Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential ⌬⌿ M contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9 -proficient cells. Interestingly, despite these alterations, the cells retained ⌬⌿ M . Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of ⌬⌿ M and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of ⌬⌿ M can be functionally separated from cytochrome c release. Caspase-9 is not only required for ⌬⌿ M loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.

Klaus Schulze-Osthoff and Marek Los share senior authorship. ABSTRACTCryopreserved cells and tissues are increasingly used for stem cell transplantation and tissue engineering. However, their freezing, storage, and thawing is associated with severe damage, suggesting the need for better cryopreservation methods. Here, we show that activation of caspase-3 is induced during the freeze-thaw process. Moreover, we demonstrate that prevention of caspase activation by the caspase inhibitor zVAD-fmk strongly improves the recovery and survival of several cryopreserved cell types and hematopoietic progenitor cells. A short preincubation with the caspase inhibitor after thawing also enhances the colony-forming activity of hematopoietic progenitor cells up to threefold. Furthermore, overexpression of Bcl-2, but not the blockade of the death receptor signaling, confers protection, indicating that cryoinjuryassociated cell death is mediated by a Bcl-2-controlled mitochondrial pathway. Thus, our data suggest the use of zVAD-fmk as an efficient cryoprotective agent. The addition of caspase inhibitors may be an important tool for the cryopreservation of living cells and advantageous in cell transplantation, tissue engineering, and other genetic technologies.Keywords: apoptosis • cryopreservation • stem cells • cathepsins • calpains W ith the recent advances in cell and tissue transplantation, tissue engineering, in vitro fertilization, and other genetic technologies, preservation of biological materials has become increasingly important in medical care and biotechnological industries. Cryopreservation is the only reliable form of long-term storage of viable cells and tissues. However, both the freeze and the thaw process result in considerable cell and tissue injury (1, 2). Various mechanisms, including oxidative stress, mechanical injury due to ice crystal formation, altered physical properties of cellular structures, osmotic injury, and disturbed ion homeostasis due to Na + /K + -ATPase inhibition are responsible for cell damage during hypothermia and freezethaw processes (3)(4)(5)(6)(7)(8). Traditional protocols rely on supplementation of the freeze medium with penetrating cryoprotectants such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, or hydroxyethyl starch (2). Their major role is the prevention of lethal ice formation and osmotic injury. Stabilization of cellular structures with small carbohydrate sugars such as trehalose has recently been shown to also markedly improve cell recovery (9). Despite significant improvements of cryopreservation protocols, clinical procedures are often not satisfactory, and up to 50% of the cryopreserved cells may die within the first 24 h after the thaw process (10). There are some indications that hypothermia is associated with apoptosis (9, 11), although the role of apoptosis in cryoinjury has not been studied in detail. Because inhibition of proteases of the caspase family protects against apoptosis in various experimental settings (12-15), we examined whether addition of the ...

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.CD95 (APO-1/Fas) is the prototype member of the death receptor family (1). Stimulation of CD95 with its cognate ligand or agonistic antibodies results in receptor oligomerization and recruitment of signaling proteins in a death-inducing signaling complex (DISC) 3 (2, 3). Essential for apoptosis signaling through death receptors are the DISC components Fas-associated death domain and caspase-8. Recruitment of caspase-8 into the DISC leads to its dimerization-induced activation and the autoproteolytic release of its active subunits into the cytosol. The active caspase-8 activates downstream caspases, such as caspase-3, -6 and-7, that are responsible for most of the morphological manifestations of cell death (1). This death receptor-mediated pathway is also called the extrinsic pathway and is distinguished from the intrinsic mitochondrial death pathway. Apoptosis induction via mitochondria is regulated by the Bcl-2 family and involves early loss of mitochondrial membrane potential (⌬⌿ M ), release of cytochrome c, and other apoptogenic factors (4). In the cytosol, cytochrome c binds to Apaf-1 leading to caspase-9 recruitment. At this apoptosome complex, caspase-9 is activated and initiates the caspase cascade and subsequent apoptosis.An involvement of mitochondria has also been demonstrated in CD95 signaling, suggesting a functional role of this organelle in death receptor-mediated apoptosis of certain cell types (5, 6). Two types of CD95 signaling pathways have been proposed (7). CD95 type I cells show efficient DISC formation and strong activation of caspase-8 at the receptor level, which directly triggers the caspase cascade. Type II cells, in contrast, form a weak DISC and produce very little active caspase-8 at the receptor level, which is insufficient to initiate the apoptotic process. The low amounts of active caspase-8 can cleave Bid, a proapoptotic Bcl-2 protein that tra...