2'-5'-Oligoadenylate Synthetase Gene Expression ... - Journal of Virology

JOURNAL OF VIROLOGY, Mar. 1989, p. 1116-1122

Vol. 63, No. 3

0022-538X/89/031116-07$02.00/0 Copyright ©D 1989, American Society for Microbiology

2'-5'-Oligoadenylate Synthetase Gene Expression in Normal and Murine Sarcoma Virus-Transformed NIH 3T3 Cells SASSON DAVID,' AHUVA NISSIM,2 JUDITH CHEBATH,2 AND SAMUEL SALZBERG1t*

Department

of Life

Sciences, Bar-llan University, Ramat-Gan 52100,1 and Department of Virology, the Weizmann Institute of Science, Rehovot 76100,2 Israel Received 22 December 1987/Accepted 17 November 1988

Mouse fibroblasts transformed by murine sarcoma virus (MSV) are highly sensitive to the antiproliferative effect of interferon (IFN) (M. Bakhanashvili, D. H. Wreschner, and S. Salzberg, Cancer Res. 43:1289-1294, 1983). To elucidate the mechanism leading to this IFN sensitivity, the expression of the 2'-5'-oligoadenylate synthetase (2-5A synthetase) gene, the presence of the 2-5A synthetase protein, and the level of its enzymatic activity were determined in IFN-treated and untreated cultures. NIH 3T3 mouse fibroblasts were compared with two different NIH 3T3 clones transformed by MSV. Cultures were treated with 300 IU of beta IFN (IFN-I) per ml for 16 to 24 h. While no detectable 2-5A synthetase-derived transcripts were seen in untreated NIH 3T3 cells, two size classes of RNA transcripts, i.e., 1.7 and 4.2 kilobases, were detected in IFN-treated cultures. Surprisingly, a similar amount of transcripts were present in untreated transformed cells. However, following IFN treatment, an eightfold increase in the level of RNA was readily detected in these cells, with no change in the size classes. Similar results were obtained with the 2-5A synthetase protein, for which three size classes of 42, 71, and 102 kilodaltons were demonstrated by immunoblotting, and with the enzymatic activity, for which again, the highest level was seen in IFN-treated MSV-transformed cultures. The basal level of 2-5A synthetase gene expression in the transformed cells has biological significance since these cells were more resistant to mengovirus infection than NIH 3T3 mouse fibroblasts. Medium collected from transformed cultures failed to induce 2-5A synthetase activity in NIH 3T3 cells. Furthermore, antibodies directed against mouse IFN-4 failed to inhibit 2-5A synthetase activity detected in transformed cultures. These results suggest that at least IFN-,I secretion is not involved in the elevated level of 2-5A synthetase gene expression in these cells. formed by the Moloney strain of murine sarcoma virus (MSV) (3). In the present study, the possibility that the genes coding for 2-5A synthetase are better expressed in transformed than in normal fibroblasts was investigated. This might shed some light on the mechanism responsible for the high sensitivity of the transformed cells to IFN. In addition, the biological significance of a high basal level of 2-5A synthetase gene expression in cells not treated with IFN was also studied.

well-known as a general regulaa variety of processes within the organism (13, 31). The mechanism leading to these multifunctional properties of IFN is not completely understood, but it has been established that IFN induces the synthesis of a number of new proteins in treated cells (21). At least, the antiviral state induced by IFN is most likely the result of the activity of these proteins, of which the best known and well-characterized is 2'-5'-oligoadenylate synthetase (2-5A synthetase) (12, 19). This enzyme in the presence of double-stranded RNA and ATP catalyzes the synthesis of oligomers of adenylic acid in a 2'-5' phosphodiester bond. The oligomers then activate a latent RNase, leading to the degradation of mRNA and rRNA molecules (35). It should be emphasized that the enzyme apparently appears in different structures, and at least four forms of 2-5A synthetase have been shown to be present in human cells (8). The question of whether this enzyme plays a role in cell growth and differentiation is still open, but it has been demonstrated that the expression of 2-5A synthetase during the differentiation of hematopoietic cells and along the cell cycle of fibroblasts correlates well with their growth characteristics (9, 20). One of the known properties of IFN is its antiproliferative effect (6, 29). IFN was shown to inhibit the growth of various cell types, and we have been able to demonstrate that it arrests specifically the growth of mouse fibroblasts trans-

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MATERIALS AND METHODS Cell cultures. NIH 3T3 mouse fibroblasts were used as a source of normal cells. Two cell clones transformed by MSV were used as a source of transformed cells. These clones were NIH 3T3(MSV) and MA10, obtained as previously described (2, 28) and derived from NIH 3T3 fibroblasts. All cultures were maintained in Dulbecco modified Eagle medium supplemented with 10% newborn calf serum (Biological Industries, Beth-Haemek, Israel). The cells were transferred every 3 to 4 days and grown at 37°C in a humidified incubator flushed with 10% Co2. In all experiments with IFN, the serum concentration was reduced to 2.5%. IFN treatment. NIH 3T3 or NIH 3T3(MSV) cells were treated with mouse beta IFN (IFN-P) (specific activity, 1.5 x 107 IU/mg of protein (Lee Biomolecular, San Diego, Calif.) at a concentration of 300 IU/ml for 16 h when RNA extraction was planned or for 24 h when protein analysis was needed. Human WISH or SV80 cells were treated with human recombinant IFN-P, at a concentration of 250 IU/ml for 12 h for RNA extraction or for 24 h for protein and enzymatic activity analyses. Analysis of 2-5A synthetase RNA transcripts. The mouse 2-5A synthetase cDNA (E-Mo L3) which was used as a

* Corresponding author. t Present address: Biological, Environmental and Medical Research Division, Argonne National Laboratory, 9700 S. Cass Ave., Argonne, IL 60439.

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VOL. 63, 1989

probe for the detection of 2-5A synthetase transcripts was isolated from a library of cDNA clones made from poly(A)+ RNAs of mouse L-929 cells treated with IFN-a/I (J. Chebath and E. M. Coccia, unpublished data). This cDNA was detected by using as a probe the human 9.21 cDNA encoding the 40-kilodalton (kDa) human enzyme (5). The L3 cDNA insert, 13 kilobases (kb) long, encodes an active enzyme and represents the copy of a 1.7-kb mRNA transcript. Its sequence is identical to the one previously published (18). Total RNA was extracted from confluent monolayers of cells either untreated or treated for 16 h with IFN. The RNA was purified by LiCl-urea precipitation and phenol-chloroform extraction (1). The poly(A)+ RNAs purified on oligo(dT)-cellulose columns were analyzed on 1.3% agarose gels in formaldehyde and transferred to nitrocellulose by blotting (22). The L3 cDNA insert was labeled with 32P by nick translation (26) and was used as a probe to detect the 2-5A synthetase transcripts on the blots. Analysis of 2-5A synthetase proteins by immunoblots. Cells in confluent monolayers were treated for 24 h with IFN. They were then washed and scraped in phosphate-buffered saline. The cell pellet was lysed in 20 mM HEPES (N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.6)-S5 mM magnesium acetate-20% glycerol-0.5% Nonidet P-40-200 mM phenylmethylsulfonyl fluoride followed by centrifugation at 15,000 x g for 15 min. The supernatant proteins were then analyzed on a sodium dodecyl sulfate7.5% polyacrylamide gel and transferred to nitrocellulose paper by electroblotting (30). The blot was incubated successively with the rabbit anti-peptide B serum, which recognizes the four forms of 2-5A synthetase, and with 1251I labeled protein A as previously described (8). As markers, we used a '4C-methylated protein mixture (catalog no. CFA 626; Amersham International, Amersham, United Kingdom). Determination of 2-5A synthetase activity in cell extracts. The enzymatic activity was determined as described elsewhere (8), using 2 jig of protein in the presence of 50 ,ug of poly(I)-poly(C) per ml. The 32P-labeled 2-SA oligomers were analyzed by high-voltage electrophoresis, and the radioactivity of the spots was determined by immersing the isolated dried spots in toluene-based scintillation fluid. Mengovirus infection. Stocks of mengovirus were prepared by growing the virus on NIH 3T3 mouse fibroblasts for 18 h. The culture medium was then collected, sedimented at 800 x g for 15 min, and titrated on NIH 3T3 cells by the agar overlay method. The stocks were stored at -70°C in small aliquots. For the determination of cell sensitivity to viral infection, the various cell lines were seeded in a 24-multiwell plate (Nunc, Roskilde, Denmark) at 4 x 105 cells per well. About 4 h later, the medium was removed and fresh medium containing twofold dilutions of mengovirus was added. The cultures were incubated overnight, and the cells were washed with phosphate-buffered saline, fixed with methanol, and stained with Giemsa stain. Determination of virus yield. Based on the results obtained on the sensitivity of the various cell lines tested to mengovirus infection, NIH 3T3 and NIH 3T3(MSV) cells were seeded in a 24-multiwell dish as described above. The cells were infected at two multiplicities with mengovirus, and after overnight incubation, the medium was removed, sedimented, and titrated on NIH 3T3 fibroblasts as described above. Preparation of culture medium for biological activity. NIH 3T3 or NIH 3T3(MSV) cells were seeded at 106 cells per

2-5A SYNTHETASE EXPRESSION IN NIH 3T3 CELLS

1117

plate on 10-cm tissue culture dishes. After 48 h, the medium was replaced with fresh medium for 18 h. The medium was then harvested and centrifuged, first at 800 x g for 15 min and then at 105,000 x g for 90 min at 4°C. No virus particles could be detected in the clarified medium, based on reverse transcriptase activity (21). This medium was filtered through a filter (0.45-,um pores; Millipore Corp., Bedford, Mass.) and added to NIH 3T3 cultures for 18 h. The susceptibility to mengovirus infection or the inducibility of 2-SA synthetase

activity was determined as described above. Treatment of cultures with antibodies. NIH 3T3(MSV) cells were seeded at 106 cells per 10-cm dish. A few hours later, the medium from all cultures was replaced with fresh medium containing 2.5% newborn calf serum. The dishes were then subdivided into two parts. Cultures from one part were incubated for 3 days, the medium was removed, and fresh medium containing either 100 IU of IFN-0 per ml or 50 U of rabbit anti-mouse IFN-P antiserum (Lee Biomolecular) per ml was added to individual dishes for 1 day. Cultures from the second part were immediately treated either with 50 U of anti-IFN antibodies per ml or with control normal pooled rabbit serum (Lee Biomolecular) at a protein concentration identical to that of the anti-IFN antibodies. The cultures were incubated for 3 days, and the medium was then replaced with fresh medium and further treated as before for one additional day. Cells from all cultures were harvested, extracts were prepared, and 2-SA synthetase activity was determined as described above. The enzymatic products were analyzed by high-voltage electrophoresis. The radioactive spots were removed from the 3-mm electrophoresis paper, dried, immersed in toluene-based scintillation fluid, and counted. In some experiments, 100 IU of IFN per ml was first incubated with 100 U of anti-IFN antibodies per ml for 30 min at room temperature before addition to cultures for 1

day. RESULTS 2-5A synthetase gene expression in NIH 3T3 and in MSVtransformed NIH 3T3 cells. We have previously demonstrated that MSV-transformed NIH 3T3 cells respond better to the antiproliferative effects of IFN and that 2-5A synthetase activity is induced to higher levels in the transformed cells compared with normal NIH 3T3 fibroblasts (3). This initial observation was extended to understand at what level the regulation of gene expression in the transformed cells differs from that which operates in the normal counterpart, particularly in response to IFN. Thus, it was important to establish whether the higher induction of 2-5A synthetase activity involves changes at the RNA or protein levels or simply is a result of some type of different regulation of the enzymatic activity. We therefore tested the levels of RNA transcripts, the protein forms, and the enzyme activity of 2-5A synthetase in normal and MSV-transformed cells. In agreement with previously published data (11), the extract of transformed cells treated with IFN contained much more

2-5A synthetase activity than that of nontransformed cells (Fig. 1C). However, the enzymatic activities of the two

extracts differed neither in their sensitivity to doublestranded RNA nor in their ionic requirements. For the same percentage of ATP incorporated, the two extracts showed the same production of 2-5A trimers and tetramers relative to dimers (data not shown). Moreover, it was striking that the extracts from noninduced NIH 3T3(MSV) cells contained comparable levels of enzyme activity as the extracts

of IFN-induced NIH 3T3 cells.

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J. VIROL.

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2 Protein FIG. 1. 2-5A synthetase gene expression in normal and MSV-transformed NIH 3T3 cells. Cultures were treated either with 250 IU of human IFN-,131 per ml or with 300 IU of mouse IFN-P per ml (+IFN). Untreated cultures served as controls (-IFN). (A) Level of 2-5A synthetase RNA transcripts. Samples containing 8 jig of total poly(A)+ RNA extracted from the various cell types were analyzed on 1.3% agarose gels, transferred to nitrocellulose, and hybridized with 2-5A synthetase cDNA probe. Lanes: 1 and 2, human WISH cells; 3 and 4, NIH 3T3 cells; 5 and 6, NIH 3T3(MSV) cells. (B) Detection of 2-SA synthetase protein by immunoblotting. Samples containing 20 ,ug of protein from the various cell extracts were analyzed on sodium dodecyl sulfate-7.5% polyacrylamide gels and transferred to nitrocellulose by electroblotting. The blot was then incubated with antiserum directed against peptide B followed by 125I-labeled protein A. Lanes: M, markers; 1 and 2, human SV80 cells; 3 and 4, NIH 3T3 cells; 5 and 6, NIH 3T3(MSV) cells. (C) 2-5A synthetase activity. Different amounts of protein from the various cells extracts were assayed for 2-5A synthetase activity. The enzymatic products were analyzed by high-voltage electrophoresis, and the radioactivity of the spots was determined. 05

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The level of 2-5A synthetase RNA transcripts was analyzed by using the mouse 2-5A synthetase cDNA probe L3 (Fig. 1A). Whereas no transcripts appeared in uninduced NIH 3T3 cells (lane 3), two size classes of 1.7- and 4.2-kb RNA were detected in IFN-induced cells (lane 4). The major signal corresponds to the 1.7-kb transcript, which codes for a protein of 43 kDa (18). In uninduced NIH 3T3(MSV) cells, however, a similar level of the same size classes of RNA was detected with no change in their relative concentrations (lane 5). Finally, in IFN-induced transformed cells, a significant elevation of the level of both RNA transcripts was evident (lane 6). Lanes 1 and 2 represent a control experiment in which poly(A)+ RNA from either uninduced (-IFN) or IFN-induced human WISH cells was analyzed with the mouse L3 probe. As expected, this probe detected a major 1.6-kb species in human cells. Recently, four different forms of human 2-5A synthetase have been described which were detected by an antiserum directed against peptide B, common to the 40- and 46-kDa forms of the enzyme (8). The coding frame of L3 cDNA predicts that 16 amino acids present in peptide B are conserved in the mouse sequence. We therefore used the antiserum against peptide B to analyze the protein forms of 2-5A synthetase in the mouse cell lines. As summarized in

Fig. 1B, we observed three sizes of IFN-induced proteins in MSV-transformed cells (lane 6). The 42-kDa protein encoded by the 1.7-kb RNA was clearly visible in uninduced cells (lane 5), but its level increased following IFN treatment (lane 6). The two larger forms, the 71-kDa protein in particular, but also the 102-kDa protein, were preferably present in IFN-induced cells. In comparison, in NIH 3T3 cultures induced with IFN (lane 4), at least the 42- and 102-kDa forms of the enzyme were visible, although in smaller amounts. In uninduced NIH 3T3 cells, no 2-5A synthetase proteins were detected (lane 3). The results indicate that the higher sensitivity of the transformed cells toward the antiproliferative effect of IFN correlates with a much stronger appearance of the three forms of 2-5A synthetase, in particular, the 71-kDa protein. Several other MSV-transformed clones have been tested for 2-SA synthetase expression. We could confirm that all the transformed clones investigated express the gene to higher levels than normal NIH 3T3 cells following IFN treatment (data not shown). Susceptibility of NIH 3T3 and MSV-transformed NIH 3T3 cells to cytolytic virus infection. Since an increased level of both gene expression and enzyme activity of 2-5A synthetase was demonstrated in MSV-transformed NIH 3T3

2-5A SYNTHETASE EXPRESSION IN NIH 3T3 CELLS

VOL. 63, 1989 5

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FIG. 2. Susceptibility of normal and transformed mouse fibroblasts to mengovirus infection. Cells were seeded at 4 x 105 cells per well and a few hours later were infected with mengovirus at the indicated multiplicities of infection. After an additional 24 h, the cells were fixed with methanol and stained with Giemsa stain. mouse fibroblasts, it was then important to establish whether these results were reflected by a difference in the susceptibility of normal or transformed NIH 3T3 cells to viral infection. Hence, NIH 3T3 cells and two MSV-transformed NIH 3T3 cell clones, NIH 3T3(MSV) and MA10, were infected with twofold dilutions of mengovirus, and 24 h later the plates were fixed and stained. The results presented in Fig. 2 clearly demonstrate that both transformed cell lines were significantly less susceptible to viral infection than normal NIH 3T3 cells. Figure 3 illustrates the difference in virus yield obtained by infection of either NIH 3T3 or NIH 3T3(MSV) cells with two multiplicities of mengovirus. With both multiplicities, a 10-fold reduction in virus yield was

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MULTIPLICITY OF INFECTION (PFU/mI) FIG. 3. Virus yield from infected normal and transformed mouse fibroblasts. NIH 3T3 (E1) and NIH 3T3(MSV) (-) cells were infected at the indicated multiplicity of infection. After 24 h, the medium was collected and clarified by centrifugation, and the virus titer was determined on NIH 3T3 cultures.

FIG. 4. Analysis of 2-5A synthetase activity induced in NIH 3T3 cells by different media. Cell extracts were prepared from NIH 3T3 mouse fibroblasts incubated with regular medium (lane A), medium supplemented with 200 IU of IFN per ml (lane B), medium collected from NIH 3T3 cultures (lane C), or medium collected from NIH 3T3(MSV) cultures (lane D). The extracts were subjected to a 2-5A synthetase assay, and the products were analyzed by high-voltage electrophoresis. The positions of 2-5A dimer and trimer are shown.

received with the transformed cells compared with the normal counterpart. Effect of culture medium on induction of 2-5A synthetase activity. As shown above, NIH 3T3(MSV) cells demonstrated an increased expression of the 2-5A synthetase gene, an increased amount of the 2-5A synthetase proteins, and an elevated level of the enzymatic activity. One possible explanation for these results would be a constitutive release of endogenous IFN by the transformed cells leading to the expression and synthesis of 2-5A synthetase, which is one of the major proteins induced by IFN. Preliminary results indicated that the medium collected from NIH 3T3(MSV) cultures failed to protect NIH 3T3 cells from the cytopathic effect of mengovirus. However, to increase the sensitivity of the IFN assay, we tested the capability of this medium to induce 2-5A synthetase activity in normal NIH 3T3 mouse fibroblasts. Thus, the cells were incubated with medium collected from either NIH 3T3 or NIH 3T3(MSV) cultures, and cell extracts were prepared. These were compared with extracts prepared from NIH 3T3 cultures grown in regular medium or in medium supplemented with 300 IU of IFN per ml. All extracts were subjected to a 2-5A synthetase assay, and the reaction products were analyzed by high-voltage electrophoresis. The results summarized in Fig. 4 demonstrate that only the extract prepared from cells incubated with IFN yielded a positive reaction, indicated by the presence of 2-5A oligomers. All other extracts, including those prepared from cells incubated with NIH 3T3(MSV) medium, failed to yield any detectable amounts of enzymatic products. We concluded from these results that if IFN is secreted by the transformed cells, it is not in an amount sufficient to explain the significant constitutive expression of 2-5A synthetase in these cells. Effect of anti-IFN antibodies on level of 2-5A synthetase activity. To further clarify whether IFN is secreted from MSV-transformed NIH 3T3 mouse fibroblasts, we used antibodies directed against IFN-P. We decided to concentrate on this type of antibody since, in previous studies, a type of IFN-3 has been shown to be endogenously produced and released from hematopoietic cells (25). Thus, cultures were treated with 50 U of anti-IFN antibodies for either 1 or

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