MOLECULAR ORDERING OF APOPTOSIS INDUCED BY ANTICANCER DRUGS IN NEUROBLASTOMA-CELLS

Apoptosis mediated by anticancer drugs may involve activation of death -inducing ligand/receptor systems such as CD95 (APO-1/Fas), cleavage o f caspases, and perturbance of mitochondrial functions. We investigate d the sequence of these events in SHEP neuroblastoma cells transfected with Bcl-2 or Bcl-X-L using two different drugs, namely, doxorubicin (Doxo), which activates the CD95/CD95 ligand (CD95-L) system, and betu linic acid (Bet A), which does not enhance the expression of CD95 or C D95-L and which, as shown here, directly targets mitochondria, Apoptos is induced by both drugs was inhibited by Bcl-2 or Bcl-X-L overexpress ion or by bongkrekic acid, an agent that stabilizes mitochondrial memb rane barrier function, suggesting a critical role for mitochondria, Af ter Doxo treatment, enhanced CD95/CD95-L expression and caspase-8 acti vation were not blocked by Bcl-2 or Bcl-X-L and were found in cells wi th a mitochondrial transmembrane potential (Delta Psi(m)) that was sti ll normal (Delta Psi(m)(high) cells). In marked contrast, after Bet A treatment, caspase-8 activation occurred in a Bcl-2- or Bcl-X-L-inhibi table fashion and was confined to cells that had lost their Delta Psi( m) (Delta Psi(m)(low) cells). Mitochondria from cells treated with eit her Doxo or Bet A induced cleavage of both caspase-8 and caspase-3 in cytosolic extracts. Thus, caspase-8 activation may occur upstream or d ownstream of mitochondria, depending on the apoptosis-initiating stimu lus. In contrast to caspase-8, cleavage of caspase-3 or poly(ADP-ribos e)polymerase was always restricted to Delta Psi(m)(low) cells, downstr eam of the Bcl-2- or Bcl-X-L-controlled checkpoint of apoptosis, Cytoc hrome c, released from mitochondria undergoing permeability transition , activated caspase-3 but not caspase-8 in a cell-free system. However , both caspases were activated by apoptosis-inducing factor, indicatin g that the mechanism of caspase-8 activation differed from that of cas pase-3 activation. Taken together, our findings demonstrate that pertu rbance of mitochondrial function constitutes a central coordinating ev ent in drug-induced cell death.