The GRIMs: a new interface between cell death regulation and interferon/retinoid induced growth suppression

Cytokines and vitamins play a central role in controlling neoplastic cell growth. The interferon (IFN) family of cytokines regulates antiviral, anti-tumor, antimicrobial, differentiation, and immune responses in mammals. Significant advances have been made with respect to IFN-induced signal transduction pathways and antiviral responses. However, the IFN-induced anti-tumor actions are poorly defined. Although IFNs themselves inhibit tumor growth, combination of IFNs with retinoids (a class of Vitamin A related compounds) strongly potentiates the IFN-regulated anti-tumor action in a number of cell types. To define the molecular mechanisms involved in IFN/retinoid (RA)-induced apoptosis we have employed a genetic approach and identified several critical genes. In this review, I provide the current picture of IFN- RA- and IFN/RA-regulated growth suppressive pathways. In particular, I focus on a novel set of genes, the genes-associated with retinoid-interferon induced mortality (GRIM). GRIMs may be novel types of tumor suppressors, useful as biological response markers and potentially novel targets for drug development.     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

Interferons and the tumor cell

Optimal use of interferons (IFNs) for the treatment of tumor disease requires experimental work in order to precisely define IFN actions. We have pointed out three modes of such actions relevant for the antitumor efficacy exerted by IFNs: effects on apoptosis, effects on genes involved in malignant transformation and effects on angiogenesis. These are but three selected areas forming a basis for the development of optimal IFN therapy. Further experimental work, undertaken in these and additional IFN areas, is mandatory for the most effective clinical use of IFNs for the treatment of tumor disease.Abbreviations IFN interferon - FGF basic fibroblast growth factor     

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.     

Cytoplasmic localization of the interferon-inducible protein that is encoded by the AIM2 (absent in melanoma) gene from the 200-gene family

While interferons (IFNs) (alpha, beta and gamma), a family of cytokines, have the ability to exert the growth-inhibitory effect on target cells, the molecular mechanism(s) by which IFNs inhibit cell growth remains to be identified. Because IFN-inducible 'effector' proteins mediate the biological activities of IFNs, characterization of IFN-inducible proteins is critical to identify their functional role in IFN action. One family (the 200-family) of IFN-inducible proteins is encoded by structurally related murine (Ifi202a, Ifi202b, Ifi203, Ifi204 and D3) and human (IFI16, MNDA and AIM2) genes. The proteins encoded by genes in the family share a unique repeat of 200-amino acids and are primarily nuclear. The AIM2 gene is a newly identified gene that is not expressed in a human melanoma cell line. Here we report that AIM2 is estimated to be a 39 kDa protein and, unlike other proteins in the family, is localized primarily in the cytoplasm. Interestingly, overexpression of AIM2 in transfected cells retards proliferation and, under reduced serum conditions, increases the susceptibility to cell death. Moreover, AIM2 can heterodimerize with p202 in vitro. Together, these observations provide support to the idea that AIM2 may be an important mediator of IFN action.     

NFkappaB negatively regulates interferon-induced gene expression and anti-influenza activity

Interferons (IFNs) are antiviral cytokines that selectively regulate gene expression through several signaling pathways including nuclear factor kappaB(NFkappaB). To investigate the specific role of NFkappaB in IFN signaling, we performed gene expression profiling after IFN treatment of embryonic fibroblasts derived from normal mice or mice with targeted deletion of NFkappaB p50 and p65 genes. Interestingly, several antiviral and immunomodulatory genes were induced higher by IFN in NFkappaB knock-out cells. Chromatin immunoprecipitation experiments demonstrated that NFkappaB was basally bound to the promoters of these genes, while IFN treatment resulted in the recruitment of STAT1 and STAT2 to these promoters. However, in NFkappaB knock-out cells IFN induced STAT binding as well as the binding of the IFN regulatory factor-1 (IRF1) to the IFN-stimulated gene (ISG) promoters. IRF1 binding closely correlated with enhanced gene induction. Moreover, NFkappaB suppressed both antiviral and immunomodulatory actions of IFN against influenza virus. Our results identify a novel negative regulatory role of NFkappaB in IFN-induced gene expression and biological activities and suggest that modulating NFkappaB activity may provide a new avenue for enhancing the therapeutic effectiveness of IFN.     

Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis

IFNs are a family of cytokines with pleiotropic biological effects mediated by scores of responsive genes. IFNs were the first human proteins to be effective in cancer therapy and were among the first recombinant DNA products to be used clinically. Both quality and quantity of life has been improved in response to IFNs in various malignancies. Despite its beneficial effects, unraveling the mechanisms of the anti-tumor effects of IFN has proven to be a complex task. IFNs may mediate anti-tumor effects either indirectly by modulating immunomodulatory and anti-angiogenic responses or by directly affecting proliferation or cellular differentiation of tumor cells. Both direct or indirect effects of IFNs result from induction of a subset of genes, called IFN stimulated genes (ISGs). In addition to the ISGs implicated in anti-viral, anti-angiogenic, immunomodulatory and cell cycle inhibitory effects, oligonucleotide microarray studies have identified ISGs with apoptotic functions. These include TNF- related apoptosis inducing ligand (TRAIL/Apo2L), Fas/FasL, XIAP associated factor-1 (XAF-1), caspase-4, caspase-8, dsRNA activated protein kinase (PKR), 2'5'A oligoadenylate synthetase (OAS), death activating protein kinases (DAP kinase), phospholipid scramblase, galectin 9, IFN regulatory factors (IRFs), promyelocytic leukemia gene (PML) and regulators of IFN induced death (RIDs). In vitro IFN-, IFN- and IFN- induced apoptosis in multiple cell lines of varied histologies. This review will emphasize possible mechanisms and the role of ISGs involved in mediating apoptotic function of IFNs.     

The latent membrane protein 1 of Epstein-Barr virus (EBV) primes EBV latency cells for type I interferon production

Epstein-Barr virus (EBV) latency has been associated with a variety of human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. We have previously shown that LMP-1 induces the expression of several interferon (IFN)-stimulated genes and has antiviral effect (Zhang, J., Das, S. C., Kotalik, C., Pattnaik, A. K., and Zhang, L. (2004) J. Biol. Chem. 279, 46335-46342). In this report, a novel mechanism related to the antiviral effect of LMP-1 is identified. We show that EBV type III latency cells, in which LMP-1 is expressed, are primed to produce robust levels of endogenous IFNs upon infection of Sendai virus. The priming action is due to the expression of LMP-1 but not EBV nuclear antigen 2 (EBNA-2). The signaling events from the C-terminal activator regions of LMP-1 are essential to prime cells for high IFN production. LMP-1-mediated activation of NF-kappaB is apparently necessary and sufficient for LMP-1-mediated priming effect in DG75 cells, a human B cell line. IFN regulatory factor 7 (IRF-7) that can be activated by LMP-1 is also implicated in the priming action. Taken together, these data strongly suggest that LMP-1 may prime EBV latency cells for IFN production and that the antiviral property of LMP-1 may be an intrinsic part of EBV latency program, which may assist the establishment and/or maintenance of viral latency.     

Host defense, viruses and apoptosis

To thwart viral infection, the host has developed a formidable and integrated defense network that comprises our innate and adaptive immune response. In recent years, it has become clear that in an attempt to prevent viral replication, viral dissemination or persistent viral infection of the cell, many of these protective measures actually involve the induction of programmed cell death, or apoptosis. An initial response to viral infection primarily involves the innate arm of immunity and the killing of infected cells with cytotoxic lymphocytes such as natural killer (NK) cells through mechanisms that include the employment of perforin and granzymes. Once the virus has invaded the cell, however, a second host defense-mediated response is also triggered which involves the induction of a family of cytokines known as the interferons (IFNs). The IFNs, which are essential for initiating and coordinating a successful antiviral response, function by stimulating the adaptive arm of immunity involving cytotoxic T cells (CTLs), and by inducing a number of intracellular genes that directly prevent virus replication/cytolysis or that facilitate apoptosis. The IFN-induced gene family is now known to comprise the death ligand TRAIL, the dsRNA-dependent protein kinase (PKR), interferon regulatory factors (IRFs) and the promyelocytic leukemia gene (PML), all of which have been reported to be mediators of cell death. That DNA array analyses indicate that numerous cellular genes, many as yet uncharacterized, may similarly be induced by IFN, further emphasizes the likely importance that these cytokines have in the modulation of apoptosis. This likelihood is additionally underlined by the elaborate strategies developed by viruses to inhibit IFN-antiviral function and the mechanisms of cell death.     

Identification and expression analysis of cDNAs encoding channel catfish type I interferons

Previously a cDNA encoding a putative interferon gene, designated CF IFN-1, was identified from a catfish EST library. However, its constitutive expression, absence of a signal peptide, and apparently low level of biological activity suggested that this cDNA likely encoded an expressed pseudogene. Since Southern blot analysis suggested the presence of two to three IFN genes, additional cDNAs were generated from catfish fibroblast and lymphoid cell lines using primers designed to conserved regions of zebrafish and catfish interferon. Using this approach, three novel CF IFN genes, two of which likely encode functional interferon molecules, were identified. At the amino acid level, similarity among CF IFNs ranged from 71% to 82%, whereas similarity to other fish IFNs ranged from 15% to 35%. Although CF IFN-3, like CF IFN-1, lacks a signal peptide, CF IFN-2 and -4 appear to encode full-length, signal sequence-bearing genes. Consistent with their putative identification as functional genes, CF IFN-2 and -4 were not expressed in unstimulated cell lines, and CF IFN-2 was rapidly upregulated in CCO cells in response to virus infection or treatment with dsRNA. Moreover, as with salmon, fugu, and zebrafish interferon genes, CF IFN-1 contained four introns whose locations were conserved not only with respect to other fish IFNs, but also with respect to mammalian IFN-lambda. While it is likely that CF IFNs represent Type I IFNs, several characteristics preclude assigning these cytokines to any particular subfamily.     

Regulation of apoptosis by type III interferons

Abstract. Objective: Two types of interferons (IFNs), type I (IFN‐α/β) and type III (IFN‐λs), utilize distinct receptor complexes to induce similar signalling and biological activities, including recently demonstrated for IFN‐λs antitumour activity. However, ability of type III IFNs to regulate cell population growth remains largely uncharacterized.Materials and methods: Intact and modified human colorectal adenocarcinoma HT29 cells were used to study regulation of apoptosis by IFN‐λs. Results and Conclusions: We report that the IFN‐λR1 chain of the type III IFN receptor complex possesses an intrinsic ability to trigger apoptosis in cells. Signalling induced through the intracellular domain of IFN‐λR1 resulted in G₁/G₀ phase cell cycle arrest, phosphatidylserine surfacing and chromosomal DNA fragmentation. Caspase‐3, caspase‐8 and caspase‐9 were activated; however, pancaspase inhibitor Z‐VAD‐FMK did not prevent apoptosis. In addition, the extent of apoptosis correlated with the level of receptor expression and was associated with prolonged IFN‐λ signalling. We also demonstrated that the ability to trigger apoptosis is a unique intrinsic function of all IFN receptors. However, more robust apoptosis was induced by signalling through type III IFN receptor than through type I or type II (IFN‐γ) receptors, suggesting higher cytotoxic potential of type III IFNs. In addition, we observed that IFN‐γ treatment sensitized HT29 cells to IFN‐λ‐mediated apoptosis. These results provide evidence that type III IFNs, alone or in combination with other stimuli, have the potential to induce apoptosis.