Abstract
Background : Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived anti-apoptotic Mcl-1 protein, but it is unclear if and how this leads to Bax or Bak activation that is required for most forms of cell death.
Aim: To study the effects of seliciclib on apoptosis gene expression and Mcl-1 and Bcl-2 protein interactions in B cell chronic lymphocytic leukemia (B-CLL), a malignancy with known aberrant apoptosis regulation.
Methods: Purified B-CLL cells (PBMC consisting of >90% B-CLL cells; n=20) and Ramos cell lines overexpressing different apoptosis regulators were used in this study. The effect of seliciclib on viability, apoptosis gene expression pattern, and protein associations was investigated via RT-Multiplex-Ligation-dependent Probe Amplification (RT-MLPA), Western blotting and co-immunoprecipitation assays. Ramos cells were transduced with retroviral vectors expressing either Noxa siRNA, Bim siRNA or control-GFP virus, and tested for different apoptosis stimuli.
Results: We found that although seliciclib resulted in proteasome-dependent Mcl-1 degradation within 4 hrs in B-CLL cells, Bax and Bak activation and apoptosis occurred with a considerable delay, i.e. at 16 hrs. During this period, there was no evidence of transcriptional changes in p53-responsive or apoptosis-related genes. In freshly isolated, viable B-CLL cells, pro-survival Mcl-1 was engaged by the pro-apoptotic proteins Noxa and Bim but not by Bak. The contribution of Noxa [Figure1] and Bim (liberated from McL-1 within 4 hours) as specific mediators of seliciclib-induced apoptosis was demonstrated via RNAi in two model systems. Interestingly, 16 hrs after seliciclib treatment, there was a clear accumulation of Bcl-2, Bim, and Bax in the detergent insoluble (mitochondria containing) fraction of B-CLL cells. This suggests that after Mcl-1 degradation, the remaining apoptosis neutralizing capacity of Bcl-2 is gradually overwhelmed, probably resulting in Bax multimerisation and pore formation in the mitochondria.
Conclusions: These data support the ’oncogene-addiction’ model in which malignant cells depend on increased Bcl-2 levels, and extend it to include Mcl-1. Furthermore, since Noxa is elevated in B-CLL, its involvement in p53-independent apoptosis suggests this BH3-only protein may be a therapeutic target.
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Disclosures: One of the co-authors, Simon R. Green, from the company Cyclacel Ltd., provided the drug seliciclib for free. (for Academic investigators group).
