Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied

When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37°C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.     

Nocodazole induces mitotic cell death with apoptotic-like features in Saccharomyces cerevisiae

We investigated the fate of budding yeast treated with nocodazole, a microtubule-depolymerizing drug. Cells died after mitotic arrest while staying in mitosis, suggesting that mitotic cell death, but not mitotic slippage, mainly occurs in nocodazole-treated cells. Nocodazole-treated cells showed features of apoptotic-like cell death, but not those of cell lysis or autophagy. Consistently, mitochondria-dependent production of reactive oxygen species was involved in the cell death. Similar cell death was also seen in cells after mitotic arrest by perturbation of the anaphase-promoting complex/cyclosome. In addition, caspase activity was found in nocodazole-treated cells, which was independent of the metacaspase, Mca1. Our results suggest that budding yeast can be a model to study mitotic cell death in cancer treatment with antimitotic drugs.     

Caspase 3, periodically expressed and activated at G2/M transition, is required for nocodazole-induced mitotic checkpoint

Caspases have been known for several years for their involvement in executing apoptosis, where unwanted or damaged cells are eliminated. Surprisingly, after analysis of the relevant data set from the Stanford microarray database, we noticed that the gene expression pattern for caspase 3, but not for caspase 1, 6, 7, 8, 9, or 10, undergoes periodic change in the HeLa cell cycle. In this study, we have demonstrated that caspase 3, but not other caspases, is upregulated and activated just prior to mitosis. Pretreatment of human hepatoma cells with a caspase 3 inhibitor z-DEVD-FMK, prior to the treatment with an antimicrotubule drug nocodazole, abrogates the mitotic arrest, suggesting that caspase 3 (or a caspase 3-like enzyme) might be involved in mitotic-spindle checkpoint. The studies not only characterize caspase 3 as a cell cycle-regulated protein, but also link the protein to nocodazole-dependent mitotic checkpoint, greatly expanding the understanding of caspase 3. These authors contributed equally.     

Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells

The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.     

Secretion of invertase in mitotic yeast cells.

In mammalian cells intracellular transport is inhibited during mitosis. Here we show that in the yeast Saccharomyces cerevisiae secretion continues uninterrupted during mitosis. S. cerevisiae cells were arrested in mitosis by treating wild-type cells with the microtubule-inhibitor nocodazole, or by incubating a temperature-sensitive cell division cycle mutant (cdc16) at the restrictive temperature. Secretion of invertase into the periplasmic space was equally efficient in mitotic and in unsynchronized cells. Electron microscopy of nocodazole-treated mitotic wild-type cells revealed stretches of rough endoplasmic reticulum, strongly fenestrated Golgi cisternae and clusters of vesicles with the diameter of 30-90 nm. Secretion of invertase was inhibited in mitotic sec7 cells at the restrictive temperature, but continued at the permissive temperature. Sec7 is a mutant strain where intracellular traffic is blocked in unsynchronized cells in the Golgi complex at the restrictive temperature. Thus, the elements of the mitotic Golgi complex appear to be able to support intracellular traffic.     

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death.     

Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction

Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1. We investigated the cause of this induction. We detected DNA damage late in mitotic arrest and also after slippage. This damage was inhibited by treatment with caspase inhibitors and by stable expression of mutant, noncleavable inhibitor of caspase-activated DNase, which prevents activation of the apoptosis-associated nuclease caspase-activated DNase (CAD). These treatments also inhibited induction of p53 after slippage from prolonged arrest. DNA damage was not due to full apoptosis, since most cytochrome C was still sequestered in mitochondria when damage occurred. We conclude that prolonged mitotic arrest partially activates the apoptotic pathway. This partly activates CAD, causing limited DNA damage and p53 induction after slippage. Increased DNA damage via caspases and CAD may be an important aspect of antimitotic drug action. More speculatively, partial activation of CAD may explain the DNA-damaging effects of diverse cellular stresses that do not immediately trigger apoptosis.     

Substrate degradation by the anaphase promoting complex occurs during mitotic slippage

Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. Knockdown of Cdc20 and Cdh1 prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.     

Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of inhibitors of caspases 1, 3, 8, 9 and pan-caspase inhibitors. We observed that caspases-3, -8, -9 and pan caspase inhibitors resulted in significant inhibition of etoposide-induced DNA fragmentation. However, caspase-1 specific inhibitor failed to prevent DNA fragmentation, suggesting that either caspases belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10), selectivity of caspase-I inhibitors can be ascertained for the above panel (caspases 2, 6, 7, 8, 9 and 10) of caspases from this single assay.     

Budding yeast Bub2 is localized at spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2.

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.     

Activation of JNK triggers release of Brd4 from mitotic chromosomes and mediates protection from drug-induced mitotic stress

Some anti-cancer drugs, including those that alter microtubule dynamics target mitotic cells and induce apoptosis in some cell types. However, such drugs elicit protective responses in other cell types allowing cells to escape from drug-induced mitotic inhibition. Cells with a faulty protective mechanism undergo defective mitosis, leading to genome instability. Brd4 is a double bromodomain protein that remains on chromosomes during mitosis. However, Brd4 is released from mitotic chromosomes when cells are exposed to anti-mitotic drugs including nocodazole. Neither the mechanisms, nor the biological significance of drug-induced Brd4 release has been fully understood. We found that deletion of the internal C-terminal region abolished nocodazole induced Brd4 release from mouse P19 cells. Furthermore, cells expressing truncated Brd4, unable to dissociate from chromosomes were blocked from mitotic progression and failed to complete cell division. We also found that pharmacological and peptide inhibitors of the c-jun-N-terminal kinases (JNK) pathway, but not inhibitors of other MAP kinases, prevented release of Brd4 from chromosomes. The JNK inhibitor that blocked Brd4 release also blocked mitotic progression. Further supporting the role of JNK in Brd4 release, JNK2-/- embryonic fibroblasts were defective in Brd4 release and sustained greater inhibition of cell growth after nocodazole treatment. In sum, activation of JNK pathway triggers release of Brd4 from chromosomes upon nocodazole treatment, which mediates a protective response designed to minimize drug-induced mitotic stress.     

Contribution of caspase(s) to the cell cycle regulation at mitotic phase

Caspases have been suggested to contribute to not only apoptosis regulation but also non-apoptotic cellular phenomena. Recently, we have reported the involvement of caspase-7 to the cell cycle progression at mitotic phase by knockdown of caspase-7 using small interfering RNAs and short hairpin RNA. Here we showed that chemically synthesized broad-spectrum caspase inhibitors, which have been used to suppress apoptosis, prevented the cell proliferation in a dose-dependent manner, and that the subtype-specific peptide-based caspase inhibitor for caspase-3 and -7, but not for caspase-9, inhibited cell proliferation. It was also indicated that the BIR2 domain of X-linked inhibitor of apoptosis protein, functioning as an inhibitor for caspase-3 and -7, but not the BIR3 domain which plays as a caspase-9 inhibitor, induced cell cycle arrest. Furthermore, flow cytometry revealed that the cells treated with caspase inhibitors arrested at G(2)/M phase. By using HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, the prevention of the cell proliferation by caspase inhibitors induced cell cycle arrest at mitotic phase accompanying the accumulation of the substrates for APC/C, suggesting the impairment of the APC/C activity at the transition from M to G(1) phases. These results indicate that caspase(s) contribute to the cell cycle regulation at mitotic phase.     

Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5

Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.     

The ability to survive mitosis in the presence of microtubule poisons differs significantly between human nontransformed (RPE-1) and cancer (U2OS, HeLa) cells

We used live cell imaging to compare the fate of human nontransformed (RPE-1) and cancer (HeLa, U2OS) cells as they entered mitosis in nocodazole or taxol. In the same field, and in either drug, a cell in all lines could die in mitosis, exit mitosis and die within 10 h, or exit mitosis and survive > or =10 h. Relative to RPE-1 cells, significantly fewer HeLa or U2OS cells survived mitosis or remained viable after mitosis: in nocodazole concentrations that inhibit spindle microtubule assembly, or in 500 nM taxol, 30% and 27% of RPE-1 cells, respectively, died in or within 10 h of exiting mitosis while 90% and 49% of U2OS and 78% and 81% of HeLa died. This was even true for clinically relevant taxol concentrations (5 nM) which killed 93% and 46%, respectively, of HeLa and U2OS cells in mitosis or within 10 h of escaping mitosis, compared to 1% of RPE-1 cells. Together these data imply that studies using HeLa or U2OS cells, harvested after a prolonged block in mitosis with nocodazole or taxol, are significantly contaminated with dead or dying cells. We also found that the relationship between the duration of mitosis and survival is drug and cell type specific and that lethality is related to the cell type and drug used to prevent satisfaction of the kinetochore attachment checkpoint. Finally, work with a pan-caspase inhibitor suggests that the primary apoptotic pathway triggered by nocodazole during mitosis in RPE-1 cells is not active in U2OS cells. Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.     

Caspase-independent apoptosis is activated by diazepam-induced mitotic failure in HeLa cells,but not in human primary fibroblasts

DZ, a benzodiazepine known to affect centrosome separation at prophase, leads to a higher degree of mitotic arrest in HeLa cells than in primary human fibroblasts. In fact, differently from fibroblasts, which undergo a transient block in prophase-to-prometaphase transition, a high proportion of tumor cells attempt to escape from the DZ-imposed mitotic block, fail to undergo complete mitosis and die by mitotic failure. DZ-treated samples showed certain biochemical hallmarks of apoptosis, such as induction of the proapototic Bax protein, mitochondrial alterations assessed by JC-1 staining and TEM analysis, PARP cleavage, and DNA fragmentation. However, in DZ-treated cells, we observed a very low or absent caspase activation as shown by immunofluorescence and immunoblot experiments with antibodies directed to activated caspases and by staining with the pancaspase inhibitor FITC-VAD-FMK. Experiments on mitochondrial depolymerization and apoptosis induction carried out in the presence of specific inhibitors of caspase-2 and caspase-3/7 indicated a caspase-independent apoptotic process induced by DZ. Accordingly, TEM analysis of treated cells revealed ultrastructural features resembling those reported for caspase-independent apoptosis. In conclusion, we hypothesize that HeLa cells override the prophase block imposed by DZ, producing a high rate of aberrant pro-metaphases, which, in turn, activates caspase-independent, apoptosis-like mitotic catastrophe.     

The inhibition of Aurora A abrogates the mitotic delay induced by microtubule perturbing agents

The spindle assembly checkpoint functions during mitosis to ensure that chromosomes are properly aligned in mitotic cells prior to the onset of anaphase, thereby ensuring an equal segregation of genetic material to each daughter cell. Defects in the function of this checkpoint lead to aneuploidy, and eventually to cell death or senescence. The Aurora-related kinases, and in particular Aurora B, have been shown to play a role in regulating the spindle assembly checkpoint. In this study, we demonstrate that Aurora A activity is required for maintainance of the spindle assembly checkpoint mediated-mitotic delay induced by microtubule perturbing agents. Inhibition of Aurora A using MLN8054, a selective small-molecule inhibitor of Aurora A, in paclitaxel- or nocodazole-treated cells induces cells to become multinucleated. Using time-lapse microscopy, we demonstrate that the multinucleation phenotype arises via mitotic slippage, which is significantly accelerated upon Aurora A inhibition. Under these conditions, the spindle assembly checkpoint protein BubR1 remains localized to kinetochores prior to mitotic slippage. Moreover, we demonstrate that Aurora B remains active in these mitotic cells, indicating that the mitotic slippage induced by MLN8054 is most likely due to the inhibition of Aurora A. This finding was corroborated by demonstrating that Aurora A depletion using RNA interference in paclitaxel-treated cells also induces multinucleation. Taken together, these results suggest that Aurora A is necessary for the maintenance of the mitotic delay induced in response to microtubule-perturbing agents.     

CDK1 inhibitors antagonize the immediate apoptosis triggered by spindle disruption but promote apoptosis following the subsequent rereplication and abnormal mitosis

Spindle-disrupting agents and CDK inhibitors are important cancer therapeutic agents. Spindle toxins activate the spindle-assembly checkpoint and lead to sustained activation of CDK1. Different published results indicate that CDK1 activity is either important or dispensable for the cytotoxicity associated with spindle disruption. Using live cell imaging and various approaches that uncoupled mitotic events, we show that apoptosis was induced by both prolonged nocodazole treatment as well as by inhibition of CDK1 activity after a transient nocodazole block. However, distinct mechanisms are involved in the two types of cell death. The massive apoptosis triggered by nocodazole treatment requires the continue activation of cyclin B1-CDK1 and is antagonized by premature mitotic slippage. By contrast, apoptosis induced by nocodazole followed by CDK inhibitors occurred after rereplication and multipolar mitosis of the subsequent cell cycle. The presence of dual mechanisms of cytotoxicity mediated by spindle disruption and CDK inhibition may reconcile the various apparent inconsistent published results. These data underscore the essential role of cyclin B1-CDK1 as the basis of apoptosis during mitotic arrest, and the role of mitotic slippage and abnormal mitosis for apoptosis at later stages.     

Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures

In an attempt to investigate the molecular mechanism that leads to apoptotic death in Chinese hamster ovary (CHO) cells in batch and fed-batch cultures, we cloned caspase-2, -8 and -9 from a CHO cDNA library. Recombinant Chinese hamster caspase-2 and -9 expressed in Escherichia coli show highest activities towards commercial peptide substrates Ac-VDVAD-pNA and Ac-LEHD-pNA, the designated commercial substrates for human caspase-2 and -9, respectively. However, Chinese hamster caspase-8 shows a broad specificity profile and it cleaves the caspase-9 substrate more efficiently than it cleaves the caspase-8 substrate. The commercially available fluoromethyl ketone type of caspase inhibitors, such as Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk and Z-DEVD-fmk, were shown to completely lack specificity in inhibiting these caspases. The reversible aldehyde form of inhibitors for human caspase-8 and -9, Ac-LEHD-CHO and Ac-IETD-CHO, are equally efficient in inhibiting Chinese hamster caspase-8. Therefore, the wildly used method of utilizing the "caspase-specific" inhibitors to track the role of individual caspases in dying cells can be inaccurate and thus misleading. As an alternative, we stably expressed dominant negative (DN) mutants of Chinese hamster caspase-2, -8 and -9 to specifically inhibit these enzymes in CHO cells. Our results showed that inhibition of either endogenous caspase-8 or caspase-9 enhanced the viability of the CHO cells in both batch and fed-batch suspension cultures, but the inhibition of caspase-2 had minimal effects. These results suggest that caspase-8 and -9 are possibly involved in the apoptotic cell death in batch and fed-batch cultures of CHO cells, whereas caspase-2 is not. These findings can be valuable in the development of strategies for genetically engineering CHO cells to counter apoptotic death in batch and fed-batch cultures.     

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.     

Oxyhemoglobin induces caspase-mediated cell death in cerebral endothelial cells

Damaged endothelium is one of the pathological changes of the cerebral vasospastic vessels following subarachnoid hemorrhage. Our recent study shows that oxyhemoglobin (OxyHb) induces apoptosis in vascular endothelial cells. Apoptosis generally requires the action of various classes of proteases, including a family of cysteine proteases, known collectively as the caspases. This study was undertaken to investigate the activation of caspases and the efficacy of caspase inhibitors, z-IETD-fmk and z-LEHD-fmk, for oxyhemoglobin-induced apoptosis in vascular endothelial cells. Cultured bovine brain microvascular endothelial cells (passages 5-9) were used for this study. OxyHb (10 micromol/L) was added during the 24-72 h incubation with and without caspase-8 or - 9 inhibitors (z-IETD-fmk and z-LEHD-fmk). Counting surviving cells, DNA laddering, western blotting of poly(ADP-ribose) polymerase, and measurement of caspase activities were employed to confirm the cytotoxic effects of OxyHb and the protective effects of the caspase inhibitors. OxyHb produced cell detachment in a time-dependent manner and increased caspase-8 and -9 activities in the cells. z-IETD-fmk and z-LEHD-fmk (100 micromol/L) attenuated OxyHb-induced cell loss, DNA laddering, and proteolytic cleavage of PARP, although a lower concentration (10 micromol/L) of caspase inhibitors showed partial effects. OxyHb activates caspase-8 and -9 in cultured vascular endothelial cells, and blocking the action of the caspases with the inhibitors efficiently prevents loss of vascular endothelial cells from OxyHb-induced apoptosis in vitro. These results suggest that the caspase cascade participates in OxyHb-induced apoptosis.