Heat shock treatment decreases E2F1-DNA binding and E2F1 levels in human A549 cells.
The transcription factor E2F1 plays a decisive role in the G1/S and G2/M checkpoint transitions of proliferating cells. Because cells are arrested at these checkpoints after heat shock it was of interest to test heat shock effects on E2F1 activity. In human A549 cells, heat shock (44 degrees C, 30 min) caused an immediate reduction of E2F1-DNA binding as determined by electrophoretic mobility shift assay (EMSA). The complex of E2F1-DNA with the retinoblastoma protein (pRB) was also reduced after heat shock. This indicates that the former effect is not caused by a lower phosphorylation and therefore a higher binding capacity of pRB. Western blot analyses showed that the lower E2F1-DNA binding is probably due to a decrease of the E2F1 level (40% of the controls) induced by heat shock. This result was confirmed by an experiment with HeLa cells in which heat shock decreased the level to 60% of the controls. In order to test whether this decrease resulted from inhibition of transcription, RT-PCR measurements were conducted and showed only a slight reduction of the E2F1 mRNA (89% of controls). This indicates that the heat shock effect is not predominantly caused by transcriptional inhibition. Six hours after heat shock the E2F1-DNA binding capacity recovered to control levels. These results provide evidence for E2F1 involvement in heat shock-induced cell cycle arrests at the G1/S and G2/M checkpoints, which also may be relevant for hyperthermic cancer therapy.
Gerullis
Doris
D
Institute of Cell Biology, Biochemistry and Biotechnology, University of Bremen, D-28334 Bremen, Germany.
Rensing
Ludger
L
Beyersmann
Detmar
D
D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D015342:DNA Probes; D018076:DNA, Complementary / Q000096:biosynthesis; D004587:Electrophoresis, Agar Gel; D024202:Electrophoretic Mobility Shift Assay; D006358:Hot Temperature; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011485:Protein Binding; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D015342:DNA Probes; D018076:DNA, Complementary; D007202:Indicators and Reagents; D009687:Nuclear Proteins; C101633:PA2G4 protein, human; D016601:RNA-Binding Proteins; D012313:RNA; D004247:DNA