Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid-mediated apoptosis through Bak activation in ovarian cancer cells.

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.

Structural variants of IFNα preferentially promote antiviral functions.

IFNα, a cytokine with multiple functions in innate and adaptive immunity and a potent inhibitor of HIV, exerts antiviral activity, in part, by enhancing apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (APOBEC3) family members. Although IFNα therapy is associated with reduced viral burden, this cytokine also mediates immune dysfunction and toxicities. Through detailed mapping of IFNα receptor binding sites, we generated IFNα hybrids and mutants and determined that structural changes in the C-helix alter the ability of IFN to limit retroviral activity. Selective IFNα constructs differentially block HIV replication and their directional magnitude of inhibition correlates with APOBEC3 levels. Importantly, certain mutants exhibited reduced toxicity as reflected by induced indoleamine 2,3-dioxygenase (IDO), suggesting discreet and shared intracellular signaling pathways. Defining IFN structure and function relative to APOBEC and other antiviral genes may enable design of novel IFN-related molecules preserving beneficial antiviral roles while minimizing negative effects.

Potent antitumor effects of combination therapy with IFNs and monocytes in mouse models of established human ovarian and melanoma tumors.

Interferon-activated monocytes are known to exert cytocidal activity against tumor cells in vitro. Here, we have examined whether a combination of IFN-α2a and IFN-γ and human monocytes mediate significant antitumor effects against human ovarian and melanoma tumor xenografts in mouse models. OVCAR-3 tumors were treated i.t. with monocytes alone, IFN-α2a and IFN-γ alone or combination of all three on day 0, 15 or 30 post-tumor implantation. Mice receiving combination therapy beginning day 15 showed significantly reduced tumor growth and prolonged survival including complete regression in 40% mice. Tumor volumes measured on day 80 in mice receiving combination therapy (206 mm(3)) were significantly smaller than those of mice receiving the IFNs alone (1,041 mm(3)), monocytes alone (1,111 mm(3)) or untreated controls (1,728 mm(3)). Similarly, combination therapy with monocytes and IFNs of much larger tumor also inhibited OVCAR-3 tumor growth. Immunohistochemistry studies showed a large number of activated macrophages (CD31(+)/CD68(+)) infiltrating into OVCAR-3 tumors and higher densities of IL-12, IP10 and NOS2, markers of M1 (classical) macrophages in tumors treated with combination therapy compared to the controls. Interestingly, IFNs-activated macrophages induced apoptosis of OVCAR-3 tumor cells as monocytes alone or IFNs alone did not mediate significant apoptosis. Similar antitumor activity was observed in the LOX melanoma mouse model, but not as profound as seen with the OVCAR-3 tumors. Administration of either mixture of monocytes and IFN-α2a or monocytes and IFN-γ did not inhibit Lox melanoma growth; however, a significant inhibition was observed when tumors were treated with a mixture of monocytes, IFN-α2a and IFN-γ. These results indicate that monocytes and both IFN-α2a and IFN-γ may be required to mediate profound antitumor effect against human ovarian and melanoma tumors in mouse models.

Clinical model: interferons activate human monocytes to an eradicative tumor cell level in vitro.

Eradicative levels of antitumor activity by cytokines and leukocytes have not yet been reached experimentally and are needed clinically. Only a limited number of human cancers respond to therapy with interferon (IFN), other cytokines, or mononuclear leukocytes despite significant antitumor activity in vitro. We studied the IFN and monocytic cell conditions that would lead to an eradicative effect using human cells in vitro. Targets of the IFN-activated monocytic cells were either four human tumor cell lines (human osteosarcoma [HOS], LOX melanoma, A549 lung tumor, and SNB-19 glioblastoma) or two diploid cell lines (WI38 and MRC5). An average of 30-90 colony-forming tumor target cells were cultured overnight in 96-well tissue culture plates prior to treatment with serially diluted IFN with or without activated elutriation-purified monocytes or lymphocytes. The target cell colonies were treated for 3 days. The colonies were then stained with crystal violet to determine the levels of antitumor activity. IFN-activated human monocytes reached an eradicative level (95%-100%) against three of four tumor cell lines. The eradicative level (1) was induced best in human monocytes activated by combined type I and II IFNs, (2) was effective against tumor cells that were growing for 24 h, (3) was specific for human tumors, as diploid human cells were not inhibited, and (4) required contact between the macrophage and the tumor cells. Also, for the first time, the minimal effective concentration (MEC) of IFNs to activate monocytes can approach those needed for antiviral activity. To our knowledge, this is the first report of near total eradication of many tumor cells, but not diploid cells, by IFN-activated monocytes. Because of its potency and specificity, the IFN-activated monocyte arm of the innate immune system may be a candidate for therapy of established tumors.

Protein engineering of interferon alphas.

Interferon (IFN)-alphas constitute a family of proteins exhibiting high degree of homology in primary, secondary, and tertiary structure and display a high level of species specificity in their biological properties. However, small structural differences in these proteins may be responsible for a significant variety of biological actions. Understanding the structure and function of human IFN-alpha is very important. Recombinant techniques are important tools for the production and modification of IFN proteins. The first IFN hybrid, IFN-alpha1/alpha2 was constructed using recombinant technology in 1981. Subsequently, a number of IFN hybrids and mutants have been constructed, expressed and characterized. These hybrids and mutants have resulted in novel IFNs that either combine different biological properties from the parental proteins or have significantly different biological activity. Therefore, IFN hybrids and mutants have provided a powerful tool for studying the structure and function of these molecules. Also, these engineered IFNs may have important new therapeutic applications and may provide greater sights into understanding of the clinical activities of these molecules.

Amino acid substitutions in loop BC and helix C affect antigenic properties of helix D in hybrid IFN-alpha21a/alpha2c molecules.

We compared the antigenic properties of human interferon-alpha2c (IFN-alpha2c), IFN-alpha21a, hybrids IFN-alpha21a/alpha2c, and their mutants, using a panel of 27 anti-IFN-alpha1, anti-IFN-alpha2, and anti-IFN-alpha8/1/8 monoclonal antibodies (mAb). After immunoanalysis by ELISA, we found parental IFN-alpha2c and IFN-alpha21a to be antigenically distinct. Lack of reactivity of anti-IFN-alpha1 mAb with IFN-alpha21a indicated an antigenic distinction between subtypes alpha1 and alpha21a. The antigenic properties of hybrid IFNs consisting of the N-terminal portion (1-75) of IFN-alpha21a and the C-terminal portion (76-166) of IFN-alpha2c were analyzed with mAb recognizing defined regions of IFN-alpha2c, IFN-alpha1, and IFN-alpha8/1/8. We found that extending the sequence of IFN-alpha21a up to position 95 in hybrid molecule decreased the immunoreactivity of mAb specific for the antigenic structure formed by residues --112-132-- (helix D) of IFN-alpha2c. Inserting the sequence 76-81 (loop BC) of IFN-alpha2c into the sequence of 1-95 of IFN-alpha21a restored the reactivity of anti-IFN-alpha2c mAb. Some amino acid substitutions at positions 86 and 90 (helix C) of hybrid IFN-alpha21a/alpha2c also affected the immunoreactivity of C-terminal-specific mAb, which recognize helix D, but did not influence the structure of C-terminus of IFN (aa 151-165). Changes in the structure of constructs affected not only their antiproliferative activity but also their antiviral activity on human cells.

New function of type I IFN: induction of autophagy.

Autophagy is a highly conserved cellular process responsible for recycling of intracellular material. It is induced by different stress signals, including starvation, cytokines, and pathogens. Type I interferons (IFN) are proteins with pleiotropic functions, such as antiviral, antiproliferative, and immunomodulatory activities. Several recent studies showed type I IFN-induced autophagy in multiple cancer cell lines as evidenced by autophagic markers, for example, the conversion of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B, also known as LC3-I) to LC3-II and the formation of autophagosomes by electron microscopy. In addition, studies suggest the involvement of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT) and mechanistic target of rapamycin, serine/threonine kinase (mTOR) pathways in the induction of autophagy. This review highlights a new function of type I IFN as an inducer of autophagy. This new function of type I IFN may play an important role in viral clearance, antigen presentation, inhibition of proliferation, as well as a positive feedback loop for the production of type I IFN.

Immunomodulatory effects of interferons in malignancies.

Investigation of the antitumor and immunomodulatory activities of interferon (IFN) began shortly after the cytokine was discovered in 1957. Early work showed a direct correlation between administration of IFN and inhibition of symptoms associated with virally induced leukemia in mice as well as an increase in their survival time. Subsequent studies with purified IFNs confirmed the direct and indirect stimulation of immune cells, resulting in antitumor activities of IFN. Clinically, IFN-alphas (αs) have been shown to have activity against a variety of tumors. Initially, the U.S. Food and Drug Administration licensed 2 recombinant IFN-αs for the treatment of hairy-cell leukemia and then later for several other cancers. The success rate seen with IFNs and certain tumors has been varied. Unfortunately, some neoplasms show no response to IFN. Monocytes/macrophages play an important role in cancer progression. Monocytes in combination with IFN may be an important therapy for several cancers. This article focuses on the role of IFN and monocytes alone or in combination in affecting malignancies.

Type I interferons induce autophagy in certain human cancer cell lines.

Autophagy is an evolutionarily conserved cellular recycling mechanism that occurs at a basal level in all cells. It can be further induced by various stimuli including starvation, hypoxia, and treatment with cytokines such as IFNG/IFNγ and TGFB/TGFß. Type I IFNs are proteins that induce an antiviral state in cells. They also have antiproliferative, proapoptotic and immunomodulatory activities. We investigated whether type I IFN can also induce autophagy in multiple human cell lines. We found that treatment with IFNA2c/IFNα2c and IFNB/IFNß induces autophagy by 24 h in Daudi B cells, as indicated by an increase of autophagy markers MAP1LC3-II, ATG12-ATG5 complexes, and a decrease of SQSTM1 expression. An increase of MAP1LC3-II was also detected 48 h post-IFNA2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines exposed to type I IFN was confirmed by electron microscopy analysis. Increased expression of autophagy markers correlated with inhibition of MTORC1 in Daudi cells, as well as inhibition of cancer cell proliferation and changes in cell cycle progression. Concomitant blockade of either MTOR or PI3K-AKT signaling in Daudi and T98G cells treated with IFNA2c increased the level of MAP1LC3-II, indicating that the PI3K-AKT-MTORC1 signaling pathway may modulate IFN-induced autophagy in these cells. Taken together, our findings demonstrated a novel function of type I IFN as an inducer of autophagy in multiple cell lines.

BID is a critical factor controlling cell viability regulated by IFN-α.

Clinical applications of human interferon (IFN)-α have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-α have not been clearly identified. Our previous study indicated that IFN (α, ß, and ω) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-α treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-α. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-α treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-α notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-α and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-α and TRAIL from collapsing the mitochondrial membrane potential (ΔΨm). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-α or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-α. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-α resistant tumors.

Near eradication of clinically relevant concentrations of human tumor cells by interferon-activated monocytes in vitro.

We have previously reported that low concentrations of interferon (IFN)-activated monocytes exert near-eradicative cytocidal activity against low concentrations of several human tumor cells in vitro. In the present study, we examined 7 human tumor cell lines and 3 diploid lines in the presence or absence of 10 ng/mL IFNα2a and monocytes. The results confirmed strong cytocidal activity against 4 of 7 tumor lines but none against 3 diploid lines. To model larger in vivo tumors, we increased the target cell concentration and determined the concentration of IFNα2a and monocytes, required for cell death. We found that increasing the tumor cell concentration from 10- to 100-fold (10(5) cells/well) required an increase in the concentration of IFNs by over 100-fold and monocytes by 10-fold. High concentrations of monocytes could sometimes kill tumor or diploid cells in the absence of IFN. We may conclude that killing of high concentrations of tumor or diploid cells required high concentrations of monocytes that could sometimes kill in the absence of IFN. Thus, high concentrations of tumor cells required high concentrations of IFN and monocytes to cause near eradication of tumor cells. These findings may have clinical implications.

Antiproliferative Properties of Type I and Type II Interferon.

The clinical possibilities of interferon (IFN) became apparent with early studies demonstrating that it was capable of inhibiting tumor cells in culture and in vivo using animal models. IFN gained the distinction of being the first recombinant cytokine to be licensed in the USA for the treatment of a malignancy in 1986, with the approval of IFN-α2a (Hoffman-La Roche) and IFN-α2b (Schering-Plough) for the treatment of Hairy Cell Leukemia. In addition to this application, other approved antitumor applications for IFN-α2a are AIDS-related Kaposi's Sarcoma and Chronic Myelogenous Leukemia (CML) and other approved antitumor applications for IFN-α2b are Malignant Melanoma, Follicular Lymphoma, and AIDS-related Kapoisi's Sarcoma. In the ensuing years, a considerable number of studies have been conducted to establish the mechanisms of the induction and action of IFN's anti-tumor activity. These include identifying the role of Interferon Regulatory Factor 9 (IRF9) as a key factor in eliciting the antiproliferative effects of IFN-α as well as identifying genes induced by IFN that are involved in recognition of tumor cells. Recent studies also show that IFN-activated human monocytes can be used to achieve >95% eradication of select tumor cells. The signaling pathways by which IFN induces apoptosis can vary. IFN treatment induces the tumor suppressor gene p53, which plays a role in apoptosis for some tumors, but it is not essential for the apoptotic response. IFN-α also activates phosphatidylinositol 3-kinase (PI3K), which is associated with cell survival. Downstream of PI3K is the mammalian target of rapamycin (mTOR) which, in conjunction with PI3K, may act in signaling induced by growth factors after IFN treatment. This paper will explore the mechanisms by which IFN acts to elicit its antiproliferative effects and more closely examine the clinical applications for the anti-tumor potential of IFN.

Attenuating mutations in the P/C gene of human parainfluenza virus type 1 (HPIV1) vaccine candidates abrogate the inhibition of both induction and signaling of type I interferon (IFN) by wild-type HPIV1.

Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (Delta) mutations in the P/C gene (r-CDelta10-15HNT553A, r-CR84G, r-CF170S and r-CDelta170), which have previously been evaluated as HPIV1 vaccine candidates, were evaluated for their effect on the type I interferon (IFN) response in vitro. HPIV1 wt infection inhibited the IFN response by inhibiting IFN regulatory factor-3 (IRF-3) activation and IFN production in A549 cells and IFN signaling in Vero cells. In contrast, r-CR84G, r-CF170S and r-CDelta170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-CDelta170 did not inhibit IFN signaling. Thus, HPIV1 antagonizes the IFN response at both the level of induction and signaling, and antagonism at both levels was disrupted by mutations in the P/C gene. Because CF170S affects C and not P, the anti-IFN function can be attributed to the C proteins. These data, in the context of previous in vivo studies, suggest that the loss of antagonism of the IFN response at both the level of induction and signaling, observed with the P/C mutants, r-CF170S and r-CDelta170, was necessary for significant attenuation in African green monkeys (AGMs).

Gateway cloning is compatible with protein secretion from Pichia pastoris.

Secretion of a recombinant protein from the yeast Pichia pastoris requires the presence of a signal peptide at the amino terminus. Maintaining the full amino acid sequence of the signal peptide is thought to be important for proper signal processing and protein secretion. We show that at least for one protein, a synthetic human interferon, the presence of a Gateway recombination site within the signal peptide is fully compatible with high levels of protein secretion. The amino termini of the secreted interferon proteins cloned with Gateway and cloned with restriction enzymes and ligase are identical, and the proteins were highly active in biological assays. Compatibility with Gateway cloning simplifies construction of plasmids directing secretion of recombinant proteins from P. pastoris.

Human interferons alpha, beta and omega.

Type I interferons (IFNs), IFN-alpha, IFN-beta, IFN-omega, IFN-delta and IFN-tau are a family of structurally related, species-specific proteins found only in vertebrates. They exhibit a variety of biological functions, including antiviral, antiproliferative, immunomodulatory and developmental activities. Human Type I IFNs interact with the human IFN alpha receptor (IFNAR), which is composed of two identified subunits (IFNAR-1 and IFNAR-2). The interaction of IFN-alpha/beta with its receptor components results in the activation of a number of signaling pathways. The regulation of specific genes and proteins contributes to the numerous biological functions of Type I IFNs.