Resistance to the antiproliferative activity of IFN-alpha: further characterization and demonstration of antagonistic effects of IFN-gamma

Mouse B16 melanoma cells have been shown to rapidly develop resistance to the antiproliferative effects of MuIFN-alpha or MuIFN-beta when exposed to these interferons. In cloning studies, the maximal antiproliferative effects of MuIFN-alpha were seen with 2-4 days treatment. This resistance has been further characterized. The level of resistance which develops in B16 melanoma cells is dependent upon the concentration of MuIFN-alpha to which the cells are exposed. In addition, B16 melanoma cells which are resistant to the antiproliferative effects of MuIFN-alpha have greatly elevated levels of the interferon-induced enzyme 2',5'-oligoadenylate (2-5A) synthetase. Since it has previously been shown that B16 melanoma cells do not develop resistance to the antiproliferative effects of MuIFN-gamma, several experiments studied the influence of MuIFN-gamma on the development of resistance to MuIFN-alpha. Combinations of IFN-gamma and IFN-alpha have previously been shown to result in a synergistic enhancement of the antiproliferative effects. Kinetic studies show that the response of the cells to the MuIFN-gamma antiproliferative effect appears to be dominant over the development of resistance since no resistance develops in response to combination treatment. Not only is MuIFN-gamma able to prevent development of resistance when it is present continuously, but also when it is used for the sequential treatment of the cells before their exposure to MuIFN-alpha. A 2-day pretreatment with MuIFN-gamma is sufficient to prevent the development of resistance during later exposure of the cells to MuIFN-alpha alone for up to 6 additional days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell killing

The effects of mouse interferon-alpha (MuIFN-alpha), -beta (MuIFN-beta), and -gamma (MuIFN-gamma) on macrophage activation for tumor cell killing were determined by using proteose peptone-elicited peritoneal macrophages from C3H/HeN and C3H/HeJ mice under conditions that either included or were free of detectable endotoxin. Alone, under the conditions used, none of the interferons was able to activate macrophages directly for tumor cell killing. However, with a second signal provided to responsive macrophages by contaminating endotoxin, added bacterial lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM), all three types of interferon induced cytolytic activity, with MuIFN-gamma approximately 500 to 1000-fold more active than either MuIFN-alpha or -beta. Thus, all three interferons were able to prime macrophages for killing but required a second signal before cytolytic activity could be expressed. When MuIFN-gamma was mixed with either MuIFN-alpha or -beta and placed on macrophages, little or no killing developed. Mixtures of MuIFN-gamma with either MuIFN-alpha or -beta did increase the sensitivity of macrophages to triggering by LPS, however, compared with macrophages treated with MuIFN-gamma alone. The results are collectively important because they i) confirm that significant quantitative differences exist between the various interferons with regard to their capacity to prime macrophages for tumor cell killing; ii) indicate that to be an efficient activator each type of interferon must be combined with a second stimulus, such as LPS or HKLM; iii) show that neither MuIFN-alpha nor -beta can provide an efficient second triggering signal for macrophages that are primed by MuIFN-gamma; and iv) document that mixtures of MuIFN-gamma with either MuIFN-alpha or -beta are most efficient at inducing priming, compared with any one of the interferons used alone.     

Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferons

The antiviral and antiproliferative effects of highly purified Escherichia coli-derived human interferons (IFNs) were examined in human melanoma cells (Hs294T). Antiproliferative activity was monitored by measuring inhibition of cell multiplication, and antiviral activity was determined by inhibition of herpes simplex virus type 1 replication. Treatment of cells with IFN-gamma in combination with IFN-alpha A or IFN-beta 1 resulted in potentiation of both antiproliferative and antiviral activities. In contrast, combination treatments composed of IFN-alpha A and IFN beta 1 yielded inconsistent results. Some combinations reflected additive responses, whereas others were antagonistic. To examine correlations between IFN-induced biological activities and interactions of the different IFNs with cell surface receptors, in vivo [35S]methionine-labeled IFN-alpha A was prepared. Binding studies indicated the presence of 2,980 +/- 170 receptors per cell, each with an apparent Kd of (8.4 +/- 1.3) X 10(-11) M. Results from competitive binding studies suggested that Hs294T cells possess at least two types of IFN receptors: one which binds IFN-alpha A and IFN-beta 1 and another to which IFN-gamma binds.     

Comparative antiproliferative and receptor binding activities of interferons alpha and beta on lymphoblastoid and melanoma cells

The relative antiproliferative and receptor binding characteristics of the hitherto little-characterized interferon alpha 4a on cells of lymphoid and epithelial origin are compared with two other type I interferons, alpha 2 a and beta. Using the lymphoblastoid cell line, Daudi, interferons alpha 4 a and alpha 2 b had similar antiproliferative activity, and were about 10-fold more active than IFN beta. By contrast, using the melanoma cell line Sk-Mel-28, IFN beta was the most active, whereas IFN alpha 2b and IFN alpha 4a were respectively 60-fold and greater than 1000-fold less active than on Daudi cells. Receptor binding did not correlate with antiproliferative sensitivities, but confirmed a shared receptor component for these three interferons. These results indicate that the antiproliferative activities of three type I IFNs differs markedly on different cell types and that this is unlikely to be due to receptor binding, but more likely a post receptor binding event.     

Regulation of MuIFN-alpha/beta and MuIFN-gamma-induced antiviral states: differential effects with different challenge viruses and lack of correlation with 2'5' oligoadenylate synthetase levels

The MuIFN-alpha/beta and MuIFN-gamma induced antiviral states which are directed against mengovirus have been shown previously to be differentially regulated. Following interferon removal, the MuIFN-alpha/beta-induced antiviral state decays rapidly, while the MuIFN-gamma-induced antiviral state increases dramatically. To determine whether these observations with mengovirus represent part of a general phenomenon, these studies have been extended using vesicular stomatitis virus and vaccinia virus, which represent two distinctly different groups of viruses. The antiviral states induced by MuIFN-gamma against all three viruses increased dramatically following interferon removal. The antiviral state induced by MuIFN-alpha/beta against vesicular stomatitis virus was stable following interferon removal, while the antiviral states induced by MuIFN-alpha/beta against mengovirus and vaccinia virus decayed rapidly. Also, levels of 2'5' oligoadenylate synthetase were determined at various times following interferon removal. MuIFN-alpha/beta was found to be a relatively strong inducer of 2'5' oligoadenylate synthetase, while MuIFN-gamma was a relatively weak inducer. Further, while the changes in 2'5' oligoadenylate synthetase levels paralleled the changes in the levels of the antiviral states induced by MuIFN-alpha/beta and MuIFN-gamma against mengovirus and vaccinia virus, the changes in 2'5' oligoadenylate synthetase levels did not parallel the changes in the antiviral state induced by MuIFN-alpha/beta against vesicular stomatitis virus. The results suggested that the 2'5' oligoadenylate synthetase levels did not correlate with the level of antiviral state.     

Antiviral and antiproliferative activity of human fibroblast interferon fractions

The affinity chromatography of Human crude beta-interferon preparations on Blue Dextran Sepharose columns resulted in isolation of several fractions with different ratio of antiviral to antiproliferative activities. The results of investigation of two of these fractions are described in this report. The first of them was eluted by 1N NaCl in 0.01 M tris buffer at pH 7.8, the second was eluted by 1 M NaCl, 50% methylethylenglycol in 0.01 M tris-HCl buffer at pH 7.8. The first of the fractions possessed presumably antiproliferative and the second presumably antiviral activity. Both fractions induced the increase of 2'5'-oligoadenylatesynthetase activity in cells although the inducing activity of the first fraction was about 6-fold higher than that of the second one as compared with their antiviral activities. The obtained results indicate that purification of interferon preparation for interferons main antiviral activity may lead to the loss of the great part of antiproliferative material.     

Antiviral and antiproliferative activity of human interferons and their induction of 2',5'-oligoadenylate synthetase

Native preparations of alpha, beta and gamma-interferons as well as recombinant beta-interferon and purified leukocyte alpha-interferon and purified leukocyte alpha-interferon exert antiviral and antiproliferative activity in CaOv cells. Native interferon preparations were shown to be more antiproliferative than purified interferons per unit of antiviral activity (with EMC as well as with less susceptible VSV used as test viruses). It was shown that level of 2'5' oligoadenylatesynthetase activity induction in general correlates with antiproliferative and pronounced antiviral activity of interferons, besides that, the earlier (by 11 hours) induction of the enzyme activity by beta-interferon correlates with more rapid expression of antiproliferative effects by this interferon in comparison with that of alpha-interferon, the latter inducing the peak of enzyme activity by 24 hours.     

Transport of gamma-interferon into the cell nucleus may be mediated by nuclear membrane receptors

Purified mouse interferon gamma (MuIFN-gamma), a lymphokine having potent antiviral, immunomodulatory, and growth inhibitory activities, is internalized (t1/2 less than 1.0 min) by mouse L929 fibroblasts via receptor-mediated endocytosis. Individual MuIFN-gamma molecules, identified by a postembedding immuno-gold technique, are then transported to the cell nucleus, perhaps through nuclear pores, into areas of dense chromatin. Purified, isolated nuclei of L929 cells bind radiolabeled MuIFN-gamma specifically and with high affinity (Kd = 2 X 10(-10) M). These nuclear membrane receptors, distinct from those for MuIFN-beta, number about 24,000/nucleus. Treatment of nuclei with trypsin prevents binding of MuIFN-gamma. The demonstration of rapid cellular uptake and transport of MuIFN-gamma into the dense chromatin, perhaps facilitated by nuclear receptors, suggests that IFN-gamma molecules, alone or bound to receptor, may directly affect genome regulation.     

Effects of phenytoin on the production of interferons: differential effects on type I and type II interferons

The relative effects of treatment with an anticonvulsant, phenytoin, on the production of interferons were determined for both the murine and human systems. Phenytoin treatment was found to have differential effects on the in vitro production of Type I and Type II interferons. Phenytoin had either no effect (HuIFN-alpha) or an enhancing effect (MuIFN-alpha/beta) on the in vitro production of Type I interferons. In contrast, phenytoin pretreatment had an inhibitory effect on the in vitro production of Type II interferons (IFN-gamma) for both the murine and human systems. Phenytoin appeared to exert its inhibitory effect directly on the IFN-gamma-producing cell and was active even when added as late as 6 h after IFN-gamma induction. This inhibition was not related to a toxic effect of the phenytoin and occurred at phenytoin concentrations which were pharmacologically relevant (10-20 micrograms/ml). The effects of phenytoin on the in vivo production of MuIFN-gamma were also examined. In parallel to the in vitro observations, phenytoin treatment of mice significantly reduced the in vivo induction of MuIFN-gamma. The results raise the possibility that phenytoin therapy in humans may significantly affect the production of HuIFN-gamma.     

Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.     

Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells

The antigrowth effects of 5 different cloned human leukocyte IFN subtypes (IFN-alpha A, B, C, D, F) and 2 molecular hybrids between them (IFN-alpha AD(Bg1II) and IFN-alpha DA(Bg1II)) were examined on 6 different human cell lines. The results indicate that the interferons sort into two distinct groups: IFN-alpha B, C and F showed comparable antiproliferative activity which was greater than that of IFN-alpha A, D, AD(Bg1II) and DA(Bg1II). The interferons could also be assigned to one of two groups on the basis of their antiviral activity. IFN-alpha A, D and AD(Bg1II) were observed to be more protective than IFN-alpha B, C and F against HSV-2 and EMCV infections, i.e. the relative antiviral efficacies of the cloned IFN subtypes were the reverse of their antiproliferative activities.     

Early plasma membrane depolarization by alpha interferon: biologic correlation with antiproliferative signal

Daudi lymphoma cells, of a line sensitive to growth inhibition by alpha interferon, showed dose-dependent plasma membrane depolarization within 10 min after exposure to natural or recombinant alpha interferons (10 to 1000 IU/ml). This biophysical change was detected flow cytometrically by measuring the intensity of fluorescent emission from cells stained with dye indicators of membrane potential. Subclones of Daudi lymphoma cells, resistant to growth inhibition by alpha interferon, showed no membrane depolarization. Parallel results were obtained in initial tests of an isologous pair of T cell and B cell lines which differ in sensitivity to growth inhibition. Thus, decreased membrane potential may herald an interferon signal for antiproliferative action.     

Characterization of the antiviral activity constitutively produced by murine conceptuses: absence of placental mRNAs for interferon alpha and beta

Antiviral activity has been found in conceptus and placental tissues in numerous species, including mice, pigs, sheep, cattle and humans. In sheep and cattle, the antiviral activity is due to an interferon alpha (IFN-alpha), but in other species the nature of the protein(s) responsible for placental activity is unknown. The objectives of this study were to determine if the constitutive antiviral activity associated with the mouse conceptus is produced as early as the peri-implantation period, and to determine if the activity is due to an IFN-alpha or -beta. Conceptus and placental tissue explants released antiviral activity from Day 4 through at least Day 16 of gestation as measured in an agar overlay bioassay employing CHO cells challenged with vesicular stomatitis virus. This activity was neutralized by antiserum against MuIFN-alpha/beta. The same antiserum failed, however, to immunoprecipitate radiolabeled proteins from medium collected from Day 4 blastocysts cultured in the presence of L-[35S]-methionine. S1 nuclease analysis of placental RNA and screening of ectoplacental cone and extraembryonic ectoderm cDNA libraries with MuIFN-alpha and -beta probes failed to detect IFN related mRNAs, even under relatively non-stringent conditions of hybridization. Thus, while antiviral activity is produced by peri-implantation conceptuses in several diverse mammalian species, it does not appear to be due to a conserved type of IFN in all these species.     

Ratio of the antiproliferative action of interferon to the degree of purity of the preparation

The antiproliferative (AP) action of human leukocytic interferon (HLI) of varying degrees of purification was studied and compared in cultures of human cells differing in interferon sensitivity. The concentrated and purified preparations of HLI suppressed the incorporation of 3H-thymidine and mitotic activities of the cultures sensitive to HLI of the cells, affecting, but insignificantly, the proliferation of the resistant cell culture. Poorly purified HLI suppressed the proliferation of both cell types. This indicates that native HLI contains impurities that have an AP action and exerts no antiviral activity.     

Time-dependent differential effects of natural and recombinant murine interferon-gamma on ornithine decarboxylase activity of tumor cells

Incubation of quiescent tumor cells with fetal calf serum induced ornithine decarboxylase (ODCase) activity concomitantly with mitogenic stimulation. Pretreatment of cells with highly purified natural or recombinant murine interferon-gamma (MuIFN-gamma) for 5 h caused a dose-dependent increase of ODCase activity induced by fetal calf serum (FCS). Pretreatment of target cells with IFN-gamma for 5 h in absence of FCS stimulation did not induce ODCase activity. When pretreatment of cells with natural or recombinant MuIFN-gamma was prolonged for 18 h both ODCase activity and DNA synthesis induced by FCS were suppressed. By contrast when a mixture of MuIFN-alpha and -beta was used, ODCase activity was significantly suppressed after 5 h pretreatment compared to untreated controls. These results suggest that IFN-gamma exerts a differential effect on mitogen-stimulated events depending on the dose and the time of addition.     

The murine IFN-gamma receptor does not transduce the activation signal to a murine promoter in human cells.

MuIFN-gamma receptor cDNA has been stably transfected in the human WISH cell line. A stable transfectant, denominated WISH-C2, expressed 46;000 receptors/cell whose affinity was similar to that observed on murine cells (Kd = 2.6 x 10(-9) M). When WISH-C2 cells were treated with Hu- or MuIFN-gamma, antiviral and antiproliferative activities were observed with HuIFN-gamma only. These findings suggest that other species-specific components associated to the binding site are required for the signal transduction. To overcome the complexity of measuring biological functions that very likely involve more than one regulatory and structural gene, we set up for the first time a system where a reporter gene driven by a murine promoter was used to directly evaluate the interaction of MuIFN-gamma receptor with an inducible promoter through the missing transducer factor.     

Therapy-induced antibodies against the antiviral and antiproliferative effects of interferons in patients with chronic hepatitis C virus infection

Sera from 86 patients with chronic hepatitis C virus (HCV) infection treated with recombinant interferons-alpha (rIFN-alpha) were screened for IFN-binding and antiviral effect-neutralizing antibodies. Out of the 61 patients treated with rIFN-alpha2b, 46% had binding and 28% had neutralizing antibodies. 44% of the 25 patients treated with rIFN-alpha2a developed binding antibodies and 24% had neutralizing antibodies. Contradictory data were observed concerning the appearance of anti-IFN antibodies and the outcome of IFN therapy. A significantly higher number of the patients with a sustained response to rIFN-alpha2b therapy formed antibodies than the number among the non-responder patients. At the same time, in the patients treated with rIFN-alpha2a, opposite data were found. The activity of the antibodies in some sera was studied against the antiproliferative effect of IFNs on Daudi cells by measuring the [3H]thymidine incorporation. The binding antibodies without neutralization of the antiviral effect of the IFNs inhibited the antiproliferative activity of the rIFNs, similarly to antibodies having both IFN-binding and antiviral effect-neutralizing capacities. At the same time, the antiproliferative effect of the natural IFN was less affected. It is suggested that the antiproliferative assay is more sensitive than the antiviral method for demonstration of the presence of antibodies exerting an inhibitory effect on the biological activities of IFN.     

Regulation of macrophage growth and antiviral activity by interferon-gamma

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.     

Inhibition of the differentiation of 3T3-L1 cells by interferon-beta and difluoromethyl ornithine

Both mouse interferon-beta (MuIFN-beta) and the inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethyl ornithine (DFMO), inhibited the differentiation of mouse 3T3-L1 fibroblasts into adipocytes in a dose-dependent manner. DFMO and MuIFN-beta added together to cultures that were induced to differentiate produced an additive anti-differentiation effect. In contrast to this additive cellular effect, DFMO reduced the antiviral activity of MuIFN-beta in both undifferentiated and differentiated cells; DFMO alone had no detectable effect on replication of encephalomyocarditis virus. Putrescine, the product of ornithine decarboxylation, when added to 3T3-L1 cultures (i) enhanced differentiation, (ii) reversed completely the inhibition of differentiation by DFMO, but (iii) had little effect on the antidifferentiation effect of MuIFN-beta. Polyamine content changed four-fold or less in cultures treated with 0.5 mM DFMO and less than two-fold in cultures treated with 100 IU/ml MuIFN-beta for seven days. Thus, it appears not only that MuIFN-beta and DFMO inhibit differentiation of 3T3-L1 cells by different mechanisms but also that the antiviral action of IFN does not involve the regulation of polyamine metabolism by ornithine decarboxylase.     

Synthesis and antiproliferative activity of thiazolidine analogs for melanoma

We have previously described 2-aryl-thiazolidine-4-carboxylic acid amides as a novel class of antiproliferative agents for prostate cancer. Screening these compounds with melanoma cell lines revealed that several of them have potent antiproliferative activity and selectivity against melanoma. To further improve the potency and selectivity, we synthesized a new series of analogs and tested them in two melanoma cell lines and fibroblast cells (negative controls). Comparison of anticancer effects of these compounds with a standard chemotherapeutic agent, sorafenib, showed that they are very effective in killing melanoma cells with low micromolar to nanomolar antiproliferative activity and provide us a new lead for developing potential drugs for melanoma.