Recovery of RNA synthesis and resynthesis of antiapoptotic proteins when the CLL cells were washed into fresh media without SNS-032. (A) Time dependence of SNS-032–induced cell death. CLL cells were incubated with 0.3 μM SNS-032, and cell death was measured every 2 hours by annexin V/PI staining (●) compared with time-matched controls (○). Data represent mean plus or minus SD of 4 CLL patient samples. (B) CLL cells were incubated with SNS-032 for 6 hours, and apoptosis was measured by annexin V/PI staining (). One portion of the cells was washed and then incubated in fresh medium without SNS-032 until 24 hours (), and cell death was compared with cells that were continuously exposed to SNS-032 for 24 hours (). Data represent mean plus or minus SD of 5 CLL patient samples. (C) Recovery of RNA Pol II phosphorylation and antiapoptotic proteins when SNS-032 was washed out. After incubating with 0.3 μM SNS-032 for 6 hours, the cells were washed into fresh media and collected for analysis every 3 hours. The phosphorylation status of RNA Pol II at Ser2 and Ser5 and protein levels of Mcl-1 and XIAP were measured by immunoblotting and compared between time-matched controls (ctrl), cells exposed to SNS-032 continuously (SNS), and cells washed at 6 hours and placed in drug-free media (wash). *Nonspecific band. (D) Recovery of RNA synthesis after washing out SNS-032. [³H]Uridine incorporation was presented as percentage of time-matched controls (■). Cells were washed at 6 hours into drug-free media (●); cells were incubated with SNS-032 (▲) continuously. Data represent mean ± SD of 3 experiments, each done in triplicate.