C243 Cells Research Articles

Isolation and characterization of a novel interferon-α-encoding gene, IFN-α11 within a murine IFN cluster

Two recombinant cosmids containing three complete murine interferon-α-encoding genes (Mu IFN-α) have been isolated from a mouse cosmid library. The cluster organization of these genes has been determined. A new Mu IFN-α gene (Mu IFN-α11) has been isolated and studied with respect to its structure and inducible transcription pattern. The nucleotide and deduced amino acid sequences of the Mu IFN-α11 gene, as compared to the two other IFN-α7 and IFN-α8 genes, show that, albeit highly homologous, these genes are all different. The transient expression of the three genes gave rise to proteins showing antiviral properties which were neutralized with murine anti-IFN-α antibodies. The transcription of the Mu IFN-α genes was studied in two uninduced or Newcastle-disease-virus-induced murine cell types. Mu IFN-α11, as well as α7 and α8, are not expressed in L929 nor in C243 cells upon viral induction and therefore constitute an interesting model to study Mu IFN-α gene repression.

Expression of the Genes of Class I Interferons and Interleukin-6 in Individual Cells

The use of a highly sensitive method of in situ hybridization capable of detecting one copy of interferon (IFN) mRNA per cell coupled with quantitative analysis of cytokine mRNA showed that the number of copies of mRNA per cell was directly proportional to the logarithm of the number of silver grains formed over that cell. More than 90% of both virus-induced human Namalwa and mouse C243 cells exhibited grain counts significantly greater than background values following in situ hybridization with riboprobes complementary to human IFN- alpha and mouse IFN- beta mRNA, respectively. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Although the large majority of cells within a population responded to induction, considerable variation was observed, however, in the content of IFN mRNA per cell: 24% of induced C243 cells contained more than 50 copies of IFN-beta mRNA per cell while 60% of the cells contained 10 copies or less. Low levels of IFN mRNA were also detected in both uninduced C243 cells and uninduced Namalwa cells. Five to 10% of peripheral blood mononuclear cells from normal donors expressed INF-alpha mRNA following induction in vitro. Approximately 1% of untreated peripheral blood mononuclear cells also exhibited low levels of IFN-alpha mRNA. Analysis of interleukin-6 (IL-6) mRNA showed that 97% of TNF-induced human MG63 cells contained IL-6 mRNA, although, again, the amount varied considerably from cell to cell.

Purification and carbohydrate structure of natural murine interferon-beta.

Mouse interferon-beta (Mu-INF-beta) induced in C-243 cells with Newcastle disease virus was purified in four steps including ammonium sulfate fractionation. DEAE-cellulose, monoclonal Mu-IFN-beta antibody affinity and Mono-S cation-exchange chromatographies. Specific activity of the purified Mu-IFN-beta ranged over 1.1-1.4 X 10(9) NIH units/mg protein. This preparation was submitted to pronase digestion and gel on Fractogel TSK HW-40. The permethylated and acetylated glycopeptide fraction was analyzed by chemical-ionization (ammonia) mass spectrometry. The major glycopeptide is composed of Gal, Man, GlcNAc and NeuAc with a molar ratio of 2.0:3.6:3.4:0.5. The GLC pattern of methyl derivatives obtained by methanolysis and acetylation of fully methylated glycopeptide identified 2,3,4,6-tetra-O-methylgalactose; 3,4,6-tri-O-methyl-mannose; 2,3,4- and 2,4,6-tri-O-methylgalactose; 2,4,di-O-methyl mannose and 3,6-di-O-methylglucosamine. These results when compared with data on N-glycans suggest the following structure for the carbohydrate moiety of Mu-INF-beta: (formula; see text).

Interactions of interferons and transforming growth factors during clonal growth of mouse or human cells in soft agar and in mice

The mouse fibroblast-like transformed cell line C-243 adapted to growth in suspension was used as a source of virus-induced interferons (MulFN-alpha, beta), and spontaneously produced active growth factors. These factors were purified from C-243 cells grown as tumors in BALB/c mice, and had properties identical to those of TGF-alpha or TGF-beta isolated by others from different tissues. Exogenous TGF-alpha, beta stimulated colony formation by C-243 cells in soft agar, whereas MulFN-alpha, beta inhibited it. Clonal growth of human lung adenocarcinoma A549 cells in soft agar was inhibited as well by human interferons (types alpha, beta, or gamma) as by TGF-beta. Inhibition was dose-related. Pure EGF, which is an analogue of TGF-alpha, diminished the antiproliferative activity of interferons alpha, beta, and gamma in A549 cells. On the other hand, the anti-mitogenic action of IFN-beta and TGF-beta was clearly synergistic. In mice bearing C-243 cell tumors, TGF-alpha, beta stimulated growth, whereas MulFN-alpha, beta inhibited it. Stimulation of tumor growth was also observed after administration of anti-IFN serum that could neutralize endogenous IFN-alpha, beta. The simultaneous administration of MulFN-alpha, beta and TGF-alpha, beta diminished anti-tumor effects of IFN in mice. Our results suggest that both TGFs and IFNs are autocrine, positive or negative growth factors modulating the rate of proliferation and the neoplastic behavior of the cells. The final effects depend on the target-cell sensitivity and on the relative concentration of the various hormone-like factors. Cancer cells overstimulated by TGF-alpha, beta or by EGF may not respond to IFNs.

Natural antibodies to interferon-alpha and interferon-beta are a common feature of inbred mouse strains.

Neutralizing antibodies to murine interferon-alpha (MuIFN-alpha) and MuIFN-beta of the immunoglobulin (Ig)G and IgM class were naturally present in sera of BALB/c and C57BL/6 mice of four different age groups, ranging from 1 to 15 mo. These antibodies not only neutralized IFN made by C-243 cells (Swiss genotype), but also isogenic IFN-alpha and IFN-beta, and can therefore be considered to be real autoantibodies. Natural anti-MuIFN antibodies were also found in the sera of mice from 12 other inbred strains, and seem to be a common feature of inbred mouse strains.

65] The detection of individual cells containing interferon mRNA by in situ hybridization with specific interferon DNA probes

This chapter describes a method used for the detection of murine interferon (IFN)-α and IFN-β messenger RNA (mRNA) in butyrate-treated mouse C-243 cells induced with Newcastle disease virus (NDV). The method of in situ hybridization permits the localization and identification of nucleic acids of specific sequences in individual cells. With the availability of complementary DNAs (cDNAs) corresponding to the different interferon (IFN) species, it has become possible to set up experimental systems to identify individual cells synthesizing IFN mRNA. To be able to study IFN production at the single cell level is of obvious importance, because it can provide answers to several questions, such as the number of IFN producing cells in vivo as a function of the inducing agent. To reduce nonspecific binding of the labeled cDNA probe to the slide surface, several procedures for coating of glass slides are described. During autoradiography, specific pairing between labeled cDNA probe, and the respective target sequence in the induced cell results in the formation of silver grains. There are, however, a variety of artifacts that produce silver grains, too, as for instance nonspecific binding of probe to the specimen or pressure exerted on the emulsion layer during manipulation.

Mapping of murine Interferon-α genes to chromosome 4

A cDNA library was constructed from polysomal poly(A) + RNA from Newcastle disease virus (NDV)-induced mouse C243 cells, and screened with a human interferon-a (HuIFN-α) cDNA probe. A cDNA clone for one of the murine interferon-a (MuIFN-α) genes was isolated, and sequencing analysis revealed that it was a partial copy which is almost identical to the published sequence for the MuIFN-α2 gene. This partial cDNA clone represents a virus-induced message as seen by Northern blot analysis of RNA from NDV-induced C243 cells, and Southern blot analysis of DNA from BALB/c mouse revealed the presence of a multiple IFN-α gene family. The MuIFN-α genes were mapped to chromosome 4 by Southern blot analysis of hamster/mouse somatic cell hybrid DNAs.

Interaction of mouse interferon and platelet-derived growth factor during multiplication of BALB/c 3T3 cells.

Mouse fibroblast-drived interferon (MuIFN) at concentrations of 0.1-0.001 U/ml enhanced the multiplication of BALB/c 3T3 cells. Experiments with the cells cultured in 5% platelet poor plasma serum (PPPS) medium in which the cells not multiply without platelet derived growth factor (PDGF), suggested that there is a positive cooperativity between small amounts of MuIFN and submitogenic doses of PDGF. The growth-promoting activity of 10,000-1,000 U/ml detected in the partially purified MuIFN from C243 cells assayed in 5% PPPS medium was probably the result of the contamination of MuIFN preparations with a PDGF-like growth factor.

Antigenic correlations between components of C243 and L cell interferons

The three components of virus-induced interferon (IFN) from C243 cells, with molecular weights of 19,000, 24,000 and 30,000, were examined for neutralization by antisera against the α (24,000) and β (36,000) types of virus-induced L cell IFN. The results indicated that the C243 19,000 component belongs to the α type, and the 24,000 and 30,000 components to the β type. The 19,000 component was also indistinguishable from L cell IFN-α in the reaction with antisera against human IFN-α.

Isotopic labeling of mouse interferon by incorporation of radioactive amino acids during synthesis

Mouse interferon produced by C-243 cells induced with Newcastle disease virus was isotopically labeled by adding either [ 35S]methionine or a 14C-labeled amino acid mixture to the culture medium. A method combining butyric acid and theophylline treatment and resulting in high interferon yields was used. Following purification by two-step affinity chromatography on poly(U) and antibody columns, the resulting material was analyzed on SDS-PAGE. The migration pattern of radioactivity and interferon coincided well and autoradiography revealed three major bands at migration distances corresponding, respectively, to 35, 28, and 22 K. Interferon represented 3.8% of all [ 35S]methionine-labeled proteins and 2.6% of all 14C-amino acid-labeled proteins released into the medium.

Molecular cloning of partial cDNA copies of two distinct mouse IFN-beta mRNAs.

Two cDNA libraries were constructed, using respectively the 12S and the 16S sucrose gradient fractions of polysomal poly (A)+ RNA from mouse C243 cells induced with Newcastle disease virus. Screening of a part of both libraries by mRNA selection hybridization assays revealed the presence of two plasmids hybridizing to an mRNA, whose translation product was characterized as mouse IFN-beta. Blot analysis of RNA indicated that mRNA hybridizing to the DNA from both plasmids could be detected in induced but not in uninduced C243 cells. The two cDNA inserts did not cross hybridize and had distinct restriction maps. Sequencing revealed that both inserts represented the end of the coding region and the entire 3' non coding region of two district mRNAs. Although different, the putative 39 AA and 65 AA carboxy termini of both Mu IFN-beta s display some homology to human IFN-beta 1. Thus there are at least two different murine IFN-beta genes.

Enhancement of mouse interferon production by combined treatment of C-243 cells with butyric acid and theophylline

Summary A method for obtaining high yields of mouse (α and β) interferon from C-243 cells is described. The presence in the culture medium of 1 mM butyric acid for 48 h before induction with Newcastle disease virus, and 3 mM theophylline during interferon production, resulted in preparations titering from 1.2 to 6.0×10 6 reference units, with a specific activity of 1.6×10 7 . Analysis by polyacrylamide gel electrophoresis, following purification on poly-U and anti-interferon antibody agarose columns, revealed 3 zones of antiviral activity peaking, respectively, at 35 K, 22 K and 15 K, plus a very minor component at 65 K.

82] Separation of molecular species of human leukocyte and mouse interferons by polynucleotide affinity chromatography

Publisher Summary This chapter discusses that mouse interferon produced by C243 cells upon induction by Newcastle disease virus (NDV) can form a binary complex with single-stranded polynucleotides, such as poly(I) and poly(U), and tRNA. Complex formation is demonstrated by (1) direct binding of interferon to polynucleotide-agarose matrix (or tRNA agarose matrix) (2) elution with polynucleotide or tRNA of interferon previously bound to Blue Dextran-agarose, a matrix that can recognize the polynucleotide binding site on proteins, (3) protection of interferon from thermoinactivation when bound to these molecules. Based on the model of polynucleotide phosphorylase, it is shown that mouse interferon (type I) bound on Blue Dextran-agarose, could not be displaced by most of the usual nucleotides but could be easily eluted by the single-stranded polyribonucleotides poly(I) and poly(U). The interaction with polynucleotide has also been demonstrated for mouse immune interferon (type 11) 13 and human leukocyte interferon. These studies suggest that the polynucleotide binding property could be a basic characteristic of native interferons. This property provides a simple means to separate different molecular forms of interferons according to the degree of their affinity for a polynucleotide ligand or to separate a given molecular form from the degraded molecule.

Electrophoretically pure mouse interferon has priming but no blocking activity in poly(I.C.)-induced cells

The effect of interferon on its own synthesis was examined by exposing cells to electrophoretically pure interferon prior to induction with poly(I.C.) In C-243 cells, pretreatment with pure interferon, at any dose, up to 160,000 units, resulted in a very pronounced priming effect and no blocking was observed. Crude interferon did not display any blocking effect either, but its priming effect was much less pronounced than that of pure interferon. In mouse embryo fibroblast cultures, low doses of pure interferon had a slight priming effect, whereas higher doses, from 500 up to 50,000 units, had neither priming nor blocking activity. At a dose of 50,000 units the crude interferon preparation had blocking activity. When NDV instead of poly(I.C.) was used as inducer, pretreatment with pure interferon of both C-243 and mouse embryo fibroblast cells resulted in inhibition of interferon synthesis.

Characterization of Two Distinct Molecular Populations of Type I Mouse Interferons

The molecular heterogeneity of acid-stable (Type I) mouse interferons induced in C243 cells by Newcastle disease virus was analysed by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions, and the profiles of antiviral activities obtained were characterized biologicaly in mouse cells and in heterologous (guinea-pig) cells. Two bands of activity, A and B, were consistently present in all interferon preparations tested: under reducing conditions, the activity in all fractions of band A (with a peak of activity at about 38000 daltons) was uniformly increased, while that of band B (with a peak at about 22000 daltons) was uniformly diminished. All the active fraction in band A had only slight activity (less than 10% of homologous titres) on guinea-pig cells, whereas all those in band B were significantly more active an guinea-pig cells than on homologous L cells. Thus, mouse type I interferon preparations contain two molecular populations of interferons that can be distinguished physically (by size), biochemically (by the effect of reduction on reactivation from SDS) and biologically (by activity in heterologous cells).