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Activity of the GR in G2 and mitosis
G Alexander Abel1, Gabriela M Wochnik, Joëlle Rüegg
1Max Planck Institute of Psychiatry, Munich D-80804, Germany.
Abstract:
To elucidate the mechanisms mediating the reported transient physiological glucocorticoid resistance in G2/M cell cycle phase, we sought to establish a model system of glucocorticoid-resistant cells in G2. We synchronized various cell lines in G2 to measure dexamethasone (DEX)-induced transactivation of either two endogenous promoters (rat tyrosine aminotransferase and mouse metallothionein I) or the mouse mammary tumor virus (MMTV) promoter stably or transiently transfected. To circumvent the need for synchronization drugs, we stably transfected an MMTV-driven green fluorescent protein to directly correlate DEX-induced transactivation with the cell cycle position for each cell of an asynchronous population using flow cytometry. Surprisingly, all promoters tested were DEX-inducible in G2. Even in mitotic cells, only the stably transfected MMTV promoter was repressed, whereas the same promoter transiently transfected was inducible. The use of Hoechst 33342 for synchronization in previous studies probably caused a misinterpretation, because we detected interference of this drug with GR-dependent transcription independent of the cell cycle. Finally, GR activated a simple promoter in G2, excluding a functional effect of cell cycle-dependent phosphorylation of GR, as implied previously. We conclude that GR itself is fully functional throughout the entire cell cycle, but GR responsiveness is repressed in mitosis due to chromatin condensation rather than to specific modification of GR.
Insights
Glucocorticoid receptor (GR) is functional throughout the cell cycle. GR responsiveness is repressed in mitosis due to chromatin condensation, not GR modification, challenging previous findings on cell cycle-dependent glucocorticoid resistance.
Area of Science:
- Molecular Biology
- Cell Biology
- Endocrinology
Background:
- Previous studies suggested transient physiological glucocorticoid resistance in the G2/M cell cycle phases.
- The underlying mechanisms for this reported resistance were not fully understood.
- Existing models relied on synchronization drugs, potentially introducing confounding factors.
Purpose of the Study:
- To elucidate the mechanisms of glucocorticoid resistance during the G2/M cell cycle.
- To establish a reliable model system for studying glucocorticoid resistance in G2-synchronized cells.
- To investigate the role of cell cycle-dependent modifications of the glucocorticoid receptor (GR).
Main Methods:
- Synchronization of cell lines in G2 phase.
- Measurement of dexamethasone (DEX)-induced transactivation of endogenous and transfected promoters (MMTV).
- Development of a drug-free system using MMTV-driven green fluorescent protein and flow cytometry for real-time cell cycle analysis.
- Assessment of drug interference (Hoechst 33342) with GR-dependent transcription.
Main Results:
- All tested promoters were dexamethasone-inducible in the G2 phase.
- Only stably transfected MMTV promoter showed repression in mitosis, while transiently transfected promoter remained inducible.
- Hoechst 33342 interfered with GR-dependent transcription independently of the cell cycle.
- GR activated a simple promoter in G2, ruling out cell cycle-dependent GR phosphorylation as the cause of resistance.
Conclusions:
- The glucocorticoid receptor (GR) is functionally active throughout the entire cell cycle.
- Glucocorticoid responsiveness is repressed in mitosis primarily due to chromatin condensation.
- Previous interpretations of cell cycle-dependent glucocorticoid resistance may have been influenced by synchronization drugs.