Retinoic acid and interferon signaling cross talk in normal and RA-resistant APL cells.

Retinoic acid (RA) and interferon (IFN) potentiate each other to induce biological responses. Their combination has shown synergistic differentiating, antiproliferative and antiviral activities in various cell lines including those derived from the acute promyelocytic leukemia (APL). IFNs have demonstrated broad applications in cancer, as well as in virologic diseases. RA has variable effectiveness in therapy. Its real success is in APL where it provides the first example of a differentiation therapy. However, complete clinical remission with RA alone is always transient as RA resistance develops in the treated patients as well as in vitro. In various cell lines, including those derived from APL, RA induces directly the expression of two transcription factors, Stat1 and IRF-1 which play central roles in the IFN signal transduction. In addition, RA induces IFN-alpha synthesis and enhances the IFN-induced Stat activation. Here, we review the molecular mechanisms by which RA and IFNs can cooperate in inducing differentiation, inhibition of cell growth or viral replication focusing on recent results derived from normal and RA-resistant APL cells.

Retinoic acid resistance in NB4 APL cells is associated with lack of interferon alpha synthesis Stat1 and p48 induction.

In the t(15;17) acute promyelocytic leukaemia (APL), all trans-retinoic (RA) treatment induces maturation leading to clinically complete but not durable remission, as RA resistance develops in the treated patients as well as in vitro. RA and interferons (IFNs) are known inhibitors of proliferation in various cells including those from APL. In this report, we show that they can act cooperatively to inhibit growth and to induce differentiation of NB4 cells but not of two RA-resistant NB4 derived cell lines, NB4-R1 and NB4-R2. However, the resistant cell lines respond to IFN. In NB4 cells, RA increases the expression of Stat1, p48 and IRF-1, three transcription factors playing a central role in the IFN response and induces the synthesis and the secretion of IFN alpha. RA-induced IFN alpha seems to play a role in inhibition of NB4 cell growth but not in their differentiation. In the resistant cells, NB4-R1 and NB4-R2, both the induction of IFN and the increase of Statl and p48 expression by RA are completely blocked. In contrast, IRF-1 mRNA and protein expressions are induced in the three cell lines. This suggests that increase of IRF-1 expression is not sufficient for IFN induction. Our results identify some defects linked to RA-resistance in APL and support the hypothesis that RA-induced Stat1 expression and IFN secretion may be one of the mechanisms mediating growth inhibition by RA.

[Interferon and retinoic acid in the treatment of human cancer: mechanisms of action]. / L'interféron et l'acide rétinoïque dans le traitement des cancers humains: mécanismes d'action.

Retinoic acid (RA) and interferons (IFN) are negative regulators of cell proliferation. A number of clinical trials were thus carried out in cancer therapy with RA and/or IFN. In vitro and in vivo, their combination leads to a more potent cell growth inhibition. Moreover, RA and IFN act cooperatively to increase the expression of many IFN-stimulated genes, leading also to a higher cell differentiation and inhibition of viral replication. However, the molecular mechanisms by which RA and IFN potentiate each other are not fully understood. The cooperative effects by RA and IFN are mediated through multiple pathways. RA causes the induction and secretion of IFN alpha. RA also stimulates the IFN regulatory factor gene expression (IRF1 and p48). Additional mechanisms could be involved as RA increases the level of signal transducing activators of transcription (Stat) proteins, and thus enhances the IFN-induced Stat activation.

Resistance to virus infection conferred by the interferon-induced promyelocytic leukemia protein.

The interferon (IFN)-induced promyelocytic leukemia (PML) protein is specifically associated with nuclear bodies (NBs) whose functions are yet unknown. Two of the NB-associated proteins, PML and Sp100, are induced by IFN. Here we show that overexpression of PML and not Sp100 induces resistance to infections by vesicular stomatitis virus (VSV) (a rhabdovirus) and influenza A virus (an orthomyxovirus) but not by encephalomyocarditis virus (a picornavirus). Inhibition of viral multiplication was dependent on both the level of PML expression and the multiplicity of infection and reached 100-fold. PML was shown to interfere with VSV mRNA and protein synthesis. Compared to the IFN mediator MxA protein, PML had less powerful antiviral activity. While nuclear body localization of PML did not seem to be required for the antiviral effect, deletion of the PML coiled-coil domain completely abolished it. Taken together, these results suggest that PML can contribute to the antiviral state induced in IFN-treated cells.

Retinoic acid enhances the expression of interferon-induced proteins: evidence for multiple mechanisms of action.

Retinoic acid (RA) and interferons (IFNs) are negative regulators of cell proliferation. In vitro and in vivo, their combination leads to a more potent growth inhibition. However, the molecular mechanisms by which RA and IFNs potentiate each other are not fully understood. As some IFN-induced gene products regulate cell growth and/or antiviral activity, we analysed the effects of RA on their expressions. RA increases the level of 2'5'oligoadenylate synthetase, p68 kinase, the promyelocytic leukemia protein (PML) and Sp100 in both HL-60 and WISH cells. Moreover, RA and IFN act cooperatively to increase the expression of these proteins. RA also inhibits vesicular stomatitis virus replication and induces a higher antiviral state and growth inhibition when combined with IFN. RA stimulates the IFN regulatory factor 1 (IRF-1) gene expression directly through the GAS motif and causes the induction and secretion of IFNalpha. Additional mechanisms could be involved as RA increases the level of signal transducing activators of transcription (STAT) proteins, and enhances the IFN-induced STAT activation, suggesting that cooperative effects by RA and IFN are mediated through multiple pathways.

Effects of serotonin and melanin on in vitro HIV-1 infection.

In this article, we describe the effect of indoleamines: serotonin (5-HT) and synthetic soluble melanin, on the multiplication of HIV-1 in T4 lymphocytic cell lines. The results show that viral production is increased when infected CEM-11 cells are incubated with 5-HT (10(-7) M and 10(-8) M) for 72 hours, whereas at higher doses (10(-3) M and 10(-4) M), there is an inhibition of viral multiplication. As well, when infected CEM cells were cultured in the presence of 5-HT at 10(-4) M, during 15 days, virus production, syncytia formation and cytolytic effect were drastically inhibited. Melanin also inhibits HIV-1 cytopathic effect on MT-2 cells, without cell toxicity, at concentrations of 0.2-10 micrograms/ml. Syncytium formation and cell lysis were also blocked by melanin at concentrations of 0.1 to 10 micrograms/ml, when uninfected MT-2 cells were mixed with HIV-1 chronically infected CEM-11 cells.

Induction of the PML protein by interferons in normal and APL cells.

PML has been identified through its fusion to the RAR alpha gene in acute promyelocytic leukemia (APL). The PML protein is specifically associated to nuclear bodies (NBs) whose alterations in APL were proposed to contribute to leukemogenesis. The role of this nuclear domain (which also harbors the Sp100 autoantigen and the NDP52 protein) is unknown. Here, we show that the PML protein, like Sp100 and NDP52, is induced by interferons (IFNs alpha, beta and gamma) in a large variety of human cells. Interestingly, the NBs that contain the three IFN-induced proteins appear to be associated to speckles labelled by the IFN-mediator Mx1. These observations link NBs to IFN response pathways, which may contribute to the elucidation of the biological role of these structures. In APL cells, IFNs induced both PML and PML/RAR alpha expression, resulting in an increased sequestration of PML and RXRs in the microspeckles induced by the fusion protein. As PML has growth suppressing properties, it may mediate some of the antiproliferative effects of IFN. In APL, inactivation of PML may result in disruption of growth control.

Transcriptional induction of the PML growth suppressor gene by interferons is mediated through an ISRE and a GAS element.

PML is a nuclear matrix protein with growth suppressing properties, whose expression is deregulated during oncogenesis. Moreover, in the t(15;17) translocation of acute promyelocytic leukaemia (APL), PML fusion to the retinoic acid receptor alpha (RAR alpha) is the likely molecular basis of leukaemogenesis. Here we show that interferons (IFNs) alpha, beta, and gamma upregulate PML mRNA expression. Analysis of 5' genomic sequences of the PML gene revealed an IFN-alpha/-beta stimulated response element (ISRE) and an IFN-gamma activation site (GAS) in the untranslated first exon. Binding of IFN signal transducers and activators of transcription (STATs) was demonstrated to be weak for the PML GAS, but strong for the PML ISRE which also seemed to contribute substantially to the IFN-gamma response. Thus, PML is a primary target gene of IFNs and would appear as a suitable candidate for mediating some of their antiproliferative effects. Abnormalities of PML structure, localisation or expression in human malignancy, constitute examples of how an IFN target gene may be altered in oncogenesis.