Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24.

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

Immune modulation alters brain opioid activities: Evidence for a role of muramyl dipeptide in the process of neuroimmunemodulation

During the last twenty years a scientific basis for the anecdotal reports of an interaction between the brain and the immune system has established neuroimmunemodulation as a new field of study in the biomedical sciences. A means for the brain to exert a regulatory influence upon various lymphoid reactions has been well established by many investigators world wide. This dissertation was geared to test the central hypothesis that the immune system, in turn, produces signals which affect CNS functions. Specifically, it is shown through several different experiments, behavioral and electrophysiologic, that the immune modifiers interferon-alpha, gamma irradiation, cyclosporine-A and muramyl-dipeptide modify brain opioid related activities. Each agent attenuates naloxone-precipitated morphine withdrawal following either systemic or intracranial injection. Each agent also has effects upon either the acute antinociceptive or hypothermic activities of morphine. Finally, each agent modifies baseline evoked electrical activity of several brain areas of awake freely-behaving rats. Later studies demonstrate that muramyl-dipeptide modifies the unit firing rate of single neurons in the brain following either systemic or localized administration within the brain. These results suggest that the immune system produces signals which affect brain activity; and thus, support the contention of a bi-directional interaction between the brain and the immune system. ^

Synthesis and properties of heterobifunctional photoaffinity reagents for the identification and isolation of receptors

A method employing isotopically- and photoaffinity-labeled probes and polyclonal and monoclonal antibody to the probes for the identification, isolation and recovery of protein receptors is described. Antibody was raised against N-(3-(p-azido-m-($\sp{125}$I) -iodophenyl)) propionate (AIPP) coupled to and photolyzed to BSA. The antibodies specifically bound AIPP-derivatized proteins. An isolation system was developed utilizing this probe and two antigenically identical reversible analogues. N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl)propionyl)amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) reacts with primary amines and N-(((3-p-azido-m-($\sp{125}$I) -iodophenyl)propionyl)amidoethyl)dithiopyridine ($\sp{125}$I-AIPP-PDA) reacts with reduced thiols. The applicability of the system was established by derivatizing known ligands (Transferrin and Interferon-alpha) with one of the probes. The ligand-probe was then allowed to interact with its receptor by incubation with SS5 lymphoma cells and cross-linked by photolysis at 300 nm. The photolyzed ligand/probe/receptor preparation was then recovered with AIPP antibody. Utilization of N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl-propionyl)-amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) allowed the components of the photolyzed complex to be separated by treatment with 2-mercaptoethanol in the SDS-PAGE solubilization buffer. Ligand and receptor labeling were then assessed by Coomassie staining and autoradiography. Results of receptor assays suggest that $\sp{125}$I-AIPP was, indeed, transferred to moieties that represent the receptors for both Transferrin and Interferon-alpha. ^

Regulation of angiogenesis by interferon-beta

The purpose of these studies was to investigate the role of interferon-beta (IFN-$\beta$) in angiogenesis. IFN-$\alpha/\beta$ have been implicated in inhibiting a number of steps in the angiogenic pathway. We examined the balance of angiogenesis-regulating molecules in several systems including human infantile hemangiomas, UV-B irradiated mice, and dorsal incisional wound healing in mice. In each system, epidermal hyperplasia and cutaneous angiogenesis were directly related to the expression of positive angiogenic factors (bFGF and VEGF) and inversely related to the expression of endogenous IFN-$\beta.$ The re-expression of IFN-$\beta$ correlated with tumor regression and/or resolution of wound healing. In contrast to control mice, UV-B-induced cutaneous angiogenesis and hyperplasia persisted in IFN-$\alpha/\beta$ receptor knock-out mice. In normal mice, endogenous IFN-$\beta$ was expressed by all differentiated epithelial cells exposed to environmental stimuli. The expression of endogenous IFN-$\beta$ was necessary but insufficient for complete differentiation of epidermal keratinocytes.^ The tumor organ microenvironment can regulate angiogenesis. Human bladder carcinoma cells growing in the bladder wall of nude mice express high levels of bFGF, VEGF, and MMP-9, have higher vascular densities, and produce metastases to lymph nodes and lungs, whereas the same cells growing subcutaneously express less bFGF, VEGF, and MMP-9, have lower vascular densities, and do not metastasize. IFN-$\alpha/\beta$ was found to inhibit bFGF and MMP-9 expression both in vitro and in vivo in human bladder carcinoma cells. Systemic therapy with human IFN-$\alpha$ of human bladder cancer cells growing orthotopically in nude mice, resulted in decreased vascularity, tumorigenicity, and metastasis as compared to saline treated mice. Human bladder cancer cells resistant to the antiproliferative effects of IFN were transfected with the human IFN-$\beta$ gene. Hu-IFN-$\beta$ transfected cells expressed significantly less bFGF protein and gelatinase activity than parental or control-transfected cells and did not grow at ectopic or orthotopic sites. Collectively the data provide direct evidence that IFN-$\alpha/\beta$ can inhibit angiogenesis via down-regulation of angiogenesis-stimulating cytokines. ^