IFN-treated Cells Research Articles

Inhibitory effects of interferon on the expression of genes regulated by platelet-derived growth factor.

The G0/G1 to S transition in quiescent BALB/c 3T3 cells stimulated by serum growth factors can be specifically blocked by the administration of interferon (IFN) to the system. In the present communication, we studied whether IFN inhibits the early events in the G0/G1 phase that are initiated by the platelet-derived growth factor (PDGF). The results show that IFN inhibits most of the PDGF-mediated increase of c-myc, ornithine decarboxylase, and beta-actin mRNAs measured 3 hr after stimulation. c-fos mRNA levels are reduced by IFN as early as 20 min after exposure of the quiescent cells to PDGF. The expression of several genes that belong to the competence gene family is, therefore, inhibited by IFN and this could account for the failure of the IFN-treated cells to enter into the S phase when growth factors present in the platelet-poor plasma are added. We also report that the PDGF-mediated increase in the uptake of deoxyglucose is not impaired by IFN, thus suggesting that the early effects of IFN on gene expression do not result from inhibition of binding of PDGF to its cell-surface receptors. Unlike the direct stimulatory effect of PDGF, which is not sensitive to cycloheximide, the inhibitory effect of IFN on c-myc mRNA levels depends in part on protein synthesis. We propose that a putative product of one of the IFN-induced genes could mediate the decrease in expression of the PDGF-regulated gene family.

Interferon-γ is a strong modulator of NK susceptibility and expression of β2-microglobulin but not of transferrin receptors of K562 cells

The human cell line K562 was treated with human natural leukocyte interferon (IFN-α) and recombinant immune interferon (IFN-γ). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-α, as little as 10 U/ml of IFN-γ caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human β2-microglobulin (β2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-α (1.4-fold) and IFN-γ (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K.562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-α and IFN-γ as did K562, when effects on NK susceptibility, β2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of β2M and TR. These results demonstrate that IFN-γ is highly efficient in modulating the NK susceptibility, and the expression of β2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.

Subcellular distribution of 2′,5′-oligoadenylate synthetase in differentiating Friend leukemia cells

The occurrence of distinct (2′–5′)(A) n-synthetase activities has recently been documented in cytoplasmic and nuclear extracts of several interferon (IFN)-treated cell lines. Since a role has been proposed for (2′–5′)(A) n synthetase in the control of cell growth and differentiation, we examined the subcellular distribution of (2′–5′)(A) n-synthetase activity both in IFN-treated undifferentiated Friend leukemia cells (FLCs) and during dimethyl-sulfoxide (DMSO)-induced erythroid differentiation of FLCs. Both the nuclear and cytoplasmic (2′–5′)(A) n activities were modulated to the same extent by IFNs and DMSO. No evidence for a causal relationship between enzyme activation and FLC differentiation was found.

Regulation of Moloney murine leukemia virus replication in chronically infected cells arrested at the G0/G1 phase

The replication of Moloney murine leukemia virus (MMuLV) in chronically infected mouse cells arrested at the G0/G1 phase of the cell cycle by different procedures was investigated. MMuLV production was inhibited in glutamine- and isoleucine (Gln-Ile)-deprived G0/G1 cells. In contrast, butyric acid treatment, which efficiently arrested the cells at the G0/G1 phase of the cell cycle, did not inhibit MMuLV production. Furthermore, the inhibition of MMuLV production caused by either Gln-Ile deprivation or by interferon (IFN) treatment was overcome by butyric acid treatment. Thus, the replication of MMuLV could be dissociated from cell proliferation. The inhibition of MMuLV production in Gln-Ile-deprived cell cultures was compared to the inhibitory effect of IFN, which is known to affect budding and release of the virus. Rates of MMuLV protein synthesis were not affected in both the IFN-treated and Gln-Ile-deprived cells. However, processing of the viral polyprotein Pre65gag into p30 was blocked in the Gln-Ile-deprived cells. Furthermore, whereas in IFN-treated cells, MMuLV accumulated on the cell surface and could be released upon treatment with trypsin, in Gln-Ile-deprived cells, no virions were released by such treatment. These results indicate that in cells arrested by Gln-Ile deprivation, MMuLV is inhibited at a posttranslation step. This step appears to precede the anti-MMuLV block induced by IFN.

Interferon-gamma requires serum retinoids to promote the expression of tissue transglutaminase in cultured human blood monocytes.

The culture of HPBM in serum-containing medium induced a large accumulation of the protein cross-linking enzyme, tissue TGase. Immune IFN enhanced the expression of tissue TGase in cultured monocytes. Enzyme-inducing activity, both in normal and IFN-treated cells, was completely blocked by depleting the serum of the lipid fraction. The readdition of retinol at a physiologic concentration (1 micron) to delipidized serum completely restored the enzyme-inducing activity in cultured monocytes. Thus, serum retinoids seem to play an important regulatory role in the expression of tissue TGase gene in differentiating human monocytes.

Binding of unglycosylated and glycosylated human recombinant interferon-gamma to cellular receptors.

Recombinant human interferons (IFNs), either unglycosylated produced in E. coli (rIFN-gamma) or glycosylated produced in CHO cells (g-rIFN-gamma), were labeled with 125I to similar specific activities to study their interaction with cell-surface receptors. When analyzed by gel electrophoresis, rIFN-gamma run as a single polypeptide of Mr 15,000-17,000, whereas g-rIFN-gamma separated into three components of Mr 20,000, 22,000, and 43,000, which corresponded to the known size of the two monomeric and one dimeric forms of glycosylated natural IFN-gamma. The binding of the two species of 125I-IFN-gamma was competed equally by rIFN-gamma in competition displacement experiments with Daudi cells, indicating that these IFNs bind with similar high affinity to the same receptors. KD values of 1.25 X 10(-10) and 2.5 X 10(-10) M were determined for g-rIFN-gamma and rIFN-gamma, respectively. This relatively small difference in KD does not apparently result in a detectable difference in biological activity, as measured by the increase in 2',5'-oligo(A) synthetase activity in IFN-treated HeLa and A549 cells. These results indicate that glycosylation of IFN-gamma does not play a significant role in its interaction with cellular receptors and in the induction of a biological response.

Leukocytes from multiple sclerosis patients respond to α- and γ-interferons

Blood mononuclear leukocytes from multiple sclerosis (MS) patients were evaluated for reactivity to alpha- or gamma-interferon (IFN), using a response whereby IFN-treated (primed) cells synthesize higher levels of IFN than untreated cells. Leukocytes were treated in vitro with natural alpha-IFN, recombinant alpha-IFN, recombinant gamma-IFN, or no IFN, then exposed to measles virus to induce IFN synthesis. With no IFN treatment, IFN production by cells from the MS patients was significantly less than normal (P less than or equal to 0.025). However, with IFN treatment, cells from MS patients were primed as well as cells from normal controls: IFN synthesis for the MS patients' cells was increased 10-11-fold with either alpha-IFN preparation and 3.6-fold with gamma-IFN. These findings verify the presence of leukocyte reactivity to alpha-IFN and provide the first demonstration of responsiveness to gamma-IFN in MS.

Characterization of a specific kinase inhibitory factor produced by vaccinia virus which inhibits the interferon-induced protein kinase

When mouse L cells are infected by vaccinia virus, a specific kinase inhibitory factor is produced which inhibits the interferon-induced, double-stranded RNA-dependent protein kinase ( P. Whitaker-Dowling and J. S. Youngner (1983) Virology 131, 128–136). This inhibitory factor appears early in vaccinia infection (90 min) and its production requires protein synthesis. It inhibits the phosphorylation of the α subunit of protein synthesis initiation factor eIF-2 and it is active in mixed extracts of IFN-treated cells and vaccinia-infected cells. The vaccinia-mediated inhibition of the IFN-induced protein kinase is not due to a specific phosphatase or a specific protease and can be reversed by the addition of excess double-stranded RNA. Evidence is presented which suggests that the specific kinase inhibitory factor interacts in a stoichiometric manner with the double-stranded RNA which is required for the activation of the interferon-induced protein kinase.

Cyclic effects of interferon and its antagonist on the saturation of 18-carbon fatty acids

Summary Treatment of tissue culture cells with interferon (IFN) induced a reversible increase in the saturation of the long-chain 18-carbon fatty acids (C18FA). The change was predominantly in the ratio of stearic acid (C18:0) to oleic acid (C18:1), which was expressed as the saturation index (S/O index). When the cells were exposed to IFN for 1 h, and then IFN was removed, there was an increase in the S/O index with a peak at 10–12 h and a return to normal values at about 20 h. This was followed by a rebound effect, a depression in the S/O index below normal cell ratios and a return to normal at 72 h. Intermittent replenishment of the overlay of IFN-treated cells with fresh medium led to a progressive, amplified increase in the S/O index and the appearance of a cytopathic effect. The overlay medium of the cells pulse-treated with IFN, then purified and concentrated as for IFN, contained a protein which was antagonistic to IFN in its effect on the saturation of C18FA. Pulse treatment of cells with this material produced a reversible decrease in the S/O index (increased desaturation in C18FA), which was kinetically a mirror image of the effect of IFN. Desaturation promotion factor (DPF) is the name suggested for this. A protein with a similar effect on the C18FA extracted from chick embryo fibroblasts was found in the allantoic fluid of embryonated hen's eggs infected with a virulent strain of Newcastle disease virus (Herts strain). It is postulated that IFN and DPF are antagonistic cellular hormones which could be involved in the regulation of C18FA saturation. The change in the saturation of the fatty acids is reflected in the change in membrane fluidity. Most of the biological effects of IFN could be related to changes in membrane fluidity induced by IFN.

Effect of interferon on integrity of vaccinia virus and ribosomal RNA in infected cells

The state of vaccinia and ribosomal RNAs in IFN-treated, vaccinia virus-infected mouse L cells grown in suspension is examined. In these cells a drastic inhibition (∼90%) of both viral and cellular protein synthesis occurs after virus infection of IFN-treated cells. The findings show that (1) primary transcription of vaccinia virus is not impaired by IFN, but is rather enhanced; (2) viral RNAs produced in IFN-treated, infected cells are predominantly of the early class; (3) these viral RNAs can be translated in a cell-free protein-synthesizing system; (4) in IFN-treated, infected cells there is extensive cleavage of 28 and 18 S rRNA at early times post infection, resulting in a characteristic cleavage pattern; (5) cleavage of rRNA is viral RNA dependent. The results indicate that in this virus-cell system the IFN-mediated inhibition of vaccinia and cellular protein synthesis is correlated with an alteration in ribosomal integrity.

Phosphoproteins altered by antiproliferative doses of human interferon-β in a human bladder carcinoma cell line

Phosphoproteins of control and IFN-β treated human bladder carcinoma cells (RT4) were labelled in vitro with [32P]-ATP and analyzed by polyacrylamide gel electrophoresis. Cells treated with antiproliferative doses of IFN had reduced levels of phosphorylated 60 Kd and 40 Kd proteins. IFN also induced within 24 hours the modification of a low molecular weight phosphoprotein doublet in the 22–24,000 molecular weight range. The ability to phosphorylate high molecular weight proteins by the in vitro procedures was generally depressed by IFN treatment. There was a dramatic shift in the phosphorylation of alkali stable phosphoamino acids associated with proteins in the 43–50,000 molecular weight range in IFN-treated cells. Preliminary studies indicate that at least some of the IFN-induced modifications of cellular phosphoproteins may result from transcriptional control of specific oncogenes.

Interferon-induced cytotoxicity and (2′–5′) oligo(A) synthetase activity in T cells. Differences in responsiveness among T cells from various individuals and among lymphoblastoid T-cell lines

Peripheral blood T lymphocytes became cytotoxic toward Daudi lymphoblastoid cells when incubated in vitro with interferon (IFN). The degree and rate of response to IFN varied markedly from one blood donor to another. “Low responders,” who reproducibly showed a slower response to IFN, as well as “high responders” could be identified. The rate of increase in (2′–5′) oligo(A) synthetase activity in IFN-treated cells also varied and appeared to correlate to the rate of increase in cytotoxicity. Three lymphoblastoid T-cell lines (Molt 4, Peer, HPB-ALL) also became cytotoxic towards Daudi cells when incubated with IFN and in these cells too the rate of induction of cytotoxicity appeared to correlate to the rate of increase in (2′–5′) oligo(A) synthetase activity. Differences in responsiveness of T cells to IFN-mediated cytotoxicity might, therefore, reflect differences in responsiveness to IFN in general. Thus, quantitation of these differences might yield useful information for assessing the dosage and frequency of IFN administration to patients undergoing therapy.

Interferon enhances prostacyclin production by cultured vascular endothelial cells.

The effects of interferon (IFN) on the arachidonate metabolism and physiological functions of cultured endothelial cells and blood platelets have been examined. Cultured bovine aortic endothelial cells were found to be sensitive to the antiviral and antiproliferative activities of human leukocyte (alpha) IFN and to increase their capacity to synthesize prostacyclin (PGI2) upon exposure to IFN. Several observations indicate that IFN stimulates PGI2 synthesis at the level of the enzymes phospholipase A2 and cyclooxygenase: (a) PGI2 production was dependent upon the supply of exogenous arachidonic acid or the liberation of endogenous cellular arachidonate by ionophore A23187, but was not observed when IFN-treated cells were exposed to the endoperoxide prostaglandin H2. (b) IFN had no effect on the spontaneous release of PGI2 into the culture medium during the incubation period (24-72 h). (c) The stimulatory effect of IFN on PGI2 production was inhibited by both glucocorticoids and indomethacin. The effect of IFN on platelet prostaglandin metabolism was also investigated. Incubation of platelet-rich plasma with IFN had no effect on platelet aggregation and thromboxane A2 production. The biological significance of the findings presented in this paper may be considered in view of the protective role of PGI2 in the vessel wall and the fact that infection with certain viruses induces endothelial damage both in man and experimental animal models.

Inhibition of cell division and antibody secretion by murine alpha/beta interferon: effects on plasmacytoma and hybridoma lymphoid cells.

Murine alpha/beta interferon (IFN) inhibited the growth of myeloma cells in vitro. Independently of the cell growth inhibition, IFN also reduced the number of antibody secreting myeloma cells as measured by haemolytic plaque assay. The sensitivity of both MOPC 315 plasmacytoma cells and 4F4 hybridoma cells varied, though not to the same extent, depending on the dose of IFN used and the duration of exposure. Inhibition of PFC activity was observed after one day of IFN treatment while inhibition of cell growth was not detected until Day 2 of incubation. A dose of 20 u/ml IFN had no effect on the growth rate of MOPC 315 cells but with 100 u/ml the inhibition of growth was virtually complete. In contrast, an inhibition of PFC activity was observed at all the concentrations tested. The cell growth and PFC activity of 4F4 hybridoma cells, on the other hand, were both inhibited by IFN when used at concentrations as low as 1.25 u/ml. Incubation with higher concentrations of IFN resulted in a progressive reduction in cell growth and PFC activity of 4F4 cells, however to a lesser degree of inhibition compared to that observed with MOPC 315 cells. For example, although virtually 70% of MOPC 315 PFC could be inhibited by culture for two days in the presence of 100 u/ml, it was necessary to use 1,250 u/ml IFN for 4 days incubation before the similar level of PFC inhibition was achieved with 4F4 cells. IFN treatment resulted in an increase in both cellular volume and protein content and this effect was prevented when IFN was previously neutralised by a specific antiserum. IFN-treated cells also showed an inhibition in the incorporation of 3[H]-thymidine but no alteration in the rate of utilization of 35[S]-methionine, when compared with an equal number of control cells.

Induction of proteins in human lymphoblastoid cells by recombinant alpha interferon.

Two-dimensional gel electrophoresis, using either silver staining or pulse labeling with 35S-L-methionine and autoradiography, was employed to determine changes in the synthesis of proteins that may be involved in the antiproliferative effects of recombinant alpha interferon (IFNrA) on Burkitt's lymphoma Daudi cells. IFNrA initiated and/or augmented the synthesis of at least 13 proteins that were distinct in molecular weights and isoelectric properties. Synthesis of several of these IFN-enhanced proteins was inhibited by actinomycin-D, an inhibitor of mRNA synthesis. Although IFN-induced antiproliferative effects were observed at 48 h, an increase in the synthesis of several proteins was observed as early as 3 h. The levels of these IFN-enhanced proteins in treated cells continued to increase through 24 h. At least two proteins of approximately 17 kD were observed to be synthesized in IFN-treated cells but not in control cells. Neither inhibition of synthesis of any particular protein nor post-synthetic modification of proteins in response to IFNrA was observed with these methods. The results of this study are compared and contrasted to those of several other laboratories.

Effect of mouse interferon on the expression of a porcine major histocompatibility gene introduced into mouse L cells.

Mouse L cells transformed with a swine genomic clone, which encodes a major histocompatibility (MHC) antigen (SLAd), stably express SLAd on their surface. Mouse interferon (IFN) markedly enhances the expression of the heterologous SLAd as well as endogenous H-2k antigens in two independent transformants, as measured by flow microfluorometry. The expression of an unrelated surface antigen, gp70, is not affected. The increased expression of SLAd antigens is the result of an increased pool of SLA mRNA in IFN-treated cells. In vitro nuclear transcription experiments suggest that this increased accumulation of SLA mRNA results largely from an enhanced transcription of the heterologous SLA gene. The effect of IFN is not restricted to the MHC coding sequences; the transcription of two DNA sequence elements flanking the SLA gene, which are constitutively expressed in one L cell transformant, is also enhanced by IFN. In contrast, in another transformant these same flanking sequences are not transcribed and do not respond to IFN. The results suggest that IFN enhances the transcription of MHC coding and the flanking sequences that are expressed in its absence, but it does not appear to induce do novo transcription of the silent flanking genes examined.

Vaccinia rescue of VSV from interferon-induced resistance: reversal of translation block and inhibition of protein kinase activity.

Coinfection with vaccinia virus rescues vesicular stomatitis virus (VSV) from the inhibitory effects of interferon (IFN) in mouse L cells. While vaccinia infection does not significantly affect VSV RNA synthesis, coinfection with vaccinia dramatically increases VSV protein synthesis in IFN-treated cells. Evidence is provided that vaccinia inhibits the activity of the IFN-induced dsRNA-dependent protein kinase.

Do cells treated with human interferon survive virus infection?

HeLa cells pretreated with human lympho-blastoid interferon (Hu IFN-α (Ly)), at concentrations up to 100 IU / ml and infected with moderate multiplicities of encephalomyocarditis (EMC) virus (10 PFU / cell) died 1 or 2 days after infection. However, if cells were repeatedly treated with high doses of IFN (800 IU / ml) they survived infection by EMC virus for at least a month. Cells survived Semliki Forest virus (SFV) infection when even lower IFN concentrations were used. By contrast infection of IFN-treated HeLa cells with other RNA-containing viruses, such as poliovirus, vesicular stomatitis virus (VSV), Newcastle disease virus (NDV) and reovirus type 3 resulted in cell death. Similarly, infection with a number of DNA-containing viruses such as adenovirus type 5, Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and vaccinia virus killed cells. The results are discussed in the light of different models for the molecular mechanism of action of interferon.

Inhibition of retrovirus DNA supercoiling in interferon-treated cells.

The effect of interferon (IFN) on the cytoplasmic synthesis of murine sarcoma virus DNA, its transport to the host nucleus, and its integration into the cellular genome were investigated. For this purpose, at various intervals after infection. DNA was extracted from the cytoplasmic fraction, nuclear Hirt supernatant, and chromosomal DNA pellet. The relative amount of viral DNA was estimated by C0t kinetics analysis of hybridization to murine sarcoma virus-specific [3H]cDNA. IFN was found to delay viral DNA synthesis by about 2.5 h, but the amount of viral DNA eventually formed in IFN-treated cells was comparable to that of the control. The transport of this DNA to the nucleus was delayed by IFN for 6 to 18 h, but once again, all the cytoplasmic viral DNA formed in IFN-treated cells was eventually transferred to their nucleus. However, although the main part of the viral DNA formed in control cells was finally integrated into the host genome, no significant integration was observed in IFN-treated cells. Alkaline sucrose gradient analysis revealed that IFN inhibited the accumulation of supercoiled viral DNA in the nucleus. Since supercoiled viral DNA is considered a precursor to integrated provirus, this observation may suggest that IFN inhibits viral DNA integration by blocking its supercoiling.