The mitochondrial serine protease HtrA2/Omi cleaves RIP1 during apoptosis of Ba/F3 cells induced by growth factor withdrawal

Interleukin-3 (IL-3) deprivation of the mouse pro-B cell line Ba/F3 induces cell death that is abrogated by B-cell lymphoma 2 (Bcl-2) overexpression, but remains unaffected by the pan-caspase inhibitor carbobenzoxy-valyl-analyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-fmk). IL-3 withdrawal causes receptor-interacting protein (RIP)1 cleavage into C-terminal fragments of 30 and 25 kDa, and only cleavage leading to the former was prevented by zVAD-fmk. siRNA experiments demonstrated that generation of the 25-kDa fragment was due to a Bcl-2-modulated release of the mitochondrial serine protease high temperature requirement protein A2 (HtrA2)/Omi. Accordingly, recombinant HtrA2/Omi efficiently cleaved mouse RIP1 in vitro, generating fragments matching those observed in IL-3-deprived Ba/F3 cells. The HtrA2/Omi cleavage site in mouse RIP1 was mapped to the intermediate domain and the corresponding N- and C-terminal fragments were impaired in their ability to activate nuclear factor-#B, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Interestingly, knockdown of HtrA2/Omi afforded pro-tection against IL-3 withdrawal-induced death in the presence of zVAD-fmk, demonstrating a role for HtrA2/Omi in caspase-independent cell death during growth factor withdrawal by cleaving RIP1

GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells

The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes