Flavopiridol induces apoptosis in chronic lymphocytic leukemia (CLL) cells through the transcriptional down-regulation of anti-apoptotic proteins Free

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Although flavopiridol induces apoptosis in vitro in CLL cells, and has recently shown clinical efficacy in advanced CLL, the mechanism remains unclear. Originally identified as a general cyclin-dependent kinase inhibitor in proliferating cells, flavopiridol has recently been shown to have a strong effect on transcription through the inhibition of P-TEFb (CDK9/cyclin T). P-TEFb phosphorylates the C-terminal domain of RNA pol II that is required for its elongation activity. Because viability of CLL cells is dependent upon the expression of anti-apoptotic proteins such as Mcl-1, XIAP, BAG-1 and Bcl-2, we hypothesized that flavopiridol induces apoptosis in CLL cells through the transcriptional down-regulation of such proteins. Blood lymphocytes from 10 CLL patients were collected and incubated in vitro in media supplemented with 10% autologous human sera with 0.1, 0.3, 1 and 3 μM of flavopiridol for 2, 4, 24 and 48 h. There was a time- and concentration-dependent inhibition of RNA synthesis as measured by the incorporation of [³H]uridine. The inhibition ranged 68% to 93% with a median of 85% at the end of 48-h incubation with 3 μM flavopiridol. The phosphorylation of the C-terminal domain of RNA pol II was strongly inhibited by flavopiridol in leukemic lymphocytes of 8 patients. The most labile targets of transcriptional inhibitors are proteins with short intrinsic half-lives such as Mcl-1, the anti-apoptotic protein that plays an important role in CLL disease progression and drug resistance. Coincident with the reduced phosphorylation of RNA pol II, Mcl-1 mRNA decreased significantly as measured by real-time PCR. The median inhibition was 54% (range 34% to 90%) with 3 μM flavopiridol at 24 h. The Mcl-1 protein level was also reduced by 82% (range 57% to 100%). There was a strong correlation between the mRNA and protein levels (p<0.0001, r=0.74, n=27), indicating that the observed decrease in Mcl-1 protein was the direct result of transcriptional inhibition. The mRNA and protein levels of anti-apoptotic proteins XIAP and BAG-1, were also decreased (44% and 31% inhibition on mRNA, 75%, 28% and 41% reduction for XIAP, BAG-1L and BAG-1S proteins, respectively). The median reduction of the mRNA level of Bcl-2 was 66% (32% to 86%). However, the protein level was reduced to a lesser extent, consistent with its longer half-life (∼24 h). Coincident with the decrease in anti-apoptotic proteins, PARP cleavage was observed by western blot, indicating activation of apoptosis. Because CLL cells are not cycling, these data support our hypothesis that flavopiridol-induced apoptosis in CLL is caused by the transcriptional down-regulation of anti-apoptotic proteins rather than by inhibition of cell cycle-related kinases. This information may contribute to the design of mechanism-based combinations using flavopiridol with other anticancer agents.