Activity of the GR in G2 and mitosis.
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.
Abel
G Alexander
GA
Max Planck Institute of Psychiatry, Munich D-80804, Germany.
Wochnik
Gabriela M
GM
Rüegg
Joëlle
J
Rouyer
Audrey
A
Holsboer
Florian
F
Rein
Theo
T
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005434:Flow Cytometry; D016195:G2 Phase; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D008938:Mitosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013698:Templates, Genetic; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured; D014444:Tyrosine Transaminase / Q000235:genetics / Q000378:metabolism
D001562:Benzimidazoles; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D014444:Tyrosine Transaminase; C017807:bisbenzimide ethoxide trihydrochloride