Bcl-2-mediated resistance to apoptosis is associated with glutathione-induced inhibition of AP24 activation of nuclear DNA fragmentation

Studies on the mechanism of apoptosis in this laboratory support a model in which signal transduction involving caspase 3 leads to activation of a ser ine protease called M-r 24,000 apoptotic protease (AP24), which then induce s internucleosomal DNA fragmentation in the nucleus, This study examined th e effect of Bcl-2 overexpression on activation of AP24 and the induction of DNA fragmentation by AP24 in isolated nuclei, It was demonstrated that ove rexpression of Bcl-2 in either HL-60 or PW leukemia cell lines suppressed a ctivation of AP24 induced by either tumor necrosis factor or UV light and p rotected cells from apoptosis, furthermore, nuclei isolated from Bcl-2-over expressing cells mere relatively resistant to internucleosomal DNA fragment ation induced by AP24 isolated from apoptotic cells. Bcl-2-overexpressing c ells that were nutritionally depleted of glutathione (GSH) became sensitive to tumor necrosis factor or UV light-induced activation of AP24 and underw ent apoptotic cell death. Moreover, nuclei isolated from Bcl-2-overexpressi ng cells that were depleted of GSH became sensitive to AP24-induced DNA fra gmentation. The addition of exogenous GSH blocked the proteolytic activity of AP24, as well as its ability to induce DNA fragmentation in normal isola ted nuclei. These results indicate that Bcl-2 can attenuate at least two ev ents in the AP24 apoptotic pathway: activation of AP24 and induction of DNA fragmentation by activated AP24. Furthermore, agents that deplete intracel lular levels of GSH may have therapeutic use in the sensitization of Bcl-2- overexpressing cancer cells to apoptotic cell death.