Epigenetic disruption of interferon-gamma response through silencing the tumor suppressor interferon regulatory factor 8 in nasopharyngeal, esophageal and multiple other carcinomas.

16q24 is frequently deleted in multiple tumors including cancers of nasopharynx, esophagus, breast, prostate and liver. By array comparative genomic hybridization (aCGH), we refined a 16q24 hemizygous deletion in nasopharyngeal carcinoma (NPC) cell lines. Semi-quantitative RT-PCR analysis revealed interferon regulatory factor 8 (IRF8) as the only downregulated gene within this deletion. IRF8 belongs to a family of interferon (IFN) regulatory factors that modulate various important physiologic processes including host defense, cell growth and differentiation and immune regulation. In contrast to the broad expression of IRF8 in normal adult and fetal tissues, transcriptional silencing and promoter methylation of IRF8 were frequently detected in multiple carcinoma (except for hepatocellular) cell lines (100% in NPC, 88% in esophageal and 18-78% in other carcinoma cell lines) and in a large collection of primary carcinomas (78% in NPC, 36-71% in other carcinomas). Methylation of the IRF8 promoter led to the disruption of its response to IFN-gamma stimulation. Pharmacological and genetic demethylation could restore IRF8 expression, indicating a direct epigenetic mechanism. Ectopic expression of IRF8 in tumor cells lacking its expression strongly inhibited their clonogenicity, confirming its tumor suppressor function. Thus, IRF8 was identified as a functional tumor suppressor, which is frequently silenced by epigenetic mechanism in multiple carcinomas.

New isoforms of VEGF are translated from alternative initiation CUG codons located in its 5'UTR.

Vascular endothelial growth factor (VEGF) has a central role in normal as well as in tumor angiogenesis. As such, VEGF is subjected to multi-level regulation at the transcriptional, post-transcriptional, translational, and post-translational levels to ensure proper expression during embryogenesis and adulthood. Its mRNA contains an exceptionally long (1038 bp) 5' untranslated region (5'UTR), which has a role in transcriptional as well as translational regulation of VEGF expression. In this communication, we provide new evidence showing that an open reading frame (ORF) present in the 5'UTR encodes for new putative isoforms of VEGF due to alternative translational initiation from CUG codons. Like VEGF, the translation of the new isoforms is not sensitive to stress signals such as anoxia. Most likely, these isoforms either possess new capabilities, which are different from the activity of the classical VEGF isoforms, or affect the efficiency and capacity of translational initiation from the canonical AUG codon.

Interplay between repressing and activating domains defines the transcriptional activity of IRF-1.

Interferon regulatory factor-1 (IRF-1) is a transcriptional activator with weak activation capacity. By defining the transcriptional activation domain of IRF-1 we identified two activator fragments located between amino acids 185 and 256 functioning in an additive manner. Another fragment of IRF-1, which has no activator function alone, acts as a strong enhancer element of these activator sequences. This enhancer element resides between the activator domains and the C-terminus. In addition, we identified a novel type of inhibitory domain in the N-terminal 60 amino acids of IRF-1 which strongly inhibits its transcriptional activity. Because this fragment is conserved in all interferon regulatory factors, we found similar repression effects in the corresponding fragments in IRF-2, IRF-3 and interferon consensus sequence binding protein (ICSBP/IRF-8). Interestingly, the corresponding sequence in p48/IRF-9 is divergent, so that it does not show this inhibitory activity. A five-amino-acid sequence distinguishes the p48/IRF-9 N-terminus from the homologous parts in other interferon regulatory factors containing the repressing function. Replacing the diverged amino acids in IRF-1 with the corresponding sequence of p48/IRF-9 resulted in a loss of inhibitory activity within IRF-1. The opposing activities within interferon regulatory factors may contribute to balanced or tuned regulation of gene activation, depending on the promoter context.

Cloning of an interferon regulatory factor 2 isoform with different regulatory ability.

Interferons (IFNs) are a family of multifunctional proteins involved in immune activation, regulation of cell growth and antiviral response. They exert their functions by induction of several IFN-stimulated genes, including IFN regulatory factors (IRFs), a family of transcriptional regulators. One of these factors, IRF-2, was initially cloned as an antagonistic counterpart to IRF-1 with oncogenic potential. Here we describe a second isoform of IRF-2, termed IRF-2s, cloned from human and murine cells. This isoform lacks two amino acids located C-terminal of the DNA-binding domain, which is conserved in all IRF family members, leading to a change in the predicted secondary structure. Both isoforms have similar binding affinities to known target sequences in electrophoretic mobility shift assays. Using reporter gene constructs with the type IV promoter region of the MHC class II transactivator (CIITA), which is the essential factor for IFN-gamma-induced MHC class II expression, we show that the short isoform IRF-2s exhibits a weaker activation ability compared to IRF-2. Thus, our data present the first evidence of two IRF-2 isoforms with different regulatory ability.

Interaction between interferon consensus sequence-binding protein and COP9/signalosome subunit CSN2 (Trip15). A possible link between interferon regulatory factor signaling and the COP9/signalosome.

Interferon consensus sequence-binding protein (ICSBP) is a member of the interferon regulatory factors (IRF) that has a pivotal role in mediating resistance to pathogenic infections in mice and in promoting the differentiation of myeloid cells. ICSBP exerts some of its transcriptional activities via association with other factors that enable its binding to a variety of promoters containing DNA composite elements. These interactions are mediated through a specific COOH-terminal domain termed IAD (IRF association domain). To gain a broader insight of the capacity of ICSBP to interact with other factors, yeast two-hybrid screens were performed using ICSBP-IAD as a bait against a B-cell cDNA library. Trip15 was identified as a specific interacting factor with ICSBP in yeast cells, which was also confirmed by in vitro glutathione S-transferase pull-down assays and by coimmunoprecipitation studies in COS7 cells. Trip15 was recently identified as a component of the COP9/signalosome (CSN) complex composed of eight evolutionary conserved subunits and thus termed CSN2. This complex has a role in cell-signaling processes, which is manifested by its associated novel kinase activity and by the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. We show that in vitro association of ICSBP with the CSN leads to phosphorylation of ICSBP at a unique serine residue within its IAD. The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity of ICSBP exerted on IRF-1. This suggests that the CSN has a role in integrating incoming signals that affect the transcriptional activity of ICSBP.

Transfer of antibodies elicited by baculovirus-derived VP2 of a very virulent bursal disease virus strain to progeny of commercial breeder chickens.

A baculovirus-derived recombinant VP2 (rVP2) subunit vaccine elicited anti-infectious bursal disease virus (IBDV) antibodies in commercial flocks. The induced antibody levels were similar to those evoked against IBDV by a commercial vaccine. The levels remained higher than that of the negative controls for at least four and a half months in commercial chickens. The antibodies were also transferred to their offspring and were detected in the blood of the progeny for at least 20 days after hatching. These results, along with former data, that show that antibodies elicited by baculovirus rVP2 confer protection to chickens from IBDV [J. Pitcovski et al. (1996), Insect cell-derived VP2 of infectious bursal disease confers protection against the disease in chickens. Avian Diseases, 40, 753-761], imply that the baculovirus-derived rVP2 subunit may serve as a successful vaccine for commercial breeding flocks.

Protein-protein and DNA-protein interactions affect the activity of lymphoid-specific IFN regulatory factors.

IFN regulatory factors (IRFs) constitute a family of transcription factors that are involved in IFN signaling and the development and differentiation of the immune system. Targeted gene disruption studies in mice assigned their primary role to the immune system. Two lymphoid-specific IRF members, IFN consensus sequence binding protein (ICSBP) and IRF-4, bind target DNA with greater efficiency following interaction with two transcription factors, PU.1 and E47, leading to transcriptional synergy. PU.1 and E47 are essential for proper differentiation and maturation of lymphoid cells. In addition, ICSBP interacts with two IRF members, IRF-1 and IRF-2, which also have central roles in the regulation of cell-mediated immunity. Previously, we identified a region in ICSBP, termed the IRF association domain (IAD), that is conserved in all IRFs (excluding IRF-1 and IRF-2) and is essential for its interactions with other IRF proteins. Here we show that the IAD is an independent module used by ICSBP and IRF-4 for protein-protein interactions. In addition, an IAD of IRF-2 (IAD2), necessary for interaction with ICSBP, was identified and found to be conserved in IRF-1. The IAD2 shares similar characteristics with the PEST domain that is essential for the interaction of PU.1 with IRF-4. We also show that the ICSBP DNA binding domain is indispensable for the formation of DNA binding heterocomplexes and transcriptional activity. Therefore, our results shed light on the molecular mechanisms that affect IRF activities in the immune system via discrete functional domains.

The use of bi-cistronic transfer vectors for the baculovirus expression system.

In this communication, we describe the construction of bi-cistronic transfer vectors for the baculovirus expression system (BVES), which are advantageous over the existing vectors. The new vectors provide a simple way to isolate recombinant viruses. More specifically, the gene of interest and the reporter gene luciferase (LUC), constitute the first and second cistrons, respectively, of the same transcript. Therefore, the LUC activity measured during infection of such a bi-cistronic virus, permits an on-line estimation of the recombinant protein level, a very useful feature for large-scale production of recombinant proteins. To achieve expression of the second cistron, the internal ribosome entry site (IRES) element of the encephalomyocarditis virus (EMCV) was employed. However, this element, which is highly efficient in mammalian systems, did not promote efficient internal translation of the second cistron in various insect cells lines originating from different insect species. The lack of efficient internal translation was not due to baculovirus propagation since the same phenomenon was also observed in a viral-free expression system. It seems that a component essential for efficient EMCV IRES activity is either missing or present in limiting amount in insect cells or not compatible. Nevertheless, LUC placed downstream to the IRES element, or immediately downstream to the first cistron, was expressed to a level that enabled the biotechnological application it was designed for.

Failure of viral protein 3 of infectious bursal disease virus produced in prokaryotic and eukaryotic expression systems to protect chickens against the disease.

In recent years, infectious bursal disease virus (IBDV) has become a serious economic problem as a result of the emergence of new and very virulent strains. Most of the antibodies produced against IBDV are for the structural proteins viral protein (VP) 2 (VP2) and VP3. The purpose of this study was to test the potential of recombinant VP3 to induce protective antibodies. The gene for VP3 was isolated from a virulent strain of the virus and cloned into prokaryotic (Escherichia coli) and eukaryotic (baculovirus) expression systems. The protein expressed by both systems was of the expected size (32 kD) and was detected by anti-IBDV antibodies. Following partial purification, the polypeptides were injected into intact birds and induced the production of high levels of anti-IBDV antibodies, as detected by immunoblot and enzyme-linked immunosorbent assay tests. These antibodies did not prevent changes in the bursa and mortality when birds were challenged with a virulent IBDV strain after vaccination with the recombinant VP3. The results show that VP3 polypeptide cannot be used as a subunit vaccine against IBDV and raise questions concerning the nature of the neutralizing epitope on this structural protein.

Jerusalem of cytokines.

The Second Joint Meeting of the International Cytokine Society and the International Society for Interferon and Cytokine Research was held on October 25-30, 1998 in Jerusalem, Israel. The nature of this Joint Meeting dictated that it was intensive and covered topics that included receptor-ligand interactions, signal transduction, transcriptional regulation, antiviral action and apoptotic pathways induced by cytokines such as interferons, interleukines and chemokines. Their roles in infectious diseases and cancers were considered. This overview is by no mean comprehensive and covers only part of the many topics and subjects that were presented in the many plenary talks, symposia and poster sessions. The meeting was held in an excellent scientific atmosphere, that was probably affected by the "divine presence" in Jerusalem, and special thanks for the excellent organization are owed to Drs. Kaempfer, Revel, Wallach and Witz.

Coding region of segment A sequence of a very virulent isolate of IBDV--comparison with isolates from different countries and virulence.

We determined the sequence of the coding region of segment A, coding for the viral proteins (VPs) VP2, VP4, and VP3, of a very virulent (vv) infectious bursal disease virus (IBDV) isolated in Israel and named IBDVks. We compared the deduced amino acid sequences of the proteins of the new isolate with those of the same proteins from several IBDV isolates, as published in recent years. The amino acid sequences of VP3 and VP4 of the Israeli isolate were 1.9%-2.3% different from the sequences of their counterparts from classical strains. Thus, the stable region of VP2 of IBDVks was very similar (0-0.68% difference) to the same region of VP2 from vv strains from Europe and Japan but distinct from that of proteins from classical strains from Europe, the United States, and Australia (up to 9.42% divergence), showing that IBDVks is more closely related to the vv strains from Europe and Japan. We found that viruses isolated in recent years resemble each other more than isolates from the same areas isolated a few years earlier. Hence, IBDVks can be categorized in one group with vv new isolates from Europe and Japan. This group has been found to be distinct from new isolates in the United States and strains isolated before the IBDV epidemic during the late 1980s.

Functional domains of interferon regulatory factor I (IRF-1).

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors among which are IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). These factors share sequence homology in the N-terminal DNA-binding domain. IRF-1 and IRF-2 are further related and have additional homologous sequences within their C-termini. Whereas IRF-2 and ICSBP are identified as transcriptional repressors, IRF-1 is an activator. In the present work, the identification of functional domains in murine IRF-1 with regard to DNA-binding, nuclear translocation, heterodimerization with ICSBP and transcriptional activation are demonstrated. The minimal DNA-binding domain requires the N-terminal 124 amino acids plus an arbitrary C-terminal extension. By using mutants of IRF-1 fusion proteins with green fluorescent protein and monitoring their distribution in living cells, a nuclear location signal (NLS) was identified and found to be sufficient for nuclear translocation. Heterodimerization was confirmed by a two-hybrid system adapted to mammalian cells. The heterodimerization domain in IRF-1 was defined by studies in vitro and was shown to be homologous with a sequence in IRF-2, suggesting that IRF-2 also heterodimerizes with ICSBP through this sequence. An acidic domain in IRF-1 was found to be required and to be sufficient for transactivation. Epitope mapping of IRF-1 showed that regions within the NLS, the heterodimerization domain and the transcriptional activation domain are exposed for possible contacts with interacting proteins.

Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription.

Vascular Endothelial Growth Factor (VEGF) is a very potent angiogenic agent that has a central role in normal physiological angiogenesis as well as in tumor angiogenesis. VEGF expression is induced by hypoxia and hypoglycemia, and thus was suggested to promote neovascularization during tumor outgrowth. Yet, the molecular mechanism that governs VEGF expression is not fully characterized. VEGF induction is attributed in part to increased levels of transcription and RNA stability. Previously, we demonstrated that the 5' Untranslated Region (5' UTR) of VEGF has an important regulatory role in its expression. VEGF has an exceptionally long 5' UTR (1038 bp) which is highly rich in G+C nucleotides. This suggests that secondary structures in the 5' UTR might be essential for VEGF expression through transcriptional and post-transcriptional control mechanisms, as demonstrated for other growth factors. In this communication, we provide evidence that a computer predicted Internal Ribosome Entry Site (IRES) structure is biologically active and is located at the 3' end of the UTR. In addition, the results demonstrate that an alternative transcriptional initiation site for VEGF exists in the 5' UTR of VEGF. This alternative initiation site is 633 bp downstream of the main transcription start site and the resulting 5' UTR includes mainly the IRES structure. Therefore, our results suggest that VEGF is subjected to regulation at either translational level through a mechanism of ribosome internal initiation and/or transcriptional level through alternative initiation.

Phosphorylation events modulate the ability of interferon consensus sequence binding protein to interact with interferon regulatory factors and to bind DNA.

Two families of transcription factors mediate interferon (IFN) signaling. The first family, signal transducers and activators of transcription (STATs), is activated within minutes of IFN treatment. Specific phosphorylation events lead to their translocation to the nucleus, formation of transcriptional complexes, and the induction of the second family of transcription factors termed interferon regulatory factors (IRFs). Interferon consensus sequence binding protein (ICSBP) is a member of IRF family that is expressed only in cells of the immune system and acts as a transcriptional repressor. ICSBP binds DNA through the association with other transcription factors such as IRF-1 or IRF-2. In this communication, the domain that is involved in protein-protein interactions was mapped to the carboxyl terminus of ICSBP. This domain is also important for mediating ICSBP-repressing activity. In vitro studies demonstrated that direct binding of ICSBP to DNA is prevented by tyrosine (Tyr) phosphorylation. Yet, Tyr-phosphorylated ICSBP can bind target DNA only through the association with IRF-2 and IRF-1. This type of phosphorylation is essential for the formation of heterocomplexes. Tyr-phosphorylated ICSBP and IRF-2 are detected in expressing cells constitutively, and Tyr-phosphorylated IRF-1 is induced by IFN-gamma. These results strongly suggest that like the STATs, the IRFs are also modulated by Tyr phosphorylation that affects their biological activities.

Structural and functional features of a specific nucleosome containing a recognition element for the thyroid hormone receptor.

The Xenopus thyroid hormone receptor betaA (TRbetaA) gene contains an important thyroid hormone response element (TRE) that is assembled into a positioned nucleosome. We determine the translational position of the nucleosome containing the TRE and the rotational positioning of the double helix with respect to the histone surface. Histone H1 is incorporated into the nucleosome leading to an asymmetric protection to micrococcal nuclease cleavage of linker DNA relative to the nucleosome core. Histone H1 association is without significant consequence for the binding of the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR/RXR) to nucleosomal DNA in vitro, or for the regulation of TRbetaA gene transcription following microinjection into the oocyte nucleus. Small alterations of 3 and 6 bp in the translational positioning of the TRE in chromatin are also without effect on the transcriptional activity of the TRbetaA gene, whereas a small change in the rotational position of the TRE (3 bp) relative to the histone surface significantly reduces the binding of TR/RXR to the nucleosome and decreases transcriptional activation directed by TR/RXR. Our results indicate that the specific architecture of the nucleosome containing the TRE may have regulatory significance for expression of the TRbetaA gene.

A dominant negative mutant of an IFN regulatory factor family protein inhibits both type I and type II IFN-stimulated gene expression and antiproliferative activity of IFNs.

Type I (alpha,beta) and type II (gamma) IFNs elicit antiproliferative and antiviral activities through two distinct transcription pathways involving 1) IRF family proteins and ISGF3, and 2) STAT1. We have employed a dominant negative strategy to study the role of IRF family proteins in eliciting the biologic activities of IFN. A truncated IRF protein retaining the DNA-binding domain (DBD) of ICSBP (a member of the IRF family) was stably transfected into U937 monocytic cells. Clones expressing DBD had markedly reduced ISRE-binding activity and were defective in expressing several type I IFN-inducible genes. STAT1 was one such type I IFN-inducible gene whose expression was also inhibited in DBD clones. As a result, the expression of several IFN-gamma-inducible genes was also inhibited in these clones, indicating functional coupling of the type I and type II IFN transcription pathways. Furthermore, DBD clones grew more slowly than control clones and were refractory to antiproliferative effects of both types of IFNs. We found that IFN treatment of U937 cells leads to a G1 arrest and an increase in underphosphorylated retinoblastoma gene product. However, IFN treatment did not change the cell cycle profile, nor retinoblastoma gene product phosphorylation state in DBD clones. These data indicate that expression of DBD disrupts cell cycle regulatory mechanisms. Combined with the previously noted failure of DBD clones to elicit antiviral activity, the present work shows that IRF family proteins play an integral part in growth control activities of IFNs.

Insect cell-derived VP2 of infectious bursal disease virus confers protection against the disease in chickens.

Infectious bursal disease virus (IBDV) has become a major problem in recent years. Conventional vaccines make use of attenuated or inactivated viral strains, but these are gradually losing their effectiveness. We investigated the possibility of using purified VP2, a subunit of IBDV structural protein expressed in insect cells, as a vaccine. The VP2 gene was cloned into pAcYM1. The cloned gene was expressed in a baculovirus system, giving rise to a high quantity of recombinant VP2 (rVP2) protein. The length of the VP2 is 453 amino acids, and it contains two additional amino acids of the baculovirus at the carboxyl terminus. The molecular mass of the protein is about 48 kD. The rVP2 protein reacted with antibodies raised against viral VP2 and had a similar molecular weight. This protein was tested in a controlled vaccination experiment and compared with an inactivated commercial vaccine. High levels of antibodies were raised by the vaccinated birds. The vaccinated birds were challenged with a pathogenic viral strain. rVP2-vaccinated chickens exhibited high resistance to the virus. No mortality or weight changes in the bursa of Fabricius were observed in the vaccinated birds, whereas in the negative control birds, vaccinated with phosphate buffer, up to 50% mortality was found. Higher levels of antibodies were found by enzyme-linked immunosorbent assay in birds vaccinated with rVP2 compared with those vaccinated with the commercial vaccine. This study suggests the potential use of the isolated rVP2 as a subunit vaccine.

Selective binding of VEGF121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells.

VEGF121 and VEGF165 are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF165 contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF165 a heparin binding capability which VEGF121 lacks. 125I-VEGF165 bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while 125I-VEGF121 bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of 125I-VEGF121 to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF165 and VEGF121 inhibited the binding of 125I-VEGF121 to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF165 by 2-3-fold. These results contrast with previous observations which have indicated that the binding of 125I-VEGF165 to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF165 is susceptible to oxidative damage caused by oxidants such as H2O2 or chloramine-T. VEGF121 was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF165 but not the receptor binding ability of damaged VEGF121. These observations suggest that alternative splicing can generate a diversity in growth factor signaling by determining receptor recognition patterns. They also indicate that the heparin binding ability of VEGF165 may enable the restoration of damaged VEGF165 function in processes such as inflammation or wound healing.