The replicon initiation burst released by reoxygenation of hypoxic T24 cells is accompanied by changes of MCM2 and Cdc7.
Although MCM2 is obviously important for the initiation of eukaryotic DNA replication, its role in O2 dependent regulation of replicon initiation is poorly understood. In this report, I analysed the changes of MCM2 during the transition from hypoxically suppressed replicon initiation to the burst of initiation triggered by reoxygenation in T24 cells. A high level of chromatin bound and nucleosolic MCM2 was found under the hypoxic replicon arrest. In contrast low cytosolic MCM2 was noticed. Recovery of O2 induced phosphorylation and diminution of chromatin bound MCM2, whereas cytosolic MCM2 increased. The level of chromatin bound Cdc7 did not change significantly upon reoxygenation. However, after reoxygenation, significant phosphorylation of Cdc7 and an increase of coimmunoprecipitation with its substrate (MCM2) were observed. This provides a hint that reoxygenation may promote the kinase activity of Cdc7. These changes might be the critical factors in O2 dependent regulation of replicon initiation. Moreover, phosphorylation of Cdc7 by Cdk2 can be observed in vitro, but seems to fail to regulate the level of chromatin bound Cdc7 as well as the changes of MCM2 in response to reoxygenation of hypoxically suppressed cells.
Martin
Leenus
L
Interfakultäres Institut für Biochemie der Universität Tübingen, Hoppe-Seyler-strasse 4, D-72076 Tübingen, Germany. lee_casmir@yahoo.com
D000098:Acetophenones / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D047468:Immunoprecipitation; D064111:Minichromosome Maintenance Complex Component 2; D009687:Nuclear Proteins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D000098:Acetophenones; D001578:Benzopyrans; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C085746:rottlerin; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D010100:Oxygen