REVIEW Noncaspase Proteases in Apoptosis

Noncaspase proteases in apoptosis DE Johnson

Departments of Medicine and Pharmacology, University of Pittsburgh, and the University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Biochemical and genetic analysis of apoptosis has determined appear to be important for apoptosis execution in different that intracellular proteases are key effectors of cell death path- cell types. ways. In particular, early studies have pointed to the primacy of caspase proteases as mediators of execution. More recently, While the importance of caspases in apoptosis is clearly however, evidence has accumulated that noncaspases, includ- established, recent studies have indicated that several other ing cathepsins, calpains, granzymes, and the proteasome com- types of proteases also may play a role in the execution pro- plex, also have roles in mediating and promoting cell death. cess. The involvement of noncaspase proteases is suggested An important goal is to understand the importance of distinct by observations that inhibition of caspase proteases generally noncaspases in various forms of apoptosis, and to determine causes delays, but does not fully block, cell death resulting whether pathways mediated by noncaspase proteases intersect with those mediated by caspases. In this review the roles from most apoptotic stimuli. In addition, implicated noncasp- of noncaspase proteases in the biochemistry of apoptosis will ases frequently are upregulated or activated during apoptosis, be discussed. Leukemia (2000) 14, 1695–1703. and their inhibition, as with caspase inhibition, can serve to Keywords: apoptosis; cathepsin B; cathepsin D; calpain; granzyme delay apoptosis. The noncaspase proteases that have been A; granzyme B most closely linked with apoptosis are cathepsins, calpains, granzymes, and the proteasome. This review will focus on the current state of knowledge regarding the involvement and role Introduction of cathepsins, calpains, and granzymes in the execution process. The role of the proteasome complex, which has been The demise of cells via apoptosis hinges on the activation of strongly implicated in apoptosis, has been recently reviewed cellular proteases. Elucidation of the specific proteases elsewhere.6 involved in apoptotic execution began in the early 1990s with genetic and biochemical studies of apoptotic cell death in the nematode C. elegans. These studies revealed that apoptosis in Cathepsins and apoptosis C. elegans was dependent on an intracellular protease bearing considerable homology to the human interleukin-1β con- The cathepsin protease family consists of at least 12 known verting enzyme, or ICE.1–3 Subsequent studies have led to the members.7,8 Cathepsins can be subdivided into three distinct identification of 13 mammalian proteases that are related to groups, based on the amino acid that comprises the active site ICE, and these proteases, along with ICE, have been termed residue: (1) serine proteases (cathepsins A and G), (2) cysteine caspase proteases (caspase-1 to caspase-13).4,5 The term cas- proteases (cathepsins B, C, H, K, L, S, and T), and (3) aspartate pase, or c-aspase, reflects the fact that these enzymes are cyst- proteases (cathepsins D and E). Like the caspases, cathepsins eine proteases that cleave substrate proteins after aspartate are synthesized as inactive zymogens, and activation involves residues. Caspases are initially synthesized in the cell as proteolytic processing.9–13 As discussed below, the involve- inactive zymogens. However, in response to an apoptotic ment of cathepsins in apoptotic cell death has primarily been stimulus, initiator caspases such as caspase-8 or -9 undergo studied in nonhematopoietic systems. Evidence from these processing to active forms.5 Active initiator caspases then systems, however, should provide the basis for investigating cleave and activate downstream executioner caspases such as the role of cathepsins in the death of both normal and malig- caspase-3 or -7, thereby initiating a cascade of caspase acti- nant hematopoietic cells.14 vation. Once activated, executioner caspases cleave cellular The most extensive evidence linking cathepsins with substrate proteins promoting the destruction of the cell. apoptosis has come from studies of the cysteine protease, The role of caspases in mammalian apoptosis has been cathepsin B, and the aspartate protease, cathepsin D. There- intensively investigated using a variety of approaches: (1) fore, this review will focus on these two members of the pharmacologic inhibition of caspases using peptide inhibitors; cathepsin family. Both cathepsin B and cathepsin D are found (2) inhibition of caspases with endogenous or viral inhibitory primarily in lysosomes or endosomes. Historically, it has been proteins; and (3) targeted gene disruption. These studies have presumed that these proteases are mainly involved in the ter- shown that inhibition of caspases significantly delays minal degradation of proteins within the lysosomal compart- apoptosis induced by a wide variety of stimuli in a variety of ment. However, both proteases can also be secreted, and different cell types. Moreover, gene knockout experiments once secreted can degrade collagen, fibronectin, laminin, and have shown that different members of the caspase family proteoglycans.9,15 The degradation of extracellular matrix components by cathepsins B and D accounts for the ability of these proteases to promote cell migration or malignant invasion.16–19 Indeed, overexpression and secretion of cathep- Correspondence: DE Johnson, Division of Hematology/Oncology, sin D, which can be induced by estrogen, closely correlates University of Pittsburgh, Biomedical Science Tower, Room BST 20–23 E1055, 211 Lothrop Street, Pittsburgh, PA, 15213–2582, USA; Fax: with high metastatic potential in breast cancer. Additional (412) 624-7794 evidence (described below) suggests that cathepsins B and D Received 20 April 2000; accepted 22 May 2000 are translocated to the cytoplasm during apoptosis. This is sig- Review DE Johnson 1696 nificant, since most of the known apoptosis execution path- were also associated with induction and processing of cathep- ways occur in the cytoplasm. Thus, the translocation of sin D protein. cathepsins to the cytoplasm may allow these proteases to Cathepsin D also participates in the execution of cells intersect with and augment other apoptosis signaling mech- treated with chemotherapy drugs or radiation. Wu et al,40 anisms. using a subtractive hybridization strategy, identified cathepsin Early evidence suggesting a role for cathepsins B and D in D mRNA as an upregulated transcript in doxorubicin-treated apoptosis came from studies of apoptotic cell death in rat cells. Induction of cathepsin D protein was observed in ML1 prostate and mammary tissues. Following androgen with- leukemic cells treated with doxorubicin, etoposide, or γ- drawal in prostate, or following lactation in breast, massive irradiation. Upregulation of cathepsin D may be impacted by apoptosis occurs in the epithelial cells of these tissues.24,25 p53, as two p53 binding sites were found in the promoter Concurrent with this apoptosis, there is upregulation of mRNA region of cathepsin D. Wu et al40 further showed that drug- and protein for both cathepsin B and D.26,27 While these initial induced apoptosis was inhibited by pepstatin A. Moreover, findings suggested a role for cathepsins in regressing prostate fibroblasts derived from cathepsin D−/− gene knockout mice and mammary gland, they did not directly demonstrate displayed enhanced resistance to adriamycin and etoposide cathepsin involvement in the execution pathways. It remained relative to fibroblasts from wild-type mice.40 Similar studies possible that cathepsins were simply needed for ‘cleanup’ in by Roberts et al29 have suggested the involvement of both the dying cells. cathepsin B and cathepsin D in camptothecin-induced More definitive experiments showing a role for cathepsins apoptosis of a hepatocyte cell line (Hep3B). Following treat- in apoptotic execution have come from studies of bile salt- ment of Hep3B cells with camptothecin, an increase in the induced apoptosis in cultured hepatocytes. This in vitro model cellular activity of cathepsin B was observed, as measured by mimics apoptosis that occurs in human hepatocytes in dis- cleavage of z-ValLeuLys-7-amino-4-chloromethylcoumarin. eases where normal bile flow is impaired. In cell culture, bile Increased activity of cathepsin D, as measured with the sub- salt-induced apoptosis was found to be markedly inhibited by strate D-PheSerPhePheAlaAla-p-aminobenzoate, was also CA-074-Me, a specific inhibitor of cathepsin B, and by pepsta- detected. Inhibition of cathepsins B and D with CA-074-Me tin A, an inhibitor of cathepsin D.28,29 Antisense-mediated and pepstatin A, respectively, markedly diminished downregulation of cathepsin B also markedly inhibited hepa- camptothecin-induced apoptosis. Also, as observed in bile tocyte apoptosis following treatment with bile salts.28 salt-induced apoptosis of hepatocytes, cathepsin D is Together, these findings showed that, similar to caspases, upstream of cathepsin B in the camptothecin-induced cell cathepsins B and D, can be important components of death pathway. apoptosis execution pathways. Interestingly, pepstatin A was While inhibition of cathepsins clearly delays some forms of found to inhibit bile salt-induced cathepsin B activation, while apoptosis, any discussion of the role of cathepsins in apoptotic CA-074-Me failed to impact cathepsin D activation.29 This execution should consider the issue of subcellular localiz- revealed that during bile salt-induced apoptosis, cathepsin D ation. Apoptosis signal transduction pathways have been is activated upstream of cathepsin B. Thus, like the caspases, found to occur in the cytoplasm, on the inner surface of the cathepsins may be activated in a cascade-like fashion during plasma membrane, in mitochondria, and in the nucleus.5,41–46 apoptosis. In support of this idea, cathepsin D has been shown In particular, caspase-mediated signaling occurs predomi- to directly cleave and activate cathepsin B.30,31 nantly in the cytoplasm.5,47,48 By contrast, cathepsins B and Cathepsins B and D exert opposing effects on apoptosis in D are localized in lysosomes, or are secreted by the cell. This serum- and neurotrophic factor-deprived neuronal cells. Dur- raises the question as to how these enzymes can facilitate ing apoptosis of serum-deprived rat PC12 cells, the levels of apoptosis signaling. An emerging line of evidence suggests cathepsin B protein decline while those of cathepsin D that during apoptosis, cathepsins B and D are translocated increase.32 Interestingly, cathepsin D levels are also elevated from lysosomes to other subcellular localizations. Three stud- in the pyramidal neurons of Alzheimers patients.33 In PC12 ies have utilized gold-labeled antibodies followed by electron cells, inhibition of cathepsin B with CA-074, or downregul- microscopic immunocytochemistry to examine cathepsin D ation of cathepsin B with antisense oligonucleotides resulted localization during apoptosis.49–51 Li et al50 found that treat- in enhanced apoptosis.34 By contrast, inhibition of cathepsin ment of the mouse macrophage cell line J-774 or human D with pepstatin A significantly inhibited PC12 cell apoptosis. macrophages with apoptosis-inducing oxidized LDL changed Similar results were seen using dorsal root ganglion neurons the pattern of cathepsin D staining from granular lysosomal to deprived of nerve growth factor.34 It will be interesting to see diffuse cytoplasmic. Similar results were seen in rat myocytes whether these findings can be extended to hematopoietic treated with the quinone naphthazarin,51 and in cells treated cells. Many hematopoietic cells are dependent on specific with hydrogen peroxide.49 In the case of bile salt-induced cytokines for survival, and undergo apoptosis when deprived apoptosis of hepatocytes, Roberts et al28 performed subcellu- of these essential factors.35,36 Future studies are needed to lar fractionation, followed by Western blotting, and found that define the role and involvement of cathepsins during cytokine preexisting cathepsin B redistributed to the nucleus in treated withdrawal-induced apoptosis.37,38 cells. This group also expressed a cathepsin B-green fluor- The involvement of cathepsin D in the execution pathways escent protein chimeric molecule, and observed translocation triggered by a variety of other stimuli has been demonstrated of the fluorescent signal to the hepatocyte nucleus following by Deiss et al.39 This group isolated antisense cathepsin D treatment with bile salts. Another group has examined the cDNA while screening an antisense library for clones that localization of cathepsin B in response to the agent atractylo- could inhibit IFN-γ-induced apoptosis in HeLa cells. In side.52 Atractyloside is known to induce mitochondrial per- addition to apoptosis caused by IFN-γ, antisense cathepsin D meability transition, and subsequent apoptosis. Surprisingly, it also markedly inhibited Fas-induced apoptosis. Moreover, was shown that atractyloside caused the release of cathepsin pepstatin A was found to inhibit both forms of cell death, as B from highly purified lysosomes. Thus, current evidence indi- well as apoptosis induced by TNF-α treatment of U937 histi- cates that cathepsins may be released from lysosomal com- ocytic lymphoma cells. IFN-γ- and TNF-α-induced apoptosis partments during some forms of apoptosis. It remains unclear,

Leukemia Review DE Johnson 1697 however, whether this is a general apoptosis phenomenon, or Another interesting feature of the calpains is their ability to is restricted to execution induced by only some apoptotic associate with membrane phospholipids. Membrane associ- stimuli. In addition, it is unclear whether release of cathepsins ation occurs in response to certain cellular stimuli, and this from lysosomes is controlled by pores or translocators, or is association may lower the intrinsic Ca++ requirements of these simply the consequence of damage to lysosomal membranes enzymes.67 A third factor influencing calpain activity may be during the apoptotic process. autolysis. Following the binding of Ca++, calpains undergo To date, relatively little is known about potential intersec- autolysis, with cleavages occurring near the amino terminii of tions of cathepsin- and caspase-mediated pathways, although the large and small subunits.68–73 Autolysis appears to increase it seems likely that these pathways will be integrated in some activity and lower the requirement for Ca++. Although it fashion. One report has shown that cathepsin G, which is remains controversial as to whether autolysis is absolutely abundantly expressed in neutrophils, can cleave and activate required for activation, Western blotting, looking for conver- procaspase-7 in vitro.53 Cathepsin G cleaves procaspase-7 sion of calpains to lower molecular weight forms, is a con- between the large and small subunits in the zymogen mol- venient and frequently used means of assessing calpain acti- ecule. Another report has demonstrated that cathepsin B can vation in cells. Finally, a fourth factor influencing calpain readily cleave caspase-1 and procaspase-11.52 In addition, activity is the presence of calpastatin, a 110 kDa endogenous cathepsin B showed weak cleavage activity towards procas- protein inhibitor.74,75 Calpastatin is commonly expressed and pase-2, -6, -7 and -14. It was not determined, however, is highly specific for calpains; it is not known to inhibit any whether cathepsin B-mediated cleavage of procaspases other proteases. The expression and function of calpastatin resulted in the activation of these enzymes. Also, while may be regulated during apoptosis by other proteases, includ- cathepsin G and cathepsin B clearly can cleave certain pro- ing caspases (discussed below). caspases in vitro, it is not known whether cathepsin-mediated Calpains have been implicated in apoptosis based on two cleavage of caspases occurs in whole cells. Thus, it is difficult types of observations: (1) the activation of calpains during cell to establish whether caspase activation may be upstream or death and (2) the inhibition of apoptotic execution by various downstream of cathepsin activation during apoptosis. Perhaps calpain inhibitors. Calpain activation has been assessed using the use of highly selective inhibitors will help to address these several different assays including, Western blotting to detect issues. In this regard, however, a caveat should be raised. Pep- calpain autolysis, Western blotting to detect cleavage of tides based on caspase cleavage sequences, and derivatized known calpain substrate proteins, and in vitro and whole cell with fluoromethyl ketone or chloromethyl ketone have com- assays to detect cleavage of the fluorogenic calpain substrate monly been used as specific inhibitors of caspases in vitro and N-succinyl-leu-leu-val-tyr-7-amido-4-methylcourmarin (N-s- in whole cells. Strikingly, a recent study found that z-VAD- LLVY-AMC). Using these and a few less common assays, cal- FMK, z-DEVD-FMK, and Ac-YVAD-CMK potently inhibited pain activation has been detected in response to a variety of cathepsin B activity both in vitro and in cells.54 Therefore, the apoptotic stimuli, in a variety of different cell types. For impact of these inhibitors on biochemical events may be due example, in murine thymocytes treated with dexamethasone, to inhibition of either caspases or cathepsins, or both. calpain activity increases rapidly, peaking within 1 h of treatment.76 Also, in freshly isolated, apoptosis-prone, human neutrophils, calpain enzymes are found to be constitutively Calpains and apoptosis active.77 Treatment of BL30A Burkitt’s lymphoma cells with radiation induces rapid calpain activation (15 min), as meas- Calpains comprise a family of cytoplasmic neutral cysteine ured by cleavage of the calpain substrate fodrin.78 By contrast, proteases.55–59 Both ubiquitously expressed and tissue-specific incubation of HL-60 cells with 9-amino-20(S)-camptothecin calpains have been identified.58,60,61 The most ubiquitous cal- causes slow autolysis/activation of calpain (8–10 h) compared pains are µ-calpain (or calpain I) and m-calpain (calpain II). with rapid activation of caspase-3 (2 h;79). Activation of cal- The µ- and m-calpains have been the focus of most studies pains has also been observed in nonhematopoietic cells. In involving calpains, and this review will focus exclusively on neuronally differentiated PC12 cells, induction of apoptosis by these two isoforms. It should be kept in mind, however, that ceramide analogs stimulates calpain-mediated cleavage of N- tissue-specific calpains may have essential in vivo roles in s-LLVY-AMC.80 Similar activation of N-s-LLVY-AMC cleavage apoptosis execution. In addition, calpain homologs have been activites are seen during apoptosis caused by reovirus infec- identified in organisms such as C. elegans, where genetic tion of cultured fibroblasts (81) or in vivo ischemia-reperfusion analyses may provide clues regarding the importance of injury in rat hepatocytes.82 these proteases.62 In addition to observations of calpain activation in experi- Both µ-calpain and m-calpain are composed of two sub- mental apoptosis, activation or overexpression of calpains has units, a large catalytic subunit of approximately 80 kDa, and also been seen in human diseases marked by excessive cell a smaller subunit of approximately 30 kDa.58,63 The 80 kDa death. For example, considerable activation of calpains is subunits specific for µ-calpain and m-calpain are encoded by seen in brain tissue from patients with Alzheimer’s disease distinct genes, while the 30 kDa subunit is shared between the when compared with normal brain tissue.83,84 Moreover, two isoforms. Both enzymes bind and require Ca++ for optimal Alzheimer’s brains also exhibit significantly reduced activity.55–57,59,64,65 Although µ- and m-calpains appear to expression of the calpain inhibitor calpastatin.84 In Parkinson’s have the same substrate specificity, they are functionally dis- disease, elevated expression of m-calpain is found in the tinquished on the basis of their Ca++ sensitivity. µ-Calpain mesencephalon region of the brain.85 Thus, it is possible that requires micromolar concentrations of Ca++ for optimal overzealous expression and/or activation of calpains may activity, while m-calpain requires millimolar concentrations. contribute to inappropriate cell loss in some important patho- The activation and activity of calpains is influenced by sev- logical conditions. eral factors. As mentioned, calpains must bind Ca++ in order While the activation of calpains during apoptosis suggests to be active, and sequences resembling EF-hand Ca++ binding an involvement of these enzymes in the execution process, it motifs are found in both the 80 and 30 kDa subunits.66 does not directly demonstrate an essential role. An under-

Leukemia Review DE Johnson 1698 standing of the importance of calpains in apoptotic execution amyloid peptide or staurosporin.98,99 Taken together, the stud- has come from studies using calpain inhibitors. A number of ies described above show that calpains can play important different calpain inhibitors have been developed. Unfortu- roles in the apoptotic execution of both hematopoietic and nately, many of the earlier inhibitors are somewhat nonspe- nonhematopoietic cell types. cific. Thus, experiments using these inhibitors must be inter- The ability of calpains to promote apoptosis in a variety of preted with caution; it is possible that actions ascribed to systems raises questions regarding the molecular calpains may actually be due to other cross-inhibited pro- mechanism(s) of calpain action. Undoubtedly, the cleavage of teases (eg the proteasome or cysteine proteases in the cathep- specific substrate proteins accounts for the proapoptotic sin family). The first generation of calpain inhibitors included activity of calpain, since the catalytic activity of the enzyme the active site inhibitors leupeptin and E64 (E64d is the cell is required. A large variety of proteins have been shown to be permeable derivative of E64).86–89 These inhibitors also exhibit calpain substrates, including actin, alpha-actinin, talin, fila- 77,100 94,101 96 102 β 103 considerable activity against the proteasome and lysosomal min, fodrin, gelsolin, FAK, integrin 3, PKC cysteine proteases. More recent active site inhibitors include α/β/γ,104 calcium/calmodulin-dependent kinase IV,105 c- calpain inhibitor I (N-acetyl-leu-leu-norleucinal (aLLnL)), cal- mos,106 c-fos, c-jun,107,108 cyclin D1,109 p53,110 procaspase-3 pain inhibitor II (N-acetyl-leu-leu-methioninal (aLLM)), and and -9,96 and Bax.111 This group of substrate proteins is clearly calpeptin (benzyloxycarbonyl-leu-norleucinal).88–90 Although very diverse. However, it should be noted that several of these more specific than leupeptin and E64, these inhibitors also proteins are cytoskeletal proteins or proteins that can associate inhibit to some degree lysosomal cysteine proteases and the with cell membranes. This has led to speculation that calpains proteasome. An even more specific calpain inhibitor is the may be particularly important in destruction of cellular archi- compound PD150606 [3-(4-iodophenyl)-2-mercapto-(Z)-2- tecture during apoptosis. The impact of calpain-mediated propenoic acid], which interacts with Ca++ binding sites in the cleavage of the apoptosis regulatory proteins p53, Bax, and calpain enzymes.91 Finally, the most specific calpain inhibitor procaspase-3 and -9 is presently unclear. In the case of known to exist is the protein calpastatin, which directly binds caspase-3 and -9, calpain cleavage neither activates nor to calpains and potently inhibits their activities.74,75 To date, inactivates the caspase enzyme.96 Unfortunately, many of the however, relatively few studies have employed calpastatin to proteins identified as calpain substrates have only been shown investigate the role of calpains in apoptotic cell death. to be cleaved using in vitro experiments. Thus, it remains As perhaps may have been expected, numerous studies unclear how many of these proteins are directly cleaved by have shown that calpain inhibitors inhibit apoptosis in calpains in vivo. Also, it is currently unknown which sub- response to some, but not all, apoptotic stimuli. Studies by strates may be priority substrates, whose cleavage is critical Squier et al76,92 showed that dexamethasone-induced to the proapoptotic action of calpain. apoptosis of mouse thymocytes was inhibited by E64d and A number of studies have indicated cross-talk between cal- calpain inhibitor I. It was also found that calpain inhibitor I pain-mediated pathways and caspase-mediated pathways. In could inhibit thymocyte apoptosis caused by low-dose radi- some cases, caspase activation has been reported to be ation, A23187 ionophore, ionomycin, and forskolin; each of upstream of calpain activation, while in other cases the these forms of apoptosis requires new RNA synthesis in the opposite has been found. Wood and Newcomb79 determined thymocyte.92 By contrast, calpain inhibitor I could not prevent that calpain activation in camptothecin-treated HL-60 cells apoptosis that is not dependent on RNA synthesis, namely can be blocked by the pancaspase inhibitor z-VAD-FMK. By apoptosis caused by heat shock or valinomycin. In human contrast, the calpain inhibitor calpeptin failed to block acti- neutrophils, calpain inhibitor I and PD150606 were shown to vation of caspases. Interestingly, two groups have reported inhibit apoptosis caused by cycloheximide treatment, but not that activated caspases cleave and inactivate calpastatin apoptosis resulting from Fas stimulation.93 During spon- inhibitor.112,113 Caspase-mediated calpastatin cleavage was taneous neutrophil apoptosis, inhibition of calpains by calpep- seen in Jurkat and U937 cells following stimulation of Fas or tin alone, or the proteasome-specific inhibitor, lactacystin, TNF receptors, or following treatment with staurosporin. It alone had no effect on the rate of cell death.77 However, when remains undetermined, however, whether cleavage of calpas- used in combination, these inhibitors displayed a synergistic tatin inhibitor is entirely responsible for the calpain activation inhibitory effect, indicating synergy between calpains and the in these cells. An opposite relationship between calpains and proteasome in spontaneous neutrophil apoptosis. Related caspases has been reported during radiation-induced studies have revealed that antisense-mediated downregulation apoptosis. Waterhouse et al78 observed that following treat- of calpastatin inhibitor protein significantly accelerates neu- ment of BL30A lymphoma cells with radiation, calpain was trophil apoptosis.93 Studies with HL-60 cells have shown that activated early, and caspases were activated significantly later. calpain inhibition by calpeptin fails to block DNA fragmen- Moreover, inhibition of calpain activity abrogated down- tation and loss of viability caused by treatment with campto- stream activation of caspase-3. In yet another study, calpain thecin.79 However, calpeptin was able to block TNF-induced activation was found to occur independently of caspase acti- U937 cell apoptosis, while calpain inhibitor I delayed U937 vation during activation of platelets.96 Given these different apoptosis resulting from calphostin treatment.94,95 In human findings, it seems likely that the relationship between caspases platelets, calpeptin blocked A23187-induced caspase frag- and calpains may vary depending on the cell type and apop- mentation, cleavage of apoptosis substrate proteins, and totic stimulus. microvesiculation.96 Studies with nonhematopoietic cells have shown that calpain inhibitor I and PD150606 blocked reovirus-induced apoptosis in murine fibroblasts, and calpain Granzyme B and apoptosis inhibitors I and II blocked TGF-β-induced DNA fragmentation and loss of viability in rat hepatocytes.81,97 In addition, calpain Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells inhibitor I blocked apoptosis in chick ciliary neurons deprived are important for the host defense against viruses, parasitic of CNTF, and the calpain inhibitor MDL 28,170 inhibited the agents, and transformed cells.114,115 CTLs and NK cells induce death of rat hippocampal pyramidal neurons treated with β- apoptosis in target cells using at least two distinct mech-

Leukemia Review DE Johnson 1699 anisms. One mechanism involves stimulation of cell surface DEVD- or VAD-containing peptides (100 µM DEVD-CHO or death receptors (such as Fas) on the target cells by death VAD peptides inhibit caspases, but not granzyme B).139,151,153–155 116,117 ligands expressed on the surface of the effector cell. This Two additional caspase substrate proteins, DNA-PKcs and leads to activation of caspase cascades in the target cell. NuMA, are also cleaved in granzyme B/perforin-treated cells, Another mechanism, termed ‘granule exocytosis’, involves the but cleavage of these proteins is insensitive to DEVD or VAD 155 vectoral transfer of the contents of effector cell cytoplasmic peptide inhibitors. Moreover, the sizes of the DNA-PKcs and granules into the target cell.118–120 Key components of these NuMA proteolytic fragments generated by granzyme B differ granules are perforin and the granzyme family of serine pro- from those resulting from caspase cleavage. This indicates that teases. during granzyme B-mediated apoptosis, important cellular Perforin is a 70 kDa protein that binds to membrane phos- substrates are cleaved in a caspase-independent fashion. The phorylcholine groups in a calcium-dependent manner.121–123 importance of these caspase-independent cleavage events Following binding, perforin inserts into the membrane and oli- remains to be determined. However, the fact that granzyme gomerizes, forming pores. This permeabilization of the mem- B/perforin-mediated DNA fragmentation and apoptotic death brane may facilitate the entry of other molecules, including is significantly delayed by 100 µm DEVD/VAD,139,153,154 granzymes, into the target cell. underscores the necessity for caspase activation during this Within the granules of CTLs and NK cells, granzymes A and form of apoptosis. B are particularly abundant.124 Granzyme B (also called fragmentin or cytotoxic T cell protease (CCP)) shares with caspases the unique characteristic of cleaving substrate proteins Granzyme A and apoptosis after aspartate residues.125–129 An important role for granzyme B in the induction of target cell apoptosis has been demon- Granzyme A is the most abundant protease found in the gran- strated using gene knockout mice. CTLs and NK cells derived ules of CTL cells. The mature granzyme A enzyme is a disul- from granzyme B−/− mice exhibit greatly reduced capacity to phide cross-linked homodimer of 50 kDa that cleaves sub- induce apoptotic DNA fragmentation in target cells.119,130 strate proteins following lysine or arginine residues.127,156,157 Earlier complementary studies showed that purified granzyme Although granzyme A is capable of inducing apoptosis after B alone did not promote apoptosis when added to target cells. loading into target cells, the mechanism of action of this pro- However, cotreatment with purified granzyme B and perforin tease differs significantly from that of granzyme B. In addition, proteins induced marked DNA fragmentation and apoptotic based on the experimental systems that have been employed features in four lymphoma target cell lines.129 Based on the thus far, it appears that the role of granzyme A in CTL-induced ability of perforin to form membrane pores, it has been the apoptosis is far more subtle than that of granzyme B. Mice general consensus that granzyme B gains entry into target cells which are deficient in granzyme A expression (granzyme A−/− through perforin pores. This mode of entry, however, is cur- mice), exhibit relatively normal CTL-mediated cytotoxicity.158 rently being called into question. Several studies have shown The only reported defect for granzyme A−/− mice is an inability that granzyme B is internalized by target cells in the absence to clear the mouse pox virus Ectromelia.159 By contrast, CTLs of added perforin.131–135 The internalized granzyme B has from granzyme B−/− mice are capable of inducing target cell been reported to reside in the cytoplasm,132,133 or in a novel death only after prolonged coincubation.130 Thus, granzyme vesicular compartment.134 The triggering of apoptosis in cells B is critically important for rapid CTL killing. The possibility that have internalized granzyme B requires further addition of that granzyme A does have some role in CTL-mediated killing, perforin to the cells.131–135 It may be that perforin is required has been suggested by recent experiments using mice that are for the release of granzyme B from internal vesicles in the deficient in both granzyme A and granzyme B. CTLs from target cell. Other studies have indicated that perforin facili- granzyme A−/−/granzyme B−/− mice are unable to induce target tates translocation of granzyme B to the nucleus, and that cell DNA fragmentation, even after prolonged coincu- nuclear localization is critical to the ability of granzyme B to bation.160 This indicates that granzyme A activity accounts for cause apoptosis.132–137 the ability of granzyme B−/− CTLs to induce target cell While the importance of granzyme B subcellular localiz- apoptosis after prolonged exposure. Therefore, granzyme A ation remains controversial, it is quite clear that granzyme B may allow CTLs to kill target cells under conditions where has the ability to impact the caspase pathway of apoptosis. granzyme B activity is inhibited (eg target cells that express Studies done in vitro, have shown that granzyme B is capable granzyme B inhibitors). of cleaving procaspase-3, -6, -7, -8, -9 and -10.138–153 In the Studies using recombinant proteins have shown that coin- case of procaspases-3, -7 and -9, granzyme B-mediated pro- cubation of granzyme A and perforin with target cells leads cessing has been shown to generate active caspase to rapid (within 2 h) accumulation of DNA single-strand enzymes.138,141,146,149 More importantly, studies with whole breaks.161,162 This contrasts with the rapid degradation of cells have shown that caspases are activated in target cells DNA to oligonucleosomal-length fragments seen in cells following coincubation with granzyme B and per- treated with granzyme B and perforin. Granzyme A/perforin forin.139,153,154 It remains to be determined, however, which treatment also leads to nuclear condensation.162 The DNA sin- caspases are the preferred in vivo substrates for granzyme B. gle-strand breakage and nuclear condensation that occurs in In any event, it is reasonable to propose that granzyme B may response to granzyme A is insensitive to caspase inhibitors, promote apoptosis simply by cleaving and activating indicating that these actions of granzyme A are caspase-inde- endogenous caspases in the target cell. pendent.162 Consistent with this, granzyme A/perforin treat- In cells undergoing granzyme B/perforin-mediated ment does not result in processing/activation of procaspase-3 apoptosis, cleavage of the caspase substrate proteins PARP, or cleavage of the caspase substrate proteins PARP, lamin B, lamin B, and U1–70 kDa is also observed.151,153–155 These or rho-GTPase.162 By contrast, granzyme B-induced DNA cleavage events appear to be due to caspases activated by fragmentation is strictly dependent on the activation of casp- granzyme B, and not the result of direct granzyme B cleavage, ases. Both granzyme A and granzyme B (in conjunction with since cleavage of all three proteins is inhibited by 100 µM perforin) also induce target cell cytolysis, and in both cases

Leukemia Review DE Johnson 1700 this is a caspase-independent event. Taken together, current 21 Tandon AK, Clark GM, Chamness GC, Chirgwin JM, McGuire WL. evidence indicates that granzyme B is the primary CTL Cathepsin D and prognosis in breast cancer. New Engl J Med mediator of target cell DNA fragmentation and apoptotic 1990; 322: 297–302. 22 Kute TE, Shao ZM, Sugg NK, Long RT, Russell GB, Case D. death, and that the apoptotic effects of this protease are Cathepsin D as a prognostic indicator of node-negative breast mediated primarily through the activation of caspases. Gran- cancer patients using both immunoassays and enzymatic assays. zyme A, on the other hand, may be more of a default or speci- Cancer Res 1992; 52: 198–203. alized mediator of target cell apoptosis, with the pathways 23 Garcia M, Platet N, Liaudet E, Laurent V, Derocq D, Brouillet J- initiated by granzyme A being distinctly different from those P, Rochefort H. 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