Clonal derivatives of the RD-114 cell line differ in their antiviral and gene-inducing responses to interferons.

The human rhabdomyosarcoma cell line RD-114 is partially responsive to interferons (IFNs). In these cells, alpha interferon (IFN-alpha) or gamma interferon (IFN-gamma) inhibits the replication of some viruses but not of others. Similarly, some of the IFN-inducible mRNAs are induced poorly, whereas others are induced well. Here we report the isolation of clonal derivatives of this line which display different spectra of responses to IFNs. Among the eight extensively characterized clonal lines, one, C10, did not respond to IFN-alpha or IFN-gamma at all. Retrovirus production by each of the seven other lines was inhibited by both IFN-alpha and IFN-gamma. Replication of vesicular stomatitis virus was inhibited strongly by IFN-alpha in clone B1 but not in others, whereas it was not appreciably affected by IFN-gamma in any clone. Replication of encephalomyocarditis virus was inhibited strongly by IFN-gamma in clones A1, A2, A3, B3, and B8 and by IFN-alpha in clone A2. Neither IFN inhibited the multiplication of these clones greatly, although their doubling times were slightly increased. Five mRNAs were induced by IFNs to varying degrees in the seven clones. mRNA 2A was most strongly induced by IFN-gamma in clone A3. mRNA 1-8 was strongly induced by IFN-alpha in clone A1 and by either IFN in clones A2 and A3. The highest concentrations of 2',5'-oligoadenylate synthetase mRNA, mRNA 561, and mRNA 6-16 were in IFN-alpha-treated clones A1 and A2. These results demonstrated the existence of clonal heterogeneity in IFN responses in a cell line and strengthened the view that IFN treatment of cells generates multiple signals leading to a variety of IFN-induced phenotypes.     

Biochemical analysis of mutants of a macrophage cell line resistant to the growth-inhibitory activity of interferon

While a multiplicity of cellular and biochemical effects are mediated by interferons on cultured cells, the mechanisms involved in the direct growth-inhibitory activity of interferons remain problematic. We have previously found that variants in cAMP metabolism in a macrophage cell line, J774.2, were at least 50-fold less sensitive to the growth inhibitory activity of interferons (IFN) than the parental clone. To test the hypothesis that cAMP mediates the growth inhibition produced by IFN in these cells, interferon-resistant variants were selected and characterized with respect to cAMP synthesis and function. Approximately one-third of the IFN-resistant clones were found to be resistant to growth inhibition produced by cholera toxin, but not 8Br-cAMP. IFN was fully able to protect all of the interferon-resistant/choleratoxin-resistant (IFNr/CTr) clones against infection by vesicular stomatitis virus and markedly stimulated 2', 5'-oligodenylate synthetase activity. These IFNr/CTr variants were shown to have a defect in adenylate cyclase. The remaining IFN-resistant clones were fully susceptible to the growth-inhibitory effects of cholera toxin because their basal and stimulated adenylate cyclase activity is similar to that of the parental clone. IFN failed to protect these IFNr/choleratoxin sensitive clones against infection by vesicular stomatitis virus and failed to stimulate 2', 5-oligodenylate synthetase, suggesting that they have defective or deficient IFN receptors. In addition, IFN failed to increase intracellular cAMP levels in both IFNr/CTr and IFNr/choleratoxin sensitive clones. These results provide firm genetic and biochemical evidence that the growth inhibitory effects of IFN on this cell line are mediated by cAMP.     

Reversibility of the antiproliferative effect of interferon

The reversibility of the antiproliferative effect of interferon (IFN) and its correlations to the induction of (2',5') oligoadenylate synthetase (2-5A synthetase) activity was studied on NIH/3T3 cells transformed by Moloney murine sarcoma virus. The cells were treated with various doses of mouse beta-IFN. At 72 h after treatment, the cultures were subdivided. While half received fresh doses of IFN, the second half received no IFN. Reversibility of the IFN effect was then followed. Three different parameters as indicators for cell proliferation were used: cell growth, protein synthesis and cloning efficiency. In parallel, the IFN-induced activity of 2-5A synthetase was determined. The data obtained led to the following conclusions. (1) The antiproliferative effect of IFN increases with increased IFN concentration (90-1,800 IU/ml) and with time of treatment, up to 72 h after treatment. (2) The induced activity of 2-5A synthetase increases with a much faster rate, reaching maximum activity at 24 h after treatment with 450 IU/ml. This means that the induction of the enzyme precedes the antiproliferative effects of IFN. (3) There is almost no recovery of the IFN antiproliferative effect following treatment for 72 h with high doses of IFN (1,200-1,800 IU/ml). However, at lower doses, recovery is evident. (4) Removal of IFN after treatment for 3 days with 450 IU/ml resulted in a gradual decrease of 2-5A synthetase activity, reaching the basal level at 72 h after removal. However, there is no reduction of enzyme activity following treatment for 72 h with 1,800 IU/ml of IFN.     

Isolation and characterization of interferon-resistant variants from S49 mouse lymphoma

S49 mouse lymphoma cells were found to be extremely sensitive to the antiproliferative activity of interferon. These characteristics were studied to select for IFN-resistant cell variants. Some 0.6% of the parental S49 cell population were resistant to the antiproliferative and cytotoxic activities of IFN. The resistant cells were cloned and analyzed for their responses to several of the activities of IFN, namely, inhibition of encephalomyocarditis (EMC) virus, murine leukemia virus (MuLV) replications, and the induction of (2'-5') oligoadenylate synthetase. Among the clones selected some were highly resistant while others demonstrated only partial responsiveness to IFN. S49 cells demonstrate tubular structures in the cytoplasm. These structures were previously reported to be antigenically related to mouse mammary tumor virus (MMTV). We report here that IFN treatment decreases the expression of these cytoplasmic viral structures as revealed by electron microscopy. To correlate this novel antiviral activity to the more established functions of IFN we utilized the above mentioned S49 IFN-resistant variants. The anti-MMTV activity of IFN correlated with the other effects of IFN in both the highly resistant and partially responsive S49 clones. Our findings indicate that a relatively high proportion of S49 cells vary in their response to IFN. The defect in the resistant cells appears to affect a primary response to IFN which is common to its diverse activities. Furthermore, the effect of IFN on MMTV-related structures involves the usual pathway of IFN action.     

Biological characteristics of interferon-r-induced resistant histiocytic lymphoma cell line U937

A brief exposure (for 6 h) of U937 cells to interferon (IFN)-gamma (500 U/ml) followed by a long term incubation of cells in normal medium for 8 or more weeks resulted in the induction of cells that were refractory to the anticellular and differentiating effects of not only IFN-gamma but also IFN-alpha and IFN-beta at concentrations up to 10(4) U/ml. In addition, the cells became insensitive to the potent differentiating effect of the phorbol ester--tumor promoting agent (TPA). However, the resistant cells retained their sensitivity to the antiviral effect of different IFNs and were fullu responsive to the induction of endonuclease 2',5'-oligoadenylate (2-5A) synthetase by IFN. Furthermore, the resistant cell population appeared to be homogeneous because clones derived from single cells from this population all exhibited the same resistant phenotype to IFN and TPA. These results suggest that induction of resistant U937 cells may involve a dedifferentiation process which results in the formation of an immature cell population that do not respond to the differentiating and/or anticellular effects of various types of IFNs.     

Induction of interferon-specific enzymes in cultures of cells sensitive and resistant to interferon

Induction of 2'-5'-oligoadenylatesynthetase (2-5A synthetase) by interferons and theophylline by means of activation of cAMP-system in interferon susceptible and resistant cell lines were studied. In interferon resistant cell lines the basal activity of 2-5A synthetase exceeded the level of the same enzyme in interferon susceptible cell lines. Activity of 2-5A synthetase is increased in interferon susceptible cell lines by interferon treatment, but the activity of the enzyme is not altered in interferon resistant cell lines. Among the studied cell lines the induction of 2-5A synthetase by theophylline was possible only in L929 cell line. The common mechanism for the absence of 2-5A synthetase induction by interferon and theophylline in interferon resistant cells is discussed.     

Studies on the role of the 2''-5''-oligoadenylate synthetase-RNase L pathway in beta interferon-mediated inhibition of encephalomyocarditis virus replication.

Interferons inhibit the replication of vesicular stomatitis virus (VSV), but not of encephalomyocarditis virus (EMCV), in mouse JLSV-11 cells. We report the isolation of clonal derivatives from this cell line in which the replication of both viruses is impaired by interferons. These clones were selected from the parental line by virtue of their rescue by interferon treatment from the cytopathic effects of EMCV infection. In one such clone, RK8, the replication of VSV and EMCV and the production of resident murine leukemia virus were inhibited by interferon. On the other hand, in clone RK6, which was isolated without any selection, the replication of VSV, but not of EMCV, was impaired by interferons. The levels of 2'-5'-oligoadenylate synthetase mRNA and enzyme activity were similarly elevated upon interferon treatment in the two clones. However, the level of RNase L, as determined by binding and cross-linking of a radiolabeled 2'-5'-oligoadenylate derivative, was much lower in RK6 cells than in RK8 cells. In accord with this observation, the introduction of 2'-5'-oligoadenylates into cells inhibited protein synthesis much less strongly in RK6 cells than in RK8 cells. These results are consistent with the notion that the 2'-5'-oligoadenylate-dependent RNase L may be a mediator of the inhibition of EMCV replication by interferons.     

Enhancement of interferon-induced 2–5 oligoadenylate synthetase activity by retinoic acid in human histiocytic lymphoma U937 cells and WISH cells

The effect of retinoic acid (RA) on the level of interferon (IFN)-induced 2-5 oligoadenylate (2-5A) synthetase activity was examined in human histiocytic lymphoma U937 cells and WISH cells** in order to ascertain the role of this polymerase in interaction between IFNs and RA. Cultures containing both IFNs (1-100 U/ml) and RA (0.1-10 microM) consistently had higher levels of enzyme activity than corresponding cells treated with IFN alone and this was true for all three types of IFNs in both cell lines. The potentiating effect of RA was dose- and time-dependent and under optimal conditions, the induction of the synthetase was synergistic between IFN-beta (10-100 U/ml) and RA (0.1-10 microM). Furthermore, pretreatment (but not posttreatment) with RA followed by subsequent treatment with IFNs preferentially induced higher levels of enzyme activity in U937 cells but not in WISH cells. In addition, our results indicated that the modulating effect of RA on IFNs did not involve interaction at the receptor level and the level of enhancement of 2-5A synthetase activity was not in parallel with either cell-growth arrest or promotion of differentiation. Lastly, the present study raises the possibility that interactions between IFNs and RA, in either a synergistic or antagonistic manner, may be mediated through amplification of the 2-5A system.     

Induction of (2'-5')oligoadenylate synthetase in serum-starved HeLa S3 cells by growth factors and its role in growth regulation

When serum-starved HeLa S3 cells were stimulated to proliferate by addition of fetal calf serum (FCS), (2'-5')oligoadenylate synthetase (2-5A synthetase) activity was induced. Although no interferon (IFN) activity was detectable in the HeLa S3 cell-conditioned culture medium after growth stimulation, addition of anti-IFN-beta monoclonal antibody inhibited both the expression of the 2-5A synthetase gene and the production of the enzyme, suggesting that endogenous IFN-beta was involved in 2-5A synthetase induction. Purified preparations of three growth factors, epidermal growth factor, platelet-derived growth factor, and insulin, also induced 2-5A synthetase through IFN-beta. When serum-starved HeLa S3 cells were treated with FCS, DNA synthesis was initiated synchronously, with peaks after 12 and 32 h, although the level of 2-5A synthetase reached a maximum after the first peak of DNA synthesis. Inhibition of 2-5A synthetase induction by anti-IFN-beta antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa S3 cells, after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.     

Expression of interferon-inducible genes in RD-114 cells.

RD-114 is a cell line which is partially responsive to interferon (IFN). Although both IFN-alpha and IFN gamma inhibit production of the resident retrovirus, they do not inhibit replication of other viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, in these cells. In the studies reported here, we studied the characteristics of induction of seven IFN-inducible mRNAs in RD-114 cells. We observed that mRNAs 561, 6-16, 1-8, 2A, and 6-26 have similar induction characteristics in RD-114 cells and in HeLa cells, a fully responsive line. mRNA 2'-5'-oligo-adenylate synthetase (2-5(A) synthetase), however, was induced more efficiently by IFN-alpha in HeLa cells than in RD-114 cells. The same was true for the induction of metallothionein II mRNA by IFN-gamma. However, the latter mRNA was induced equally strongly in both lines when ZnCl2 was used as the inducer, suggesting that the gene is not defective in RD-114 cells. Although IFN-alpha induced 2-5(A) synthetase mRNA poorly and IFN-gamma did not induce it at all in these cells, a mixture of IFN-alpha and IFN-gamma induced this mRNA quite effectively, to a level of induction comparable to that in HeLa cells. Only 1 U of IFN-gamma per ml was sufficient to elicit this synergism, and the data suggested that an IFN-gamma-inducible protein was needed for this process. Induction of mRNA 561 by IFN-alpha in RD-114 cells, unlike that in HeLa cells, did not need ongoing protein synthesis. Once induced, this mRNA turned over rapidly in both cell lines, and this turnover could be slowed down by inhibiting protein synthesis in either cell line. IFN-induced mRNAs, such as 561 and 1-8, were polysome associated in IFN-treated RD-114 cells, suggesting that they were actively translated. Therefore, it is unlikely that the products of these IFN-inducible genes, by themselves, mediate the inhibition of replication of those viruses which are insensitive to IFN action in RD-114 cells.     

Effect of interferon on the production of HBsAg and induction of an antiviral state in human hepatoma cell line PLC/PRF/5

The effects of human alpha and beta interferons (IFN) on the production of HBsAG by PLC/PRF/5 cells, an HBsAg-producing human hepatoma cell line, were studied in the exponential and stationary phases of cell growth. When exponential phase cells were treated with 100 or 1,000 U of IFN per ml for 48 hr. the amount of HBsAg in the culture medium decreased. The number of cells and the synthesis of DNA and proteins were also reduced by the IFN treatment. These results suggested that IFN did not affect the production of HBsAg specifically in exponential phase cells. When cells in the stationary phase were similarly treated with IFN, HbsAg production was not inhibited nor did the number of cells decrease. To examine the antiviral state induced by IFN in PLC/PRF/5, induction of 2'5'-oligo (A) synthetase and susceptibility to two kinds of viruses were examined. The 2'5'-oligo (A) synthetase activity was increased in an IFN-dose dependent manner. Susceptibility to vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) was decreased by treatment with 10 and 100 U of IFN per ml for 20 hr. It was concluded that IFN-alpha and IFN-beta induce 2'5'-oligo (A) synthetase and the antiviral state, but do not inhibit HBsAg production by PLC/PRF/5 cells.     

Pharmacokinetic study of a human recombinant interferon (Re-IFN-alpha A) in cynomolgus monkeys by 2'-5' oligoadenylate synthetase assay

We evaluated 2'-5'Oligoadenylate (2-5 A) synthetase assay for pharmacokinetic study of human interferon (IFN) in cynomolgus monkeys. The enzyme was induced in primary cultures of cynomolgus monkey kidney (PMK) cells as well as in FL cells in response to human recombinant IFN-alpha A treatment. The enzyme activity increased with IFN dose and, in parallel with the enzyme elevation, developed the antiviral state of the cells. The enzyme activity induced in the peripheral blood lymphocytes peaked at 6 to 12 hr after iv or im administration. The peak level of the enzyme activity depended on the IFN concentration of the blood and the activity rapidly decreased as serum IFN was cleared from the blood. These results indicate that human recombinant IFN-alpha A induces 2-5 A synthetase in monkey cells both in vitro and in vivo, and that the enzyme assay can be used to quantitatively monitor the host response after IFN administration.     

Expression of interferon-induced genes in different tissues of mice.

In vivo responses to interferon (IFN) in mice were determined by measuring the steady-state levels of induced mRNAs following injection of IFN and poly(I)-poly(C). With cDNA probes for mouse 2'-5' oligoadenylate synthetase (2-5A synthetase) and 1-8, constitutive expression of the corresponding mRNA was detectable in different organs of normal C3H/He mice. These mRNA levels were increased by as much as 15-fold over control levels in various tissues, including the brain, after IFN and poly(I)-poly(C) treatment, coincident with increases in 2-5A synthetase enzyme activity. The basal activity level of this enzyme could be reduced in normal mice by treatment with anti-mouse IFN (alpha + beta) antibody. This treatment also reduced the levels of 2-5A synthetase and 1-8 mRNAs. Thus, physiological levels of circulating IFN maintain elevated levels of IFN-induced mRNAs in mice. Furthermore, changes in 2-5A synthetase enzyme activity reflect the changes in gene expression in vivo.     

In vivo and in vitro induction of (2'-5') oligoadenylate synthetase by human interferons in leukocytes from healthy donors and patients with renal cancer and hairy cell leukemia

The effect of in vivo administered interferon-alpha (IFN-alpha) on 2-5-oligoadenylate (A) synthetase activity of peripheral blood mononuclear cells (PBMC) was compared in patients with hairy cell leukemia and renal cell cancer. Basic levels of this enzyme varied from donor to donor, but mean levels were not significantly different in patients with renal cell cancer or hairy cell leukemia compared to healthy donors. After a single injection of 3 x 10(6) IU IFN, these basic levels rose 2- to 8-fold within 12-24 h post-injection and reverted to pretreatment levels after 48 h. The extent of this in vivo stimulation by IFN-alpha was similar in patients with hairy cell leukemia and renal cell carcinoma, and was correlated with down-regulation of IFN-alpha receptors. The in vitro effects of IFN-alpha, -beta and -gamma were compared after 18 h treatment with 10, 10(2) and 10(3) IU/ml of each IFN. Unlike IFN-alpha and -beta, IFN-gamma did not induce 2-5 A synthetase activity in either normal PBMC or hairy cells; these results were related to the effects of the three IFN on proliferative response of normal PBMC to phytohemagglutinin. Our data support the idea that 2-5 A synthetase activity is a marker of biological response to interferon treatment in human cancers.     

VIP induces in HT-29 cells 2'5'oligoadenylate synthetase and antiviral state via interferon beta/alpha synthesis.

Vasoactive intestinal peptide (VIP), composed of 28 amino acids, is a multifunctional neurotransmitter. We have demonstrated here that its action on human transformed colonic epithelial (HT-29) cells is mediated through the induction of interferon (IFN) synthesis. We have found that these cells have a functional receptor for IFN alpha 2; binding was specific to either IFN alpha 2 or IFN beta but not to IFN gamma. VIP induced the 2'5'oligoadenylate synthetase (2'5'A synthetase) and the antiviral state with the same efficiency as poly (I).poly (C). The induction of 2'5'A synthetase activity required cellular RNA and protein synthesis, and the maximum induction occurred with 10(-7) M VIP at 24 h. VIP, like some IFN inducers, induced the synthesis of the 70 hsp which, however, preceded the expression of 2'5'A synthetase. VIP treatment caused the induction and secretion of IFN, having a titer value of 32 international units/ml. This IFN has been identified as type beta/alpha, because both 2'5'A synthetase and the antiviral activities were abolished by anti-human IFN beta/alpha antibodies, but not by anti-IFN gamma antibodies. Thus the pathway of VIP action on HT-29 cells may be outlined as 1) binding of VIP, 2) synthesis of 70 hsp, 3) induction of IFN synthesis and its secretion, 4) binding of the secreted IFN to cell surface receptors and 5) turning on the induction of 2'5'A synthetase and antiviral activities.     

PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2alpha phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2alpha phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-alpha is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2alpha phosphorylation is induced by IFN-alpha in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2alpha phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2alpha phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3' untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.