Accumulation of NFkB1 (p105) and NFkB2 (p100) Is Essential for Apoptosis Induced by Proteasome Inhibition in a Lymphoma Model.

The proteasome is a novel target in the therapeutic approach against different subtypes of lymphomas due to its role in the degradation of many proteins involved in cell cycle progression and apoptosis. This multi-enzyme complex controls gene expression by degrading transcription factors such as NF-kB, p53, c-Jun, c-Myc, HIFla, and MATa2. Among these, the best characterized is the NFkB family of transcription factors. Mammals express five NFkB proteins including REL-A (p65), c-REL, REL-B, p50 and p52. All NFkB proteins contain a highly conserved REL-homology domain (RHD), which is responsible for DNA binding, dimerization, nuclear translocation, and interaction with the IkB proteins. Active NFkB is present in the nucleus as heterodimers of p65 and p50 or p52 and REL-B subunits. In contrast, inactive NFkB dimers are sequestered in the cytoplasm due to their interaction with inhibitory proteins such as the IkB’s or the large NFkB subunits p105 and p100. Activation of the NFkB pathway is mediated by proteasome processing of p105 and p100 to produce p50 and p52, respectively, and proteasome degradation of the IkB proteins. Therefore, the proteasome plays a central role in the equilibrium between anti-apoptotic signals derived from p50 and p52, and the NFkB inhibitory signals of p105 and p100. However, it is still undetermined whether modulation of the inhibitory signals or the anti-apoptotic signals by proteasome inhibition is more important for the induction of apoptosis in lymphomas. To address this question we tested the apoptotic effect of the proteasome inhibitor PS341 in two lymphoma cell lines (Daudi and SC) after knocking down p105 and/or p100, using lentivirus expressing siRNA’s. After 24 hours of treatment with titration doses of PS341 (5, 10, 50, and 100nM), we measured apoptosis and NFkB inhibition by Annexin V-Alexa fluorescence and an NFkB luciferase reporter assay, respectively. Interestingly, co-expression of siRNA against both p100 and p105 rendered cells resistant to the induction of apoptosis at clinical doses of PS341 (5 and 10nM) compared to the controls (see table). Similar results were obtained after selective knock down of either p105 or p100. Western Blot analysis showed much lower accumulation of p105 and p100 in siRNA expressing cells than in control cells. However, IkB levels remain stable or increase during treatment in the p105 and/or p100 siRNA expressing cells, despite the observed reduction in apoptosis. These results suggest that the accumulation of p105 and p100, rather than IkB, contributes to the induction of apoptosis. In addition, the expression of NFkB anti-apoptotic signals, such as BCL-xL, increased in all cell lines, including controls. Therefore, the presence of anti-apoptotic signals has no impact on the apoptotic effect produced by PS341. In conclusion, the current study in our lymphoma model demonstrates that the accumulation of p105 and p100 is essential for the induction of apoptosis produced by proteasome inhibition. siRNA Against p100 and/or p105 Reduces PS341 Induction of Apoptosis ps314(nM) 5 10 50 100 SC Luciferase siRNA 61.2±3.2 70±3.6 72.5±3.6 79.5±5 Both siRNA’s 23.5±1.5 33±2.9 59.2±3.9 72.1±5.7 p100 siRNA 27.2±0.4 29±0.5 56±2.7 66.3±6.3 p105siRNA 32.2±7.3 45±2.4 55.4±7.7 78.4±4.1 DAUDI Luciferase siRNA 45.6±3.3 49.5±5.1 61.5±7.1 69.9±5 Both siRNA’s 21.4±4.6 29.3±1.7 34.5±2.4 55.3±7.7 p100 siRNA 17.3±4.2 24.4±9.9 44.5±3.3 56.6±4.7 p105 siRNA 19.7±1.6 34.3±3.3 31.3±2.6 46.3±3.4