KU135, a novel novobiocin-derived C-terminal inhibitor of the 90-kDa heat shock protein, exerts potent antiproliferative effects in human leukemic cells.

The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Consequently, there is considerable interest in developing chemotherapeutic drugs that specifically disrupt the function of Hsp90. Here, we investigated the extent to which a novel novobiocin-derived C-terminal Hsp90 inhibitor, designated KU135, induced antiproliferative effects in Jurkat T-lymphocytes. The results indicated that KU135 bound directly to Hsp90, caused the degradation of known Hsp90 client proteins, and induced more potent antiproliferative effects than the established N-terminal Hsp90 inhibitor 17-allylamino-demethoxygeldanamycin (17-AAG). Closer examination of the cellular response to KU135 and 17-AAG revealed that only 17-AAG induced a strong up-regulation of Hsp70 and Hsp90. In addition, KU135 caused wild-type cells to undergo G(2)/M arrest, whereas cells treated with 17-AAG accumulated in G(1). Furthermore, KU135 but not 17-AAG was found to be a potent inducer of mitochondria-mediated apoptosis as evidenced, in part, by the fact that cell death was inhibited to a similar extent by Bcl-2/Bcl-x(L) overexpression or the depletion of apoptotic protease-activating factor-1 (Apaf-1). Together, these data suggest that KU135 inhibits cell proliferation by regulating signaling pathways that are mechanistically different from those targeted by 17-AAG and as such represents a novel opportunity for Hsp90 inhibition.

Caspase-9 activation by the apoptosome is not required for fas-mediated apoptosis in type II Jurkat cells.

Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-x(L) were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-x(L)-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells.

Cleavage of Bid by executioner caspases mediates feed forward amplification of mitochondrial outer membrane permeabilization during genotoxic stress-induced apoptosis in Jurkat cells.

The extent to which the BH3-only protein Bid is important for intrinsic (mitochondria-mediated) apoptotic cell death induced by genotoxic stress remains controversial. In the present study, we examine this issue using a panel of gene-manipulated Bax-deficient Jurkat T-lymphocytes. Cells stably depleted of Bid were far less sensitive than control-transfected cells to etoposide-induced apoptosis. In particular, drug-induced Bak activation, cytochrome c release, loss of mitochondrial membrane potential, and caspase activation were all decreased in cells lacking Bid. Reconstitution experiments using recombinant proteins and permeabilized Bid-deficient cells demonstrated that truncated Bid (tBid), but not full-length Bid, potently induced Bak activation and the release of cytochrome c. Further, caspase-8-deficient Jurkat cells efficiently cleaved Bid and were sensitive to drug-induced apoptosis. By comparison, Apaf-1-deficient cells, as well as cells overexpressing full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP, failed to cleave Bid in response to genotoxic stress. These data suggest that tBid plays an important regulatory role in the execution of DNA damage-induced cytochrome c release and apoptosis. However, the fact that cleavage of Bid to tBid is mediated by executioner caspases suggests that a self-amplifying feed forward loop involving caspases, Bid, and mitochondria may help determine irreversible commitment to apoptosis.

Caspase-mediated Bak activation and cytochrome c release during intrinsic apoptotic cell death in Jurkat cells.

Mitochondrial outer membrane permeabilization and the release of intermembrane space proteins, such as cytochrome c, are early events during intrinsic (mitochondria-mediated) apoptotic signaling. Although this process is generally accepted to require the activation of Bak or Bax, the underlying mechanism responsible for their activation during true intrinsic apoptosis is not well understood. In the current study, we investigated the molecular requirements necessary for Bak activation using distinct clones of Bax-deficient Jurkat T-lymphocytes in which the intrinsic pathway had been inhibited. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were equally resistant to apoptosis induced by the DNA-damaging anticancer drug etoposide as determined by phosphatidylserine externalization and caspase activation. Strikingly, characterization of mitochondrial apoptotic events in all three drug-resistant cell lines revealed that, without exception, resistance to apoptosis was associated with an absence of Bak activation, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, we found that etoposide-induced apoptosis and mitochondrial events were inhibited in cells stably overexpressing either full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP. Combined, our findings suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway.