mediated knockdown of CcnA2 and Cdk2 resulted in growth inhibition in both cell lines. These results indicate that the resistance of OSCC to IFNγ is not due simply to the deficiency in STAT1-dependent signaling but results from a defect in the signaling component that mediates this IFNγ-induced down-regulation of CcnA2 and Cdk2 expression at the transcriptional and post-transcriptional levels. Interferon-γ (IFNγ) has an antiproliferative effect on a variety of tumor cells. However, many tumor cells resist treatment with IFNs. Here, we show that IFNγ fails to inhibit the growth of some types of oral squamous cell carcinoma (OSCC) cells that possess a fully functional IFNγ/STAT1 (signal transducer and activator of transcription-1) signaling pathway. IFNγ inhibited the growth of the HSC-2, HSC-3, and HSC-4 OSCC cell lines. However, Ca9–22 cells were resistant to IFNγ despite having intact STAT1-dependent signaling, such as normal tyrosine phosphorylation, DNA binding activity, and transcriptional activity of STAT1. The growth inhibition of HSC-2 cells resulted from S-phase arrest of the cell cycle. IFNγ inhibited cyclin A2 (CcnA2)-associated kinase activity, which correlated with the IFNγ-mediated down-regulation of CcnA2 and Cdk2 expression at both the transcriptional and post-transcriptional level in HSC-2 cells but not in Ca9–22 cells. RNAi-mediated knockdown of CcnA2 and Cdk2 resulted in growth inhibition in both cell lines. These results indicate that the resistance of OSCC to IFNγ is not due simply to the deficiency in STAT1-dependent signaling but results from a defect in the signaling component that mediates this IFNγ-induced down-regulation of CcnA2 and Cdk2 expression at the transcriptional and post-transcriptional levels. Interferon-γ (IFNγ) 2The abbreviations used are:IFNinterferonJAK-STATJanus kinase-signal transducer activation of transcriptionGASγ-IFN activation sequenceIRFinterferon regulatory factorOSCCoral squamous cell carcinomaCcnA2cyclin A2Cdkcyclin-dependent kinaseTKthymidine kinaseqRT-PCRreal-time quantitative RT-PCRAREadenylate/uridylate (AU)-rich elementssiRNAsmall interfering RNAkbkilobase(s)PI3Kphosphoinositide 3-kinase. is a cytokine produced by activated T cells and natural killer cells. It exhibits a number of biological activities in host-defense systems and immunoregulation, including anti-viral and anti-tumor responses (1Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1588) Google Scholar, 2Boehm U. Klamp T. Groot M. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2451) Google Scholar). The antiproliferative activity of IFNs has been well documented in a variety of tumor cell types. Multiple studies have shown that both type I (IFNα/β) and type II (IFNγ) IFNs induce cell cycle arrest at G0/G1, which is mediated by the up-regulation of the cyclin-dependent kinase inhibitors p21WAF1/Cip1 and p27Kip1 after IFN treatment (3Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (724) Google Scholar, 4Sangfelt O. Erickson S. Einhorn S. Grandér D. Oncogene. 1997; 14: 415-423Crossref PubMed Scopus (100) Google Scholar, 5Mandal M. Bandyopadhyay D. Goepfert T.M. Kumar R. Oncogene. 1998; 16: 217-225Crossref PubMed Scopus (92) Google Scholar, 6Kominsky S. Johnson H.M. Bryan G. Tanabe T. Hobeika A.C. Subramaniam P.S. Torres B. Oncogene. 1998; 17: 2973-2979Crossref PubMed Scopus (62) Google Scholar, 7Gooch J.L. Herrera R.E. Yee D. Cell Growth Differ. 2000; 11: 335-342PubMed Google Scholar). The p21 protein has been shown to inhibit cyclin/ Cdk activity, which phosphorylates the retinoblastoma (Rb) tumor suppressor and then activates members of the E2F transcription factor family (8El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7869) Google Scholar, 9Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5201) Google Scholar). IFNγ-induced signal transducer and activator of transcription 1 (STAT1) has been shown to induce transactivation of the p21WAF1/Cip1 gene (3Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (724) Google Scholar). In some tumor cells, however, the arrest of IFNγ-mediated, cyclin-dependent kinase inhibitor-independent cell growth has been reported (10Harvat B.L. Jetten A.M. Cell Growth Differ. 1996; 7: 289-300PubMed Google Scholar, 11Vivo C. Lévy F. Pilatte Y. Fleury-Feith J. Chrétien P. Monnet I. Kheuang L. Jaurand M.C. Oncogene. 2001; 20: 1085-1093Crossref PubMed Scopus (27) Google Scholar, 12Kortylewski M. Komyod W. Kauffmann M.E. Bosserhoff A. Heinrich P.C. Behrmann I. J. Invest. Dermatol. 2004; 122: 414-422Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, IFNγ-mediated growth inhibition appears to be mediated by multiple pathways, depending on the cell type, and the molecular mechanisms by which IFNγ inhibits tumor cell growth remain to be fully elucidated. interferon Janus kinase-signal transducer activation of transcription γ-IFN activation sequence interferon regulatory factor oral squamous cell carcinoma cyclin A2 cyclin-dependent kinase thymidine kinase real-time quantitative RT-PCR adenylate/uridylate (AU)-rich elements small interfering RNA kilobase(s) phosphoinositide 3-kinase. Although IFN exhibits a potent antiproliferative and proapoptotic effects on many tumor cells, some types of tumor cells resist IFN treatment (13Xu B. Grandér D. Sangfelt O. Einhorn S. Blood. 1994; 84: 1942-1949Crossref PubMed Google Scholar, 14Wong L.H. Krauer K.G. Hatzinisiriou I. Estcourt M.J. Hersey P. Tam N.D. Edmondson S. Devenish R.J. Ralph S.J. J. Biol. Chem. 1997; 272: 28779-28785Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 15Sun W.H. Pabon C. Alsayed Y. Huang P.P. Jandeska S. Uddin S. Platanias L.C. Rosen S.T. Blood. 1998; 91: 570-576Crossref PubMed Google Scholar, 16Kaplan D.H. Shankaran V. Dighe A.S. Stockert E. Aguet M. Old L.J. Schreiber R.D. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 7556-7561Crossref PubMed Scopus (1141) Google Scholar, 17Dovhey S.E. Ghosh N.S. Wright K.L. Cancer Res. 2000; 60: 5789-5796PubMed Google Scholar). Several studies have demonstrated the molecular mechanisms underlying this resistance to IFN. Defects in components of the IFN signaling pathway, such as the expression of the IFNγ receptor, Janus kinase (JAK), STAT1, STAT2, and interferon regulatory factor-9 (IRF-9/p48), have been identified in resistant cells (13Xu B. Grandér D. Sangfelt O. Einhorn S. Blood. 1994; 84: 1942-1949Crossref PubMed Google Scholar, 14Wong L.H. Krauer K.G. Hatzinisiriou I. Estcourt M.J. Hersey P. Tam N.D. Edmondson S. Devenish R.J. Ralph S.J. J. Biol. Chem. 1997; 272: 28779-28785Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 15Sun W.H. Pabon C. Alsayed Y. Huang P.P. Jandeska S. Uddin S. Platanias L.C. Rosen S.T. Blood. 1998; 91: 570-576Crossref PubMed Google Scholar, 16Kaplan D.H. Shankaran V. Dighe A.S. Stockert E. Aguet M. Old L.J. Schreiber R.D. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 7556-7561Crossref PubMed Scopus (1141) Google Scholar, 17Dovhey S.E. Ghosh N.S. Wright K.L. Cancer Res. 2000; 60: 5789-5796PubMed Google Scholar). Furthermore, reduced expression of ISGF-3 (a tetramer complex with STAT1, STAT2, and IRF-9) has been detected in skin squamous carcinoma cells from surgical specimens (18Clifford J.L. Walch E. Yang X. Xu X. Alberts D.S. Clayman G.L. El-Naggar A.K. Lotan R. Lippman S.M. Clin. Cancer Res. 2002; 8: 2067-2072PubMed Google Scholar). However, some types of tumor cells have been reported to resist IFNs despite having a normal JAK-STAT pathway (7Gooch J.L. Herrera R.E. Yee D. Cell Growth Differ. 2000; 11: 335-342PubMed Google Scholar, 12Kortylewski M. Komyod W. Kauffmann M.E. Bosserhoff A. Heinrich P.C. Behrmann I. J. Invest. Dermatol. 2004; 122: 414-422Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 19Chawla-Sarkar M. Leaman D.W. Jacobs B.S. Tuthill R.J. Chatterjee-Kishore M. Stark G.R. Borden E.C. J. Interferon Cytokine Res. 2002; 22: 603-613Crossref PubMed Scopus (23) Google Scholar, 20Lei H. Furlong P.J. Ra J.H. Mullins D. Cantor R. Fraker D.L. Spitz F.R. Cancer Biol. Ther. 2005; 4: 709-715Crossref PubMed Scopus (20) Google Scholar). Thus, both JAK-STAT-dependent and-independent mechanisms appear to explain IFN resistance. However, the mechanism of JAK-STAT-independent IFN resistance remains poorly understood. To gain insight into the molecular mechanisms responsible for the antiproliferative effect of IFNγ and the resistance to the IFNγ-mediated effect in human oral squamous cell carcinomas (OSCC) cells, we examined the effect of IFNγ on the growth of human OSCC cell lines. We also explored the mechanisms underlying the antiproliferative effect of IFNγ and the unresponsiveness of cells to this molecule. We demonstrated that IFNγ inhibits the growth of the HSC-2, HSC-3, and HSC-4 human OSCC cell lines, whereas Ca9–22 cells are resistant to IFNγ despite the presence of intact STAT1-dependent signaling. IFNγ inhibited the expression of cyclin A (CcnA2) and cyclin-dependent kinase 2 (Cdk2) in HSC-2 cells, but not in Ca9–22 cells, and knockdown of either CcnA2 or Cdk2 by siRNA inhibited cell growth in both cell types. Furthermore, IFNγ suppressed the promoter activity of the CcnA2 and Cdk2 genes and destabilized CcnA2 and Cdk2 mRNAs in HSC-2 cells but not in Ca9–22 cells. These results suggest that the resistance of OSCC cells to the antiproliferative effect of IFNγ is not because of a deficiency in STAT1-dependent signaling but, instead, results from a defective signaling component that mediates the IFNγ-induced down-regulation of CcnA2 and Cdk2 expression. Recombinant human IFNγ was purchased from BioSource International Inc. (Camarillo, CA). A cell counting kit was obtained from Dojin Laboratories (Tokyo, Japan). Antibodies against CcnA2 (H432), Cdk2 (M-2), Cdk6 (C-21), p21 (C-19), STAT1 (E23), STAT3 (C-20), histone H1 (AE-4), and β-actin (I-19) were obtained from Santa Cruz Biotechnology (Hercules, CA). Antibodies against cyclin D1 (#2926), cyclin D2 (#2924), cyclin D3 (#2936), cyclin E (#4129), Cdk4 (#2906), phospho-STAT1 Tyr-701 (#9171S), phospho-STAT3 Tyr-705 (#9131S), phospho-Akt Thr-308 (#2965), phospho-Akt Ser-473 (#4060), pan-Akt (#4691), and phospho-PDK1 Ser-241 (#3061) were obtained from Cell Signaling Technology (Danvers, MA). Anti-α-tubulin antibody was obtained from Sigma. FuGENE transfection reagent and histone H1 were purchased from Roche Diagnostics. StealthTM Select RNAi oligonucleotides for CcnA2, Cdk2, and green fluorescent protein were obtained from Invitrogen. The HSC-2, HSC-3, HSC-4, and Ca9–22 human OSCC cell lines were described previously (21Kamata N. Chida K. Rikimaru K. Horikoshi M. Enomoto S. Kuroki T. Cancer Res. 1986; 46: 1648-1653PubMed Google Scholar, 22Momose F. Araida T. Negishi A. Ichijo H. Shioda S. Sasaki S. J. Oral. Pathol. Med. 1989; 18: 391-395Crossref PubMed Scopus (153) Google Scholar, 23Hiroi M. Ohmori Y. Biochem. J. 2003; 376: 393-402Crossref PubMed Scopus (46) Google Scholar). These cells were originally isolated from metastatic OSCC cells derived from the oral cavity (21Kamata N. Chida K. Rikimaru K. Horikoshi M. Enomoto S. Kuroki T. Cancer Res. 1986; 46: 1648-1653PubMed Google Scholar). For the cell proliferation assays, cells were seeded in 96-well plates (3 × 103 cells/well) and grown for 20 h before being treated with IFNγ. After treatment with IFNγ, the viable cell number was determined using the Cell Counting kit (Dojin) according to the manufacturer's protocol. After incubation with the reagent, the optical density at 450 nm was measured using a microplate reader. The cell number after treatment with IFNγ was also counted using a hemocytometer after trypsinization. Nuclear extracts were prepared as described previously (24Ohmori Y. Schreiber R.D. Hamilton T.A. J. Biol. Chem. 1997; 272: 14899-14907Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) using a modification of the method described by Dignam et al. (25Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). Nuclear extracts (5 μg of total protein) were incubated in 12.5 μl of 20 mm HEPES, pH 7.9, containing 50 mm KCl, 0.1 mm EDTA, 1 mm dithiothreitol, 5% glycerol, 200 μg/ml bovine serum albumin, and 1.25 μg of poly(dI-dC). A 32P-labeled, double-stranded oligonucleotide from the IRF-1 gene (5′-tcgaGCCTGATTTCCCCGAAATGAGGC-3′) (26Sims S.H. Cha Y. Romine M.F. Gao P.Q. Gottlieb K. Deisseroth A.B. Mol. Cell Biol. 1993; 13: 690-702Crossref PubMed Scopus (249) Google Scholar) was then added to the reaction mixture. The reaction products were analyzed by electrophoresis in a 5% polyacrylamide gel. Total cell lysates were resolved in SDS-PAGE sample buffer (62.5 mm Tris, pH 6.8, containing 2% SDS, 20% glycerol, 5% β-mercaptoethanol, and 0.2% bromphenol blue) and separated by SDS-PAGE in a 7.5% polyacrylamide gel, as described previously (23Hiroi M. Ohmori Y. Biochem. J. 2003; 376: 393-402Crossref PubMed Scopus (46) Google Scholar). siRNA transfection was performed with Lipofectamine RNAiMaxTM according to the manufacturer's instructions (Invitrogen). Briefly, 5 × 104 cells were seeded in a 6-well plate. One day after plating, the siRNA (final concentration, 10 nmol) was suspended in 1 ml of RNAiMAX and added to each well. Cells were harvested 48 h after transfection and either used to determine the efficiency of knockdown or reseeded into a 96-well plate for the cell proliferation assays. Sequences encoding the 5′-flanking promoter/enhancer region of the human CcnA2 and Cdk2 genes were cloned from human genomic DNA using PCR with high fidelity Platinum PCR SuperMix (Invitrogen) and a set of primers corresponding to the human CcnA2 (from −881 to +216) (GenBankTM accession number X68303) (27Henglein B. Chenivesse X. Wang J. Eick D. Bréchot C. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 5490-5494Crossref PubMed Scopus (243) Google Scholar) and the human Cdk2 (from −767 to +19) genomic sequences (GenBankTM accession number U50730) (28Shiffman D. Brooks E.E. Brooks A.R. Chan C.S. Milner P.G. J. Biol. Chem. 1996; 271: 12199-12204Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The amplified PCR fragments were individually subcloned into a luciferase reporter construct (pGL3-Basic, Promega), and the nucleotide sequence was confirmed. The resulting plasmids were designated as pGL-CcnA2–881 (CcnA2) and pGL-Cdk2–767 (Cdk2). The luciferase reporter plasmid pDel-1, which contains a 2.5-kb sequence from the 5′-flanking region of the human c-myc gene (29He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4026) Google Scholar), was obtained from Addgene (Cambridge, MA). The luciferase reporter construct pGL-IRF-1, which contains a 1.3-kb sequence from the 5′-flanking region of the IRF-1 gene (26Sims S.H. Cha Y. Romine M.F. Gao P.Q. Gottlieb K. Deisseroth A.B. Mol. Cell Biol. 1993; 13: 690-702Crossref PubMed Scopus (249) Google Scholar, 30Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (339) Google Scholar), and pTK GASluc, which contains four copies of the IFNγ activation site (GAS) motif from the IRF-1 gene upstream of the herpes simplex virus-thymidine kinase (TK) promoter, were described previously (31Ohmori Y. Hamilton T.A. J. Immunol. 1997; 159: 5474-5482PubMed Google Scholar). The transfection procedure and luciferase reporter assay were described previously (23Hiroi M. Ohmori Y. Biochem. J. 2003; 376: 393-402Crossref PubMed Scopus (46) Google Scholar). To standardize the transfection efficiencies, the firefly luciferase activity from pGL luciferase reporter plasmids was normalized to the Renilla luciferase activity from the pRL-TK plasmid (Promega), and the relative luciferase activities in the different OSCC cells were normalized to the activity of the pCMVluc or the pGL3-Control plasmid. Cells were cultured in 6-cm dishes 20 h before stimulation and were then treated with IFNγ for the indicated periods. After trypsinization, the cells were collected by centrifugation, washed in phosphate-buffered saline and fixed with cold 70% ethanol. Cells were then washed with phosphate-buffered saline and treated with 250 μg/ml of RNase at 37 °C for 40 min. Cellular DNA was stained with 50 μg/ml of propidium iodide, and 5 × 104 cells were analyzed on a FACScan flow cytometer (EPICS ALTRA, Beckman Coulter, Fullerton, CA). The proportions of cells in different stages of the cell cycle were determined using WinCycle software (Beckman Coulter). Cells were cultured in 24-well plates for 20 h in complete medium before treatment with IFNγ. After treatment with or without IFNγ for varying periods, cells were pulse-labeled with 1 μCi of [3H]thymidine (PerkinElmer Life Sciences) for the last hour of the cultivation. The cells were then washed with phosphate-buffered saline, fixed in cold 5% trichloroacetic acid, and washed with 5% trichloroacetic acid. Incorporated [3H]thymidine was extracted with 0.2% SDS and 0.5 n NaOH and measured in a liquid scintillation counter (Aloka, Tokyo, Japan). CcnA2-dependent kinase activity was assessed in vitro by the phosphorylation of histone H1. Cells were lysed in an ice-cold kinase lysis buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 0.1% Nonidet P-40, 0.1% Triton X-100, 1 mm EDTA, 2.5 mm EGTA, 10 mm glycerophosphate, 50 mm NaF, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and proteinase inhibitor mixture (Sigma)). After centrifugation, 1 μg of anti-CcnA2 antibody was added, and the sample was agitated for 1 h at 4 °C. After incubation, protein A-agarose beads were added and incubated for an additional hour. The immunoprecipitates were washed with Cdk kinase buffer (50 mm Hepes, pH 7.4, 10 mm MgCl2, 10 mm MnCl2, and 1 mm dithiothreitol) and mixed in a 50-μl reaction containing 50 mm Hepes, pH 7.4, 40 mm MgCl2, 25 mm ATP, 2.5 μCi of [γ-32P]ATP (3000 Ci/mmol), and 1 μg of histone H1 at 37 °C for 30 min. Reactions were terminated by the addition of SDS sample buffer, and the samples were subjected to 10% SDS-PAGE. The gels were dried, and phosphorylation of histone H1 was detected with autoradiography. The preparation of total RNA by the guanidine isothiocyanate-cesium chloride method and northern hybridization analyses were carried out as described previously (24Ohmori Y. Schreiber R.D. Hamilton T.A. J. Biol. Chem. 1997; 272: 14899-14907Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). The cDNA fragments for human CcnA2 and Cdk2 were prepared by reverse transcriptase-PCR using a set of primers corresponding to sequences for human CcnA2 (GenBankTM accession number NM001237) and Cdk2 (GenBankTM accession number NM001798). The PCR products were subcloned into pBluescript (Stratagene, La Jolla, CA), and the nucleotide sequences were confirmed. Some northern blots were quantified using a Molecular Imager (Bio-Rad). qRT-PCR was performed on the LightCycler 480 real-time PCR system (Roche Diagnostics) using the TaqMan® probe (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Briefly, total RNA (500 ng) was reverse-transcribed with random hexamers using the High Capacity cDNA reverse transcription kit (Applied Biosystems) in a 20-μl reaction volume. After the reverse transcriptase reaction, qRT-PCR was performed using the TaqMan® Gene Expression Master Mix reagents, the TaqMan® gene expression assay (c-myc, assay ID Hs00153408_m1), and the endogenous control (18 S rRNA) in a final volume of 20 μl. Reactions were performed and analyzed using the LightCycler 480 system (Roche Diagnostics). Each mRNA level was normalized to that of the 18 S rRNA, and the relative expression level was determined using the standard curve method for multiplex PCR. The transcript abundance was calculated as the percentages of the control or experimental sample values normalized to the 18 S rRNA. IFNγ has been shown to inhibit the growth of a wide variety of tumor cells. We initially examined the effects of various doses of IFNγ on the growth of four asynchronously proliferating human OSCC lines (Fig. 1A). Treatment of HSC-2, HSC-3, and HSC-4 cells with IFNγ inhibited proliferation in a dose-dependent fashion. However, IFNγ had only a marginal effect on the proliferation of Ca9–22 cells, even at high concentrations (100 ng/ml). In separate experiments, a higher concentration of IFNγ (300 ng/ml) similarly showed no significant inhibitory effect on the proliferation of Ca9–22 cells (data not shown). The differential sensitivity of cell proliferation to IFNγ was also determined by direct cell counting at different times during culture (Fig. 1B). IFNγ inhibited the growth of HSC-2, HSC-3, and HSC-4 cells, with a significant inhibition observed after 48 h of IFNγ treatment. By contrast, IFNγ failed to inhibit the growth of Ca9–22 cells at any of the time points examined. IFNγ-mediated biological activities are mediated by the STAT1-dependent signaling pathway (32Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver-Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1377) Google Scholar, 33Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1275) Google Scholar). To determine whether the differential antiproliferative effect of IFNγ is because of a defect in STAT1-dependent function, we analyzed the functional integrity of IFNγ-induced STAT1 activation in these oral carcinoma cells. Initially, we assessed tyrosine phosphorylation of STAT1 (Try-701) in the OSCC cells by Western blotting (Fig. 2A). IFNγ caused phosphorylation of STAT1 on Tyr-701, and all of the OSCC cells showed similar levels of phosphorylated STAT1. We next analyzed the DNA binding activity of STAT1 in nuclear extracts from cells treated with IFNγ (Fig. 2B). Gel-shift assays using nuclear extracts from OSCC cells showed that in all cell lines IFNγ induced the formation of a prominent DNA binding complex on the probe for the IRF-1 GAS, which contains a high affinity STAT1 binding motif (26Sims S.H. Cha Y. Romine M.F. Gao P.Q. Gottlieb K. Deisseroth A.B. Mol. Cell Biol. 1993; 13: 690-702Crossref PubMed Scopus (249) Google Scholar, 30Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (339) Google Scholar, 34Decker T. Kovarik P. Meinke A. J. Interferon Cytokine Res. 1997; 17: 121-134Crossref PubMed Scopus (332) Google Scholar). The presence of STAT1 in the DNA binding complexes was demonstrated by a supershift assay with anti-STAT1 antibody. We further analyzed IFNγ-induced STAT1-dependent transcriptional activity using luciferase reporter constructs containing 1.3 kb of the 5′-flanking sequence from the IRF-1 gene (Fig. 2C), which is regulated by STAT1 (26Sims S.H. Cha Y. Romine M.F. Gao P.Q. Gottlieb K. Deisseroth A.B. Mol. Cell Biol. 1993; 13: 690-702Crossref PubMed Scopus (249) Google Scholar, 30Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (339) Google Scholar), and a heterologous promoter construct containing four copies of the GAS motif from the IRF-1 gene placed upstream of the herpes simplex virus-TK promoter (31Ohmori Y. Hamilton T.A. J. Immunol. 1997; 159: 5474-5482PubMed Google Scholar) (Fig. 2D). IFNγ markedly induced the luciferase activity driven by the IRF-1 promoter and the TK heterologous promoter in all OSCC cells. These results indicate that IFNγ-induced STAT1-dependent transcriptional activity is intact in these OSCC cells and that activation of STAT1 by itself may be insufficient to mediate the antiproliferative effect of IFNγ. Recent studies have shown that IFNγ-activated STAT3 functions as a component of the STAT1-independent signaling pathway (35Qing Y. Stark G.R. J. Biol. Chem. 2004; 279: 41679-41685Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). To determine whether the differential antiproliferative effect of IFNγ is because of IFNγ-induced STAT3, we analyzed the levels of tyrosine-phosphorylated STAT3 (Tyr-705) in the OSCC cells by Western blotting (supplemental Fig. 1). Although constitutively active tyrosine-phosphorylated STAT3 was observed in all OSCC cells, IFNγ failed to further enhance the phosphorylated Tyr-705 STAT3. These results indicate that STAT3 is not involved in the differences observed in the antiproliferative effect of IFNγ on the different OSCC cells. IFNγ has been shown to activate the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, which mediates some biological activities of IFNγ (36Nguyen H. Ramana C.V. Bayes J. Stark G.R. J. Biol. Chem. 2001; 276: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). To examine whether IFNγ differentially affects the PI3K/Akt signaling pathway in HSC-2 and Ca9–22 cells, we measured the levels of the phosphorylated forms of Akt over time using Western blotting techniques (supplemental Fig. 2). Both HSC-2 and Ca9–22 cells constitutively expressed phosphorylated forms of Akt on Thr-308 and Ser-473. However, IFNγ failed to up-regulate these phosphorylated forms of Akt. Consistent with these results, phosphorylation of PDK1 (3-phosphoinositide-dependent protein kinase-1) (37Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 38Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (907) Google Scholar), an upstream kinase responsible for the phosphorylation of Akt Thr-308, was also unchanged in the cells treated with IFNγ. These results do not implicate the PI3K/Akt pathway in the observed differential sensitivity to IFNγ. To understand the underlying mechanism of the IFNγ-induced differential growth inhibition of OSCC cells, we next analyzed the cell-cycle distribution in response to IFNγ treatment using flow cytometry (Fig. 3). Quantitative analysis of the distribution of cells in the cell cycle indicates that treatment of HSC-2 cells with IFNγ blocks S-phase progression concomitant with a decrease in the relative proportion of cells in the G0/G1 phase (Fig. 3A). By contrast, Ca9–22 cells showed only a slight change in cell cycle distribution after IFNγ treatment. To confirm that the S-phase cell cycle arrest in HSC-2 cells correlated with the antiproliferative effect of IFNγ, we assessed the rate of DNA synthesis by measuring [3H]thymidine incorporation into DNA (Fig. 3B). A marked inhibition of [3H]thymidine incorporation was observed in HSC-2 cells after 48 h of IFNγ treatment. Because DNA replication and S-phase progression are regulated by CcnA2-associated kinase (39Girard F. Strausfeld U. Fernandez A. Lamb N.J. Cell. 1991; 67: 1169-1179Abstract Full Text PDF PubMed Scopus (742) Google Scholar), we analyzed the activity of this kinase using histone H1 as a substrate (Fig. 4A). Although CcnA2-associated kinase activity decreased in a time-dependent fashion in untreated cultures (lanes 1 and 3), treatment of HSC-2 cells with IFNγ markedly inhibited the kinase activity (lanes 2 and 4). By contrast, no inhibitory effect of IFNγ on the kinase activity was observed in Ca9–22 cells. These results indicate that IFNγ inhibits CcnA2-associated kinase activity in HSC-2 cells but not in Ca9–22 cells. CcnA2-associated kinase activity is regulated by Cdk inhibitors such as p21WAF1/Cip1 (8El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7869) Google Scholar, 9Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5201) Google Scholar, 40Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3141) Google Scholar), and IFNγ is known to up-regulate p21WAF1/Cip1 expression in a wide variety of cell types (3Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (724) Google Scholar, 4Sangfelt O. Erickson S. Einhorn S. Grandér D. 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• W2074443441 title "Mechanisms of Resistance to Interferon-γ-mediated Cell Growth Arrest in Human Oral Squamous Carcinoma Cells" @default.
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