Pharmacological inter ventions aimed at rescuing the mutant

6% cells and a decrease in total RNA per cell of 54%, This is an especially important finding, as Pyronin Ylow cells are enriched for dormancy rather than terminal differentiation as demon strated by their KU-55933 臨床試験 engraftment capacity in both normal haematopoietic cells and tumour initiating cells, We also observed a corresponding decrease in cell size, We noted that formazan production from XTT, an indicator of mitochondrial metabolism, was reduced by 34% in dormancy enriched cells, We also noted a 32% ROS decrease in dormancy enriched cells, Superiority of transcriptional CDK RP2 inhibitors in targeting dormancy enriched cells As nucleoside analogues and topoisomerase inhibitors are the mainstay of AML therapy, we examined the toxicity of these drug classes as well as that of RP2 inhibitors against unmanipulated and dormancy enriched KG1a cells.<br><br> We derived dose response curves for the topoisomerase targeting agents daunorubicin, etoposide and buy Linifanib irinotecan, nucleoside analogues ara C, 5 azacytidine and clofarabine and the transcriptional CDK RP2 inhibitors flavopiridol, roscovitine and TG02 in proliferating and dormancy enriched KG1a cells. We also used the specific RNA polymerase 2 inhibitor 5,6 dicholoro 1 B D ribofuranoslybenzimidazole as positive control for RP2 targeting, Figure 3A dem onstrates that dose response curves from dormancy enriched cells have a greater tendency than those from unmanipulated cells to flatten out and there are more residual cells under the flattened part of the curve.<br><br> We therefore asked a novel question: namely, how can we measure the difficulty for a drug to target further cells after the initial IC50 has been passed For this measure, the parameter we used was cell reduction at 2 × IC50. Thus, using an example from Figure 3B, roscovitine re duces LY3009104 1187594-09-7 unmanipulated cell number by 94% at 2 × IC50, i. e. by doubling the IC50 concentration roscovitine man aged to deplete a futher 44% of cells, whereas araC man ages to deplete only a further 9% of cells when the IC50 concentration is doubled, We established that the RP2 inhibitor group of drugs were significantly more effective at reducing cell number at 2 × IC50 than the topoisomerase targeting agents or the nucleoside an alogues, Targeting of RNA polymerase II and RNA sythesis by RP2 inhibitors Serine 2 of the elongation factor RNA Polymerase II is a molecular target of CDK9, Flavopiridol, roscovitine and TG02 have multiple and diverse targets in addition to RP2.<br><br> We therefore measured whether RP2S2 and RNA synthesis were being targeted at each drugs IC50. The existing literature, including our own previous work with TG02 indicated that in vestigation of these parameters after 6 hours of treat ment would show optimal effects. At this timepoint, RP2S2 was significantly downregulated in dormancy enriched KG1a cells treated with RP2 inhibitors, RNA synthesis was greatly reduced at the same timepoint, A number of molecules with short message and protein half lives are depleted by RNA polymerase II inhibitors, including several sur vival and cycle related proteins, Moreover TG02, flavopiridol and roscovitine are all documented to induce cell cycle arrest in G0 G1, which we confirmed in the KG1a model, so it was important to establish whether the decreases in cell numbers relative to untreated controls were solely due to growth inhibition or whether, as would be necessary for dormant cell targeting, they also undergo apoptosis.