Molecular Journal

In addition, mM staurosporine was unable to abrogate G arrest in the topotecantreated cells, as a control because SB does have weak PKC inhibitory activity.Control cells reached G hafter release from a doublethymidine block, progressed through mitosis by h, and had returned to a normal cell cycle profile by h.The minor GM delay could be due to partial inhibition of cdccyclin B at the concentration used.Additional studies demonstrated that concentrations of SB at mM or higher caused significant G arrest. This was followed by decondensation between and has cells exited from mitosis.hafter release from doublethymidine block were at condensation at h.This increase over the control cells is likely due to some spontaneous premature chromatin condensation caused by the radiation as reported previously. However, the irradiated cells did not have any significant progression in chromatin condensation between and h.Data were generated in triplicate and are shown as the average value; bars, SE.or mM SB, the cell cycle profile was very similar to that of the control cells, suggesting that they were unable to arrest in G in response to the DNA damage.mM, the cells remained arrested in G.It is important to note, however, that complete abrogation of G arrest may not be necessary to enhance the effects of DNA damage on the cells.Thus, lower concentrations of this compound could have effects in other cellular assays.A topoisomerase I inhibitor can cause inhibition of DNA replication as well as DNA damage.We also treated cells with girradiation, which activates only the damage checkpoint.Additional experiments were conducted to confirm that the cell cycle profile observed when cells were treated with SB and topotecan was due to abrogation of G arrest and not an alternative effect such as a G delay.Ten thousand events were captured for each treatment, and the data are shown as histograms.The gain was adjusted so that cells with a G DNA content would have a fluorescence value to and G cells would have a value of. Panel shows the majority of cells synchronized at the GS transition.hafter release from thymidine block.Cells were incubated for hfollowing release, and DNA content was measured as described above.We examined the survival of cells treated with the combination of topotecan and SB in a h cytotoxicity assay.On the basis of studies using a wide range of drug concentrations, we chose concentrations of topotecan and SB that had little effect alone to test whether they would have a synergistic effect in combination.However, when these treatments were combined, survival was decreased to. SB has significantly better selectivity versus cdc, and is therefore a more useful pharmacological tool for checkpoint modulation.Another recent report identified isogranulatimide as a G checkpoint inhibitor by use of a specific cellbased screening assay. We observed that SB was able to significantly enhance the cytotoxicity of topotecan at concentrations where either agent alone had no significant effect. The concentration of SB that caused this effect was nM, which is fold lower than that required for complete abrogation of G arrest by topotecan in a day assay.Cells were incubated and harvested at various times over the course of h.

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