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.     

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.     

Growth regulation of A431 cells. Modulation of expression of transforming growth factor-alpha mRNA and 2',5'-oligoadenylate synthetase activity

To explore bidirectional regulatory interactions between interferons and autocrine polypeptide factors, we examined the modulation of expression of transforming growth factor-alpha and 2',5'-oligoadenylate synthetase activity in A431 epidermoid carcinoma cells after treatment with interferon-gamma and transforming growth factor-alpha. Treatment of A431 cells with interferon-gamma increased steady state levels of transforming growth factor-alpha mRNA by 4-fold and increased the levels of transforming growth factor-alpha in the culture medium. There were additive growth inhibitory effects upon coaddition of exogenous transforming growth factor-alpha and interferon-gamma to the cultures. Addition of transforming growth factor-alpha to A431 cell cultures in the absence of interferon could stimulate the induction of 2',5'-oligoadenylate synthetase activity by more than 2-fold. These findings demonstrate that the induction of transforming growth factor-alpha in interferon-gamma-treated A431 cells could act to regulate interferon-induced gene(s), e.g. 2',5'-oligoadenylate synthetase, suggesting interactions between a potential autocrine growth factor and the interferon system in the growth regulation of A431 cells.     

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 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.     

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.     

2',5'-Oligoadenylate synthetase expression is induced in response to heat shock

Hyperthermia (45 degrees C) induced the synthesis of a characteristic heat-shock protein of 70,000 daltons (70 hsp) in Madin-Darby bovine kidney (MDBK) cells. In addition, subsequent to heat shock, there was a substantial increase in the 2',5'-oligoadenylate synthetase (2',5'-A synthetase) activity in both MDBK and human WISH cells. However, in contrast to 70 hsp synthesis, which reached its maximum 3 h after cell transfer from 45 to 37 degrees C, increase in 2',5'-A synthetase expression, conspicuous after 6 h, attained its maximum only 18 h after transfer. Another interesting observation is that, during recovery at 37 degrees C, the cells released into the medium heat-shock-induced factor(s) (HSIF) capable of inducing an increase in 2',5'-A synthetase activity in fresh MDBK cells. HSIF behaves as a polypeptide with a molecular weight of more than 5,000; it is relatively heat stable and sensitive to acidic treatment. HISF seems different from interferon (IFN) since: 1) no detectable antiviral state developed after infection in cells treated with HSIF; 2) antibovine IFN antibodies did not abolish the inducing capacity of HSIF; 3) IFN had an additive effect on the inducing capacity of HSIF, and 4) HSIF released from bovine cells induced a net enhancement of 2',5'-A synthetase activity in human WISH cells. The first three of these observations applied also to heat-shocked MDBK cells.     

Interleukin-6 induces the (2'-5') oligoadenylate synthetase gene in M1 cells through an effect on the interferon-responsive enhancer

Interleukin-6 (IL-6) activates (2'-5') A synthetase (2'-5' AS) gene expression in differentiating myeloleukemic M1 cells. Antibodies to type I interferon (IFN) inhibit 2'-5' AS induction but not differentiation. Analysis of the mechanism of 2'-5' AS induction shows that it does not result from increased IFN formation, but from a synergism between IL-6 and endogenously secreted IFN. IL-6 can activate expression of a CAT construct fused to the interferon response sequence (IRS) of the 2'-5' AS gene. In extracts of IL-6-treated M1 cells, changes in protein binding to IRS DNA can be demonstrated. One of the effects of IL-6 on M1 cells is, therefore, to induce DNA binding factors, some of which act on the same enhancer sequence as IFNs, resulting in a synergistic gene activation. M1 variants resistant to differentiation by IL-6 have lost the ability to induce the 2'-5' AS gene.     

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.     

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.     

Induction of 2,3-bisphosphoglycerate synthase in Friend leukemia cells

Friend leukemia cells (clone 745A) induced to differentiate with dimethylsulfoxide showed at least a 10-fold increase of 2,3-bisphosphoglycerate synthase and concomitant accumulation of 2,3-bisphosphoglycerate. These changes paralelled that of the number of hemoglobin-positive cells. Accumulation of 2,3-bisphosphoglycerate was also induced by dimethylsulfoxide in the other clone C-10-6, but not in C-9-6 which is resistant to differentiation with dimethylsulfoxide. Induced activity of 2,3-bisphosphoglycerate synthase in clone 745A was neutralized by antiserum prepared from a rabbit which was immunized with human erythrocyte 2,30bisphosphoglycerate synthase. By using this antiserum, biosynthesis of 2,3-bisphosphoglycerate synthase was detected in Friend cells only after induction by dimethylsulfoxide.     

Effects of cyclic AMP on the growth of differentiating and undifferentiated Friend erythroleukemic cells.

Elevated concentrations of cyclic AMP elicit only minor reductions in growth rate and saturation density in undifferentiated Friend erythroleukemic cells. During the course of dimethylsulfoxide (DMSO)-induced differentiation, Friend cells convert from a cyclic AMP-tolerant state to a phenotype characterized by a high degree of sensitivity to cyclic AMP-mediated growth arrest. Conversion to cyclic AMP sensitivity is detectable after 30 hours growth in medium containing 2% DMSO, and either 0.5 mM 8-Br-cyclic AMP or 5 nM cholera toxin. Cultures of differentiating Friend cells achieved a stationary phase density that was approximately 8-fold higher than the cell density observed in parallel, differentiating cultures treated with 0.5 mM 8-Br-cyclic AMP. Temporally, the appearance of cyclic AMP-sensitivity corresponds to the early expression of in vitro erythroid differentiation (Ross et al., '74), but growth arrest does not alter the subsequent accumulation of hemoglobin in non-dividing DMSO-induced cells. Since growth arrest is preceded by a round of cell division, these observations are consistent with the concept that DMSO must be present during DNA replication for the subsequent expression of hemoglobin synthesis (McClintock and Papaconstantinou, '74; Levy et al., '75; Harrison, '76).     

Cloning of a novel 2',5'-oligoadenylate synthetase-like molecule, Oasl5 in mice.

The 2',5'-oligoadenylate synthetase (2-5OAS) is a enzyme that catalyzes synthesis of 2',5'-oligoadenylates (2-5A) in a dsRNA-dependent manner, and known as a major component of the IFN-induced host defense mechanisms against microbial infections. Here, we report the presence of a novel 2-5OAS-like molecule, termed Oasl5, in mice. The size of Oasl5 cDNA was about 2 kb and encoded a protein consisting of 362 aa. The amino acid sequence showed 76% similarity to the mouse 2-5OAS, however, several motifs being important for the enzyme activity were not conserved. The Oasl5 mRNA was most significantly expressed in the brain, and relatively weak expression was found in other organs such as the spleen, kidney, ovary and testis. It was also expressed in embryonic stem (ES) cells. The Oasl5 mRNA expression in ES cells was elevated 5-fold after treatment with IFN and about 2-fold in the brain when stimulated with IFN inducer, polyinosinic-polycytidylic acid (poly[I:C]). In situ hybridization analysis revealed that Oasl5 is expressed in neurons in the central nervous system in adult mice. When Oasl5 was expressed in E. coli, it yielded 42 kDa protein that binds to dsRNA, but it did not show oligoadenylate synthetase activity. These findings suggest a novel function of Oasl5, which are independent of oligoadenylate synthetase activity, in the brain and developing embryos.     

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.     

Growth regulation of melanoma cells by interferon and (2''-5'')oligoadenylate synthetase.

We report that endogenous, as well as exogenous, interferon (IFN) regulates the growth of human melanoma cells in culture. When antibodies directed against human fibroblast IFN were incorporated into the media of high-density cells stimulated to proliferate with serum, the cells entered the cell cycle earlier than did the controls. In investigating the biochemical basis for this finding, we have found that there is an inverse relationship between the (2'-5')oligoadenylate synthetase levels and the percentage of cells in S in untreated cultures. Upon IFN treatment, the relationship is obliterated and (2'-5')oligoadenylate synthetase levels increase throughout all phases of the cell cycle. This increase in enzyme levels correlates well with the decreased probability of the IFN-treated cells to cycle. These findings suggest a biological role for IFN as a negative growth factor for cells in culture.     

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.