Regulation of 2'',5''-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor.

In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2',5'-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta interferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2',5'-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2',-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.     

Induction and maintenance of 2'',5''-oligoadenylate synthetase in interferon-treated chicken embryo cells.

Treatment of primary cultures of chicken embryo cells with homologous interferon results in a substantial increase in the level of 2',5'-oligoadenylate synthetase activity that can be detected in cell extracts. This increase can be prevented by inhibitors of RNA or protein synthesis and is thus thought to represent the induction of an interferon-inducible gene, perhaps the 2',5'-oligoadenylate synthetase gene itself. To examine this response in greater detail, we studied its kinetics under the following conditions: (i) cessation of interferon treatment after different lengths of time, (ii) delayed inhibition of RNA or protein synthesis, and (iii) combinations of these treatments. The results showed that in cells treated continuously with interferon, the enzyme level reached a peak after 9 h of treatment and then decreased with a half-life of about 30 h, despite the continued presence of interferon. Removal of interferon during induction reduced the peak level of activity that was attained and somewhat accelerated its decline but did not otherwise affect the time-course of the response. On the other hand, removal of interferon after maximum induction clearly accelerated the decay of enzyme activity. This process could be delayed by inhibitors of protein synthesis, which effectively stabilized the induced enzyme. This behavior is reminiscent of other inducible enzymes, such as the steroid-induced tyrosine aminotransferase, and suggests that the level of 2',5'-oligoadenylate synthetase, which is also inducible by steroid hormones in some cell types, is subject to similar control mechanisms.     

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.     

Induction of poly(I):poly(C)-binding 50 K protein and 2',5'-oligoadenylate synthetase in interferon-treated mouse L929 cells

A new protein retained by poly(I):poly(C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2',5'-oligoadenylate synthetase (2,5A synthetase), in interferon-treated mouse L929 cells; it had an apparent molecular weight of 50,000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly(I):poly(C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein. When the interferon preparation was heated to 100 degrees C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2',5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a de novo synthesis of the enzyme.     

Chloroquine impairs the interferon-induced antiviral state without affecting the 2',5'-oligoadenylate synthetase

Chloroquine, a weak base which raises the pH in acidic cellular compartments such as lysosomes and endosomes, counteracts the induction by interferon of the antiviral state but not that of the 2',5'-oligoadenylate synthetase in three different types of cell lines (MDBK, WISH, and L929). Active interferon is recovered in crude extracts of cells which have been treated with interferon and chloroquine together, but not in extracts of cells treated with interferon alone, indicating that chloroquine has inhibited the intralysosomal proteolysis of interferon. A low pH-dependent event in the intracellular fate of interferon (perhaps its intralysosomal degradation) is, therefore, necessary for the establishment of the antiviral state but not for the induction of the 2',5'-oligoadenylate synthetase.     

Inhibitor of 2',5'-oligoadenylate synthetase induced in human T lymphoblastoid cell line treated with deoxyadenosine, deoxycoformycin and interferon

The effect of deoxyadenosine (dAdo) with deoxycoformycin on the induction of 2',5'-oligoadenylate synthetase by interferon was investigated. After semi-purification through poly(I):poly(C) gel, the activity was similar in control and dAdo-treated cells. However, the activity in the crude extract decreased with rising concentrations of dAdo. On the other hand, the level of 2'-phosphodiesterase, which is also induced by interferon and degrades 2',5'-oligoadenylate, showed no significant change after dAdo treatment. Thus, the crude extract was speculated to contain an inhibitor of 2',5'-oligoadenylate synthetase. Further characterization of the inhibitor revealed that inhibition was not due to dATP accumulation in cells.     

Activation of 2'',5''-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.     

Initial characterization of a spontaneous interferon secreted during growth and differentiation of Friend erythroleukemia cells

A gradual increase in the level of 2',5'-oligoadenylate synthetase takes place in Friend erythroleukemia cells after a shiftdown in the rate of cell growth. The increase is about 5-fold after entry of cells into the stationary phase of growth, but much higher (25-fold) when reduction in growth accompanies cell differentiation. In the latter case, the enzyme increase is similar to that which can be induced in these cells by exogenous interferon (IFN). The increase in 2',5'-oligoadenylate synthetase was shown to be due to a spontaneous secretion of IFN by the cells themselves: it is completely abolished if antiserum to murine type I IFN is added to the culture medium. In attempts to isolate some of this spontaneously secreted IFN, we show that it is stable at pH 2, not neutralized by antiserum to type II IFN, and that it also differs from the known IFN species induced by Sendai virus in Friend cells. The major component of this spontaneously secreted IFN is 20,000 M(r) and differs from the corresponding virus-induced 20,000-M(r) IFN by its lower affinity for antiserum to type I IFN and its antigenic characterization as beta-murine IFN. The major component of the spontaneous IFN also exhibits a higher ratio of antigrowth to antiviral activity than the Sendai-induced IFNs. We suggest that Friend cells produce this specific type of IFN for the regulation of their growth and differentiation.     

Ethanol induces 2',5'-oligoadenylate synthetase and antiviral activities through interferon-beta production.

We demonstrate here that ethanol, in contrast to heat shock (Chousterman, S., Chelbi-Alix, M.K., and Thang, M.N. (1987) J. Biol. Chem. 262, 4806-4811), induces interferon (IFN) synthesis and its related activities in Madin-Darby bovine kidney (MDBK) cells. The induced IFN is secreted maximally at 6 h, whereas the induction of 2',5'-oligoadenylate synthetase mRNA peaks between 9 and 12 h and its activity at 15 h. The appearance of both 2',5'-oligoadenylate synthetase activity and the antiviral state upon ethanol treatment is prevented by anti-bovine recombinant IFN-beta antibodies. Bovine diarrhea virus infection-free MDBK cells cultured in medium supplemented with serum substitute also gave similar results, thus indicating that IFN synthesis induced by ethanol is not mediated by the activation of bovine diarrhea virus. Together, these results show that: 1) ethanol induces the 2',5'-oligoadenylate synthetase and antiviral activities through IFN-beta production; and 2) the IFN produced does not act directly from inside the cells, but has to be first secreted to bind to its receptor. In MDBK cells, ethanol induces the synthesis of the 70-kDa protein, which precedes the expression of 2',5'-oligoadenylate synthetase; moreover, the transient nature of the synthesis of the hsp 70 in these cells is similar after both heat shock and ethanol treatment.     

The dynamics of 2',5'-oligoadenylate synthetase activity in the nuclear and cytoplasmic fractions of human fibroblasts treated by double-stranded RNAs, by their complexes with DEAE-dextran and by type-I recombinant interferons. The ratio of double-stranded RNA-activated and -nonactivated froms of the enzyme

Novel original preparations of double-stranded RNAs (dsRNAs), i.e. larifan, ridostin and rifastin, and recombinant alpha 2- and beta-interferons promising for the clinical use were studied. The size and morphology of the dsRNAs in the preparation composition, the dynamics of their induction of interferon and the antiviral state in human fibroblasts and the effect of the DEAE dextran polycation on the activity of the dsRNAs were specified. For the first time the dynamics of 2',5'-oligoadenylate synthetase activity in the nuclei and cytoplasm of the human fibroblasts treated with the dsRNAs of different origin and their complexes with DEAE dextran was defined. To elucidate the specific features of the mechanism of antiviral action of dsRNAs and interferon, the relation of the 2',5'-oligoadenylate synthetase activity to dsRNAs was investigated. In the cells treated with dsRNAs and DEAE dextran there were an early activation of the enzyme and predominance of the enzyme activated forms requiring no addition of poly I.poly C to the reaction mixture. The results were indicative of possible intracellular activation of its isoforms, similar to that in the cells treated with interferon and contaminated with viruses. All the tested preparations of dsRNAs and interferons induced an increase in the activity of 2',5'-oligoadenylate synthetase both in the cytoplasm and the nuclei of human fibroblasts. The same ability was observed in DEAE dextran which is likely to be one of the causes of the increase in dsRNAs antiviral activity under its effect.     

2',5'-Oligoadenylate synthetase activity in lymphocytes from normal mouse.

The activity of 2',5'-oligoadenylate synthetase, an enzyme recently discovered in interferon-treated cells, was found in lymphocytes from normal mouse spleen that had received neither exogenous interferon nor its inducers. The oligoadenylate synthesized by lymphocyte cell extracts inhibited protein synthesis in rabbit reticulocyte lysates. The oligomers were composed mainly of trimer and were resistant to digestion by T2 ribonuclease. The level of the enzyme in lymphocytes was about 20 to 30% of that in L929 cells treated with interferon. The activity of the enzyme was further enhanced in lymphocytes in vitro by addition of interferon. The 2',5'-oligoadenylate synthetase was distributed among several lymphoid tissues, but was not detected in cell extracts from brain or liver. The enzyme may play an important role in the regulation of the immune system.     

Inhibition of mouse fibroblast interferon by gangliosides. Differential effects on biological activity and on induction of (2'--5')oligoadenylate synthetase

Gangliosides are potent inhibitors of the antiviral activity of mouse fibroblasts and other beta-interferons. We have compared the effects of gangliosides on antiviral and antigrowth activities of mouse fibroblast interferon and on the induction of (2'--5')oligoadenylate synthetase, one of the enzymes implicated in the antiviral state induced by interferon. Whereas both biological effects appear to be inhibited by gangliosides in an analogous fashion, inhibition of induction of (2'--5')oligoadenylate synthetase does not correlate with inhibition of vesicular stomatitis virus replication. Ganglioside concentrations that inhibit the interferon-induced (2'--5')oligoadenylate synthetase to levels close to those of uninduced cells, still allow for a 100--1000-fold reduction of viral yield. Significantly higher ganglioside concentrations are required to prevent completely the antiviral effect. This biphasic relationship between (2'--5')oligoadenylate synthetase levels and inhibition of viral yield suggests that no or very small increases in synthetase levels are involved in inhibition of virus by between two and three orders of magnitude.     

Interferon action: binding of viral RNA to the 40-kilodalton 2''-5''-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus.

The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by double-stranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2'-5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A)n synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN-treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa (2'-5') (A)n synthetase protein in complex with both strands of EMCV RNA. The immunoprecipitate was active in (2'-5') (A)n synthesis even without addition of double-stranded RNA, whereas the immunoprecipitate from IFN-treated, uninfected cells was not. These and other results demonstrate that in IFN-treated, EMCV-infected cells, viral RNA is bound to the (2'-5') (A)n synthetase and suggest that the agent activating the (2'-5') (A)n synthetase is the bound viral RNA.     

Differential induction of 2',5'-oligoadenylate synthetase by IFN-beta ser and IFN-alpha 2 in serum-supplemented and serum-free HL-60 leukemic cell cultures

The influence of cell culture conditions on the induction of 2',5'-oligoadenylate (2-5A) synthetase by recombinant interferons IFN-beta ser and IFN-alpha 2 has been investigated in human HL-60 leukemic cells. Cells maintained either in the fetal bovine serum-supplemented medium (FBS-SM) or in a serum-free, chemically defined Nutridoma-supplemented medium (SFN-SM) are treated with different concentrations of the two types of IFN and the extent of 2-5A synthetase induction compared. While cells in FBS-SM show a substantially greater increase in 2-5A synthetase by IFN-beta ser than cells in SFN-SM, the latter culture condition is significantly more effective in elevating synthetase activity with the addition of IFN-alpha 2. These data suggest that there are growth modulators and other "factors" in the fetal bovine serum which may interact specifically with each type of IFN to coordinate the optimal expression of the 2-5A synthetase protein.     

Interferon and phorbol esters down-regulate sIgM expression by independent pathways

We studied the effects of recombinant interferon, 12-0-tetradecanoylphorbol-13-acetate (TPA), and phorbol 12, 13 dibutyrate (PDB) on surface immunoglobulin expression by Daudi cells. Incubation of cells with recombinant alpha 2 interferon (IFN-alpha 2) caused a 2.5-fold (60%) decrease in sIgM expression as measured by relative fluorescence index (RFI) using a flow cytometer. This decrease in sIgM expression was independent of inhibitory effects on proliferation and cell cycle progression. TPA or PDB also caused a threefold (67%) decrease in sIgM expression, while enhancing proliferation and cell cycle progression. Coincubation of cells with IFN-alpha 2 and TPA decreased sIgM expression by more than fourfold (greater than 75%), which was greater than the decrease induced by the optimal concentration of either agent alone. Molecular studies demonstrated that the treatment of cells with IFN-alpha 2 or TPA decreased the steady-state levels of mRNA for the heavy chain of IgM (c mu), suggesting that down-regulation of sIgM occurred at a pretranslational level. Activation of the cell membrane sodium/proton antiport did not play an integral role in the IFN-alpha 2 or phorbol-ester-induced pathway of sIgM down-regulation. Whereas IFN-alpha 2 induced an increase in the activity of 2',5'-oligoadenylate (2-5A) synthetase, the addition of TPA to IFN-alpha 2 caused a significant decrease in the activity of this enzyme. Although IFN-alpha 2 and TPA exhibited additive effects on sIgM expression, they had opposing effects on cell proliferation, cell cycle progression, and induction of 2-5A synthetase activity, suggesting that these agents down-regulate sIgM expression through independent pathways.     

A misaligned double-stranded RNA, poly(I).poly(C12,U), induces accumulation of 2',5'-oligoadenylates in mouse tissues

2',5'-Oligoadenylate synthetase was induced 3-2000-fold in spleen, liver, kidney and brain of NIH Swiss mice injected intravenously with 2-200 micrograms of the misaligned dsRNA, poly(I).poly(C12,U). Levels of 2',5'-oligoadenylates extracted from these tissues were also elevated, although the amount of 2',5'-oligoadenylates extracted did not correlate directly with the amount of enzyme present. These results suggest that double-stranded portions of the misaligned polymer survived intracellularly and activated the 2',5'-oligoadenylate synthetase, and that the level of dsRNA may contribute to the control of 2',5'-oligoadenylate metabolism.     

Interferon-induced 2',5'-oligoadenylate system: key components and biological functions

The current data about the 2',5'-oligoadenylate system is reviewed. Its role in interferon signaling and cell metabolism regulation is discussed. The interferon system is known to be characterized by a wide range of biological functions such as antiviral defense, control of cell growth and differentiation, oncogenic stability, apoptosis, immune activation, etc. The biological role of interferon that is the multifunctional cytokine is discussed more in detail. The structure of main components of interferon signal transduction cascade (2',5'-oligoadenylate, 2',5'-oligoadenylate-synthetase and ribonuclease L) is reviewed. The interferon-induced 2',5'-oligoadenylate system is considered as the component of common regulatory system coordinating cell metabolism.