Interestingly, the decatena tion activities sellckchem of both immunoprecipitates were higher than their corresponding DMSO treated immunopreci pitates. However, because it was proposed earlier that PHA767491 could also target cell division kinase 9, a kinase involved in the phosphorylation of RNA polymerase II and in the transcriptional regulation of gene expression, we investigated whether gene expression is altered in HME and MDAMB231 cells treated with 10 uM PHA767491 for 24 hours by using a reverse transcriptase polymerase chain reaction assay. In both control and PHA767491 treated HME cells, we detected similar levels of normal expression of cell cycle regulators such as cyclin D1, A and B1, Inhibitors,Modulators,Libraries growth factors such as epidermal growth factor and basic fibroblast growth factor, cell surface receptors such as epidermal growth factor receptor and human epidermal growth factor receptor 2, and housekeeping genes such as glyceraldehyde 3 phosphate dehydrogen ase, 18S and actin mRNA.
Similar results were obtained using MDAMB231 cells. Thus, we concluded that, at least in HME or MDAMB231 cells, the effect of PHA767491 on Cdk9 kinase is minimal. Geminin Inhibitors,Modulators,Libraries overexpression reduces TopoIIa level on chromosomes and induces etoposide resistance A very low Inhibitors,Modulators,Libraries level of TopoIIa was immunoprecipitated from the chromatin of control treated MDAMB231 cells that were exposed to 10 uM etoposide for 24 hours. This immunoprecipitate showed only about 30% decatenation activity. Interestingly, a signifi cantly higher level of TopoIIa was immunoprecipitated from the chromatin of geminin silenced MDAMB231 cells that were exposed to 10 uM etoposide during the preceding 24 hours.
These immunoprecipitates showed significantly higher decatenating activity. Inhibitors,Modulators,Libraries These data show that endogenous geminin overexpression in breast cancer cell lines such as MDAMB231 prevents the persistence of TopoIIa on chromosomes in a CKI�� dependent and Cdc7 dependent manner, thus reducing the ability of the TopoIIa poison to covalently bind TopoIIa to DNA. This action perhaps decreases these drugs killing effect. Indeed, geminin overexpression in Gem9 overexpressing geminin led to low TopoIIa levels that could be detected on the chro matin following treatment of these cells with 10 uM eto poside or 10 uM doxorubicin as compared to control HME cells Inhibitors,Modulators,Libraries treated the same way.
Further more, when uninduced Gem9 cells were exposed to 10 uM TopoIIa selleck Ruxolitinib drugs for 24 hours, their viability deceased by about 50%. The same treatment had no effect on induced Gem9 cells, except when Cdc7 expression or activity was decreased, although only partially. These data suggest that geminin overexpression prema turely releases TopoIIa from chromosomes before these drugs can induce binding to DNA. This leads to the fail ure of these drugs to poison the enzyme and thus sup presses their cellular toxicity.