Giovanna Romeo

Review: IRF-1 as a Negative Regulator of Cell Proliferation

Numerous evidence has demonstrated the involvement in growth control of interferon (IFN) regulatory factor-1 (IRF-1), which shows tumor suppressor activity. IRF-1 is a well-studied member of the IRF transcription factors that reveals functional diversity in the regulation of cellular response by activating expression of a diverse set of target genes, depending on the cell type and on the specific stimuli. IRF-1 gene rearrangements may be a crucial point in the pathogenesis of some cancer types. Furthermore, different aspects of the tumor suppressor function of IRF-1 may be explained, at least in part, by the observations that IRF-1 is a regulator of cell cycle and apoptosis and that its inactivation accelerates cell transformation. Studies on gene knockout mice contributed greatly to the clarification of these multiple IRF-1 functions. We summarize our current knowledge of the antigrowth effect of IRF-1, focusing also on a more general involvement of IRF-1 in mediating negative regulation of cell growth induced by numerous cytokines and other biologic response modifiers.

In Vitro Treatment of Human Monocytes/Macrophages with Myristoylated Recombinant Nef of Human Immunodeficiency Virus Type 1 Leads to the Activation of Mitogen-Activated Protein Kinases, IκB Kinases, and Interferon Regulatory Factor 3 and to the Release of Beta Interferon

ABSTRACT The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-κB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the α and β subunits of the IκB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.

Modulation of ferritin H-chain expression in Friend erythroleukemia cells : transcriptional and translational regulation by hemin

The mechanisms that regulate the expression of the H chain of the iron storage protein ferritin in Friend erythroleukemia cells (FLCs) after exposure to hemin (ferric protoporphyrin IX), protoporphyrin IX, and ferric ammonium citrate (FAC) have been investigated. Administration of hemin increases the steady-state level of ferritin mRNA about 10-fold and that of ferritin protein expression 20-fold. Experiments with the transcriptional inhibitor actinomycin D and transfection studies demonstrate that the increment in cytoplasmic mRNA content results from enhanced transcription of the ferritin H-chain gene and cannot be attributed to stabilization of preexisting mRNAs. In addition to transcriptional effects, translational regulation induces the recruitment of stored mRNAs into functional polyribosomes after hemin and FAC administration, resulting in a further increase in ferritin synthesis. Administration of protoporphyrin IX to FLCs produces divergent transcriptional and translational effects. It increases transcription but appears to suppress ferritin mRNA translation. FAC treatment increases the mRNA content slightly (about twofold), and the ferritin levels rise about fivefold over the control values. We conclude that in FLCs, hemin induces ferritin H-chain biosynthesis by multiple mechanisms: a transcriptional mechanism exerted also by protoporphyrin IX and a translational one, not displayed by protoporphyrin IX but shared with FAC.

Human immunodeficiency virus type 1 (HIV-1) Nef activates STAT3 in primary human monocyte/macrophages through the release of soluble factors: involvement of Nef domains interacting with the cell endocytotic machinery

Increasing evidence indicates that the expression of the human immunodeficiency virus‐1 (HIV‐1) Nef protein significantly influences the activation state of the host cell. Here we report that Nef specifically activates STAT3 in primary human monocyte‐derived macrophages (MDM). This was demonstrated by both single‐cycle infection experiments driven by Vesicular Stomatitis virus glycoprotein (VSV‐G) pseudotyped HIV‐1 and treatment with exogenous recombinant Nef. The analysis of the effects of Nef mutants revealed that domains of the C‐terminal flexible loop interacting with the cell endocytotic machinery are involved in the STAT3 activation. In particular, our data suggest that the Nef‐dependent STAT3 activation relies on the targeting of Nef to the late endosome/lysosome compartment. In addition, we found that Nef activates STAT3 through a mechanism mediated by the release of soluble factor(s), including MIP‐1α, that requires de novo protein synthesis but appears independent from the activation of src tyrosine kinases. The results presented here support the idea that the first intervention of Nef in the intracellular signaling of monocyte‐macrophages could generate, by means of the release of soluble factor(s), a secondary wave of activation that could be of a potential pathogenetic significance.

2–5A synthetase activity does not increase in interferon-resistant friend leukemia cell variants treated with α/β interferon despite the presence of high-affinity interferon receptor sites

The presence of interferon (IFN) receptors on mouse Friend leukemia cells (FLC) has been investigated in binding experiments with highly purified 125I-labeled mouse alpha/beta IFN. Both IFN-resistant clones and wild-type IFN-sensitive FLC showed a specific saturable binding site for mouse IFN with a similar affinity constant. In contrast to IFN-sensitive FLC, IFN-resistant FLC variants were not inducible by IFN for double-stranded RNA-dependent 2-5A synthetase activity.

In vitro treatment of human monocyte/macrophages with myristoylated recombinant Nef of HIV-1 leads to the activation of MAPKs, IκB kinases and Interferon Regulatory Factor 3 and to the release of Beta Interferon

ABSTRACT The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-κB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the α and β subunits of the IκB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.

Senescence and Cell Death Pathways and Their Role in Cancer Therapeutic Outcome

Anticancer drug-induced tumor suppression may involve mechanisms of protection against neoplastic transformation that are normally latent in mammalian cells and consist in a genetic program implemented during anti-tumoral defense. This defense program results in the self elimination of cells harboring potentially dangerous mutations by triggering cell death through apoptosis and/or autophagy or in the execution of a program that leads to a permanent growth arrest known as senescence. These responses are considered crucial tumor suppressive mechanisms and their study appears to be essential to develop therapeutical procedures based on the enhancement of the different responses. This review summarizes fundamental knowledge on the underlying mechanisms able to limit excessive or aberrant cellular proliferation and on the prognostic value of both apoptosis and senescence detection. In addition, interesting evidence showing that different drugs induce senescence or cell death depending on the genetic features of the tumor cells as well as on the integrity of the relative pathways is reported.

Interferons in cell growth and development

Interferons (IFNs), besides inducing an antiviral state in uninfected cells, are also natural regulatory molecules. They play a key role in the regulation both of cell growth and differentiation, and of development. Up- or down-regulation of oncogenes by IFNs may be one of the mechanisms by which these molecules affect cell physiology. The list of IFN-inducible proteins continues to grow rapidly and future research should identify among these the mediators of the biological effects of IFNs.

HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2

Background HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3. Methodology/Principal Findings Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. Conclusions Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ.

Antiproliferative activity of interferon α and retinoic acid in SiHa carcinoma cells: The role of cell adhesion

Several lines of evidence have demonstrated that IFNs could be relevant in the treatment of certain neoplastic diseases such as carcinomas. In particular, IFN‐α, in addition to the anti‐proliferative and cytostatic effects, was demonstrated to be capable of inducing cell death by apoptosis both in vivo and in vitro. Numerous protocols have also been proposed which consider the association of IFN‐α with other drugs. Among these are retinoids, a class of compounds capable of inducing inhibition of cell growth and differentiation. We address the question here by analyzing the role of cell adhesion in susceptibility to IFN‐α, RA and their combination of a human cell line derived from a squamous carcinoma of the cervix, the Bcl‐2‐negative SiHa cell line. In this context, cytoskeleton components and several surface molecules playing a role in cell substrate and cell‐to‐cell relationships have been evaluated. We found that RA treatment is capable of improving stress fiber formation, decreasing cell detachment and increasing cell‐adhesion capability. However, no variations in the ability to adhere to specific extracellular‐matrix molecules were found in RA‐treated cells. No quantitative changes were detected in integrins involved as receptors for extracellular matrix molecules (VLA1‐VLA5) or in other cell‐adhesion‐associated molecules (e.g., CD44). By contrast, 2 important molecules involved in cell‐adhesion processes appeared to be up‐regulated by RA exposure: focal adhesion kinase and E‐cadherin, involved in adhesion plaque formation and cell‐to‐cell contacts, respectively. Keeping in mind the importance of adhesion properties in the cell‐growth pathway, our findings could be of interest in the study of carcinoma‐cell proliferation and metastatic potential. Int. J. Cancer 76:531–540, 1998.© 1998 Wiley‐Liss, Inc.