The classical pathway of p53-mediated apoptosis involves transcriptional upregulation of pro-apoptotic proteins including Puma and Noxa. However, recent studies have identified a novel non-transcriptional mechanism mediated by direct binding of p53 to Bcl-2 or Bcl-XL at the mitochondrial surface and consequent neutralization of their anti-apoptotic function (

Chipuk and Green,
Cell Cycle
). Induction of apoptosis of chronic lymphocytic leukaemia (CLL) cells by cytotoxic agents is strongly dependent on a functional p53 system and genetic changes which compromise p53 function result in drug resistance. We are therefore attempting to identify novel therapeutic agents which induce selective apoptosis of CLL cells by mechanisms independent of p53. Here we present data suggesting that 2-phenylacetylenesulfonamide (PAS; trivial name pifithrin μ) is a potential therapeutic agent for CLL. PAS was toxic to CLL cells, with a median LC50 of 10.72 μM (SD 3.8 μM n=20) as measured by a dye reduction cytotoxicity assay. Killing of CLL cells was by apoptosis, documented by the ability of 10–20 μM PAS to induce cytochrome c release from mitochondria (Fn2) to the cytosol (Fn1; Figure 1, upper), conformational change of Bax to a pro-apoptotic conformation, translocation of Bax from the cytosol to mitochondria (Figure 1, lower) and cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP). PAS toxicity was p53-independent, since p53-deleted CLL isolates were susceptible to killing. Furthermore, PAS was toxic towards leukemic cell lines with either inducible (Daudi), non-functional (Raji) or deleted (K562) p53. PAS treatment of CLL cells resulted in upregulation of Noxa and the subsequent displacement by Noxa of the pro-apoptotic Bim long (L) and extra-long (EL) isoforms from Bcl-2, as determined by western blot analysis of Bcl-2 immunoprecipitates (Figure 2). These observations contrast with those of Strom et al (
Nat Chem Biol
), who reported that PAS caused the displacement of p53 from mitochondria to the cytosol and thereby protected the cells from p53-dependent apoptosis. These differences may result from ectopic viral expression of p53 in the latter study whereas this protein was under normal cellular regulation in CLL cells. Normal T lymphocytes were relatively resistant to PAS, with IC50 values four times greater than those observed for CLL cells. In conclusion, our data suggest that PAS represents a novel class of agent which is toxic towards CLL cells via a mechanism involving p53-independent upregulation of Noxa and the subsequent unleashing of pro-apoptotic Bim from anti-apoptotic Bcl-2. This p53-independent toxicity may be of particular value in the treatment of drug-resistant CLL patients with compromised p53 pathways.

Author notes

Disclosure: No relevant conflicts of interest to declare.