Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death

Abstract

Flavopiridol is active against chronic lymphocytic leukemia (CLL) cells in vitro and in the treatment of advanced stage disease, but the mechanisms of these actions remain unclear. Originally developed as a general cyclin-dependent kinase inhibitor, flavopiridol is a potent transcriptional suppressor through the inhibition of positive transcription elongation factor b (P-TEFb; CDK9/cyclin T). P-TEFb phosphorylates the C-terminal domain (CTD) of RNA polymerase II to promote transcriptional elongation. Because most CLL cells are not actively cycling, and their viability is dependent upon the continuous expression of antiapoptotic proteins, we hypothesized that flavopiridol induces apoptosis in CLL cells through the transcriptional down-regulation of such proteins. This study demonstrated that flavopiridol inhibited the phosphorylation of the CTD of RNA polymerase II in primary CLL cells and reduced RNA synthesis. This was associated with a decline of the transcripts and the levels of short-lived antiapoptotic proteins such as myeloid cell leukemia 1 (Mcl-1), and resulted in the induction of apoptosis. The B-cell lymphoma 2 (Bcl-2) protein level remained stable, although its mRNA was consistently reduced, suggesting that the outcome of transcriptional inhibition by flavopiridol is governed by the intrinsic stability of the individual transcripts and proteins. The dependence of CLL-cell survival on short-lived oncoproteins may provide the biochemical basis for the therapeutic index in response to flavopiridol.

Figure 1.

Flavopiridol inhibited RNA synthesis in CLL cells. (A) The impact on the potency of flavopiridol was compared between 10% autologous patient plasma and fetal bovine serum in the culture medium. [³H]uridine incorporation was measured in the presence of 1 μM (♦) and 3 μM (•) flavopiridol in media with autologous plasma, or in the presence of 1 μM flavopiridol in fetal bovine serum (▪). Data are presented as percentages of time-matched controls (mean ± SE; n=4 patients each performed in triplicate). The disintegrations per minute (DPM) values for untreated samples in plasma or fetal bovine serum averaged 70 057 and 39 159, respectively. (B) Time and concentration dependence of inhibition on [³H]uridine incorporation by flavopiridol. Data are presented as percentages of time-matched controls (mean ± SE, triplicate samples) of results from CLL cells from 11 patients incubated with 0.1 (▪), 0.3 (▾), 1 (♦), and 3 μM (•) flavopiridol.

Figure 2.

Flavopiridol inhibits the phosphorylation of RNA polymerase II CTD. CLL lymphocytes were incubated with 3 μM flavopiridol for 24 or 48 hours. The phosphorylation of RNA polymerase II was analyzed by immunoblotting, using antibodies towards the phosphorylated Ser2 or Ser5 sites of the CTD, as well as total RNA polymerase II. (A) Representative blot from patient no. 7. (B) Action of flavopiridol on the phosphorylation status of RNA polymerase II. The immunoblots were quantified by densitometry. Levels of phosphorylation were normalized to the loading control β-actin, and then expressed as percentage of controls incubated with DMSO. Five CLL samples were incubated with 3 μM flavopiridol for 48 hours as well, and 4 samples incubated with flavopiridol for 24 hours only are shown. • indicates 24 hours; ▴, 48 hours.

Figure 3.

Flavopiridol reduced the mRNA and protein levels of antiapoptotic proteins. The total RNA and protein of CLL cells from patient no. 7 were isolated after 4, 24, and 48 hours of incubation with or without 3 μM of flavopiridol. (A) The mRNA levels of Mcl-1 (▴), Bcl-2 (▪), BAG-1 (▾), and XIAP (•) were measured by real-time RT-PCR, each performed in duplicate. After normalizing to 18s ribosomal RNA, the relative level expressed as percentage of time-matched controls incubated in DMSO were calculated for each mRNA. (B) Immunoblots of Mcl-1, BAG-1, XIAP, and Bcl-2 and PARP from the same samples described in panel A.

Figure 4.

Relationship between Mcl-1 transcripts, proteins, and RNA polymerase II phosphorylation status. (A) Mcl-1 protein levels were related to Mcl-1 mRNA in CLL cells after incubation with 3 μM flavopiridol for 2, 4, 24, and 48 hours (n = 25, P < .001, r = 0.75). The mRNA and protein levels were expressed as percentages of controls incubated with DMSO. (B) Mcl-1 mRNA levels were related to the phosphorylation of RNA polymerase II CTD at Ser2 (pSer2-Pol II) in CLL cells after incubation with 3 μM flavopiridol for 2, 4, 24, and 48 hours (n = 25, P = .006, r = 0.54). (C) Mcl-1 protein levels were related to the phosphorylation of RNA polymerase II CTD at Ser2 (pSer2-Pol II) in CLL cells after incubation with 3 μM flavopiridol for 2, 4, 24, and 48 hours (n = 25, P = .004, r = 0.56). Solid line indicates linear regression of data points (•).