Oligo-isoadenylate Synthetase Research Articles

Clinical and laboratory changes induced by alpha interferon in chronic lymphocytic leukemia--a pilot study.

Ten chronic lymphocytic leukemia patients were treated with partially pure alpha interferon (IFN-alpha) at doses ranging from 3-9 X 10(6) units administered intramuscularly daily. Of these patients, three patients with disease stages 0, 1, and 3 (and prolymphocytic leukemia), respectively, responded with partial remissions lasting from 10 to 24+ months; four additional patients had minor responses. Among those responding, modulation of B cell chronic lymphocytic leukemia, surface immunoglobulins, and surface antigens were observed in four patients. These changes included a decline in the proportion of B cells bearing surface immunoglobulin without change in the number of the B cells. Likewise, a decline in the proportion of B cells bearing T-1 antigen was observed in three patients. Increase in the percentage of T cells, primarily the T helper cells, was seen in two of the responding patients. Specific binding of alpha interferon was demonstrated prior to therapy in CLL cells of all patients. This binding declined rapidly following treatment with IFN-alpha. Induction of the enzyme 2'5' oligoisoadenylate synthetase was examined in CLL patients prior to and during therapy with IFN-alpha. Variable levels of the enzyme were induced in all patients on therapy; however, only minimal induction was observed in two of three patients failing therapy.

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

Activated interferon system in healthy homosexual men

More than 50% of a group of healthy homosexuals in Israel were found to have an activated interferon (IFN) system as evidenced by markedly elevated blood IFN levels, increased in vitro production of IFN by unstimulated peripheral blood mononuclear cells and HuIFN-α and HuIFN-γ production by appropriately stimulated cells, and a surprisingly high incidence of an antiviral state of cells. This pattern resembles that found in persons with acute viral illness, and is unlike that found in normal healthy controls. The type of IFN in the blood was found to be unusual in that it was mainly HuIFN-α, pH 2-labile, a type of IFN found in certain collagen diseases as well as in homosexual men suffering from Kaposi's sarcoma or lymphadenopathy. Natural killer (NK) cytotoxic activity was found to be somewhat lower than that found in normal controls, although no correlation was found between blood IFN levels and NK activity. Mean (2′–5′)-oligoisoadenylate synthetase levels in cell extracts were intermediate between normal controls and patients with viral illness. Likewise no correlation was found between enzyme levels and blood IFN levels. The highly activated IFN system found in certain homosexuals, as well as the increased spontaneous production of IFN by unstimulated mononuclear cells, suggest the possibility of the presence of a virus, active or latent, in these individuals. This virus could be a retrovirus such as HTLV-III or LAV which have recently been isolated from AIDS patients. The special type of IFN present could be the response to a novel virus in an unusual situation. On the basis of recent reports, we speculate that homosexuals with highly activated IFN systems who produce pH 2-labile HuIFN-α could be at increased risk for developing AIDS.

Identification of viral infections in pregnancy by assay of (2'-5')-oligo-isoadenylate synthetase.

Interferons are produced in response to viral infections. Among the biochemical changes they cause in cells is the induction of the enzyme (2'-5')-oligo-isoadenylate synthetase. The activity of this enzyme can be measured and this can indicate exposure and response of cells to interferon. The efficacy of such an assay of peripheral blood of pregnant women may aid in establishing screening guidelines for potentially teratogenic viral infections. The blood of 44 primigravidas with complaints of fever, myalgia, cough, vaginal discharge and/or costovertebral angle tenderness was assayed for activity of the enzyme (2'-5')-oligo-isoadenylate synthetase and compared to assays of the enzyme activity in a group of 37 healthy primigravidas which served as a control group. It was found that the group with viral infections had an increase in enzyme activity from twice to 15 times the normal value, with characteristic rises of enzyme activity in several viruses known or suspected to cause human defects. Several general guidelines are proposed to assist the obstetrician in determining a viral etiology of acute illness in pregnancy, It is suggested that the assay of enzyme activity of (2'-5')-oligo-isoadenylate synthetase may provide a simple tool for rapid diagnosis of viral infections in pregnancy.

Antibodies to chromosome 21 coded cell surface components block binding of human α interferon but not λ interferon to human cells

Antisera raised against a human × mouse hybrid cell line containing human chromosome 21 as its only human chromosome, block induction of an antiviral state by human α interferon (IFN-α), block induction of (2′-5′)oligoisoadenylate synthetase ((2′-5′)A synthetase), and block binding of 125I-labeled and 35S-labeled recombinant, human IFN-αA, but not 125I-labeled IFN-ψ, to cell surface receptors. The data presented clearly demonstrate that the cell surface receptors for IFN-α and IFN-λ are different, and provide independent evidence of the role of a chromosome 21 coded cell surface molecule in the pathway to the generation of the antiviral state.

Interferon induction of (2??5?) oligoisoadenylate synthetase in diploid and trisomy 21 human fibroblasts: Relation to dosage of the interferon receptor gene (IRFC)

Trisomy 21 human fibroblasts are more sensitive to human interferon-alpha (IFN-alpha) than are diploid controls, consistent with the location of the gene (IFRC) which codes for the IFN-alpha receptor on chromosome 21. When compared in the antiviral assay, the difference in sensitivity is five- to tenfold, much greater than the 50% difference in IFRC gene dosage. An understanding of the mechanism by which this amplification of gene dosage occurs is relevant to the specific pathology of Down's syndrome and as a model system for studying the pathogenic effects of chromosomal aneuploidy. The enzyme (2'-5') oligoisoadenylate synthetase (2-5A synthetase), which is believed to be central to the interferon-induced antiviral response, is induced 50% more in trisomy 21 fibroblasts than in diploid controls. Thus the amplification in response occurs subsequent to the binding of IFN-alpha to its receptor and the triggering of the first set of intracellular events, the latter exemplified by the induction of 2-5A synthetase. Similar results were obtained with IFN-gamma, consistent with other evidence which indicates that a gene coding for a separate IFN-gamma receptor is also located on chromosome 21.

Computer-assisted analysis demonstrates that polypeptides induced by natural and recombinant human interferon-α are the same and that some have related primary structures

The biological effects on diploid and trisomy 21 human fibroblasts of pure human interferon IFLrA, a single IFN-α species produced from cloned DNA, were compared with those of partially purified natural IFN-α. Twelve interferon-induced polypeptides were visualized by two-dimensional gel electrophoresis and autoradiography. Seven of these were shown to have related primary structures and are therefore products of related genes or are related through post-translational modification. Qualitative visual comparisons and computer-aided quantitation of autoradiograms revealed to differences in the patterns of polypeptide induction following treatment with the two types of IFN-α, and the two interferons also induced (2′–5′) oligoisoadenylate synthetase equally. By these criteria, the activities of the two interferons are qualitatively and quantitatively indistinguishable. In addition, the effects of trisomy 21 on IFLrA-induced polypeptide synthesis and on antiviral response were similar to those previously demonstrated with natural IFN-α.

Increased levels of interferon-induced (2'--5') oligo-isoadenylate synthetase in mature T-lymphocytes and in differentiated Friend-erythroleukemic cells.

The level of (2'--5') oligo-isoadenylate synthetase was measured in lymphocytes at different stages of maturation within the T cell lineage and in Friend-erythroleukemic cells induced to differentiate by 1.5% Me2SO. The level of (2'--5') oligo-isoadenylate synthetase was higher in the peripheral mature T-lymphocytes than in the thymocytes. Further fractionation of the thymic population by peanut agglutinin showed higher levels of the synthetase activity in the mature PNA- cells than in the immature PNA+ cells. The activity of the enzyme in the immature thymic subpopulation could be stimulated in vitro after treatment with interferon. On the other hand, these cells did not produce the mitogen-induced immune interferon (gamma-type). Human peripheral mononuclear cells exhibited high levels of the (2'--5') oligo-isoadenylate synthetase, as well as the leukemic cells from patients with chronic lymphatic leukemia; extremely low levels of the enzyme were detected in some of the patients with acute lymphatic leukemia. In cultures of Friend cells, induced to differentiate by Me2SO, the level of the (2'--5') oligo A synthetase was low in rapidly growing cells and increased by a factor of 8--10 as the cells reached the stationary phase. This increase was completely prevented if anti-interferon antiserum was added to the medium concomitant with the Me2SO, suggesting that the increase in the level of (2'--5') oligo A synthetase is mediated by secretion of interferon by the cells. The inhibition of induction of the synthetase during differentiation, caused by the antiserum, was accompanied by a reduction in hemoglobin accumulation, whereas adding (2'--5') ApApA core to the cells together with Me2SO increased slightly hemoglobin accumulation.

The interferon-induced enzyme oligo-isoadenylate synthetase: Rapid determination of its in vitro products

A procedure for rapid isolation of the products of the interferon-induced enzyme, oligoisoadenylate synthetase, is described. After incubation of [α- 32P]ATP with the poly(rI): (rC)-adsorbed fraction of cellular proteins, the products are treated with phosphatase. Aliquots of the nucleotides are then applied on small columns which contain 300 μl of alumina powder and eluted with 3 ml of 1 m glycine-HCl buffer, pH 2.0. The labeled free phosphate, released by the phosphatase treatment, is efficiently adsorbed by the alumina, while the phosphatase-resistant cores of the oligo-isoadenylates, up to the length of pentamer, are eluted. Larger oligomers are only partially recovered. We successfully applied this method for determination of the level of the enzyme in multiple samples of cell homogenates.

Monitoring of Interferon Therapy by Assay of (2′-5′) Oligo-Isoadenylate Synthetase in Human Peripheral White Blood Cells

With the increasing number of patients undergoing experimental interferon therapy, there is a need for a method for monitoring how the patients respond to the drug. Interferon induces in cells exposed to it the accumulation of several enzymes, among them the (2'-5') oligo-A synthetase. A simple method previously developed was used to measure this enzyme in extracts of a small number of peripheral white blood cells. In 60 healthy volunteers, the enzyme level in the mononuclear cell fraction was found to be relatively high and constant. Daily intramuscular injections of 1--2 x 10(6) units leukocyte type interferon (IFN-alpha) increased the enzyme level 7--8 fold in mononuclear cells and in granulocytes. The enzyme stayed high until IFN injections were stopped. Patients with defective response to IFN were identified. We propose that this method be used for continuous monitoring of the patient's response whenever interferon therapy is undertaken.

Interferon-dependent induction of mRNA activity for (2'-5')oligo-isoadenylate synthetase.

At least three different enzymes involved in the regulation of protein synthesis are induced in a variety of cells by interferon (IFN). Sensitive assays for these enzymes have been developed and used to establish the specificity, dose dependence and time course of their induction by IFN. One of these enzymes, the oligo-isoadenylate synthetase E, whose product (2'-5')pppApApA activates the latent ribonuclease F, is increased over 50-fold after IFN treatment. We describe here the assay for an mRNA from IFN-treated mouse L cells, that produces oligo-isoadenylate synthetase activity when injected into Xenopus oocytes. This mRNA is found in the cells only after exposure to IFN. The mRNA increases in mouse L cells with the same time course as the enzyme activity itself. In particular, there is a 3-h lag period between IFN addition and the onset of enzyme and mRNA accumulation. Using anti-IFN antibodies, we show that during this lag period the continued interaction of IFN with the cells is necessary for the full induction of the oligo-isoadenylate synthetase.

An interferon-induced cellular enzyme is incorporated into virions.

The mechanisms by which interferon inhibits viral growth are only partially understood. Several enzymatic activities increase in cells shortly after treatment with interferon. One of these enzymes, oligo-isoadenylate synthetase, synthesizes (2'-5') isoadenylate oligomers which strongly stimulate the activity of a cellular ribonuclease, RNase F (ref. 7). Interferon also significantly increases the activity of a protein kinase which phosphorylates the initiation factor eIF-2 and can inhibit in vitro protein synthesis. Such interferon-induced enzymes, which affect RNA and protein metabolism, might be responsible for many of its effects on viruses. Indeed, inhibition of viral protein and RNA synthesis appears to have a major role in the antiviral state. We have now investigated possible interactions of the two enzymes with viral constituents during the course of infection and found that in two different membrane-coated RNA viruses, vesicular stomatitis virus (VSV) and Moloney murine leukaemia virus (M-MuLV), there is an accumulation of the 2'-5') oligo-isoadenylate synthetase (E) in the virions. Most of the enzyme is bound to the virion ribonucleoprotein core. The incorporation of E into the virions suggests a direct involvement of the enzyme in regulation of virus functions.

Abnormal behavior of interferon-induced enzymatic activities in an interferon-resistant cell line.

Interferon induces two double-stranded RNA-dependent enzymatic activities: an oligoisoadenylate synthetase that converts ATP to ppp(A2'p)n5'A, and a protein phosphokinase. We have explored the level and inducibility of these two enzymes in a human cell line (HEC-1) totally insensitive to both the antiviral and the anticellular actions of interferon. The activities of both enzymes are high in untreated cells and only minor changes occur after treatment with interferon, even at high concentrations. Interferon-treated HEC-1 cells do not contain an inhibitor of the oligoisoadenylate synthetase activity. The products of this HEC-1 oligoisoadenylate synthetase consist mainly of dimers, trimers, and tetramers as found in other cell lines after interferon treatment. The synthetase level is unaffected by treating the cells with anti-interferon antiserum, indicating that the results cannot be explained by a spontaneous low production of interferon by these cells. Furthermore, virus multiplication is not inhibited, even after treatment with interferon. These observations suggest that either the two enzymatic activities do not suffice for the establishment of an antiviral state in vivo or that a regulatory control mechanism, lost in these cells and common for both enzymes, is required for the expression of the antiviral action of interferon. This might explain both the constitutivity of the two enzymes and the interferon resistance observed.

Kinetics of the induction of three translation-regulatory enzymes by interferon.

Three enzymes that cause inhibition of mRNA translation, eukaryotic initiation factor 2 protein kinase PK-i, oligoisoadenylate synthetase E, and phosphodiesterase 2'-PDi, have been recently isolated from interferon-treated cells. We show that the rise in these three enzyme activities may be used to study the response of uninfected cells to interferon. For each enzyme, a specific microassay that can be carried out on extracts from 2-5 x 10(4) monolayer cells from mouse, monkey, or man was developed. With these assays, the kinetics of induction of the three enzymes in mouse L cells are compared. The dose dependence for protein kinase PK-i induction is shown to be similar to that for the development of the antiviral state. Actinomycin D and anti-interferon serum block enzyme induction if added to the cells early after interferon treatment. The quantitative measurements of the intracellular level of these enzymes provide a new and convenient model to study the cell's response to interferon.

Interferon action: Isolation of Nuclease F, A translation inhibitor activated by interferon-induced (2′–5′) oligo-isoadenylate

Cell cultures treated by interferon become unable to support viral replication. At least one of interferon’s antiviral effects is to inhibit viral protein synthesis [l-3]. Interferon induces several biochemical mechanisms which could mediate this inhibition: (i) A double-stranded RNA (dsRNA) and ATP-dependent phosphorylation of initiation factor eIF2 and possibly other ribosome-associated proteins [4-71; (ii) The dsRNA ATP-dependent synthesis of an unusual oligo-adenylate isomer with 2’-5 phosphodiester bonds [7-91; (iii) A mechanism which affects polypeptide chain elongation, that does not require dsRNA and is reversed by tRNAs [lo-l 21. We have reported [3,9] the separation and isolation, from extracts of interferon-treated L cells, of the dsRNA ATP-activated protein kinase PKi, and of the (2’-5’) oligo-isoadenylate synthetase E. Here, we demonstrate that the oligonucleotide produced by E requires, to inhibit mRNA translation, an additional protein F already present constitutively in untreated cells. The purified F is shown to be an oligo-isoadenylate-dependent ribonuclease which degrades the mRNA template. Our data show that the dsRNA ATP-dependent nuclease activity reported [ 13,141 to be increased in extracts of interferon-treated cells, can be explained as the activation of a constitutive ribonuclease, F, by the product of the interferon-induced and dsRNA-dependent oligo-isoadenylate synthetase E. This is in

Isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase.

Large-scale purification of translational inhibitors present in interferon-treated mouse L cells, but not in untreated cells, led to the isolation of two interferon-induced activities. One is a protein kinase system that is activatable by double-stranded RNA and ATP and that phosphorylates a Mr 67,000 protein and the smallest subunit of eukaryotic initiation factor-2. The purified protein kinase is a strong translational inhibitor. The second activity is an enzyme that, with double-stranded RNA, slowly polymerizes ATP into oligoadenylate with a 2'-5' phosphodiester linkage. The oligo-isoadenylate in turn activates a potent inhibitor of mRNA translation.