TNFalpha signaling in the pro B cell line FL5.12 results in mitochondrial depolarization and cell death that can only be blocked synergistically by Bcl-xL and the caspase inhibitor ZVAD-fmk. Preliminary data show that vector control FL5.12 Neo undergo a loss of the mitochondrial transmembrane potential in the presence and absence of ZVAD-fmk during TNFalpha induced apoptosis. FL5.12 Bcl-xL can also experience a loss of the mitochondrial transmembrane potential when downstream caspases are activated during TNFalpha induced programmed cell death but not when they are inhibited by ZVAD-fmk. Cyclosporin A (CsA) can block mitochondrial depolarization and in the presence of caspase inhibitors can block TNFalpha induced cell death. We propose that mitochondrial depolarization in TNFalpha induced apoptosis proceeds through two mechanisms. One is caspase dependent and Bcl-xL and CsA insensitive and the other is caspase independent and Bcl-xL and CsA inhibitable. The proposed experiments are designed to determine whether blocking mitochondria) depolarization is required in order to prevent cell death and to determine how Bcl-xL maintains deltapsim. Finally, we will determine the targets of activated caspases that result in mitochondria) depolarization.
