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Transcriptional regulation of interferon-responsive genes is closely linked to interferon receptor occupancy
Abstract:
We show that human glioblastoma cells, moving from exponential growth into a state of density-dependent growth arrest, demonstrate a 7-fold drop in the total number of alpha-IFN-receptors expressed per cell. This loss of receptor activity was not seen when cells were grown in the presence of anti-alpha-IFN-monoclonal antibody. The active binding sites expressed on the arrested cell population were of reduced affinity for IFN, relative to the high-affinity sites expressed on the growing cells, resulting in a 3-fold lower initial rate of IFN-binding to the arrested cells. We exploited this difference to investigate the relationship between IFN receptor binding and induced gene transcription. As determined by nuclear run-off assays, the transcriptional response of both the gene family 1-8 and 2-5A synthetase to IFN treatment also showed a 3-fold reduction in density-arrested cells. Longer-term (0-8 h) induction and down-regulation of gene transcription in IFN-treated cells closely followed the binding to, and down-regulation of, cell surface-localized IFN receptors. Furthermore, inhibition of the intracellular breakdown of IFN did not affect transcriptional responses to IFN. Thus transcription of these IFN-induced genes is closely linked to surface receptor occupancy and is most likely mediated by transmembrane signals alone.
Insights
Glioblastoma cells in growth arrest show significantly fewer interferon-alpha (IFN-alpha) receptors, reducing IFN-alpha binding and gene transcription. This suggests IFN-alpha
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Glioblastoma cells exhibit density-dependent growth arrest.
- Interferon-alpha (IFN-alpha) is a cytokine involved in cellular responses.
- Understanding IFN-alpha receptor dynamics is crucial for cancer therapy.
Purpose of the Study:
- To investigate changes in IFN-alpha receptor expression and function during glioblastoma cell growth arrest.
- To determine the relationship between IFN-alpha receptor binding and IFN-alpha-induced gene transcription.
- To elucidate the signaling mechanism of IFN-alpha in glioblastoma cells.
Main Methods:
- Quantification of alpha-IFN receptors on glioblastoma cells in exponential growth versus density-arrested states.
- Measurement of IFN-alpha binding affinity and rate to cell surface receptors.
- Nuclear run-off assays to assess the transcriptional response of IFN-alpha-induced genes (gene family 1-8 and 2-5A synthetase).
- Investigation of IFN-alpha's intracellular breakdown impact on transcription.
Main Results:
- Density-dependent growth arrest in glioblastoma cells led to a 7-fold decrease in total alpha-IFN receptors per cell.
- Arrested cells displayed reduced-affinity IFN-alpha binding sites, resulting in a 3-fold lower initial IFN-alpha binding rate.
- The transcriptional response of IFN-alpha-induced genes (1-8 and 2-5A synthetase) was reduced 3-fold in arrested cells.
- Gene transcription changes correlated with cell surface IFN receptor occupancy and down-regulation, independent of intracellular IFN breakdown.
Conclusions:
- Glioblastoma cell density-dependent growth arrest is associated with a significant reduction in functional IFN-alpha receptors.
- IFN-alpha-induced gene transcription is directly linked to cell surface receptor occupancy.
- Transmembrane signaling, mediated by IFN-alpha receptor binding, appears to be the primary mechanism for transcriptional regulation.