Bak functionally complements for loss of Bax during p14ARF-induced mitochondrial apoptosis in human cancer cells

Abstract

In contrast to the initial notion that the biological activity of p14 ARF strictly depends on a functional mdm-2/p53 signaling axis, we recently demonstrated that p14 ARF mediates apoptosis in a p53/Bax-independent manner. Here, we show that p14 ARF induces breakdown of the mitochondrial membrane potential and cytochrome c release before triggering caspase-9-and caspase-3/7-like activities in p53/Bax-deficient DU145 prostate cancer cells expressing wild-type Bak. Re-expression of Bax in these cells failed to further enhance p14 ARF -induced apoptosis, suggesting that p14 ARF -induced apoptosis primarily depends on Bak but not Bax in these cells. To further define the role of Bak and Bax in p14 ARF -induced mitochondrial apoptosis, we employed short interference RNA for the knockdown of bak in isogeneic, p53 wildtype HCT116 colon cancer cells either proficient or deficient for Bax. There, combined loss of Bax and Bak attenuated p14 ARF -induced apoptosis whereas single loss of Bax or Bak was only marginally effective, as in the case of DU145. Notably, HCT116 cells deficient for Bax and Bak failed to release cytochrome c and showed attenuated activation of caspase-9 (LEHDase) and caspase-3/caspase-7 (DEVDase) upon p14 ARF expression. These data indicate that p14 ARF triggers apoptosis via a Bax/Bakdependent pathway in p53-proficient HCT116, whereas Bax is dispensable in p53-deficient DU145 cells. Nevertheless, a substantial proportion of p14 ARF -induced cell death proceeds in a Bax/Bak-independent manner. This is also the case for inhibition of clonogenic growth that occurs, at least in part, through an entirely Bax/Bakindependent mechanism.

Betulinic acid induces cytochrome c release and apoptosis in a Bax/Bak-independent, permeability transition pore dependent fashion

“…This suggests that BetA-induced apoptosis is activated in a Bax/Bak-independent, but mitochondriadependent fashion. To rule out the possibility that these findings are selective for MEFs we also obtained HCT116 colon cancer cells that were made deficient for both Bax and Bak [27,28]. Similar to the MEFs, we observed that BetA-induced apoptosis was readily induced in cells lacking Bax and Bak (Fig.…”

Dynamin-2-Dependent Targeting ofMannheimia haemolyticaLeukotoxin to Mitochondrial Cyclophilin D in Bovine Lymphoblastoid Cells

“…1D). Oligomerization of proapoptotic proteins belonging to the Bcl-2 family, such as Bax and Bak, is responsible for formation of PTPs in mitochondria (7,20,22,25,37,39,40,50). These PTPs release cytochrome c, AIF, Smac and EndoG from the intermembrane space of mitochondria, leading to apoptosis of the cells (2,11,23,43,48).…”

Differential regulation of the proapoptotic multidomain protein Bak by p53 and p73 at the promoter level

“…HCT116/wt, HCT116/p53 À/À , HCT116/bax À/À (kindly provided by B Vogelstein), 31 and HCT116 cells stably expressing Bak siRNA (HCT116/bak knockdown and HCT116/bax À/À /bak knockdown ) 32 were maintained in McCoy's 5A medium, supplemented with 10% heat-inactivated fetal calf serum, 10 mM glutamine and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) (PAA Laboratories, Linz, Austria). HeLa cells were cultured in RPMI-1640 (PAA Laboratories) with identical supplements.…”