Impaired class II transactivator expression in mice lacking interferon regulatory factor-2.

Class II transactivator (CIITA) is required for both constitutive and inducible expression of MHC class II genes. IFN-gamma induced expression of CIITA in various cell types is directed by CIITA type IV promoter. The two transactivators, STAT1 and IRF-1, mediate the IFN-gamma activation of the type IV promoter by binding to the GAS and IRF-E of the promoter, respectively. In addition to IRF-1, IRF-2, another member of the IRF family, also activates the human CIITA type IV promoter, and IRF-2 cooperates with IRF-1 to activate the promoter in transient transfection assays. IRF-1 and IRF-2 can co-occupy the IRF-E of the human CIITA type IV promoter. To understand the effect of loss of IRF-2 on the endogenous CIITA expression, we assayed for CIITA expression in IRF-2 knock-out mice. Both basal and IFN-gamma induced CIITA expression were reduced in IRF-2 knock-out mice. At least half of the amount of inducible CIITA mRNA depends on IRF-2. The reduction of IFN-gamma induced CIITA mRNA in IRF-2 knock-out mice was due to the reduction of the type IV CIITA mRNA induction. The reduction of basal CIITA mRNA was apparently due to the reduction of CIITA mRNA originating from other promoters. These data indicate that IRF-2, like IRF-1, plays a critical role in the regulation of the endogenous CIITA gene. The implications in understanding the previously described phenotypes of IRF-2 defective mice are discussed.     

The interferon-alpha regulation of interferon regulatory factor 1 (IRF-1) and IRF-2 has therapeutic implications in carcinoid tumors.

Background:Interferon regulatory factor 1 (IRF-1) has beendemonstrated to possess antiproliferative and tumor suppressor functions, onthe contrary, IRF-2 has been suggested to induce oncogenetic effect in somecell lines, but not evaluated in tumor patients. Patients and methods:In 35 carcinoid tumor patients, expressionsof IRF-1 and IRF-2 were investigated by immunohistochemistry and their valueswere analyzed with clinical treatment response. In carcinoid tumor cell line,Bon1, effects of IFN- on the expression of both IRF-1 and IRF-2 mRNAs andproteins were determined by Northern blot, RNase protection assays and Westernblot analysis. Results:IFN- up-regulated the expression of IRF-1 and IRF-2both in vivoand in vitro. In carcinoid tumors, IFN-treatment led to a significant increase in the expression of IRF-1 (P< 0.001) and IRF-2 (P < 0.001). Moreover, the IRFs inductionwas correlated with the clinical response of IFN- treatment, althoughtheir baseline values were not predictive. In addition, expressions of IRF-1and IRF-2 were significantly correlated with the p68kinaseexpression (P = 0.032 and P = 0.0176, respectively) and theexpression of IRF-1 protein was positively correlated with that of IRF-2(r = 0.671,P = 0.0001) tested in the same specimens. Conclusions:IRF-1 as well as IRF-2 have therapeutic implicationsin carcinoid tumors during treatment with interferon- and IRFs inductionmight be used as indicators of response to treatment with interferon-.     

Growth suppression of the hepatocellular carcinoma cell line Hepa1-6 by an activatable interferon regulatory factor-1 in mice

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis and few therapeutic options. The aim of the study was to evaluate the potential of IFN regulatory factor-1 (IRF-1) for cytokine gene therapy of HCC using an IRF-1/human estrogen receptor fusion protein (IRF-1hER), which is reversibly activatable by beta-estradiol (E2). IRF-1hER stably expressing murine Hepa1-6 HCC cells (HepaIRF-1hER) were characterized by lowMHC 1, highCD54, and lack of MHC II, CD80, and CD86 expression. Activation of HepaIRF-1hER cells induced a highMHC I, lowMHC II, and highCD54 phenotype. Furthermore, they were characterized by IFN-beta secretion, decreased anchorage-independent growth in a soft agar assay, and diminished cell growth. Tumor growth in E2-treated syngeneic C57L/J mice, but not in E2-untreated mice, was suppressed. These E2-treated mice were protected against rechallenge with HepaIRF-1hER and wild-type Hepa1-6 tumors even in the absence of E2, suggesting induction of tumor specific immunity. In fact, significant CTL activity against Hepa1-6 tumors and the endogenously expressed HCC-specific self antigen alpha-fetoprotein was observed. Antitumoral effects, however, were only partially dependent on both CD4+ and CD8+ T cells. IRF-1 treatment of mice bearing HepaIRF-1hER tumors resulted in growth arrest of tumors, and a significant survival benefit was observed in comparison to E2-untreated mice. In conclusion, our data demonstrate that IRF-1 suppresses HCC growth through both a direct antitumor growth effect and enhanced immune cell recognition of the tumor and is a promising candidate for gene therapy of HCC.     

Involvement of interferon regulatory factor 1 and S100C/A11 in growth inhibition by transforming growth factor beta 1 in human hepatocellular carcinoma cells

Growth inhibition by transforming growth factor (TGF)-beta 1 has been attributed to the induction of cyclin-dependent kinase inhibitors, among which p21/Waf1 plays a major role in many biological contexts. In the present study, two new intracellular mediators for the induction of p21/Waf1 by TGF-beta 1 were identified in a human hepatocellular carcinoma cell line (JHH-5) expressing mutant-type p53. After addition of TGF-beta 1 to JHH-5 cells, a marked increase of the p21/Waf1 expression preceded the inhibition of DNA synthesis. Expression of IFN regulatory factor (IRF)-1, a known transacting factor for p21/Waf1 promoter, was elevated just before or in parallel with the increase of p21/Waf1. Transduction of antisense IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-beta 1 and partially released the cells from the growth arrest by TGF-beta 1. Expression of S100C/A11, a member of the Ca(2+)-binding S100 protein family, also markedly increased after addition of TGF-beta 1. S100C/A11 protein was translocated to and accumulated in nuclei of TGF-beta 1-treated JHH-5 cells, where p21/Waf1 was concomitantly accumulated. When a recombinant S100C/A11 protein was introduced into nuclei of JHH-5 cells, DNA synthesis was markedly inhibited in a dose-dependent manner in the absence of TGF-beta 1. Prior transfection of p21/Waf1-targeted small interfering RNA efficiently blocked decrease of DNA synthesis in JHH-5 cells caused by TAT-S100C/A11 or TGF-beta 1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that IRF-1 and S100C/A11 mediate growth inhibition by TGF-beta 1 via induction of p21/Waf1.     

Forced expression of the interferon regulatory factor 2 oncoprotein causes polyploidy and cell death in FDC-P1 myeloid hematopoietic progenitor cells

The IFN regulatory factor-2 (IRF-2) oncoprotein controls the cell cycle-dependent expression of histone H4 genes during S phase and may function as a component of an E2F-independent mechanism to regulate cell growth. To investigate the role of IRF-2 in control of cell proliferation, we have constructed a stable FDC-P1 cell line (F2) in which expression of IRF-2 is doxycycline (DOX)-inducible, and a control cell line (F0). Both the F2 and F0 cell lines were synchronized in the G1 phase by isoleucine deprivation, and IRF-2 was induced by DOX on release of cells from the cell cycle block. Flow cytometric analyses indicated that forced expression of IRF-2 has limited effects on cell cycle progression before the first mitosis. However, continued cell growth in the presence of elevated IRF-2 levels results in polyploidy (>4n) or genomic disintegration (<2n) and cell death. Western blot analyses revealed that the levels of the cell cycle regulatory proteins cyclin B1 and the cyclin-dependent kinase (CDK)-inhibitory protein p27 are selectively increased. These changes occur concomitant with a significant elevation in the levels of the FAS-L protein, which is the ligand of the FAS (Apo1/CD95) receptor. We also found a subtle change in the ratio of the apoptosis-promoting Bax protein and the antiapoptotic Bcl-2 protein. Hence, IRF-2 induces a cell death response involving the Fas/FasL apoptotic pathway in FDC-P1 cells. Our data suggest that the IRF-2 oncoprotein regulates a critical cell cycle checkpoint that controls progression through G2 and mitosis in FDC-P1 hematopoietic progenitor cells.     

The ambiguous role of interferon regulatory factor-1 (IRF-1) immunoexpression in myelodysplastic syndrome

Recent investigations postulate a participation of the interferon regulatory factor-1 (IRF-1) in the development of myelodysplasia (MDS) and in the pathogenesis of autoimmune manifestations (AIMs) in patients with this disease. The aim of this prospective study was to compare the IRF-1 immunoexpression in MDS patients with or without AIMs and to investigate its prognostic relevance. Fifty consecutive MDS patients entered this prospective study. There was no difference in overall survival between patients with or without autoimmune manifestations. In a multivariate Cox regression “IRF-1 expression in immature myeloid cells”, Hb, and the IPSS risk group stratification were independent prognostic parameters. Bootstrap resampling confirmed these data. In a multivariate logistic regression older patients with, higher platelet count and increased IRF-1 expression had a higher risk to develop autoimmune-like phenomena. Thus our study shows that IRF-1 plays an ambiguous role in MDS patients. Whereas high levels of IRF-1 in myeloid cells are a favorable prognostic factor for overall survival, they increase the probability of the manifestation of autoimmune phenomena, with a diminished quality of life.