Induction of (2′–5′) oligoadenylate synthetase activity during granulocyte and monocyte differentiation

Summary Variations in the (2′–5′) oligoadenylate synthetase (2–5 A synthetase) level were examined prior to and during the differentiation in culture of the human monocyte cell line U937 and the promyelocytic cell line HL60 in an attempt to reveal whether the enzyme is actively involved in hematopoietic cell maturation. The basal level of this enzyme was much higher in U937 than in HL60 cells. The activity of 2–5 A synthetase was enhanced in both cell lines in response to α, β interferons. During cell differentiation, ten markers were measured. The level of the enzyme rose during the process of cellular maturation in both cell lines. The 2–5 A synthetase activity observed in differentiated HL60 and U937 cells was comparable to that observed in mature normal granulocytes and monocytes respectively. Induction of U937 differentiation by chemicals was associated with detectable production of IFN. The increase in enzyme activity observed was mostly dependent on endogenous production of interferon, since it was inhibited by interferon antibodies. Kinetic studies showed that in U937 cells 2–5 A synthetase was expressed prior to several of the differentiation markers. The rise in the enzyme's level observed during the differentiation of HL60 cells was independent of endogenous production of interferon, since it was not inhibited by the addition of anti-interferon antibodies. These results suggest that different biochemical and molecular mechanisms are responsible for the induction of 2–5 A synthetase observed during the differentiation of hematopoietic cells. In any case, 2–5 A synthetase can be considered as a biochemical marker of cell status and differentiation in hematopoietic cells.     

Isolation and characterization of an interferon-resistant cell line deficient in the induction of (2''-5'')oligoadenylate synthetase activity.

To screen for cells with different sensitivities to interferon (IFN), NIH 3T3 mouse fibroblasts were subcloned and examined for their response to IFN treatment. Of 30 clones tested, 2 appeared to be relatively resistant to IFN, since the replication of both vesicular stomatitis virus and mengovirus was not inhibited, even in the presence of 1,000 U of IFN per ml. One resistant (A10) and one sensitive (A5) clone were further analyzed. In both clones, murine leukemia virus replication was equally inhibited by IFN, indicating the presence of functional receptors for IFN in the resistant clone. Using the (2'-5')oligoadenylate (2-5A) radiobinding assay, we could demonstrate that both clones contained the RNase L protein. Furthermore, this enzyme appears to be active, since a similar reduction in the rate of protein synthesis was evident after the introduction of exogenous 2-5A to the cells. We also analyzed the activity of another enzyme in the 2-5A pathway, namely, 2-5A synthetase. In the sensitive cells (A5), the induction of enzyme activity was proportional to the IFN concentration used, reaching a maximum of more than a 10-fold increase over the background of untreated cells. However, little if any induction over the basal activity was observed in the resistant cells (A10) when similar doses of IFN were used. It is thus probable that the lack of induction of 2-5A synthetase activity by IFN in A10 cells is at least partly responsible for their relative resistance to IFN treatment.     

Demonstration of 2-5A synthetase in human circulating mononuclear cells after oral administration of an interferon inducer isolated from a fraction of Escherichia coli and Klebsiella pneumoniae

Evaluation of the activity of an enzyme activated by interferon (2'-5' oligo-isoadenylate synthetase or 2-5A synthetase) has been used to determine, in man, the interferon inducing capacity of a fraction isolated from Escherichia coli and Klebsiella pneumoniae, SL04, an immunomodulating agent administrated per os. An increase of the activity of this enzyme has been shown in four out of five volunteers following a single oral administration of 100 mg of SL04. This activation demonstrated in humans confirms the pharmacological results of the interferon induction obtained with SL04 in vivo in mice and in vitro in human cell cultures.     

Induction and function of 2',5'-oligoadenylate synthetase in differentiation of mouse myeloid leukemia cells

The activity of 2′,5′-oligoadenylate synthetase (2-5A synthetase), known to be induced by interferon, was detected in mouse myeloid leukemic M1 cells only when they differentiated to phagocytic cells after incubation with conditioned medium (CM) from rat embryo cells. However, no interferon activity occurred in culture fluids of CM-treated M1 cells, although some activity was detected in the cell extracts. When anti-interferon serum was added to M1 cell cultures, the induction of 2-5A synthetase by CM was suppressed. These results suggest that CM stimulated the M1 cells to produce a minute amount of interferon, which was reponsible for induction of the 2-5A synthetase activity. On the other hand, development of the phagocytic activity of M1 cells could not be influenced by addition of antiserum. Interferon added exogenously per se neither induced phagocytic activity of M1 cells, nor did it enhance the CM-induced differentiation of the cells. Moreover, dexamethasone, which induced differentiation of M1 cells, was not capable of inducing 2-5A synthetase. These results indicate that interferon and/or 2-5A synthetase plays no essential part in the differentiation of M1 cells.     

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.     

(2'-5') oligoadenylate synthetase in chicken embryo erythrocytes and immature red blood cells

The appearance and induction of (2'-5')oligoadenylate synthetase (2-5A synthetase) in chicken embryo erythrocytes during development, and the activity and molecular size of this enzyme in immature red blood cells from anemic chickens were studied. Enzyme activity first appeared in the embryos on the 15th day of incubation, a marked increase being seen 1 or 2 days after hatching. In erythrocytes from early embryos without 2-5A synthetase activity, chicken interferon (5 IU/ml at most) induced the production of a large amount of the enzyme. In immature red blood cells from anemic chickens, only a small amount of 2-5A synthetase was detected in the nuclear fraction. The cytoplasmic fraction contained the smaller enzyme (about 45 kilodaltons), but the larger enzyme (85-120 kilodaltons) was scarcely detected in either fraction. The larger enzyme may be synthesized during the maturation of red blood cells.     

Effect of modulation of the production of interferon by L-929 cells treated with theophylline

The specific inhibitor of cAMP phosphodiesterase theophylline has been shown to evoke in L929 cells 2.3-fold induction of 2-5A-synthetase activity and 3.5-fold superinduction of the same enzyme activity while acting in combination with actinomycin D. It has been shown also that temporal coincidence of 2-5A-synthetase induction with the active period of interferon production resulted in 8-16 times decrease in the level of interferon production. The result was supported by the experiments of superinduced cells (containing the high stable level of 2-5A-synthetase) fusion with monolayer of poly(I).poly(C)-induced L929 cells (taken at the start of interferon production). In this case the production of interferon was dramatically decreased in comparison with the control. Possible role of 2-5A-synthetase in regulation of interferon production is discussed.     

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.     

2'-Phosphodiesterase activity in human cell lines treated or untreated with human interferon

2'-Phosphodiesterase activity was investigated, by measuring either the disappearance of (2',5')oligo(adenylate) or the release of 5'AMP, in several human cell lines (RSa, IFr, HEC-1, WGAr and HeLa) possessing different sensitivities to interferon, and treated or untreated with human interferon. In various cell lines whose (2'-5')oligo(adenylate) synthetase was normally induced by interferon treatment, both kinetic studies and measurements at different enzyme concentrations indicated that 2'-phosphodiesterase activity remained unchanged after interferon treatment. This observation was confirmed over a broad range of substrate concentrations (1-25000 nM). The activity of 2'-phosphodiesterase was dependent on Mg(OAc)2. Our results indicate that in various human cell lines the modulation of (2'-5')oligo(adenylate) metabolism by interferon does not involve an increase of 2'-phosphodiesterase activity.     

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.     

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.     

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.     

Oligo-2',5'-adenylate synthetase activity in cells persistently infected with human T-lymphotropic virus type I (HTLV-I).

Spontaneous production of interferon-gamma (IFN-gamma) was shown in several T-lymphoblastoid cell lines persistently infected with human T-lymphotropic virus (HTLV-1). However, the produced IFN-gamma was not always associated with the induction of the antivirus state. The induction of oligo-2',5'-adenylate synthetase (2-5AS) by IFN was studied in five human T-cell lines persistently infected with HTLV-I (MT-1, MT-2, SMT-1, HUT 102 and OKM-2). Four cell lines are able to produce IFN-gamma spontaneously, while the OKM-2 cell line is not. Poor induction of 2-5AS was recognized in three (MT-1, MT-2 and SMT-1) of the four cell lines producing IFN-gamma, though the poor induction was improved after long-term cultivation of cells with IFN-alpha. On the contrary, in the OKM-2 cell line, significant activity of the enzyme was induced by IFN-alpha. Induction of 2-5AS was not correlated with cell growth inhibition, but with the antivirus state. Furthermore, an inverse relationship between IFN-gamma production and 2-5AS induction was demonstrated in these cell lines with the exception of HUT 102 cells.     

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.     

Interferon-induced 2-5A synthetase activity in human peripheral blood mononuclear cells after immunization with influenza virus and rubella virus vaccines.

The interferon-induced enzyme 2-5A synthetase can be a sensitive indicator of activation of the human interferon system during viral infection or interferon therapy. To determine the response of the human interferon system to viral antigens, the level of 2-5A synthetase activity was monitored in peripheral blood mononuclear cells of healthy adults before and after immunization with influenza or rubella virus vaccine. The influenza virus-vaccinated individuals demonstrated increases in enzyme activity on days 1 and 11 in vivo, whereas those vaccinated with rubella virus vaccine showed an increase only on day 11. The difference in the day 1 in vivo 2-5A synthetase response in the two vaccinated groups could be demonstrated by in vitro incubations of peripheral blood mononuclear cells isolated approximately 90 days postvaccination with the two vaccines. The day 11 increase of enzyme activity in the rubella virus group showed a positive correlation with an increase in serum antibody titer, suggesting activation of the interferon system during antibody production in vivo after human exposure to virus antigens. The demonstration of increased 2-5A synthetase activity at specific times postimmunization in this investigation indicates that the interferon system is involved in the human in vivo response to virus vaccination.     

Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon

We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.     

Hormonal regulation of fatty acid synthetase in chick embryo liver.

Fatty acid synthetase activity in chick embryonic liver is negligible compared to that in newly hatched, fed chicks. The enzyme activity is prematurely induced 5–50-fold in 20-day-old embryos and in newly hatched chicks by the administration of insulin, hydrocortisone, growth hormone, glucagon or dibutyryl cyclic AMP. The induction of the enzyme activity is blocked by the administration of cycloheximide, indicating that new protein synthesis is required. Immunochemical titrations of different enzyme preparations from 5-day-old chicks, adult chicken and various inducer-treated embryos gave an identical equivalence point, indicating that the changes in synthetase activity after hormonal induction in embryos are related entirely to changes in content of enzyme. The increase in liver synthetase content after administration of insulin, glucagon or dibutyryl cyclic AMP is directly related to an increase in the rate of synthetase synthesis. The induction of the synthetase activity by suboptimal doses of glucagon or cyclic AMP is potentiated by the phosphodiesterase inhibitory theophylline. There is a very rapid decay of synthetase activity, with a half-life of about 4 h after elevation to higher levels following administration of insulin, glucagon or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP induction of the synthetase activity is observed early in the embryonic development, whereas insulin induction is noted 2 days before hatching. Insulin, glucagon and cyclic AMP are potentially capable of altering the levels of glycolytic intermediates which may be involved in the induction of synthetase.