Interferon-γ-Mediated Growth Regulation of Melanoma Cells: Involvement of STAT1-Dependent and STAT1-Independent Signals
2004; Elsevier BV; Volume: 122; Issue: 2 Linguagem: Inglês
10.1046/j.0022-202x.2004.22237.x
ISSN1523-1747
AutoresAnja‐Katrin Bosserhoff, Marcin Kortylewski, Waraporn Komyod, Maria-Elisabeth Kauffmann, Peter C. Heinrich, Iris Behrmann,
Tópico(s)interferon and immune responses
ResumoInterferon-γ, a known inhibitor of tumor cell growth, has been used in several protocols for the treatment of melanoma. We have studied the molecular events underlying interferon-γ-induced G0/G1 arrest in four metastatic melanoma cell lines with different responsiveness to interferon-γ. The growth arrest did not result from enhanced expression of cyclin-dependent kinase inhibitors p21 and p27. Instead, it correlated with downregulation of cyclin E and cyclin A and inhibition of their associated kinase activities. We show that interferon-γ-induced growth inhibition could be abrogated by overexpression of dominant negative STAT1 (signal transducer and activator of transcription 1) in the melanoma cell line A375, suggesting that STAT1 plays a crucial part for the anti-proliferative effect. Erythropoietin stimulation of a chimeric receptor led to a concentration-dependent STAT1 activation and concomitant growth arrest when it contained the STAT recruitment motif Y440 of the interferon-γ receptor 1. In contrast, dose–response studies for interferon-γ revealed a discrepancy between levels of STAT1 activation and the extent of growth inhibition; whereas STAT1 was activated by low doses of interferon-γ (10 U per mL), growth inhibitory effects were only visible with 100-fold higher concentrations. Our results suggest the presence of additional signals emanating from the interferon-γ receptor, which may counteract the anti-proliferative function of STAT1. Interferon-γ, a known inhibitor of tumor cell growth, has been used in several protocols for the treatment of melanoma. We have studied the molecular events underlying interferon-γ-induced G0/G1 arrest in four metastatic melanoma cell lines with different responsiveness to interferon-γ. The growth arrest did not result from enhanced expression of cyclin-dependent kinase inhibitors p21 and p27. Instead, it correlated with downregulation of cyclin E and cyclin A and inhibition of their associated kinase activities. We show that interferon-γ-induced growth inhibition could be abrogated by overexpression of dominant negative STAT1 (signal transducer and activator of transcription 1) in the melanoma cell line A375, suggesting that STAT1 plays a crucial part for the anti-proliferative effect. Erythropoietin stimulation of a chimeric receptor led to a concentration-dependent STAT1 activation and concomitant growth arrest when it contained the STAT recruitment motif Y440 of the interferon-γ receptor 1. In contrast, dose–response studies for interferon-γ revealed a discrepancy between levels of STAT1 activation and the extent of growth inhibition; whereas STAT1 was activated by low doses of interferon-γ (10 U per mL), growth inhibitory effects were only visible with 100-fold higher concentrations. Our results suggest the presence of additional signals emanating from the interferon-γ receptor, which may counteract the anti-proliferative function of STAT1. cyclin-dependent kinase erythropoietin electrophoretic mobility shift assay interferon-γ receptor signal transducer and activator of transcription; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide Interferons (IFN), initially identified as anti-viral agents, are implicated in a variety of biologic functions concerning anti-proliferative effects, induction of cell differentiation, and modulation of the immune response. They are divided into two classes: type I IFN (comprising IFN-α and IFN-β) and type II IFN (represented by IFN-γ). Each class is characterized by distinct intracellular signaling mechanisms. IFN-γ, produced by activated T lymphocytes and natural killer cells, exerts its pleiotropic effects via a receptor complex consisting of two chains, the ligand-specific IFNGR1 and IFNGR2, expressed on nearly all cell types (Stark et al., 1998Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. How cells respond to interferons.Annu Rev Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3375) Google Scholar). Binding of IFN-γ leads to the oligomerization of the receptor subunits and initiates signal transduction. Jak1 and Jak2 protein tyrosine kinases of the Janus family that are constitutively associated with IFNGR1 and IFNGR2 receptor subunits, respectively, become activated and in turn are thought to phosphorylate tyrosine residue 440 of the cytoplasmic part of IFNGR1 among other cellular substrates. The phosphotyrosine pY440 within the sequence Y440DKPH plays a major part in receptor activity as it serves as a docking site for the transcription factor STAT1 (signal transducer and activator of transcription 1). Subsequently, STAT1 also undergoes tyrosine phosphorylation, dissociates from the receptor, forms dimers, and translocates to the nucleus where it regulates transcription of target genes (Darnell, 1997Darnell Jr., J.E. STATs and gene regulation.Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3375) Google Scholar;Stark et al., 1998Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. How cells respond to interferons.Annu Rev Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3375) Google Scholar). In addition to the ubiquitous way of IFN-γ-responsive STAT1-mediated signaling, cell-type-specific activation of two other STAT factors was recently reported. Coactivation of STAT1 and STAT3 after IFN-γ stimulation has been described in adipocytes (Stephens et al., 1998Stephens J.M. Lumpkin S.J. Fishman J.B. Activation of signal transducers and activators of transcription 1 and 3 by leukemia inhibitory factor, oncostatin-M, and interferon-gamma in adipocytes.J Biol Chem. 1998; 273: 31408-31416Crossref PubMed Scopus (79) Google Scholar) and hematopoietic cells (Sato et al., 1997Sato T. Selleri C. Young N.S. Maciejewski J.P. Inhibition of interferon regulatory factor-1 expression results in predominance of cell growth stimulatory effects of interferon-gamma due to phosphorylation of Stat1 and Stat3.Blood. 1997; 90: 4749-4758Crossref PubMed Google Scholar). IFN-γ-induced monocyte or macrophage differentiation was shown to coincide with the activation of STAT5 but not STAT1 (Meinke et al., 1996Meinke A. Barahmand-Pour F. Wohrl S. Stoiber D. Decker T. Activation of different Stat5 isoforms contributes to cell-type-restricted signaling in response to interferons.Mol Cell Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar), which is also mediated by Y440 of the IFNGR1 (Woldman et al., 2001Woldman I. Varinou L. Ramsauer K. Rapp B. Decker T. The Stat1 binding motif of the interferon-gamma receptor is sufficient to mediate Stat5 activation and its repression by SOCS3.J Biol Chem. 2001; 276: 45722-45728Crossref PubMed Scopus (27) Google Scholar). Moreover, activation of several other signaling pathways has been described, including the extracellular signal-regulated kinase (Erk) pathway induced via protein tyrosine kinase Pyk2 (Takaoka et al., 1999Takaoka A. Tanaka N. Mitani Y. et al.Protein tyrosine kinase Pyk2 mediates the Jak-dependent activation of MAPK and Stat1 in IFN-gamma, but not IFN-alpha, signaling.EMBO J. 1999; 18: 2480-2488Crossref PubMed Scopus (127) Google Scholar) or via the serine/threonine kinase Raf-1 (David et al., 1995David M. Petricoin 3rd, E. Benjamin C. Pine R. Weber M.J. Larner A.C. Requirement for MAP kinase (ERK2) activity in interferon alpha- and interferon beta-stimulated gene expression through STAT proteins.Science. 1995; 269: 1721-1723Crossref PubMed Scopus (529) Google Scholar;Stancato et al., 1998Stancato L.F. Yu C.R. Petricoin 3rd, E.F. Larner A.C. Activation of Raf-1 by interferon gamma and oncostatin M requires expression of the Stat1 transcription factor.J Biol Chem. 1998; 273: 18701-18704Crossref PubMed Scopus (36) Google Scholar), the p38 mitogen-activated protein kinase (MAPK) pathway and the recently reported C3G/Rap1 pathway, a small G protein signaling cascade (Alsayed et al., 2000Alsayed Y. Uddin S. Ahmad S. Majchrzak B. Druker B.J. Fish E.N. Platanias L.C. IFN-gamma activates the C3G/Rap1 signaling pathway.J Immunol. 2000; 164: 1800-1806Crossref PubMed Scopus (63) Google Scholar). Both types of IFN are known for strong anti-proliferative and immunomodulatory effects on melanoma cells. IFN-γ was also reported to induce expression of ISGF3 components, including STAT1, which significantly enhances responsiveness to IFN-α/β of primed cells (Carson, 1998Carson W.E. Interferon-alpha-induced activation of signal transducer and activator of transcription proteins in malignant melanoma.Clin Cancer Res. 1998; 4: 2219-2228PubMed Google Scholar;Wong et al., 1998Wong L.H. Hatzinisiriou I. Devenish R.J. Ralph S.J. IFN-gamma priming up-regulates IFN-stimulated gene factor 3 (ISGF3) components, augmenting responsiveness of IFN-resistant melanoma cells to type I IFN.J Immunol. 1998; 160: 5475-5484PubMed Google Scholar). As the downstream mechanisms of IFN-γ-mediated effects on growth and survival of human melanoma cells are largely unknown we investigated the molecular events underlying the anti-proliferative effect of IFN-γ on human melanoma cells of metastatic origin. Cell cycle analysis revealed that the growth arrest does not result from accumulation of cyclin-dependent kinase inhibitors. Rather, growth inhibition was correlated with the complex downregulation of several proteins necessary for cell cycle progression, primarily cyclin E, cyclin A, and the kinases cdk4 and cdk2. In this study, we demonstrate that the activity of IFN-γ on melanoma cells is STAT1 dependent. Most interestingly, under conditions of comparable STAT1 activation, a chimeric receptor containing the isolated tyrosine motif Y440 is much more effective at mediating growth inhibition than the endogenous IFN-γ receptor. Our results imply that divergent signals may emanate from the IFN-γ receptor, which could contribute to IFN-γ resistance. To study the anti-proliferative effect of IFN-γ on human melanoma cells, four cell lines derived from metastatic tumor stages were chosen. After 4 d of incubation in the presence of IFN-γ, a concentration-dependent growth inhibition was observed Figure 1a. In three of the tested cell lines this inhibition was evident when concentrations of IFN-γ exceeded 1000 U per mL. A375 cells exhibited the greatest inhibition in growth, whereas the effect on WM239 and Mel-Im cells was more moderate. In contrast, WM9 cells were relatively resistant to IFN-γ, reacting only at doses greater than 10,000 U per mL. These findings were confirmed by cell cycle analyses. After 2 d of treatment with IFN-γ, the number of A375, WM239, and Mel-Im cells in G0/G1 phase was increased, whereas WM9 cells remained unaffected Figure 1b. This G0/G1 arrest in A375 and WM239 cells was paralleled by a marginal increase in apoptotic cells; however, the percentage of counted sub-G1 events did not exceed 4% of total counts (data not shown). A known mechanism underlying the IFN-γ-induced G0/G1 arrest in many cells is the upregulation of cyclin-dependent kinase inhibitors (Yamada et al., 1995Yamada H. Ochi K. Nakada S. Takahara S. Nemoto T. Sekikawa T. Horiguchi-Yamada J. Interferon modulates the messenger RNA of G1-controlling genes to suppress the G1-to-S transition in Daudi cells.Mol Cell Biochem. 1995; 152: 149-158Crossref PubMed Scopus (25) Google Scholar;Chin et al., 1996Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1.Science. 1996; 272: 719-722Crossref PubMed Scopus (728) Google Scholar;Mandal et al., 1998Mandal M. Bandyopadhyay D. Goepfert T.M. Kumar R. Interferon-induces expression of cyclin-dependent kinase-inhibitors p21WAF1 and p27Kip1 that prevent activation of cyclin-dependent kinase by CDK-activating kinase (CAK).Oncogene. 1998; 16: 217-225Crossref PubMed Scopus (93) Google Scholar). None of the cell lines whose growth was inhibited by IFN-γ (A375, Mel-Im, WM239), however, showed an upregulation of p21/Waf-1/Cip-1, p27/Kip-1, or p18/INK4c. In fact, a decrease in the protein level of p21 and, to a lesser extent, of p27 and p18 was observed Figure 2. It is also noteworthy that the basal levels of cyclin-dependent kinase inhibitor, particularly p21, were not consistent in the cell lines studied Figure 2 and did not correlate with their normal growth rate in cell culture (not shown). Growth arrest can also be mediated by the downregulation of positive regulators of the cell cycle. Therefore, we analyzed the protein expression of cyclins D1, E, and A, and the cyclin-dependent kinases cdk4 and cdk2. Cyclin D1 expression did not significantly change after stimulation with IFN-γ in any of the melanoma cell lines Figure 2. Protein levels of cyclin E or cyclin A, however, were downregulated in all melanoma cell lines after IFN-γ treatment with the exception of the IFN-γ-resistant WM9 cells. We also observed a reduction in the expression of the cyclin-dependent kinases cdk4 and cdk2 in the IFN-γ-sensitive A375, Mel-Im, and WM239 cells but not in the IFN-γ-resistant WM9 cells. These findings suggest that IFN-γ inhibits growth of melanoma cells by a mechanism that does not involve cyclin-dependent kinase inhibitor upregulation, but rather entails a complex downregulation of G1/S cyclins and cyclin-dependent kinases. We next examined STAT1 expression in the melanoma cell lines, because reduced responsiveness to IFN may result from a lack of STAT1 expression (Wong et al., 1997Wong L.H. Krauer K.G. Hatzinisiriou I. et al.Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma.J Biol Chem. 1997; 272: 28779-28785Crossref PubMed Scopus (216) Google Scholar). The basal protein levels of STAT1 in untreated cells were comparable and did not correlate with their responsiveness to IFN-γ Figure 3a. Moreover, all the cell lines, including WM9 cells, exhibited strong upregulation of STAT1 protein expression within 24 h after stimulation, whereas Erk levels remained unchanged. In all the cell lines tested, including WM9 cells, IFN-γ induced strong phosphorylation of STAT1 after 15 min of stimulation. STAT1 phosphorylation was still pronounced in A375 and Mel-Im cells after 24 h of stimulation, whereas it was considerably reduced in WM239 and WM9 cells Figure 3a. STAT1 phosphorylation was paralleled by the appearance of corresponding DNA-binding activities in EMSA analysis using the SIE-oligo Figure 3b. This probe binds both STAT1 and STAT3. The presence of anti-STAT1 antibodies but not of anti-STAT3 antibodies significantly reduced the binding activity in nuclear extracts of IFN-γ-treated A375-cells Figure 3c. Moreover, the mobility of the IFN-γ-induced DNA-binding activity was clearly different from the STAT3 signals observed upon OSM stimulation Figure 3c. To study the role of STAT1 in IFN-γ induced growth inhibition we employed tripartite chimeric receptor constructs (Gerhartz et al., 1996Gerhartz C. Heesel B. Sasse J. et al.Differential activation of acute phase response factor/STAT3 and STAT1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. I. Definition of a novel phosphotyrosine motif mediating STAT1 activation.J Biol Chem. 1996; 271: 12991-12998Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar;Strobl et al., 2001Strobl B. Arulampalam V. Is'harc H. et al.A completely foreign receptor can mediate an interferon-gamma-like response.EMBO J. 2001; 20: 5431-5442Crossref PubMed Scopus (32) Google Scholar). These constructs contain the extracellular region of the mouse erythropoietin receptor (EPOR) fused to the transmembrane and truncated membrane-proximal box1/box2 region (i.e., the Jak binding region) of the interleukin-6 signal transducer gp130, without or with the seven amino acid STAT1 recruitment motif (Y440) from the IFNGR1 (EGΔB and EGΔBY440, respectively, Figure 4a, b, left upper panels). Thus, as the endogenous IFN-γ receptor, EGΔBY440 has the ability to activate STAT1. Except for the STAT recruitment motif, however, both receptors are different in sequence. Stably transfected A375 clones with similar levels of surface receptor expression were isolated (Figure 4a, b, left lower panels). As previously reported (Kortylewski et al., 1999Kortylewski M. Heinrich P.C. Mackiewicz A. et al.Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1.Oncogene. 1999; 18: 3742-3753Crossref PubMed Scopus (124) Google Scholar), clones expressing EGΔB did not exhibit STAT activation after stimulation with EPO (Figure 4a, right upper panel) and proliferated unrestrained (Figure 4a, right lower panel). In contrast, clones expressing EGΔBY440 (Figure 4b, left panels) demonstrated a strong EPO-induced STAT1 activation (Figure 4b, right upper panel). In addition, growth of these clones was efficiently growth inhibited by EPO (Figure 4b, right lower panel) as a result of a G0/G1 arrest (data not shown). Like IFN-γ, EPO decreased the expression of cyclin E, cyclin A and, to a lesser extent, of cdk4 and cdk2 within 24 h of treatment in EGΔBY440 cells Figure 5. Whereas p21 and p18 protein levels remained relatively stable, levels of p27 decreased. The downregulation of cyclin E and cyclin A, together with their kinase partner cdk2, was also evident by reduced levels of kinase activity of the respective cyclin/cdk complexes (data not shown). We conclude that the presence of the STAT1 recruitment module Y440 within the chimeric receptor was sufficient to mediate an IFN-γ-like growth arrest of A375 cells. In a separate attempt to elucidate the role of STAT1 in IFN-γ-induced growth inhibition, we examined stable A375 transfectants expressing STAT1F, a dominant negative form of STAT1 in which the tyrosine residue Y701, necessary for STAT1 phosphorylation and dimerization, is replaced by phenylalanine (Nakajima et al., 1996Nakajima K. Yamanaka Y. Nakae K. et al.A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells.EMBO J. 1996; 15: 3651-3658Crossref PubMed Scopus (521) Google Scholar;Kortylewski et al., 1999Kortylewski M. Heinrich P.C. Mackiewicz A. et al.Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1.Oncogene. 1999; 18: 3742-3753Crossref PubMed Scopus (124) Google Scholar). As presented in Figure 6, A375 cells overexpressing STAT1F were almost completely resistant to IFN-γ when compared with mock transfectants, suggesting that STAT1 is a crucial mediator for the growth arrest elicited by IFN-γ. While examining the responsiveness of the stable transfectants expressing EGΔBY440 in more detail, we observed a striking difference between the signal transduction of IFN-γ (via the endogenous receptor) and EPO (via the transfected receptor): stimulation of EGΔBY440 transfectants with increasing doses of EPO induced DNA-binding activity of STAT1, even at concentrations as low as 0.1 U per mL (Figure 7a, right panels). This increase in STAT1 DNA binding activity was paralleled by a proportionally enhanced inhibition in the growth of transfectants (Figure 7a, lower panel on the right). Similar to the parental A375 cells Figure 1, IFN-γ-induced growth inhibition of EGΔBY440 transfectants was detectable at concentrations as low as 1000 U per mL. Gel shift assays indicated that, although the activation of STAT1 by IFN-γ was evident at doses as low as 10 U per mL and was near maximal at 100 U per mL, this dose was too low to affect cell growth (Figure 7a, left panels). The induction of STAT1 protein expression by both EPO and IFN-γ paralleled the respective dose–response of STAT1 activity to these factors Figure 7b. To study further this apparent discrepancy between IFN-γ-induced STAT1 activity and growth inhibition, we examined in detail the kinetics of STAT1 activation with respect to its tyrosine and serine phosphorylation and DNA binding. Doses of IFN-γ and EPO known to induce similar STAT1 activity but affecting cell growth with minimal (1000 U per mL IFN-γ) or maximal (10 U per mL EPO) efficiency were compared. The short-term kinetics of STAT1 tyrosine and serine phosphorylation were comparable for both stimuli Figure 7c. Two longer-term assays also showed no differences between IFN-γ and EPO stimulation: addition of IFN-γ and EPO resulted in comparable levels of GAS-element-mediated reporter gene activity after 18 h; stimulation of gene activity was not additive, as the addition of both IFN-γ and EPO did not further increase gene activity Figure 7d. Moreover, both agents induced similar STAT1 DNA-binding activities at time points from 14 to 48 h Figure 7e. Therefore, factors other than STAT1 are likely to account for the differential response of EGΔBY440 cells to the two cytokines. No difference could be observed for STAT5 and IRF-1 as monitored by EMSA using probes for the respective transcription factors Figure 7e. The time course of STAT5 activation paralleled the observed STAT1 activation, whereas DNA binding of IRF-1 was prominent 14 h after stimulation and slowly declined afterwards. C/EBPβ and c-myc, two other transcription factors known to be induced by IFN-γ in several cell types, were slightly downregulated by both stimuli after 24 h Figure 7f, whereas STAT1 was dramatically upregulated. The growth inhibitory effect of IFN-γ on melanoma cells has long been known (Brown et al., 1987Brown T.J. Lioubin M.N. Marquardt H. Purification and characterization of cytostatic lymphokines produced by activated human T lymphocytes. Synergistic antiproliferative activity of transforming growth factor beta 1, interferon-gamma, and oncostatin M for human melanoma cells.J Immunol. 1987; 139: 2977-2983PubMed Google Scholar;Garbe and Krasagakis, 1993Garbe C. Krasagakis K. Effects of interferons and cytokines on melanoma cells.J Invest Dermatol. 1993; 100: 239S-244SAbstract Full Text PDF PubMed Google Scholar). IFN-γ has been used in adjuvant cytokine therapy studies (Agarwala and Kirkwood, 1996Agarwala S.S. Kirkwood J.M. Interferons in melanoma.Curr Opin Oncol. 1996; 8: 167-174Crossref PubMed Scopus (75) Google Scholar) and in gene therapeutic approaches in mouse models (Yu and Thomas-Tikhonenko, 2001Yu D. Thomas-Tikhonenko A. Intratumoral delivery of an interferon gamma retrovirus-producing cells inhibits growth of a murine melanoma by a non-immune mechanism.Cancer Lett. 2001; 173: 145-154Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) as well as in clinical trials (Nemunaitis et al., 1999Nemunaitis J. Fong T. Robbins J.M. et al.Phase I trial of interferon-gamma (IFN-gamma) retroviral vector administered intratumorally to patients with metastatic melanoma.Cancer Gene Ther. 1999; 6: 322-330Crossref PubMed Scopus (50) Google Scholar). The mechanism of IFN-γ-induced growth arrest of melanoma cells, however, has not been examined in greater detail. This study indicated that IFN-γ leads to G0/G1 growth arrest in A375, WM239, and Mel-Im cells, whereas WM9 cells are resistant to IFN-γ. This growth arrest correlated with lower levels of certain cyclins (cyclin A and cyclin E) and cyclin-dependent kinases (cdk2 and cdk4). Levels of cyclin-dependent kinase inhibitors, however, were not affected or even drastically decreased (e.g., p21 in Mel-Im and WM239 cells, Figure 2). This finding was somewhat unexpected as IFN-γ induces p21 in a number of cell types, including multiple myeloma (Urashima et al., 1997Urashima M. Teoh G. Chauhan D. et al.Interleukin-6 overcomes p21WAF1 upregulation and G1 growth arrest induced by dexamethasone and interferon-gamma in multiple myeloma cells.Blood. 1997; 90: 279-289Crossref PubMed Google Scholar), glioblastoma (Kominsky et al., 1998Kominsky S. Johnson H.M. Bryan G. Tanabe T. Hobeika A.C. Subramaniam P.S. Torres B. IFNgamma inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin dependent kinase inhibitor p21WAF1/CIP1.Oncogene. 1998; 17: 2973-2979Crossref PubMed Scopus (62) Google Scholar), colon adenocarcinoma (Hobeika et al., 1999Hobeika A.C. Etienne W. Torres B.A. Johnson H.M. Subramaniam P.S. IFN-gamma induction of p21 (WAF1) is required for cell cycle inhibition and suppression of apoptosis.J Interferon Cytokine Res. 1999; 19: 1351-1361Crossref PubMed Scopus (58) Google Scholar), and breast cancer cells (Gooch et al., 2000Gooch J.L. Herrera R.E. Yee D. The role of p21 in interferon gamma-mediated growth inhibition of human breast cancer cells.Cell Growth Differ. 2000; 11: 335-342PubMed Google Scholar), as well as in normal bone marrow macrophages (Xaus et al., 1999Xaus J. Cardo M. Valledor A.F. Soler C. Lloberas J. Celada A. Interferon gamma induces the expression of p21waf-1 and arrests macrophage cell cycle, preventing induction of apoptosis.Immunity. 1999; 11: 103-113Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Upregulation could be attributed to direct STAT1-mediated activation of the p21 gene promoter (Chin et al., 1996Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1.Science. 1996; 272: 719-722Crossref PubMed Scopus (728) Google Scholar). An upregulation of p27 by IFN-γ was observed together with an increase in protein levels of other inhibitors, such as p21 (Mandal et al., 1998Mandal M. Bandyopadhyay D. Goepfert T.M. Kumar R. Interferon-induces expression of cyclin-dependent kinase-inhibitors p21WAF1 and p27Kip1 that prevent activation of cyclin-dependent kinase by CDK-activating kinase (CAK).Oncogene. 1998; 16: 217-225Crossref PubMed Scopus (93) Google Scholar), p18/INK4c (Yamada et al., 1995Yamada H. Ochi K. Nakada S. Takahara S. Nemoto T. Sekikawa T. Horiguchi-Yamada J. Interferon modulates the messenger RNA of G1-controlling genes to suppress the G1-to-S transition in Daudi cells.Mol Cell Biochem. 1995; 152: 149-158Crossref PubMed Scopus (25) Google Scholar), or with a concomitant rapid downregulation of positive cell cycle regulators, such as cyclin A, cdk2, cdc2, and c-myc (Harvat and Jetten, 1996Harvat B.L. Jetten A.M. Gamma-interferon induces an irreversible growth arrest in mid-G1 in mammary epithelial cells which correlates with a block in hyperphosphorylation of retinoblastoma.Cell Growth Differ. 1996; 7: 289-300PubMed Google Scholar;Harvat et al., 1997Harvat B.L. Seth P. Jetten A.M. The role of p27Kip1 in gamma interferon-mediated growth arrest of mammary epithelial cells and related defects in mammary carcinoma cells.Oncogene. 1997; 14: 2111-2122Crossref PubMed Scopus (77) Google Scholar). It should be noted that p27 and p21 inhibitors are upregulated after treatment with interleukin-6 or upon γ-irradiation at least in A375 and WM239 cells (Kortylewski et al., 1999Kortylewski M. Heinrich P.C. Mackiewicz A. et al.Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1.Oncogene. 1999; 18: 3742-3753Crossref PubMed Scopus (124) Google Scholar), and data not shown). IFN-γ-mediated cell cycle arrest without concomitant upregulation of p21 and p27, however, is not without precedent: in human mesothelioma cells IFN-γ appears to be able to induce a G0/G1 block independent of p21 and p27 inhibitors, with a concurrent reduction of cyclin A expression and its associated kinase activity (Vivo et al., 2001Vivo C. Levy F. Pilatte Y. et al.Control of cell cycle progression in human mesothelioma cells treated with gamma interferon.Oncogene. 2001; 20: 1085-1093Crossref PubMed Scopus (28) Google Scholar). Cytokine resistance of melanoma cells is often correlated with diminished Jak/STAT signaling (Wong et al., 1997Wong L.H. Krauer K.G. Hatzinisiriou I. et al.Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma.J Biol Chem. 1997; 272: 28779-28785Crossref PubMed Scopus (216) Google Scholar,Wong et al., 1998Wong L.H. Hatzinisiriou I. Devenish R.J. Ralph S.J. IFN-gamma priming up-regulates IFN-stimulated gene factor 3 (ISGF3) components, augmenting responsiveness of IFN-resistant melanoma cells to type I IFN.J Immunol. 1998; 160: 5475-5484PubMed Google Scholar;Pansky et al., 2000Pansky A. Hildebrand P. Fasler-Kan E. Baselgia L. Ketterer S. Beglinger C. Heim M.H. Defective Jak-STAT signal transduction pathway in melanoma cells resistant to growth inhibition by interferon-alpha.Int J Cancer. 2000; 85: 720-725Crossref PubMed Scopus (97) Google Scholar;Böhm et al., 2001Böhm M. Schulte U. Funk J.O. et al.Interleukin-6-resistant melanoma cells exhibit reduced activation of STAT3 and lack of inhibition of cyclin E-associated kinase activity.J Invest Dermatol. 2001; 117: 132-140Crossref PubMed Google Scholar). Wong et al, 1997 proposed that a deficiency in ISGF3 components may be responsible for the resistance of melanoma cells to IFN. Indeed, reduced levels of STAT1 were found in certain IFN-resistant cells (Wong et al., 1997Wong L.H. Krauer K.G. Hatzinisiriou I. et al.Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma.J Biol Chem. 1997; 272: 28779-28785Crossref PubMed Scopus (216) Google Scholar;Pansky et al., 2000Pansky A. Hildebrand P. Fasler-Kan E. Baselgia L. Ketterer S. Beglinger C. Heim M.H. Defective Jak-STAT signal transduction pathway in melanoma cells resistant to growth inhibition by interferon-alpha.Int J Cancer. 2000; 85: 720-725Crossref PubMed Scopus (97) Google Scholar), and the restoration of normal STAT1 levels by IFN-γ induction or by overexpression or STAT1 or IRF-1 is sufficient to augment the responsiveness to IFN (Wong et al., 1997Wong L.H. Krauer K.G. Hatzinisiriou
Referência(s)