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2'-5'-oligoadenylate synthetase gene expression in normal and murine sarcoma virus-transformed NIH 3T3 cells
Abstract:
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-beta) 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-beta failed to inhibit 2-5A synthetase activity detected in transformed cultures. These results suggest that at least IFN-beta secretion is not involved in the elevated level of 2-5A synthetase gene expression in these cells.
Insights
Murine sarcoma virus-transformed cells show high interferon sensitivity due to elevated 2-5A synthetase gene expression. This basal expression confers resistance to mengovirus infection, independent of secreted interferon-beta.
Area of Science:
- Molecular Biology
- Virology
- Immunology
Background:
- Murine sarcoma virus (MSV)-transformed mouse fibroblasts exhibit heightened sensitivity to interferon's (IFN) antiproliferative effects.
- Understanding the molecular mechanisms underlying this IFN sensitivity is crucial for cellular response studies.
Purpose of the Study:
- To investigate the mechanism of IFN sensitivity in MSV-transformed mouse fibroblasts.
- To analyze the expression of the 2'-5'-oligoadenylate synthetase (2-5A synthetase) gene, protein, and enzymatic activity in response to IFN-beta.
Main Methods:
- Comparison of NIH 3T3 mouse fibroblasts with two MSV-transformed NIH 3T3 clones.
- Treatment with beta interferon (IFN-beta) and analysis of 2-5A synthetase transcripts, protein levels via immunoblotting, and enzymatic activity.
- Assessment of resistance to mengovirus infection and investigation of potential autocrine IFN-beta secretion.
Main Results:
- IFN-beta treatment induced 2-5A synthetase transcripts in NIH 3T3 cells, but these transcripts were already present at basal levels in untreated MSV-transformed cells.
- MSV-transformed cells showed an eightfold increase in RNA transcripts and elevated 2-5A synthetase protein and activity upon IFN-beta treatment.
- Basal 2-5A synthetase expression in transformed cells correlated with increased resistance to mengovirus infection, independent of secreted IFN-beta.
Conclusions:
- MSV-transformed cells possess a higher basal level of 2-5A synthetase gene expression compared to normal fibroblasts.
- This elevated basal expression contributes to cellular resistance against viral infections like mengovirus.
- The observed IFN sensitivity and elevated 2-5A synthetase levels in transformed cells are not mediated by autocrine IFN-beta secretion.