Interferon-alpha induces transient upregulation of c-FLIP through NF-kappaB activation

Interferon-alpha (IFN-alpha) induces apoptosis in some cell types and promotes cell survival in other cell types, but the molecular mechanisms underlying distinct IFN-alpha-induced cell behaviours remain poorly understood. In the present study, we show that IFN-alpha induced the cellular FLICE (FADD-like interleukin-1 beta-converting enzyme) inhibitory protein (c-FLIP), which serves as a promoter of cell survival in human B lymphoma cells. IFN-alpha induction of transient upregulation of c-FLIP was partially abrogated by the NF-kappaB inhibitor BAY11-7082 (BAY). Pretreatment with BAY sensitized both Daudi and U266 cells to the IFN-alpha-induced loss of mitochondrial membrane potential (DeltaPsi(m)). IFN-alpha phosphorylated the PKC isoform PKCalpha at a threonine residue, and the PKCalpha/betaI inhibitor Go6976 abrogated upregulation of IFN-alpha-induced NF-kappaB activity, leading to sensitization of cells to IFN-alpha-induced apoptosis. To analyze the role of PKCalpha in the IFN-alpha-induced signaling, Daudi cells overexpressing a constitutively active mutant of PKCalpha (caPKCalpha) were used. The caPKCalpha-expressing Daudi cells were partially resistant to the IFN-alpha-induced loss of DeltaPsi(m), concomitant with elevated levels of c-FLIP protein. Together, these results demonstrate that IFN-alpha causes a transient upregulation of c-FLIP expression, at least through PKCalpha-mediated activation of NF-kappaB. The balance between IFN-alpha-induced pro-apoptotic and survival signals determines the cell fate. Thus, therapeutic intervention in this balance may be effective for treatment of patients with IFN-alpha-refractory tumours.     

Synergistic effects of phorbol ester and interferon-alpha: target cell class I HLA antigen expression and resistance to natural killer and lymphokine-activated killer cell-mediated cytolysis

This study was undertaken to investigate whether target cell class I HLA antigen expression induced by phorbol ester and interferon-alpha (IFN-alpha) was associated with resistance to natural killer (NK) cells and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Class I antigen expression on the surface of the K562 erythroleukemia cell line was enhanced by either IFN-alpha or phorbol ester (PDBu). Addition of PDBu together with IFN-alpha had a synergistic effect on class I antigen expression on the cells. Furthermore, synergism between IFN-alpha and PDBu was also found in class I antigen expression by MOLT-3 cells. This synergistic effect on class I antigen expression was blocked by the protein synthesis inhibitor (cycloheximide). Pretreatment of K562 cells with PDBu and IFN-alpha made them more resistant to lysis by NK and LAK cells than did either PDBu or IFN-alpha. In contrast to PDBu, 4 alpha PDD, a biologically inactive phorbol analogue, alone or combination with IFN-alpha, had no effect on class I antigen expression and susceptibility to lysis by NK and LAK cells. Kinetic experiments showed an inverse relationship between the expression of class I antigens and susceptibility to NK cell-mediated cytolysis. Using cold target competition analysis, target cells pretreated with PDBu and IFN-alpha clearly competed less effectively than did untreated cells for lysis of untreated target cells. These results demonstrate that target cells pretreated with PDBu and IFN-alpha decrease their sensitivity to natural killer and lymphokine-activated killer cells inversely with target cell class I HLA antigen expression.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA

The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.     

Interferon-alpha (Intron A) upregulates urokinase-type plasminogen activator receptor gene expression

The regulation of urokinase plasminogen activator receptor (uPAR) gene expression by interferon-alpha (IFN-alpha, or Intron A) and interferon-gamma (IFN-gamma) was studied in a HCT116 colon cancer cell line. uPAR mRNA levels were increased in a dose- and time-dependent manner in cells stimulated with IFN-alpha or IFN-gamma. uPAR protein levels reflected IFN-alpha and IFN-gamma induction of uPAR mRNA production. Cycloheximide, a protein synthesis inhibitor, also induced uPAR mRNA accumulation either alone or in combination with IFN-alpha or IFN-gamma, suggesting that the effect on uPAR mRNA levels activated by IFN-alpha or IFN-gamma does not require de novo protein synthesis. Both sodium butyrate and amiloride inhibited the uPAR mRNA levels induced by IFN-alpha or IFN-gamma. These results may provide useful information for the treatment of patients receiving IFN-alpha or IFN-gamma.     

Apo2L/TRAIL is an indirect mediator of apoptosis induced by interferon-alpha in human myeloma cells

Interferon-alpha (IFN-alpha) is currently used for the therapy of multiple myeloma (MM) though it is only effective in some patients. IFN-alpha induces apoptosis in some MM cell lines and it has been proposed to occur through an autocrine loop involving Apo2L/TRAIL. We have analysed the sensitivity to IFN-alpha and Apo2L/TRAIL of five MM cell lines and found no correlation between the apoptosis inducing ability of both cytokines. IFN-alpha-induced apoptosis in MM cells was not prevented by a caspase-8 selective inhibitor (Z-IETD-fmk) or blocking Apo2L/TRAIL. However, human monocytes treated with IFN-alpha release bioactive Apo2L/TRAIL to culture media which was cytotoxic for MM cells resistant to IFN-alpha. We propose that Apo2L/TRAIL released from IFN-alpha-stimulated blood monocytes would be a major mediator of the anti-myeloma effect of IFN-alpha in vivo.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Inhibition of CDK2, CDK4 and cyclin E and increased expression of p27Kip1 during treatment with interferon-alpha in carcinoid tumor cells

IFN-alpha has presented antitumor effect in about 50% of carcinoid tumor patients, though the antitumor mechanism of IFN-alpha is still to be elucidated. In this study we demonstrated that IFN-alpha could result in accumulation of S-phase population and upregulation of cyclin-dependent kinase inhibitor (CKI), p27. Moreover, IFN-alpha inhibits DNA synthesis assessed by [3H] thymidine incorporation and colony formation on soft agar. Immunodepletion of p27 increased CDK2- and cyclin E-associated kinase activities. These data suggest that IFN-alpha exerts its antiproliferative effects in the neuroendocrine differentiated cell lines by: 1) inhibition of DNA synthesis and colony formation, 2) upregulation on the mRNA and protein expressions of the CKI, p27.     

Inhibition of alpha interferon signaling by hepatitis B virus

Alpha interferon (IFN-alpha) and pegylated IFN-alpha (pegIFN-alpha) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-alpha treatment are not known. pegIFN-alpha is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-alpha signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-alpha signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-alpha resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-alpha signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.     

Antiviral activity of alpha interferon in Sindbis virus-infected cells is restored by anti-E2 monoclonal antibody treatment.

Pretreatment of AT3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2 (AT3-bcl-2 cells) with alpha interferon (IFN-alpha) affected replication of a virulent strain of Sindbis virus (SV) but did not protect against virus-induced cell death. Treatment of cells with IFN-alpha late during infection affected ongoing SV replication very little. Previous studies have shown that cross-linking of the viral glycoprotein E2 with antibody delays the inhibition of K+ influx by improving the function of Na+K+ATPase and the Na(+)-K(+)-2Cl-cotransport system in SV-infected cells (P. Després, J. W. Griffin, and D. E. Griffin, J. Virol. 69:7006-7014, 1995). In these studies, we have shown that treatment of infected cells with anti-E2 monoclonal antibody also restored the ability of IFN-alpha to induce antiviral activity in infected cells late during infection. The very low rate of virus release in SV-infected cells treated simultaneously with anti-E2 monoclonal antibody and IFN-alpha was postulated to be linked to inhibition of virus maturation. Synergistic effects of antibody and IFN-alpha are likely to be important for control of SV replication in vivo.     

Interferon-alpha promotes survival of human primary B-lymphocytes via phosphatidylinositol 3-kinase

Signaling pathways for the antiviral and antiproliferative biological effects of type I interferons (IFN) are well established. In this report we demonstrate a novel signaling pathway for IFN-alpha, as it induced rapid phosphorylation of both PKB/Akt and its substrate forkhead. The PI3-kinase inhibitor LY294002 abolished these phosphorylations. PI3-kinase has been implicated in cell survival mediating its effect through the second messenger PIP3 and the subsequent activation of PKB/Akt. We could show that IFN-alpha inhibited spontaneous apoptosis of primary B-lymphocytes, in the absence of a mitogenic stimulus. This effect was inhibited by LY294002. Thus, our data suggests that IFN-alpha promotes survival of peripheral B-lymphocytes via the PI3-kinase-PKB/Akt pathway. In addition, IFN-alpha stimulation of anti-IgM activated cells resulted in downregulated expression of the cell cycle inhibitor p27/Kip1.     

Regulation of synthesis and turnover of an interferon-inducible mRNA.

Regulation of synthesis and turnover of an interferon (IFN)-inducible mRNA, mRNA 561, in HeLa monolayer cells was studied. Cytoplasmic levels of this mRNA were estimated by hybridization analyses with a cDNA clone that we have isolated as a probe. IFN-alpha A induced a high level of this mRNA in a transient fashion, whereas no induction was observed in response to IFN-gamma. Surprisingly little mRNA 561 was induced in cells treated simultaneously with IFN-alpha A and an inhibitor of protein synthesis, suggesting that in addition to IFN-alpha A, an interferon-inducible protein was needed for induction of this mRNA. Apparently this putative protein could be induced by IFN-gamma as well. Thus, although little mRNA 561 was synthesized in cells treated either with IFN-gamma alone or with IFN-alpha A and cycloheximide, a large quantity of this mRNA was induced in cells which had been pretreated with IFN-gamma and then treated with IFN-alpha A and cycloheximide. Once mRNA 561 was induced by IFN-alpha A, it turned over rapidly. This rapid turnover could be blocked by actinomycin D or cycloheximide indicating that another IFN-inducible protein may mediate this process.     

Mechanism of apoptosis induced by IFN-alpha in human myeloma cells: role of Jak1 and Bim and potentiation by rapamycin

Interferon-alpha (IFN-alpha) has been used for the last 20 years in the maintenance therapy of multiple myeloma (MM), though it is only effective in some patients. Congruent with this, IFN-alpha induces apoptosis in some MM cell lines. Understanding the mechanism of IFN-alpha-induced apoptosis could be useful in establishing criteria of eligibility for therapy. Here we show that IFN-alpha-induced apoptosis in the MM cell lines U266 and H929 was completely blocked by a specific inhibitor of Jak1. The mTOR inhibitor rapamycin mitigated apoptosis in U266 but potentiated it in H929 cells. IFN-alpha induced PS exposure, DeltaPsi(m) loss and pro-apoptotic conformational changes of Bak, but not of Bax, and was fully prevented by Mcl-1 overexpression in U266 cells. IFN-alpha treatment caused the release of cytochrome c from mitochondria to cytosol and consequently, a limited proteolytic processing of caspases. Apoptosis induced by IFN-alpha was only slightly prevented by caspase inhibitors. Levels of the BH3-only proteins PUMA and Bim increased during IFN-alpha treatment. Bim increase and apoptosis was prevented by transfection with the siRNA for Bim. PUMA-siRNA transfection reduced electroporation-induced apoptosis but had no effect on apoptosis triggered by IFN-alpha. The potentiating effect of rapamycin on apoptosis in H929 cells was associated to an increase in basal and IFN-alpha-induced Bim levels. Our results indicate that IFN-alpha causes apoptosis in myeloma cells through a moderate triggering of the mitochondrial route initiated by Bim and that mTOR inhibitors may be useful in IFN-alpha maintenance therapy of certain MM patients.     

The p38 MAPK pathway mediates the growth inhibitory effects of interferon-alpha in BCR-ABL-expressing cells

The mechanisms by which interferon-alpha (IFN-alpha) mediates its anti-leukemic effects in chronic myelogenous leukemia (CML) cells are not known. We determined whether p38 MAPK is activated by IFN-alpha in BCR-ABL-expressing cells and whether its function is required for the generation of growth inhibitory responses. IFN-alpha treatment induced phosphorylation/activation of p38 in the IFN-alpha-sensitive KT-1 cell line, but not in IFN-alpha-resistant K562 cells. Consistent with this, IFN-alpha treatment of KT-1 (but not K562) cells induced activation of the small GTPase Rac1, which functions as an upstream regulator of p38. In addition, IFN-alpha-dependent phosphorylation/activation of p38 was induced by treatment of primary granulocytes isolated from the peripheral blood of patients with CML. To define the functional role of the Rac1/p38 MAPK pathway in IFN-alpha signaling, the effects of pharmacological inhibition of p38 on the induction of IFN-alpha responses were determined. Treatment of KT-1 cells with the p38-specific inhibitors SB203580 and SB202190 reversed the growth inhibitory effects of IFN-alpha. On the other hand, the MEK kinase inhibitor PD098059 had no effects, further demonstrating the specificity of these findings. To directly determine the significance of IFN-alpha-dependent activation of p38 in the induction of the anti-leukemic effects of IFN-alpha, we evaluated the effects of p38 inhibition on leukemic colony formation in bone marrow samples of patients with CML. IFN-alpha inhibited leukemic granulocyte/macrophage colony formation in a dose-dependent manner, whereas concomitant treatment with p38 inhibitors reversed such an inhibition. Thus, the Rac1/p38 MAPK pathway is activated by IFN-alpha in BCR-ABL-expressing cells and appears to play a key role in the generation of the growth inhibitory effects of IFN-alpha in CML cells.     

Ebola virus VP24 binds karyopherin alpha1 and blocks STAT1 nuclear accumulation

Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-alpha/beta) and the ability of cells to respond to IFN-alpha/beta or IFN-gamma. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-alpha/beta production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-alpha/beta and IFN-gamma signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-alpha/beta and IFN-gamma signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with karyopherin alpha1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin alpha2, alpha3, or alpha4. Overexpression of VP24 results in a loss of karyopherin alpha1-PY-STAT1 interaction, indicating that the VP24-karyopherin alpha1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.     

Conventional protein kinase C inhibition prevents alpha interferon-mediated hepatitis C virus replicon clearance by impairing STAT activation

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. The only treatment available for HCV infections, alpha interferon (IFN-alpha), is effective in a limited percentage of patients. The mechanisms by which IFN-alpha interferes with the HCV life cycle and the reasons for limited effectiveness of IFN-alpha therapy have not yet been fully elucidated. Using a cell-based HCV replication system and specific kinase inhibitors, we examined the role played by various signaling pathways in the IFN-alpha-mediated HCV clearance. We reported that conventional protein kinase C (cPKC) activity is important for the effectiveness of IFN-alpha treatment. In cells treated with a cPKC-specific inhibitor, IFN-alpha failed to induce an efficient HCV RNA degradation. The lack of cPKC activity leads to a broad reduction of IFN-alpha-stimulated gene expression due to a significant impairment of STAT1 and STAT3 tyrosine phosphorylation. Thus, modulation of cPKC function by either host or viral factors could influence the positive outcome of IFN-alpha-mediated antiviral therapies.