Amplification loop cascade for increasing caspase activity induced by docetaxel

The hierarchy of events accompanying induction of apoptosis by the microtubule inhibitor docetaxel was investigated in HL-60 human leukemia cells. Treatment of HL-60 cells with docetaxel resulted in the production of reactive oxygen species (ROS), activation of caspase-3 (-like) protease, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation, bcl-2 phosphorylation and apoptosis. Docetaxel elicited ROS production from NADPH oxidase as demonstrated by specific oxidase inhibitor diphenylene iodonium (DPI). ROS mediated the caspase-3 activation and apoptosis in HL-60 cells. The caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) effectively inhibited JNK/SAPK activation, bcl-2 phosphorylation and partially attenuated the ROS production induced by docetaxel. Docetaxel-induced bcl-2 phosphorylation was completely blocked by expression of dominant negative JNK or the JNK/SAPK inhibitor SP600125. Overexpression of bcl-2 partially prevented docetaxel-mediated ROS production and subsequent caspase-3 activation, thereby inhibiting apoptotic cell death. It is thus conferred that such sequent events as ROS production, caspase activation, JNK/SAPK activation, bcl-2 phosphorylation and the further generation of ROS should be parts of an amplification loop to increase caspase activity, thereby facilitating the apoptotic cell death process.     

Cooperative Interception of Neuronal Apoptosis by BCL-2 and BAG-1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

STAT1 affects lymphocyte survival and proliferation partially independent of its role downstream of IFN-gamma

Lymphocytes derived from mice deficient in STAT1 showed reduced apoptosis and enhanced proliferation in vitro. To understand the involvement of STAT1 in the observed reduction in apoptosis, we examined the levels of caspase and bcl-2 family genes that are involved in cell survival and/or apoptosis. The levels of caspase 1 and 11, two enzymes involved in both cytokine protein processing and induction of apoptosis, were reduced in STAT1-/- cells compared with wild-type. However, the levels of bcl-2 genes were comparable in both mice. STAT1-/- cells also displayed an enhanced proliferation following TCR stimulation. This hyperproliferation could not be ascribed completely to the loss of IFN-gamma-mediated antiproliferation. First, similar phenotypes were also observed in fibroblasts and pre-B cells derived from STAT1-/- mice, which do not produce IFN-gamma. Second, comparisons with cells lacking the gene for IFN-gamma or with cells treated with neutralizing Abs to IFN-gamma only partially mimicked the STAT1-/- phenotype. Interestingly, the kinetics of degradation of p27kip1, a CDK inhibitor, following TCR ligation were faster, and, concomitantly, the up-regulation of CDK2 kinase activity and protein levels were increased in stimulated T cells of STAT1-/- mice relative to those of wild-type mice. Furthermore, STAT1-/- animals were more susceptible to carcinogen-induced thymic tumors, a possible consequence of altered T cell growth and/or survival. These results demonstrate an essential role for STAT1 for lymphocyte survival and proliferation that is only partially dependent on IFN-gamma signaling.     

Differentiation-associated apoptosis of neural stem cells is effected by Bcl-2 overexpression: impact on cell lineage determination

Apoptosis is an integral part of neural development. To elucidate the importance of programmed cell death on cell lineage determination we utilized murine PCC7-Mzl cells, a model system for neural differentiation. Treatment of pluripotent PCC7-Mzl stem cells with 0.1 microM all-trans retinoic acid (RA) causes a cease of proliferation and an initiation of differentiation into neurons, glial cells and fibroblasts. Simultaneously, a fraction of the cell culture (ca. 25%) dies within 24 h by apoptosis. We transfected PCC7-Mzl cells with the human bcl-2 cDNA and generated PCC7-Mz-Bcl-2 cell lines expressing two- to tenfold higher levels of Bcl-2 than parental cells. Overexpression of Bcl-2 resulted in hypophosphorylation of the retinoblastoma (Rb) protein and consequently prolonged the doubling time of the culture from 18 h to 23 h. RA-induced apoptosis was drastically reduced to 3 to 15% depending on the level of Bcl-2 expression. RA-induced caspase activation, cytochrome c release from the mitochondria to the cytosol and DNA fragmentation was completely blocked. Furthermore, treating Bcl-2 cultures with ceramide (10 microM), a second messenger mediating the RA-initiated death signal in parental cells, no longer caused DNA laddering. Bcl-2 overexpression did not interfere with the potential of PCC7-Mz cells to develop into neurons, glial cells and fibroblasts. However, the relative distribution of cell types in the culture was shifted such that the fraction of neurons was reduced to half (from 60 to 30%) with a concomitant increase in the number of glial and fibroblastoid cells. Furthermore, Bcl-2-overexpressing neurons, but not neurons of parental or mock-transfected PCC7-Mzl cultures, were able to grow as single cells.     

Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-crystallin gene

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.     

Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway

Surfactin has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in surfactin-induced apoptosis remain poorly understood. The present study was undertaken to elucidate the underlying network of signaling events in surfactin-induced apoptosis of human breast cancer MCF-7 cells. In this study, surfactin caused reactive oxygen species (ROS) generation and the surfactin-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) and catalase, suggesting involvement of ROS generation in surfactin-induced cell death. Surfactin induced a sustained activation of the phosphorylation of ERK1/2 and JNK, but not p38. Moreover, surfactin-induced cell death was reversed by PD98059 (an inhibitor of ERK1/2) and SP600125 (an inhibitor of JNK), but not by SB203580 (an inhibitor of p38). However, the phosphorylation of JNK rather than ERK1/2 activation by surfactin was blocked by NAC/catalase. These results suggest that the action of surfactin on MCF-7 cells was via ERK1/2 and JNK, but not via p38, and the ERK1/2 and JNK activation induce apoptosis through two independent signaling mechanisms. Surfactin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade reaction. The NAC and SP600125 blocked these events induced by surfactin. Moreover, the general caspase inhibitor z-VAD-FMK inhibited the caspase-6 activity and exerted the protective effect against the surfactin-induced cell death. Taken together, these findings suggest that the surfactin induces apoptosis through a ROS/JNK-mediated mitochondrial/caspase pathway.     

BCL-2 and MCL-1 Expression in Chinese Hamster Ovary Cells Inhibits Intracellular Acidification and Apoptosis Induced by Staurosporine

Multiple physiological and pharmacological stimuli induce cells to die by apoptosis. In many cases, this apoptosis is inhibited by BCL-2, suggesting the involvement of a common regulatory pathway. One frequent characteristic of apoptosis is the digestion of DNA into oligonucleosome-length fragments. Intracellular acidification and increased intracellular calcium have been variously implicated in activating the endonuclease responsible for this DNA digestion. To explore the involvement of these potential signals in endonuclease activation, we have analyzed three Chinese hamster ovary cell lines: a parental line, one expressing a cDNA encoding BCL-2, and the third expressing the BCL-2 family member MCL-1. Apoptosis was induced with the protein kinase inhibitor staurosporine and intracellular pH and calcium were measured by flow cytometry. We found that both MCL-1 and BCL-2 inhibited DNA digestion compared to the parent cell line, although BCL-2 was more potent in this regard. Concurrent with DNA digestion, we observed intracellular acidification; MCL-1 and BCL-2 inhibited intracellular acidification to an extent commensurate with their ability to inhibit DNA digestion. In contrast, staurosporine caused a dose-dependent increase in intracellular calcium in all three cell lines, demonstrating that intracellular free calcium levels did not correlate with the induction of apoptosis. These results suggest that BCL-2 and MCL-1 may regulate a pathway for intracellular pH homeostasis during apoptotic cell death.     

Transforming growth factor-beta induces apoptosis in activated murine T cells through the activation of caspase 1-like protease

Transforming growth factor-beta (TGF-beta) has been known as a potent immunosuppressive cytokine that can induce apoptosis in lymphoid cells. We established an IL-2-independent cell line, CTLL-2A, from murine T cell line CTLL-2. CTLL-2A expressed higher levels of CD95, CD69, and CD18 molecules than CTLL-2 did, suggesting a more activated state in CTLL-2A than in the CTLL-2 by phenotype. Exposing both CTLL-2 and CTLL-2A to TGF-beta results in differential apoptosis patterns defined by DNA fragmentation and plasma membrane alteration. Among the bcl-2 family members, bcl-2, bcl-w, and bcl-x(L) were also differently expressed in these two cell lines. In CTLL-2A, bcl-x(L) was amplified as a major anti-apoptotic molecule, and TGF-beta-induced cell death was more enhanced than in the original cell line. Caspase 1-like protease was activated by TGF-beta treatment and consequently it cleaved bcl-x(L) in CTLL-2A. TGF-beta-induced DNA fragmentation and cleavage of bcl-x(L) were inhibited by pretreatment with tetra peptide caspase 1 inhibitor, YVAD.cmk. These findings suggest that TGF-beta induces cell death in activated murine T cells through cleavage of bcl-x(L) via activated caspase 1-like protease, which may act as an important executor in that process.     

Crm-A, bcl-2 and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8 processing

Susceptibility to CD95 (Fas/APO-1)-mediated apoptosis in human glioma cells depends on CD95 expression and unknown factors that regulate signal transduction. Thus, LN-18 cells are highly sensitive to CD95 ligand (CD95L) whereas LN-229 cells require coexposure to inhibitors of RNA or protein synthesis for induction of apoptosis. Here, we report that caspase 8 and 3 activation, poly(ADP-ribose)polymerase cleavage and apoptosis are inhibited by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or ectopic expression of crm-A or bcl-2. CD95L-induced glioma cell apoptosis does not involve ceramide generation. Apoptosis induced by exogenous ceramide resembles CD95-mediated apoptosis in that bcl-2 is protective but differs in that NDGA and crm-A have no effect and in that cycloheximide (CHX) inhibits rather than potentiates ceramide-induced cell death. We conclude that caspase 8 and caspase 3 activation, but not ceramide generation, are required for CD95 ligand-induced apoptosis of glioma cells and that bcl-2, crm-A and NDGA all act upstream of caspases to inhibit apoptosis.     

Involvement of c-Jun NH<Subscript>2</Subscript>-terminal kinase and nitric oxide-mediated mitochondria-dependent intrinsic pathway signaling in cardiotoxin-induced muscle cell death: role of testosterone

To test the hypothesis that c-Jun NH₂-terminal kinase (JNK) and nitric oxide (NO)-mediated signaling plays an important role in muscle cell apoptosis, we examined the contribution of these molecules in muscle cell apoptosis during cardiotoxin (ctx)-induced muscle injury in mice. Compared to controls, where no apoptosis was detected, the percent of muscle cell apoptosis rose significantly (P < 0.05) at 4 h (27%) after ctx-treatment and increased further progressively up to 16 h posttreatment (80%), before it fell again at 24 h posttreatment (38%). Initiation of apoptosis was preceded by JNK activation and elevated levels of B-cell lymphoma-2 (BCL-2) in the mitochondrial fractions (BAX levels remained unaffected). Ctx treatment also resulted in the inactivation of BCL-2 through phosphorylation at serine 70, thereby perturbing the BAX/BCL-2 rheostat, and the subsequent activation of the cytochrome c-mediated death pathway. Concomitant administration of SP600125, a selective JNK inhibitor, or aminoguanidine (AG), a selected inducible nitric oxide synthase (iNOS) inhibitor, effectively diminished BCL-2 phosphorylation, suppressed cytochrome c release from mitochondria and caspase activation, and significantly prevented ctx-induced muscle cell apoptosis. In additional studies, we examined the role of testosterone in preventing such ctx-induced muscle cell apoptosis. Collectively, the present study emphasizes the role of a new signal transduction pathway involving JNK and iNOS that promotes ctx-induced myocyte apoptosis by provoking BCL-2 phosphorylation, leading to its inactivation, and subsequent activation of the intrinsic pathway signaling. Testosterone therapy has no protective effect in acute muscle injury associated with increased muscle cell death after ctx-treatment.     

Down-regulation of Bcl-2 and Bcl-xL expression with bispecific antisense treatment in glioblastoma cell lines induce cell death

The functions of the antiapoptotic proteins Bcl-2 and Bcl-xL were examined in glioblastoma cells. Expression of both Bcl-2 and Bcl-xL were found to be elevated in protein lysates from seven early passage cell lines derived from human glioblastoma tumors compared with non-neoplastic glial cells. Down-regulation of both bcl-2 and bcl-xL expression in glioblastoma cell lines U87 and NS008 with bcl-2/bcl-xL bispecific antisense oligonucleotide resulted in spontaneous cell death. The mechanism of cell death was partially caspase-dependent. Executioner caspase 6 and caspase 7, but not caspase 3, were involved in apoptosis induced by bcl-2/bcl-xL antisense treatment. Interestingly, western blots failed to demonstrate expression of caspase 3 in two of the seven glioblastoma cell lines examined. The data support the hypothesis that Bcl-2 and Bcl-xL are important in preventing cell death in glioblastoma cells. It also suggests that there are functional pathways capable of successful completion of caspase-dependent cell death in gliomas. These findings support a potential role of bcl-2/bcl-xL bispecifc antisense oligonucleotide therapy as a treatment strategy to enhance caspase-dependent cell death in patients with glioblastoma.     

Co-targeting MCL-1 and ERK1/2 kinase induces mitochondrial apoptosis in rhabdomyosarcoma cells

The RAS/MEK/ERK genetic axis is commonly altered in rhabdomyosarcoma (RMS), indicating high activity of downstream effector ERK1/2 kinase. Previously, we have demonstrated that inhibition of the RAS/MEK/ERK signaling pathway in RMS is insufficient to induce cell death due to residual pro-survival MCL-1 activity. Here, we show that the combination of ERK1/2 inhibitor Ulixertinib and MCL-1 inhibitor S63845 is highly synergistic and induces apoptotic cell death in RMS in vitro and in vivo. Importantly, Ulixertinib/S63845 co-treatment suppresses long-term survival of RMS cells, induces rapid caspase activation and caspase-dependent apoptosis. Mechanistically, Ulixertinib-mediated upregulation of BIM and BMF in combination with MCL-1 inhibition by S63845 shifts the balance of BCL-2 proteins towards a pro-apoptotic state resulting in apoptosis induction. A genetic silencing approach reveals that BIM, BMF, BAK and BAX are all required for Ulixertinib/S63845-induced apoptosis. Overexpression of BCL-2 rescues cell death triggered by Ulixertinib/S63845 co-treatment, confirming that combined inhibition of ERK1/2 and MCL-1 effectively induces cell death of RMS cells via the intrinsic mitochondrial apoptotic pathway. Thus, this study is the first to demonstrate the cytotoxic potency of co-inhibition of ERK1/2 and MCL-1 for RMS treatment.     

Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.     

Apoptosis Induced by Differentiation or Serum Deprivation in an Immortalized Central Nervous System Neuronal Cell Line

Abstract: To characterize the nature of programmed cell death (PCD) induced in neuronal cells during development, three regulators of apoptosis were investigated: one, the bcl-2-related genes, modulate cell survival, and the other two, the interleukin-1β converting enzyme (ICE)-related enzymes and the tumor suppressor protein p53, have been implicated as mediators of apoptosis. These regulators were studied in H19-7 cells, an SV40 T^ts-immortalized rat hippocampal neuronal cell line that can be differentiated with basic fibroblast growth factor at the nonpermissive temperature, resulting in a rapid attrition of cells by apoptosis. PCD occurred by two mechanisms in H19-7 cells: The first was initiated by removal of serum from undifferentiated cells, and the second was a consequence of neuronal differentiation. In differentiated H19-7 cells, the survival time was increased by both human bcl-2 and bcl-x_L, and this could be reversed by bcl-x_S.Addition of a peptide inhibitor of the ICE enzyme family to H19-7 cells resulted in a transient protection against differentiation-associated apoptosis, whereas no further protection was observed in the BCL-2- or BCL-X_L-expressing cells. Shifting the differentiated cells to 33°C to inactivate p53 did not significantly affect the apoptotic process, indicating that apoptosis induced by neuronal differentiation is not dependent on the continued presence of p53. By contrast, in undifferentiated cells, cell loss induced by transfer to serum-free media occurred more rapidly on inactivation of large T, consistent with p53 involvement. This medium-induced decrease in cell survival could not be rescued by the ICE inhibitor but was partially rescued by BCL-2 or BCL-X_L. Furthermore, studies involving expression of BCL-2 and BCL-X_L alone or together revealed differences in the survival dependent on the cellular environment. These results suggest that apoptosis of neuronal cellsoccurs by at least two processes: one in undifferentiated cells initiated by removal of serum and one linked to differentiation. The data implicate the ICE enzyme family but not p53 in apoptosis induced by differentiation and demonstrate that either BCL-2 or BCL-X_L can prolong the survival of differentiated neuronal cells.     

Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2

Cell lines derived from the hemopoetic lineages are widely used as hosts for the production of biologicals. These cell lines have been demonstrated to undergo high levels of the active death program commonly referred to as apoptosis. The effects of overexpression of the apoptosis suppressor gene bcl-2 on the properties of a Burkitt lymphoma were compared with the control cell line (transfected with a negative control plasmid) under a variety of conditions relevant to cell culture production technology. In stationary batch cultures, there was a clear reduction in both the rate of total cell death and the level of apoptosis during the decline phase of the bcl-2 transfected cell cultures as compared with that of the control cell cultures. Nutrient analysis revealed that the onset of death during the control cell cultures occurred following complete exhaustion of glutamine. However, the bcl-2 transfected cell cultures continued to grow even though glutamine had been exhausted, and a significant decline in viability only occurred when glucose had also been completely exhausted.When cells were cultured in suspension without prior adaptation, the bcl-2 transfected cells grew significantly better, suggesting that the bcl-2 gene protected the cells from apoptosis triggered by either the lack of substrate or the hydrodynamic environment. Fluorescence microscopy revealed that death of the control cells was almost entirely by apoptosis, whereas death was almost exclusively by necrosis in the delayed decline phase of the transfected cell cultures. In both instances, death occurred before total exhaustion of glucose and glutamine.The induction of apoptosis following growth arrest is a major impediment to the development of culture strategies that optimize specific productivity by reducing the growth rate. Results presented here suggest that suppression of apoptosis by bcl-2 under the condition of excess thymidine allows the maintenance of cells in a growth-arrested state for much longer than would otherwise be possible.When cells were transferred to a range of commercial serum-free media, cell growth was, in all cases, much better for the bcl-2 transfected cell line. Moreover, when cells were cultivated in glutamine-free medium, the control cells exhibited a decrease in viable cell number within the first 24 h whereas, for the bcl-2 transfected cell cultures, viable cell number did not exhibit any clear decrease until after 75 h. Clearly, these results indicate that the metabolic engineering approach can be used to alter advantageously the survival and proliferative capacity of cells in cell culture environments. (c) 1996 John Wiley & Sons, Inc.     

Stress-induced apoptosis is impaired in cells with a lysosomal targeting defect but is not affected in cells synthesizing a catalytically inactive cathepsin D

The role of cathepsin D in stress-induced cell death has been investigated by using ovine fibroblasts exhibiting a missense mutation in the active site of cathepsin D. The cathepsin D (lysosomal aspartic protease) deficiency did not protect cells against toxicity induced by doxorubicin and other cytotoxic agents, neither did it protect cells from caspase activation. Moreover, the cathepsin D inhibitor, pepstatin A, did not prevent stress-induced cell death in human fibroblasts or lymphoblasts. The possible role of lysosomal ceramide or sphingosine-mediated activation of cathepsin D in apoptosis was also excluded by using human cells either overexpressing or deficient in acid ceramidase. However, a normal lysosomal function seems to be required for efficient cell death, as indicated by the finding that fibroblasts from patients with mucolipidosis II were partially resistant to staurosporine, sphingosine and TNF-induced apoptosis, suggesting a key role of lysosomes in cell death.