Constitutive IFNα/ß signaling maintains expression of signaling intermediaries for efficient cytokine responses.

Interferons (IFNs) are a family of immunoregulatory cytokines with important roles in anti-viral and anti-tumor responses. Type I and II IFNs bind distinct receptors and are associated with different stages of the immune response. There is however, considerable crosstalk between these two cytokines with enhancement of IFNγ responses following IFNα/ß priming and loss of IFNα/ß receptor (IFNAR) resulting in diminished IFNγ responses. In this study, we sought to define the mechanism of crosstalk between the type I and II IFNs. Our previous reports demonstrated reduced expression of the canonically activated transcription factor signal transducer and activator of transcription (STAT)1, in cells lacking the IFNAR α chain (IFNAR1). Therefore, we used microarray analysis to determine whether reconstitution of STAT1 in IFNAR1-deficient cells was sufficient to restore IFNγ responses. We identified several biological pathways, including the MHC class I antigen presentation pathway, in which STAT1 reconstitution was able to significantly rescue IFNγ-mediated gene regulation in Ifnar1 (-/-) cells. Notably, we also found that in addition to low basal expression of STAT1, cells lacking the IFNAR1 also had aberrant expression of multiple other transcription factors and signaling intermediaries. The studies described herein demonstrate that basal and regulated expression of signaling intermediaries is a mechanism for crosstalk between cytokines including type I and II IFNs.

Molecular and biologic analysis of histone deacetylase inhibitors with diverse specificities.

Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited.

Modulation of antitumour immune responses by intratumoural Stat1 expression.

Signal transducer and activator of transcription 1 (Stat1) mediates anti-viral responses and cytokine-driven anti-proliferative, apoptotic and immunomodulatory activities. As de-regulated Stat1 function can affect tumour progression we sought to elucidate the effects of tumour cell-intrinsic Stat1 expression on immunosurveillance. Knockout of Stat1 enhanced the development of sarcomas induced by the chemical carcinogen 3-methylcholanthrene (MCA). Growth of transplanted MCA-induced Stat1⁻/⁻ sarcomas was suppressed in wild-type mice compared to growth in Stat1⁻/⁻ and immunocompromised recipients. Co-depletion of NK and CD8⁺ T cells from wild-type mice facilitated Stat1-deficient tumour growth whereas depletion of CD4⁺ T cells and CD8⁺ T cells did not. In vitro and in vivo analysis of the tumours implicated a role for NK cell-mediated, perforin-dependent killing of Stat1-deficient tumours. Interestingly, restoration of Stat1 expression in Stat1⁻/⁻ tumours resulted in diminished involvement of NK cells and increased contribution of CD8⁺ T cells in anti-tumour responses. Therefore, Stat1 expression within tumour cells modulated anti-tumour immune responses by altering the dominant immune effector cell involvement from NK cells to CD8⁺ T cells in the absence or presence of Stat1 respectively.

Constitutive type I interferon modulates homeostatic balance through tonic signaling.

Interferons (IFNs) were discovered as cytokines induced during and protecting from viral infection. They have been documented to play essential roles in numerous physiological processes beyond antiviral and antimicrobial defense, including immunomodulation, cell cycle regulation, cell survival, and cell differentiation. Recent data have also uncovered a potentially darker side to IFN, including roles in inflammatory diseases, such as autoimmunity and diabetes. IFN can have effects in the absence of acute infection, highlighting a physiologic role for constitutive IFN. Type I IFNs are constitutively produced at vanishingly low quantities and yet exert profound effects, mediated in part through modulation of signaling intermediates required for responses to diverse cytokines. We review evidence for a yin-yang of IFN function through its role in modulating crosstalk between multiple cytokines by both feedforward and feedback regulation of common signaling intermediates and postulate a homeostatic role for IFN through tonic signaling in the absence of acute infection.

Functional crosstalk between type I and II interferon through the regulated expression of STAT1.

Autocrine priming of cells by small quantities of constitutively produced type I interferon (IFN) is a well-known phenomenon. In the absence of type I IFN priming, cells display attenuated responses to other cytokines, such as anti-viral protection in response to IFNgamma. This phenomenon was proposed to be because IFNalpha/beta receptor1 (IFNAR1) is a component of the IFNgamma receptor (IFNGR), but our new data are more consistent with a previously proposed model indicating that regulated expression of STAT1 may also play a critical role in the priming process. Initially, we noticed that DNA binding activity of STAT1 was attenuated in c-Jun(-/-) fibroblasts because they expressed lower levels of STAT1 than wild-type cells. However, expression of STAT1 was rescued by culturing c-Jun(-/-) fibroblasts in media conditioned by wild-type fibroblasts suggesting they secreted a STAT1-inducing factor. The STAT1-inducing factor in fibroblast-conditioned media was IFNbeta, as it was inhibited by antibodies to IFNAR1, or when IFNbeta expression was knocked down in wild-type cells. IFNAR1(-/-) fibroblasts, which cannot respond to this priming, also expressed reduced levels of STAT1, which correlated with their poor responses to IFNgamma. The lack of priming in IFNAR1(-/-) fibroblasts was compensated by over-expression of STAT1, which rescued molecular responses to IFNgamma and restored the ability of IFNgamma to induce protective anti-viral immunity. This study provides a comprehensive description of the molecular events involved in priming by type I IFN. Adding to the previous working model that proposed an interaction between type I and II IFN receptors, our work and that of others demonstrates that type I IFN primes IFNgamma-mediated immune responses by regulating expression of STAT1. This may also explain how type I IFN can additionally prime cells to respond to a range of other cytokines that use STAT1 (e.g., IL-6, M-CSF, IL-10) and suggests a potential mechanism for the changing levels of STAT1 expression observed during viral infection.

Inducible activation of IFI 16 results in suppression of telomerase activity, growth suppression and induction of cellular senescence.

Expression of the human HIN-200 family member IFI 16 has been reported to suppress cell growth and contribute to the onset of cellular senescence. However the molecular events involved in this process have not been fully characterised. We fused IFI 16 to the estrogen receptor ligand-binding domain to establish an inducible model for studying the molecular events that cause these phenomena. In cells induced to express the ER-IFI 16 within the nucleus there was a decrease in cellular proliferation and concomitant growth arrest in the G1 phase of the cell cycle. Unlike previous reports, this did not appear to involve the p53-p21(WAF1/CIP1)-cdk2-pRb pathway. Following nuclear expression of ER-IFI 16 we noted senescence-like morphological changes and expression of senescence-associated beta-galactosidase in growth arrested cells. Importantly, we also found a marked reduction in telomerase activity in arrested cells compared to controls. Moreover, IFI 16 and hTERT co-localised within the nucleus and these two proteins physically interacted in vivo and in vitro. Together, these data suggest that IFI 16 may act as an endogenous regulator of telomerase activity and, through its interaction with hTERT, contributes to the inhibition of proliferation and induces a senescence-like state.

The molecular basis for perforin oligomerization and transmembrane pore assembly.

Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into perforin's mechanism of action.

Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist.

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.

The role of p202 in regulating hematopoietic cell proliferation and differentiation.

HIN-200 proteins are interferon (IFN)-inducible proteins that can regulate cell proliferation and differentiation in vitro. Characterization of the lineage and cell type-dependent expression of Ifi202 revealed little or no expression of Ifi202 in the Lin(-)/c-Kit+ fraction enriched for immature hematopoietic progenitor cells (HPCs) but higher levels in more differentiated Lin(-)/c-Kit(-) and Lin+ populations. The highest levels of Ifi202 expression were observed in CD11b+/Gr-1dim immature granulocytes in the bone marrow. In the peripheral blood, Ifi202 was expressed only in the myeloid lineage, with the highest level of expression seen in CD11b+/Gr-1dim immature granulocytes. Constitutive expression of p202 in primary HPCs delayed proliferation of these cells in vitro, caused a reduction in the number and size of myeloid colonies growing on methylcellulose, and affected the ability of the cells to reconstitute irradiated mice but did not significantly affect cell differentiation. Thus, p202 plays a role in regulating the proliferative capacity of hematopoietic cells.

Cloning and characterisation of Ifi206: a new murine HIN-200 family member.

HIN-200 proteins are interferon-inducible proteins capable of regulating cell growth, senescence, differentiation and death. Using a combination of in silico analysis of NCBI EST databases and screening of murine C57BL/6 cDNA libraries we isolated novel murine HIN-200 cDNAs designated Ifi206S and Ifi206L encoding two putative mRNA splice variants. The p206S and p206L protein isoforms have a modular domain structure consisting of an N-terminal PAAD/DAPIN/Pyrin domain, a region rich in serine, threonine and proline residues and a C-terminal 200 B domain characteristic of other HIN-200 proteins. Ifi206 mRNA was detected only in the spleen and lung of BALB/c and C57BL/6 mice and expression was up-regulated by both types I and II IFN subtypes. p206 protein was predominantly expressed in the cytoplasm and addition of LMB, a CRM1 dependent nuclear export inhibitor, caused p206 to accumulate in the nucleus. Unlike other human and mouse HIN-200 proteins that contain only a single 200 amino acid domain, overexpression of p206 impaired the clonogenic growth of tumour cell lines. Thus, p206 represents the newest HIN-200 family member discovered. It has distinct and restricted pattern of expression however maintains many of the hallmarks of HIN-200 proteins including the presence of a characteristic 200 X domain, induction by interferon and an ability to suppress tumour cell growth.

A novel c-Jun-dependent signal transduction pathway necessary for the transcriptional activation of interferon gamma response genes.

The biological effects of interferon gamma (IFNgamma) are mediated by interferon-stimulated genes (ISGs), many of which are activated downstream of Janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) signaling. Herein we have shown that IFNgamma rapidly activated AP-1 DNA binding that required c-Jun but was independent of JAK1 and STAT1. IFNgamma-induced c-Jun phosphorylation and AP-1 DNA binding required the MEK1/2 and ERK1/2 signaling pathways, whereas the JNK1/2 and p38 mitogen-activated protein kinase pathways were dispensable. The induction of several ISGs, including ifi-205 and iNOS, was impaired in IFNgamma-treated c-Jun-/- cells, but others, such as IP-10 and SOCS3, were unaffected, and chromatin immunoprecipitation demonstrated that c-Jun binds to the iNOS promoter following treatment with IFNgamma. Thus, IFNgamma induced JAK1- and STAT1-independent activation of the ERK mitogen-activated protein kinase pathway, phosphorylation of c-Jun, and activation of AP-1 DNA binding, which are important for the induction of a subset of ISGs. This represents a novel signal transduction pathway induced by IFNgamma that proceeds in parallel with conventional JAK/STAT signaling to activate ISGs.

The HIN-200 family: more than interferon-inducible genes?

The HIN-200 family was initially grouped together based on their hemopoietic expression, interferon-inducibility, nuclear localization, and characteristic 200 amino-acid domains. In this review, we performed a comprehensive search of genome databases and determined the location of previously characterized and predicted genes within the human, mouse, and rat HIN-200 loci. Several novel proteins were predicted in the mouse and rat. We also discuss recent advances in our understanding of this family of proteins and highlight the most important findings. In addition to a role in interferon biology, there is now good evidence supporting a role for these proteins as regulators of cell proliferation and differentiation. The activity of HIN-200 proteins is not restricted to the hemopoietic system as they are expressed and can function in a variety of other cells and tissues. The importance of HIN-200 proteins in disease now is beginning to be understood as they appear to be involved in autoimmunity and may act as tumor suppressor proteins.

Biochemical and growth regulatory activities of the HIN-200 family member and putative tumor suppressor protein, AIM2.

The human HIN-200 family member AIM2 was originally identified in a screen for suppressors of melanoma tumorigenicity following introduction of chromosome 6 into the UACC903 human melanoma cell line. Although the AIM2 protein contained many of the conserved structural motifs common to other HIN-200 proteins, the biochemical characteristics of AIM2 and the ability of overexpressed AIM2 to phenocopy the effect of introduction of chromosome 6 in the UACC903 cells had not been assessed. Herein we demonstrated that AIM2 was localised within the nucleus of transfected or interferon-treated human cells. In addition, AIM2 could homodimerise via the amino-terminal (PAAD/DAPIN) region and heterodimerise with the related IFI 16 protein. However, overexpressed AIM2 did not significantly affect the growth or survival of UACC903 cells or another human melanoma cell line. These data indicate that AIM2 has many of the biochemical and structural characteristics of HIN-200 proteins, however, its expression is not sufficient to induce a tumor-suppressor-like phenotype.

Expression of IFI 16 in epithelial cells and lymphoid tissues.

IFI 16 is a member of the HIN-200 protein family named for their haemopoietic expression, interferon-inducibility and nuclear localisation. These proteins have been characterised as transcriptional regulators that modulate the cell cycle. IFI 16 is expressed in some haemopoietic lineages including CD34+ progenitor cells, mature lymphocytes and monocytes, but is absent from granulocytes, erythrocytes and megakaryocytes. We present a wider study of IFI 16 expression in normal human tissues using a monoclonal antibody specifically recognising the C-terminus of IFI 16. As expected, IFI 16 was detected in the nuclei of lymphocytes in the spleen, thymus, lymph node and palatine tonsil, but was also found in epithelial cells in these tissues. Interestingly, IFI 16 protein was also expressed in non-lymphoid tissues including trachea, gastrointestinal tract, skin and testis, but was absent from others including heart and brain. In each tissue, IFI 16 was predominantly expressed in surface epithelial cells and staining was strongest in basal epithelial layers. Therefore, IFI 16 expression is not restricted to cells of the immune system, but is also expressed in epithelial cells. In contrast to the perceived role of HIN-200 proteins as suppressors of cell growth, maximal expression of IFI 16 was in cells with high proliferative potential.