The p90 ribosomal S6 kinase (RSK) inhibitor BI-D1870 prevents gamma irradiation-induced apoptosis and mediates senescence via RSK- and p53-independent accumulation of p21WAF1/CIP1.

The p90 ribosomal S6 kinase (RSK) family is a group of highly conserved Ser/Thr kinases that promote cell proliferation, growth, motility and survival. As they are almost exclusively activated downstream of extracellular signal-regulated kinases 1 and 2 (ERK1/2), therapeutic intervention by RSK inhibition is less likely to produce such severe side effects as those observed following inhibition of the upstream master regulators Raf, MEK and ERK1/2. Here, we report that BI-D1870, a potent small molecule inhibitor of RSKs, induces apoptosis, although preferentially, in a p21-deficient background. On the other hand, BI-D1870 also induces a strong transcription- and p53-independent accumulation of p21 protein and protects cells from gamma irradiation (γIR)-induced apoptosis, driving them into senescence even in the absence of γIR. Although we identified p21 in in vitro kinase assays as a novel RSK substrate that specifically becomes phosphorylated by RSK1-3 at Ser116 and Ser146, RNA-interference, overexpression and co-immunoprecipitation studies as well as the use of SL0101, another specific RSK inhibitor, revealed that BI-D1870 mediates p21 accumulation via a yet unknown pathway that, besides its off-site targets polo-like kinase-1 and AuroraB, also does also not involve RSKs. Thus, this novel off-target effect of BI-D1870 should be taken into serious consideration in future studies investigating the role of RSKs in cellular signaling and tumorigenesis.

Pifithrin-alpha protects against DNA damage-induced apoptosis downstream of mitochondria independent of p53.

Pifithrin-alpha (PFT-alpha) was shown to specifically block transcriptional activity of the tumor suppressor p53 and was therefore proposed to be useful in preventing the severe side effects often associated with chemo- and radiotherapy. We report here that although PFT-alpha efficiently protected different cell types from DNA damage-induced apoptosis, it mediated this effect regardless of the presence or absence of p53. Interestingly, PFT-alpha blocked the apoptosome-mediated processing and activation of caspase-9 and -3 without interfering with the activation of mitochondria. Neither the DNA damage-induced activation of Bax or Bak nor the loss of the mitochondrial membrane potential or the final release of cytochrome c were inhibited by this compound. Instead, the ability of PFT-alpha to protect p53-deficient cells from DNA damage-induced caspase activation and apoptosis was greatly diminished after siRNA-mediated downregulation of cyclin-D1 expression. In contrast, downregulation of other proteins involved in cell-cycle progression, such as the retinoblastoma protein, cyclin D3, as well as the cyclin-dependent kinases, 2, 4 and 6, could not abolish this protection. Thus, our data show that PFT-alpha protects cells from DNA damage-induced apoptosis also by a p53-independent mechanism that takes place downstream of mitochondria and that might involve cyclin D1.

Activation of the mitochondrial death pathway is commonly mediated by a preferential engagement of Bak.

Among the members of the Bcl-2 family, the multidomain proteins Bax and Bak are crucial for the activation of mitochondria. However, it is still unclear whether they act in a unique and distinct manner or whether they exhibit redundant functions. To systematically investigate their activation on a single-cell level, we established MCF-7 cell lines stably expressing GFP-fusion variants of these proteins. We found that MCF-7/GFP-Bak cells showed an increased sensitivity to apoptosis induction by staurosporine, actinomycin D, TRAIL and overexpression of Puma compared to GFP-Bax-expressing cells. Independently of the death stimulus used, oligomerization of endogenous and exogenous Bak was mostly detected prior to an activation of Bax, whereas cells displaying oligomerized Bax in the absence of Bak clusters were not observed. In addition, activation of Bax but not Bak was attenuated by a caspase inhibitor. Consistent with this, caspase-3-deficient MCF-7 cells displayed a significantly reduced activation of endogenous Bax than caspase-3-proficient MCF-7 cells. Thus, our data strongly suggest that diverse apoptotic stimuli preferentially engage the Bak pathway, whereas the triggering of Bax occurs, at least partially, downstream of mitochondrial caspase activation, most likely constituting a positive feedback loop for the amplification of the death signal.