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.     

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.     

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.     

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.     

An improved method for purifying 2',5'-oligoadenylate synthetases

We describe a new, rapid, and convenient procedure for purifying 2',5'-oligoadenylate synthetases, employing precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) X poly(C) cellulose, and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 X g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. We have purified the enzymes from two sources to apparent homogeneity, about 2500-fold from the cytoplasm of HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2',5'-oligoadenylate synthetase from rabbit reticulocytes, mouse L cells, and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) X poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) X poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) X poly(C) paper and activates the bound enzyme about three times better.     

2',5'-Oligoadenylates and related 2',5'-oligonucleotide analogues. 1. Substrate specificity of the interferon-induced murine 2',5'-oligoadenylate synthetase and enzymatic synthesis of oligomers

The substrate specificity of the interferon-induced mouse L-cell enzyme, 2',5'-oligoadenylate synthetase, was determined with a number of nucleoside 5'-triphosphate analogues. Selected nucleoside 5'-triphosphates were converted to 2',5'-oligonucleotides with the following order of efficiency for the nucleoside: 8-azaadenosine greater than adenosine = 2-chloroadenosine greater than sangivamycin greater than toyocamycin greater than formycin greater than 3-ribosyladenine greater than ribavirin greater than tubercidin greater than adenosine 1-oxide greater than 2-beta-D-ribofuranosylthiazole-4-carboxamide greater than inosine = 1,N6-ethenoadenosine greater than guanosine greater than 8-bromoadenosine = uridine greater than cytidine. Adenosine 5'-((beta, gamma-imidotriphosphate) did not seem to be a recognizable substrate since no detectable product resulted. Either the 2',5'-oligoadenylate synthetase is not as specific as had been previously thought, or there may be more than one 2',5'-oligonucleotide synthetase. The 2',5'-oligonucleotide analogue products in which the adenosine of ppp(A2'P5')nA was replaced by the various nucleoside analogues were separated by DEAE-cellulose column chromatography and the chain length and number of 5'-phosphate residues analyzed by a rapid, efficient high-performance liquid chromatographic (HPLC) system involving ion-pairing C18 reversed-phase column chromatography. Separation of the 5'-mono-, 5'-di-, and 5'-triphosphorylated forms of the 2',5'-oligonucleotide analogue dimers, trimers, tetramers, and pentamers was readily achieved by this useful HPLC system. No 5'-nonphosphorylated forms were detected for any of the 2',5'-oligonucleotide analogue products.     

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.     

Expression and characterization of recombinant 2',5'-oligoadenylate synthetase from the marine sponge Geodia cydonium

2',5'-oligoadenylate (2-5A) synthetases are known as components of the interferon-induced cellular defence mechanism in mammals. The existence of 2-5A synthetases in the evolutionarily lowest multicellular animals, the marine sponges, has been demonstrated and the respective candidate genes from Geodia cydonium and Suberites domuncula have been identified. In the present study, the putative 2-5A synthetase cDNA from G. cydonium was expressed in an Escherichia coli expression system to characterize the enzymatic activity of the recombinant polypeptide. Our studies reveal that, unlike the porcine recombinant 2-5A synthetase, the sponge recombinant protein associates strongly with RNA from E. coli, forming a heterogeneous set of complexes. No complete dissociation of the complex occurs during purification of the recombinant protein and the RNA constituent is partially protected from RNase degradation. We demonstrate that the sponge recombinant 2-5A synthetase in complex with E. coli RNA catalyzes the synthesis of 2',5'-phosphodiester-linked 5'-triphosphorylated oligoadenylates from ATP, although with a low specific activity. Poly(I).poly(C), an efficient artificial activator of the mammalian 2-5A synthetases, has only a minimal effect (an approximate two-fold increase) on the sponge recombinant 2-5A synthetase/bacterial RNA complex activity.     

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.     

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.     

Lectins modulate the internalization of recombinant interferon-alpha A and the induction of 2',5'-oligo(A) synthetase

After binding to specific cell surface receptors, interferon-alpha (IFN-alpha) along with its receptor is internalized by the cells. However, the physiological significance of the internalization of IFN is not known. We have found that the lectin concanavalin A (ConA), which does not inhibit the binding of 125I-rIFN-alpha A, inhibits both the internalization of 125I-rIFN-alpha A and the rIFN-alpha A-induced increase in the levels of 2',5'-oligo(A) synthetase mRNA and enzymatic activity in the B lymphoblastoid cell line Daudi. The reduced level of IFN-induced 2',5'-oligo(A) synthetase in ConA-treated cells was due neither to direct inhibition of the enzymatic activity nor to generalized inhibition of protein or RNA synthesis. The dose-response curves were similar for the effect of ConA to inhibit 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction. The correlation between the ConA-mediated inhibition of both 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction suggests that internalization of rIFN-alpha A plays a role in the responses to rIFN-alpha A. However, since ConA inhibits protein mobility in the plasma membrane, it is possible that ConA is also preventing aggregation of IFN receptors or interactions between IFN receptors and signal transducing proteins in the plasma membrane that may be necessary for responses to IFN.     

A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.     

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.     

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.     

Multiple molecular forms of interferon display different specific activities in the induction of the antiviral state and 2'5' oligoadenylate synthetase

Both Hu IFN-alpha A and Hu IFN-alpha D, produced by two independent recombinant bacterial clones, are mixtures of monomers, dimers and trimers. These forms, when assayed individually in heterologous MDBK cells, induced different degree of antiviral and 2'5' oligoadenylate synthetase (2'5' A synthetase) activities: the antiviral activity of the monomer is greater than that of the dimer and the trimer, whereas the activity of 2'5' A synthetase induction is lower with the monomer than with the dimer or the trimer. Similar differences are also observed on human cells. Compared to the mononeric form, the dimeric and the trimeric forms of Hu IFN-alpha A show higher antiviral inducing activity on heterologous MDBK cells than on homologous WISH cells, whereas the 2'5' A synthetase inducing activity in these two cell lines is about the same. Thus for the same antiviral activity, the trimer or the dimer compared to the monomer are much better inducers of the 2'5' A synthetase on human than on MDBK cells.     

Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.