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.     

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.     

Association of interferon-gamma induced growth inhibition and modulation of epidermal growth factor receptor gene expression in squamous cell carcinoma cell lines

Interferons (IFN) possess the ability to inhibit proliferation of certain transformed cell lines. Down modulation of the abnormal expression of certain oncogenes by IFN has been associated with phenotypic reversion of src, myc, or ras transformed cells. It has already been shown that some squamous cell carcinoma (SCCa) cell lines express elevated levels of the epidermal growth factor receptor (EGFR). Previously, in A431, an SCCa cell line, it was shown that IFN-gamma-induced growth inhibition was associated with both enhanced expression of EGFR and terminal differentiation. This study examines the effect of IFN-beta or IFN-gamma on five additional cervical SCCa cell lines. One cell line was shown to have amplification of the EGFR gene. An IFN-gamma induced antiproliferative response, observed in four of the five cell lines, was associated with increased expression of EGFR mRNA and induction of the IFN-inducible genes, HLA-A3 class I antigen and 2-5 oligoadenylate synthetase. These data suggest that the increased expression of the EGFR gene in a particular SCCa may predict response to IFN-gamma.     

Progesterone antagonists and progesterone receptor modulators in the treatment of breast cancer

Progesterone antagonists (PAs) (antiprogestins) or progesterone receptor modulators (PRMs) form an interesting category of new hormonal agents in the treatment of breast cancer. In vitro, antiproliferative effects of different PAs are mainly observed in estrogen-stimulated growth of PR-positive tumor cell lines. Both progestin agonist/antagonist actions on mammary tumor cells are dependent on the type of cell line, culture medium and concentrations of the PAs used, and type of biologic response measured. In various experimental animal tumor models, different PAs showed a greater antitumor activity than tamoxifen or high-dose progestins. Most interestingly, combination treatment of different PAs (mifepristone, ORG 31710, onapristone) or PRMs with different antiestrogens (tamoxifen, droloxifen, ICI 164384) or with an aromatase inhibitor (atemestane) showed greater antitumor efficacy than treatment with each single type of drug alone. These additive antiproliferative effects were demonstrated in various experimental in vitro and in vivo models. In some studies, these effects were accompanied by additive effects on several cell biologic parameters. In pretreated postmenopausal patients with metastatic breast cancer, objective responses have been observed in 10-12%, and stable disease in 42-46% of the patients; in previously untreated patients objective response rates of 11 and 56% have been reported. The clinical development of onapristone was stopped because of liver toxicity. At the present time, apart from development of new pure potent PAs, clinical investigation of combined therapy of PAs with antiestrogens are urgently needed.     

Cortisol induces (2′–5′)oligoadenylate synthetase in cultured chick embryo tendon fibroblasts

The changes in the (2′–5′)oligoadenylate synthetase activity after cortisol administration were studied in chick embryo tendon fibroblasts. The enzyme activity correlated with the cortisol concentration of the growth medium. The results indicate that the decrease in the cellular concentration of some mRNA species, such as procollagen type I mRNA species, after cortisol administration may at least partly be due to activation of ribonuclease L.     

2′–5′ oligoadenylate synthetase and its relationship to HLA and genetic markers of insulin-dependent diabetes mellitus

Cytokines and their related enzyme pathways may play a part in the development of insulin-dependent diabetes mellitus (IDDM). We have therefore studied the activity of the enzyme 2′–5′ oligoadenylate synthetase (which is induced by both interferon and the tumour necrosis factors) in circulating mononuclear cells from 40 subjects with IDDM and 32 healthy control subjects. There was no difference in mean basal enzyme activity between the two groups. A polymorphism of the 2′–5′ oligoadenylate synthetase gene, not previously described, was found using the restriction enzymeBam HI. There was no association of 2′–5′ oligoadenylate synthetase genotypes with IDDM, but there was a significant correlation between basal 2′–5′ oligoadenylate synthetase activity and 2′–5′ oligoadenylate synthetase genotypes. Significantly higher mean basal levels of 2′–5′ oligoadenylate synthetase activity were associated with HLA-DQA 4.6 phenotype (determined using the restriction enzymeTaq 1 and a DQA probe) and HLA-DR3 (determined serologically), whereas significantly lower mean levels of enzyme activity were associated with HLA-DQA 5.5 and HLA-DR7, in both IDDM and control subjects. An analysis of variance confirmed that these associations were independent 2′–5′ oligoadenylate synthetase genotype. Likewise, a significantly higher mean level of enzyme activity was associated with the heterozygous 1/3 insulin-related genotype in the IDDM subjects only. This study therefore suggests that the possession of certainHLA haplotypes might be associated with differing levels of basal 2′–5′ oligoadenylate synthetase activity.     

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.     

Regulation of MuIFN-alpha/beta and MuIFN-gamma-induced antiviral states: differential effects with different challenge viruses and lack of correlation with 2'5' oligoadenylate synthetase levels

The MuIFN-alpha/beta and MuIFN-gamma induced antiviral states which are directed against mengovirus have been shown previously to be differentially regulated. Following interferon removal, the MuIFN-alpha/beta-induced antiviral state decays rapidly, while the MuIFN-gamma-induced antiviral state increases dramatically. To determine whether these observations with mengovirus represent part of a general phenomenon, these studies have been extended using vesicular stomatitis virus and vaccinia virus, which represent two distinctly different groups of viruses. The antiviral states induced by MuIFN-gamma against all three viruses increased dramatically following interferon removal. The antiviral state induced by MuIFN-alpha/beta against vesicular stomatitis virus was stable following interferon removal, while the antiviral states induced by MuIFN-alpha/beta against mengovirus and vaccinia virus decayed rapidly. Also, levels of 2'5' oligoadenylate synthetase were determined at various times following interferon removal. MuIFN-alpha/beta was found to be a relatively strong inducer of 2'5' oligoadenylate synthetase, while MuIFN-gamma was a relatively weak inducer. Further, while the changes in 2'5' oligoadenylate synthetase levels paralleled the changes in the levels of the antiviral states induced by MuIFN-alpha/beta and MuIFN-gamma against mengovirus and vaccinia virus, the changes in 2'5' oligoadenylate synthetase levels did not parallel the changes in the antiviral state induced by MuIFN-alpha/beta against vesicular stomatitis virus. The results suggested that the 2'5' oligoadenylate synthetase levels did not correlate with the level of antiviral state.     

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.     

Modulation of glucocorticoid receptor activity by cyclic nucleotides and its implications on the regulation of human skin fibroblast growth and protein synthesis

The modulation of glucocorticoid receptor activity by cyclic nucleotides was studied in cultured human skin fibroblasts. The receptors appeared to be activated in the presence of dibutyryl-cAMP and inactivated by dibutyryl-cGMP. Significantly, the cGMP content of the fibroblasts increased during cell growth, with a concomitant decrease in the glucocorticoid receptor activity, while when the cells reached early confluency the decrease in cGMP content was accompanied by an increase in cAMP and increased activity of the glucocorticoid receptors. In addition, cortisol induced (2'-5')oligoadenylate synthetase in these cells and raised the cellular (2'-5')oligoadenylate concentrations. This resulted in a decrease in both DNA and protein synthesis activity in the cells, a response which correlated with the (2'-5')oligoadenylate concentration. The combination of cortisol and dibutyryl-cAMP had a synergetic stimulatory effect on the (2'-5')oligoadenylate concentration and a synergetic inhibitory effect on protein synthesis. In conclusion, it is demonstrated here that cyclic nucleotides can modulate glucocorticoid receptor activity in cultured human skin fibroblasts, and thus these compounds may indirectly affect cellular metabolism by regulating the cellular responses to glucocorticoids.     

A mouse cell line, which is unprotected by interferon against lytic virus infection, lacks ribonuclease F activity

A mouse cell line, NIH 3T3, does not respond to some of the activities of interferon. Even after treatment with high concentrations of interferon the replication of lytic viruses, such as encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) is not inhibited in these cells. In contrast, interferon treatment of these same cells results in the inhibition of Moloney murine leukemia virus (MMuLV) production. We have analyzed enzymatic pathways which are induced by interferon in these cells. After interferon treatment, the level of the (2'-5')oligoadenylate [(2'-5)An] synthetase activity and the phosphorylation of the 67000-dalton protein (P1) are enhanced in NIH 3T3 cells to approximately the same level as interferon-sensitive mouse L-cells. Moreover, NIH 3T3 and L-cells, contain approximately the same levels of enzymes which inactivate (2'-5')An. Both exogenously added (2'-5')A3 or double-stranded RNA (dsRNA) failed to inhibit protein synthesis in NIH 3T3 extracts even though they were potent inhibitors of L-cell extract-directed protein synthesis. Direct measurements of the (2'-5')An-dependent ribonuclease F (RNase F) failed to detect such activity in NIH 3T3 cells. Our results, therefore, suggest that the presence of RNase F activity is necessary for the interferon-induced antiviral activity against EMCV and against VSV. The induction of protein kinase activity by interferon treatment of NIH 3T3 cells appears to have no direct effect on EMCV and VSV replication.     

Cloned human interferons alpha: differential affinities for polyinosinic acid and relationship between molecular structure and species specificity

The HuIFN-alpha A and HuIFN-alpha D interferons, produced by two independent recombinant bacterial clones, have different affinities for polyinosinic acid (poly I). The monomeric form HuIFN-alpha A (FMM), but not the HuIFN-alpha D, binds to poly (I)-agarose and is protected by poly (I) from thermal inactivation. Other subtypes of HuIFN-alpha A including the monomer SMM and oligomers have no affinity for this polynucleotide. In addition, these interferons show different target cell preferences in agreement with our previous suggestion (23) that the polynucleotide binding domain may be responsible for species specificity. Two significant observations are 1) the fractions of HuIFN-alpha D and HuIFN-alpha A unbound on poly (I)-agarose show higher antiviral inducing activity on heterologous (MDBK) than on homologous (WISH) cells, whereas they induce about the same activity of 2'5' oligoadenylate synthetase in these two cell lines. These fractions are also active on L929 cells. 2) The bound fraction of HuIFN-alpha A induces almost the same antiviral and 2'5' oligoadenylate synthetase activities in MDBK and in WISH cells but neither activity in L929 cells.     

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.     

Phenobarbital enhances 2',5'-oligoadenylate synthesis in rat liver nuclei

Treatment of rats with phenobarbital for three days greatly increases the activity of 2,5 oligoadenylate synthetase in liver nuclei. Analysis of 2',5'-oligoadenylates synthesized in vitro showed that nuclei from both phenobarbital-treated and control rats synthesized 2',5'-oligoadenylates ranging from di- to hexamers. However, nuclei from drug treated rats showed a two fold increase in trimer and tetramer synthesis and a three-four fold increase in longer chained oligoadenylates. There was no change in the nuclear 2'-phosphodiesterase activity as the result of phenobarbital treatment, This activity remained low in nuclei from either the treated or the control rats. To our knowledge, this is the first report on phenobarbital affecting the liver 2',5'-oligoadenylate system.     

Substituted phenanthrenes with basic amino side chains: a new series of anti-breast cancer agents

In the course of our search for new anti-breast cancer agents, substituted phenanthrenes with basic amino side chains were synthesized and some of them showed remarkable antiproliferative activity against ER +ve MCF-7 cell line with IC(50) in the range of 3.53-22.25 microM. One of the compounds 15 ca showed anti-breast cancer activity in 7,12-dimethylbenz[a]anthracene (DMBA) induced hormone-dependent mammary tumor in rat and the activity was comparable to that shown by tamoxifen.     

M1204, a novel 2',5' oligoadenylate synthetase with a ubiquitin-like extension, is induced during maturation of murine dendritic cells.

A novel molecule expressed by spleen dendritic cells (DC) was isolated using a subtractive hybridization approach. The full-length M1204 clone has 3063 bp, with 1415 bp spanning a single open reading frame, coding for a protein of a predicted size of about 50 kDa. This sequence has strong homology to 2', 5' oligoadenylate synthetase and contains a ubiquitin-like domain. In Northern blot analyses the mRNA is strongly expressed in spleen DC, whereas, in bone marrow-derived DC, the amount of mRNA increases during the maturation process. None of the other leukocytes nor several hemopoietic cell lines tested express this mRNA, but clear expression occurs in many organs, the highest levels being in thymus, lung, and bone marrow. In situ hybridization, combined with immunocytochemical staining of tissue sections of lung and spleen, shows colocalization of M1204 with the 2A1 and NLDC DC markers. In Western blot experiments, an antiserum raised against the recombinant M1204 recognizes a single band in bone marrow-derived DC and in the lung. The expressed oligoadenylate synthetase domain is active in synthesizing 2',5' diadenylate, which by itself may inhibit viral protein synthesis and may also function as a substrate for 2',5' oligoadenylate synthetase. Since the oligoadenylate/RNase L system provides early protection against virus infection, we hypothesize that M1204 prevents virus-induced cell death in DC.     

Isolation and characterization of interferon-resistant variants from S49 mouse lymphoma

S49 mouse lymphoma cells were found to be extremely sensitive to the antiproliferative activity of interferon. These characteristics were studied to select for IFN-resistant cell variants. Some 0.6% of the parental S49 cell population were resistant to the antiproliferative and cytotoxic activities of IFN. The resistant cells were cloned and analyzed for their responses to several of the activities of IFN, namely, inhibition of encephalomyocarditis (EMC) virus, murine leukemia virus (MuLV) replications, and the induction of (2'-5') oligoadenylate synthetase. Among the clones selected some were highly resistant while others demonstrated only partial responsiveness to IFN. S49 cells demonstrate tubular structures in the cytoplasm. These structures were previously reported to be antigenically related to mouse mammary tumor virus (MMTV). We report here that IFN treatment decreases the expression of these cytoplasmic viral structures as revealed by electron microscopy. To correlate this novel antiviral activity to the more established functions of IFN we utilized the above mentioned S49 IFN-resistant variants. The anti-MMTV activity of IFN correlated with the other effects of IFN in both the highly resistant and partially responsive S49 clones. Our findings indicate that a relatively high proportion of S49 cells vary in their response to IFN. The defect in the resistant cells appears to affect a primary response to IFN which is common to its diverse activities. Furthermore, the effect of IFN on MMTV-related structures involves the usual pathway of IFN action.