Kathryn C. Zoon

Clinical proteomics: translating benchside promise into bedside reality

The ultimate goal of proteomics is to characterize the information flow through protein networks. This information can be a cause, or a consequence, of disease processes. Clinical proteomics is an exciting new subdiscipline of proteomics that involves the application of proteomic technologies at the bedside, and cancer, in particular, is a model disease for studying such applications. Here, we describe proteomic technologies that are being developed to detect cancer earlier, to discover the next generation of targets and imaging biomarkers, and finally to tailor the therapy to the patient.

Regulation of interferon production by human monocytes : requirements for priming for lipopolysaccharide-induced production

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS‐stimulated macrophages is interferon (IFN‐α/β). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter‐current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly‐l,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte‐macrophage colony stimulating factor (GM‐CSF) or interferon‐γ (IFN‐γ). The IFN secreted is of the α subtype. Monocytes primed with GM‐CSF or IFN‐γ also maintained LPS responses for production of tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1). M‐CSF did not prime monocytes for LPS‐induced IFN production, although it did enhance production of TNF‐α and promoted monocyte survival. Northern analysis indicated that the induction of IFN‐α by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN‐α by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.

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.

Human interferon alpha enters cells by receptor-mediated endocytosis

A small number of Escherichia coli-derived human interferon alpha A molecules (up to approximately 800/cell) bind specifically to high-affinity cell surface receptors on bovine kidney cells at 4 degrees. When the cells are subsequently warmed to 37 degrees, the amount of surface-bound interferon alpha (IFN-alpha) as recognized by a radiolabeled monoclonal antibody rapidly decreases. Using 125I-IFN-alpha and treatment of cells with acetic acid/sodium chloride to remove surface-bound IFN, a similar decrease of surface-bound IFN and a corresponding increase in intracellular radiolabeled material is observed following the temperature shift. An analysis of the trichloroacetic acid precipitability of the radiolabeled material in the medium, removed by acid treatment or internalized, shows that IFN is degraded intracellularly and that its degradation products are then released into the medium. The process of uptake, degradation, and release of degraded material can be inhibited by the lysosomotropic agent chloroquine. A stable conjugate of IFN with 5-nm colloidal gold was prepared without a detectable loss of antiviral activity. As shown by transmission electron microscopy, the conjugate, bound to cells at 4 degrees, was found in clathrin-coated pits and later in receptosomes following a temperature shift to 37 degrees. Morphometric quantitation showed that an excess of native IFN added during binding of the conjugate in the cold reduced the appearance of conjugate in receptosomes by 80%. These studies demonstrate that at least a portion of receptor-bound IFN enters cells by receptor-mediated endocytosis.

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.

IRF9 is a key factor for eliciting the antiproliferative activity of IFN-α

A number of tumors are still resistant to the antiproliferative activity of human interferon (IFN)-α. The Janus kinases/Signal Transducers and Activators of Transcription (JAK-STAT) pathway plays an important role in initial IFN signaling. To enhance the antiproliferative activity of IFN-α, it is important to elucidate which factors in the JAK-STAT pathway play a key role in eliciting this activity. In human ovarian adenocarcinoma OVCAR3 cells sensitive to both IFN-α and IFN-γ, only IFN regulatory factor 9 (IRF9)-RNA interference (RNAi) completely inhibited the antiproliferative activity of IFN-α among the intracellular JAK-STAT pathway factors. Conversely, Stat1-RNAi did not inhibit the antiproliferative activity of IFN-α, whereas it partially inhibited that of IFN-γ. As a cell death pathway, it is reported that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through TRAIL-receptor (R) 1 and TRAIL-R2. In IFN-α-treated OVCAR3 cells, IRF9-RNAi inhibited transcription of TRAIL whereas Stat1-RNAi did not, suggesting that the transcription of TRAIL induced by IFN-α predominantly required IRF9. Furthermore, IFN-stimulated response element-like motifs of TRAIL bound to IFN-stimulated gene factor 3 (ISGF3) complex after IFN-α treatment. Subsequently, TRAIL-R2-RNAi inhibited both antiproliferative activities of IFN-α and TRAIL, suggesting that TRAIL-R2 mediated both IFN-α and TRAIL signals to elicit their antiproliferative activities. Finally, IRF9 overexpression facilitated IFN-α-induced apoptosis in T98G (human glioblastoma multiforme) cells, which were resistant to IFN-α. Thus, this study suggests that IRF9 is the key factor for eliciting the antiproliferative activity of IFN-α and TRAIL may be one of the potential mediators.

Large-Scale Production and Concentration of Human Lymphoid Interferon

A stable and predictable production system is described for pilot plant quantities (milligram) of human lymphoid interferon, using suspension culture of an African Burkitts lymphoma derived cell line Namalva with induction by Newcastle disease virus, B-1 strain. Cell cultures were grown in impeller-driven 50-liter fermentors with dilution of the postinduction culture using serum-free medium. High levels of dissolved oxygen were necessary for optimum cell growth. A total of 4,207 liters of interferon culture was produced in a series of 116 fermentor runs. An average yield of 3.5 log10 international units of interferon per ml was realized before processing. Trichloroacetic acid was used to precipitate the interferon. An average of 3.35 log10 international units of interferon per ml was recovered in the final nonpurified product.

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.

Interferon Fever: Absence of Human Leukocytic Pyrogen Response to Recombinant α‐Interferon

A sensitive in vitro assay for generation of human leukocytic pyrogen has been used to study the pathogenesis of fever accompanying administration of human α‐interferon. Unlike other potent pyrogens, two recombinant interferon preparations tested over a wide concentration range did not stimulate release of leukocytic pyrogen. This result suggests that interferon may cause fever by a novel mechanism not dependent on leukocytic pyrogen.

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

Clinical applications of human interferon (IFN)-&agr; have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-&agr; have not been clearly identified. Our previous study indicated that IFN (&agr;, &bgr;, and &ohgr;) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-&agr; treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-&agr;. 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-&agr; treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-&agr; 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-&agr; and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-&agr; and TRAIL from collapsing the mitochondrial membrane potential (&Dgr;&PSgr;m). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-&agr; or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-&agr;. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-&agr; which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-&agr; resistant tumors.