Interferon Induction

Conceptually, it is important to distinguish IFN production and action (Figure 56-1). Viruses remain the prototypic inducers of IFN expression. All body cells probably have the capacity to produce IFN-α and IFN-β. IFN-γ was identified after exposure of lymphocytes to mitogens or sensitized lymphocytes to specific antigens. Interleukin-2 (IL-2), TNF (tumor necrosis factor), IL-12, IL-15 and a 15-kilodalton (kDa) protein induced by IFN-α and IFN-β are also potent inducers of IFN-γ—under some circumstances resulting in substantial titers.^5–7 Production of IFNs are part of host defense mechanisms for resistance to neoplasia; experimental suppression of IFN in mice results in increased lethality from tumors.⁸

Figure 56-1

Production of IFN-α or IFN-β is stimulated by viruses through a final common pathway of double-stranded RNA or, in the case of IFN-γ, exposure to specific antigens. Action of IFNs is mediated via binding to a specific receptor (more...)

The first chemically defined inducers of IFNs were double-stranded polyribonucleotides, which are potent IFN inducers and immunomodulators in mice but not in humans. Low-molecular-weight inducers of IFNs have been identified in different animal species and several low-molecular-weight inducers have been introduced into clinical trial.^9–12 Orally active inducers, such as the acridines, halopyrimidinones, or substituted quinolones, would be convenient and useful not only for therapeutic purposes but also as chemopreventive agents.¹³ Halopyrimidinones are clinically effective for low-grade transitional carcinomas of the bladder.¹²

Receptors

The cellular response to IFNs requires the interaction of only a small number of molecules with high affinity, species-specific multimeric cell surface receptors. The gene for the receptor for human IFN-α and IFN-β is on chromosome 21, whereas a gene on chromosome 6 codes for the human IFN-γ receptor. IFN receptors confer almost absolute species restrictions. IFN-α and IFN-β share and compete for the same receptor, although IFN-β binds with higher affinity and forms a more stable complex.^14,15 Although downregulation of receptors occurs with IFN-α2 administration, neither this event nor receptor number has clearly correlated with therapeutic effects in hematologic malignancies.^16,17

Studies on binding and cross-linking of radiolabeled IFNs to the cell surface suggest the existence of a multiunit structure for the receptor for IFNs-α and IFN-β. To elicit a cellular response, IFN-α and IFN-β require two subunits, IFNAR-1 and IFNAR-2. Human IFNAR-1 is a glycoprotein of 557 amino acids.¹⁸ A 515-amino-acid form of human IFNAR-2 has been identified as the universal ligand binding subunit of the IFN-α and IFN-β receptors. The solution structure of other members of the human cytokine receptor II family has enabled the three-dimensional structure of the extracellular domains of IFNAR-1 and -2 to be modeled. IFN-β shares only 30% identity with the IFN-α subtypes and appears to engage the receptor heterodimer differently. This can result in the greater activation by IFN-β of subsets of genes,^19–23 possibly through serine phosphorylation of STAT (signal transducers and activators of transcription)-1 and/or activation of other transcription factors.²¹

Signal Transduction

After receptor binding by IFN-α or IFN-β, specific tyrosine kinases, tyk2 (not part of the receptor structure per se), together with one or more additional tyrosine kinases, Janus-activated kinase (JAK)-1 and JAK-2, are phosphorylated. These activated tyrosine kinases activate the signal-transducing peptides²⁴ (Figure 56-2) and induce the formation of a complex of protein subunits (interferon-stimulated gene factor [ISGF]-3α) consisting of STAT-1α or STAT-1β and STAT-2. The proteins STAT-1α and STAT-1β are alternatively spliced products of the same gene. The STAT-1α protein contains at its carboxyl end 39 additional amino acids. The phosphorylated ISGF-3α complex is translocated to the nucleus and forms (with the addition of a fourth subunit p48 or IRF-9), a DNA-binding complex specific for the IFN-stimulated response element (ISRE). IFN-γ receptor activation results in a similar sequence of events, although the transcriptional regulatory complex consists of a homodimer of STAT-1 that binds to DNA elements termed gamma activated sites (GAS).

Figure 56-2

Pathway of signal transduction by IFNs. After binding to specific receptors, tyrosine kinases (JAK-1, JAK-2, Tyk2) are activated. These tyrosine kinases phosphorylate inactive transcription factors (STAT-1α, STAT-1β, STAT-2), which form (more...)

Other signaling cascades are also activated by IFNs, including activation of phosphatidylinositol-3-kinase and the mitogen-activated protein (MAP) kinase cascades.^21,25,26 For example, treatment of cells with IFN-α or IFN-β causes the transient release of arachidonic acid.^27,28 Inhibition of this enzyme blocks the formation of the transcription factor complex ISGF-3. Inhibitory also for action of IFNs are a family of suppressor of cytokine signaling (SOCS) proteins.^29,30 SOCS may be effectors of a negative feedback mechanism that shuts down response following IFN stimulation.

Induced Genes and Antitumor Actions

IFNs are pleiotropic cellular modulators (Table 56-3). Antitumor effects are postulated to result from either a direct effect on viability and proliferation or antigenic composition of tumor cells, or from an indirect effect on modulation of immune effector cell populations with tumor cell specificities. IFNs regulate gene expression, modulate expression of proteins on the cell surface, and induce synthesis of new enzymes. Alterations in gene expression result in modulation of levels of receptors for other cytokines, concentration of regulatory proteins on the surface of immune effector cells, and activities of enzymes that modulate cellular growth and function. On a cellular basis, these effects translate into alterations of the state of differentiation, rate of proliferation and cell death, and functional activity of many cell types.

Table 56-3

Pleiotropic Biologic Effects of Interferons.

A problem in attributing the effects of IFNs to specific gene products has been the evidence that they induce hundreds of genes. This has been confirmed by determining messenger RNA (mRNA) profiles from IFN-α, IFN-β, or IFN-γ treatments of the human fibrosarcoma cell line, HT1080, by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes.¹⁹ Until overexpression or deletion can manipulate these genes, delineation of their specific role in cellular response to IFNs can only be speculative. However, a number of genes induced by type I IFNs are involved in apoptosis, including protein kinase R (PKR), promyelocytic leukemia (PML), RAP46/Bag-1, phospholipid scramblase, and TNF-related apoptosis-inducing ligand (TRAIL).^19,20 Consequently, it is likely that apoptosis will be augmented by combinations of IFNs with apoptosis-inducing agents.³¹

The enzymes 2′5′-oligoadenylate synthetase (2′5′-A synthetase), a PKR, and indoleamine 2,3-dioxygenase (IDO) are among the enzymes induced by IFNs (see Table 56-3). 2′5′-A synthetases, a multiple isoform product of a three-gene family,³² are relatively specific markers of IFN system activation. A latent ribonuclease is activated by 2′5′-A synthetase; the result in part of induction of these enzymes is inhibition of protein synthesis. The level of the ribonuclease L (RNaseL) increased in growth-arrested cells and during cellular differentiation.³³ Apoptosis was suppressed in RNaseL null mice treated with different apoptotic agents.³⁴ Occurrence of elevated levels of RNaseL mRNA and enzymatic activity were detected in human colorectal carcinomas compared with the corresponding normal mucosa,³⁵ suggesting RNA turnover may be an important step in tumor progression. Germ line mutations in RNaseL are associated with familial prostate carcinoma, further validating the postulate that influence on RNA levels by RNaseL may be important in oncogenesis.³⁶

A constitutive serine-threonine kinase with a unique requirement for double-stranded RNA (dsRNA) (PKR) undergoes induction and activation by IFNs and dsRNAs.³⁷ The activated enzyme phosphorylates several cellular proteins including protein synthesis initiation factor 2a, which results in cessation of peptide chain initiation³⁷ and IκB, the inhibitor of the transcription factor, NFκB, resulting in activation of the latter.³⁸ The major function of this kinase is growth control; it can also induce apoptosis.³⁹ Cells expressing PKR mutants formed colonies in soft agar and upon injection into nude mice produced large tumors.⁴⁰ PKR and STAT-1 were both required for IFN-mediated downregulation of c-myc.⁴¹

PKR expression may be partially controlled by an IFN-induced transcription factor, IRF-1.⁴² Because IRF-1 increases rapidly in growth-arrested cells, it may influence expression of genes involved in negative control of cell growth and may mediate antiproliferative effects of IFNs. A second transcription factor, IRF-2, identified in murine cells, has sequence homology to IRF-1 and is a functional antagonist of IRF-1. Upon constitutive expression, IRF-2 can result in cell transformation, which is inhibited by IRF-1.⁴²

There are at least three families of GTPases (guanosine triphosphatases) that are induced by IFNs. The best characterized is the Mx family. The Mx proteins belong to the dynamin superfamily of large GTPases, proteins involved in endocytosis and vesicle transport.⁴³ Other families of GTPases are some of the most abundant proteins in IFN-treated human cells.⁴⁴

Several additional interferon-induced genes may influence apoptosis. Among these is the gene PML. PML knockout mice were used to demonstrate that PML is necessary for programmed cell death induced by IFN-α and IFN-β.⁴⁵ Phospholipid scramblase flips phosphatidylserine from the inner layer of the plasma membrane to the outer layer.⁴⁶ The appearance of phosphatidylserine on the outer layer of the cell membrane has been observed in apoptotic cells. In IFN-sensitive but not IFN-resistant melanoma cells, TRAIL/Ap02L was induced, as were other proteins implicated in apoptosis.^31,47 These IFN-stimulated proteins initiated the apoptotic cascade with alterations in mitochondrial membrane potential and changes in bcl-2 levels.^31,47,48 Molecular interactions between TRAIL and IFN suggest that these two molecules may synergize in induction of apoptosis.^47,49 IFNs downregulate expression of the multidrug resistance gene (mdr1) in human colon carcinoma cells, suggesting they might increase apoptosis after other cellular stresses.⁵⁰

Induced proteins and their products can be identified on cells and in serum of treated patients (Table 56-4). Their measurement or the quantitation of immune effector cell function can be used to define biologically active molecules, doses, schedules, and routes of administration. Most biologic response modulatory effects peak at 24 to 48 h, which contrasts with maximal serum levels in pharmacokinetic studies.^51,52 After intravenous bolus administration, the t _½ α of IFN-α2 is short (< 60 min); mean terminal half-life is 4 to 5 h with no serum levels measurable at 12 h. After intramuscular or subcutaneous administration, peak levels are 6 to 10 h.⁵³ The pharmacologic hallmark of IFN-β is virtual absence of serum levels with subcutaneous or intramuscular administration; yet, biologic response modulatory and therapeutic effects occur.⁵¹

Table 56-4

Modulation of Gene Expression by Interferons ^a.

Pegylated IFNs have markedly different kinetics than do unmodified IFNs.^54–56 Once-weekly administration has resulted in measurable serum levels at 7 days in excess of that required for gene induction and antiproliferative effects in vitro and has resulted in tumor responses in metastatic renal carcinoma and chronic myelogenous leukemia (CML). Although pegylated IFNs may be modestly better tolerated, it is their greater therapeutic effectiveness that has resulted in regulatory approvals in chronic active hepatitis.^57,58 Clinical effectiveness against a hepatitis C virus serotype previously unresponsive to unmodified IFN-α2,^57,58 suggest that the altered pharmacokinetics and pharmacodynamics result in a drug with different clinical actions. This greater effectiveness may be important in decreasing frequency of development of hepatocellular carcinoma.^59,60