In particular, cyclin A2 mRNA levels demonstrated an attenuated variation dur ing combination treatments, which is consistent with the cell cycle distribution as observed by flow cytometry. At the protein level, the combination of Roscovitine with Doxorubicin resulted in an inversion Vorinostat HDAC3 of the Doxorubicin induced molecular switch between cyclin A1 and cyclin A2. Unlike cyclin A1 mRNA levels, treatment with Ros covitine alone also resulted in a decrease in cyclin A1 protein expression over time, suggest ing that, aside from transcriptional regulation, Roscov itine may also regulate cyclin A1 on a post transcriptional level. To confirm this hypothesis we treated A549 cells with Doxorubicin and Roscovitine respectively as well as 10 uM of the proteosome inhibi tor MG Inhibitors,Modulators,Libraries 132.

Inclusion of MG 132 significantly prevented the downregulation of cyclin A1 protein levels after treatment with Inhibitors,Modulators,Libraries 20 uM Roscovitine. The transcriptional and post transcriptional regula tion of cyclin A1 by Roscovitine was Inhibitors,Modulators,Libraries confirmed in a panel of NSCLC, breast and prostate cancer cell lines. Combined treatment with Roscovitine and Doxorubicin results in a downregulation of NHEJ capability Cyclin A1 knockout MEFs have shown a reduced NHEJ capability. To determine if Roscovitine may have a comparable effect on NHEJ mechanisms, we incubated untreated A549 cell lysates with 20 uM Roscovitine, DMSO, or Wortmannin for 15 minutes prior to incubation with linearized plasmid. While Wortmannin was able to almost completely Inhibitors,Modulators,Libraries inhibit NHEJ activity, DMSO had no effect and Roscovitine resulted in an approximate 25% diminution in plasmid re ligation, which can be accounted for by a direct inhibition of CDK activity and eventual off target effects of the drug.

However, when lysates from A549 cells treated for 12 hours with 20 uM Roscovitine were assayed for NHEJ capability, they demonstrated Inhibitors,Modulators,Libraries an approximate 45% reduction in plas mid re ligation as a result of an additional biological mechanism to the pharmacological inhibition of CDK2. Roscovitine enhances Doxorubicin induced DSBs and delays DNA damage repair over time To determine if the inhibition of NHEJ activity led to an overall increase in DNA DSBs we analyzed the quantity of phosphorylated gH2AX by western blot.

After six hours of incubation with respective drug treat ments, we removed the drug containing medium and analyzed A549 cells for gH2AX phosphorylation imme diately following the six hour treatment, then six and 24 hours after drug removal with respect to control cells. Doxorubicin treatment induced an activa tion of gH2AX, which was significantly augmented selleck chemical SB203580 following combined treatment with Roscovitine over time, even though Roscovitine alone did not significantly activate gH2AX as shown by western blot and immunofluorescence staining.