Lysosome-dependent Cell Death Research Articles

Typical and Atypical Inducers of Lysosomal Cell Death: A Promising Anticancer Strategy

Lysosomes are conservative organelles with an indispensable role in cellular degradation and the recycling of macromolecules. However, in light of recent findings, it has emerged that the role of lysosomes in cancer cells extends far beyond cellular catabolism and includes a variety of cellular pathways, such as proliferation, metastatic potential, and drug resistance. It has been well described that malignant transformation leads to alterations in lysosomal structure and function, which, paradoxically, renders cancer cells more sensitive to lysosomal destabilization. Furthermore, lysosomes are implicated in the regulation and execution of cell death in response to diverse stimuli and it has been shown that lysosome-dependent cell death can be utilized to overcome apoptosis and drug resistance. Thus, the purpose of this review is to characterize the role of lysosome in cancer therapy and to describe how these organelles impact treatment resistance. We summarized the characteristics of typical inducers of lysosomal cell death, which exert its function primarily via alterations in the lysosomal compartment. The review also presents other anticancer agents with the predominant mechanism of action different from lysosomal destabilization, the activity of which is influenced by lysosomal signaling, including classical chemotherapeutics, kinase inhibitors, monoclonal antibodies, as well as photodynamic therapy.

Ferroptosis is a lysosomal cell death process

Ferroptosis is a form of regulated cell death resulting from iron accumulation and lipid peroxidation. While impaired ferroptosis is tightly linked to human diseases and conditions, the mechanism and regulation of ferroptosis remain largely unknown. Here, we demonstrate that STAT3 is a positive regulator of ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cell lines. Activation of the MAPK/ERK pathway, but not inhibition of system Xc−, was required for STAT3 activation during erastin-induced ferroptosis. Importantly, pharmacological inhibition and genetic silencing of STAT3 through small molecules (e.g., cryptotanshinone and S3I-201) or siRNA blocked erastin-induced ferroptosis in PDAC cells. Mechanically, STAT3-mediated cathepsin B expression was required for ferroptosis. Consequently, inhibition of lysosome-dependent cell death by pharmacological blockade of cathepsin activity (using CA-074Me) or vacuolar type H+-ATPase (using bafilomycin A1) limited erastin-induced ferroptosis. These studies indicate that ferroptosis is a lysosomal cell death process.

Lysosome-dependent cell death and deregulated autophagy induced by amine-modified polystyrene nanoparticles.

Nanoparticles (NPs) typically accumulate in lysosomes. However, their impact on lysosomal function, as well as autophagy, a lysosomal degradative pathway, is still not well known. We have previously reported in the 1321N1 cell line that amine-modified polystyrene (NH2-PS) NPs induce apoptosis through damage initiated in the lysosomes leading ultimately to release of lysosomal content in the cytosol, followed by apoptosis. Here, by using a combination of biochemical and cell biological approaches, we have characterized in a mouse embryonic fibroblast cell line that the lysosomal alterations induced by NH2-PS NPs is progressive, initiating from mild lysosomal membrane permeabilization (LMP), to expansion of lysosomal volume and intensive LMP before the summit of cell death. Though the cells initially seem to induce autophagy as a surviving mechanism, the damage of NH2-PS NPs to lysosomes probably results in lysosomal dysfunctions, leading to blockage of autophagic flux at the level of lysosomes and the eventual cell death.

Modern ideas about cell death

Cell death is an important part of normal (physiological) and pathological histogenesis. In the past two decades, our knowledge of the processes of non-programmed and programmed cell death significantly enriched. The International Nomenclature Committee on Cell Death is constantly working, it regularly updates information on the terminology and development mechanisms recommended for this or that type of death, but the general principle of classification of cell death has not yet been worked out. In this review, the principle of separation according to which the mechanisms involved in physiological histogenesis include the rooting, the external pathway of apoptosis, anoikis, macroautophagy and lysosome-dependent cell death. The mechanisms involved in pathological histogenesis include: the internal pathway of apoptosis, necroptosis, pyroptosis, netosis, mitotic catastrophe, partanatosis, entosis, mitochondrial-driven necrosis, ferroptosis, immunogenic cell death, necrosis and oncosis.

Lysosomal membrane permeabilization as a cell death mechanism in cancer cells.

Lysosomes are acidic organelles that contain hydrolytic enzymes that mediate the intracellular degradation of macromolecules. Damage of these organelles often results in lysosomal membrane permeabilization (LMP) and the release into the cytoplasm of the soluble lysosomal contents, which include proteolytic enzymes of the cathepsin family. This, in turn, activates several intracellular cascades that promote a type of regulated cell death, called lysosome-dependent cell death (LDCD). LDCD can be inhibited by pharmacological or genetic blockade of cathepsin activity, or by protecting the lysosomal membrane, thereby stabilizing the organelle. Lysosomal alterations are common in cancer cells and may increase the sensitivity of these cells to agents that promote LMP. In this review, we summarize recent findings supporting the use of LDCD as a means of killing cancer cells.

Lysosomal membrane stability plays a major role in the cytotoxic activity of the anti-proliferative agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT)

The potent and selective anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), localizes in lysosomes and forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) and cell death. Herein, the role of lysosomal membrane stability in the anti-tumor activity of Dp44mT was investigated. Studies were performed using molecules that protect lysosomal membranes against Dp44mT-induced LMP, namely heat shock protein 70 (HSP70) and cholesterol. Up-regulation or silencing of HSP70 expression did not affect Dp44mT-induced LMP in MCF7 cells. In contrast, cholesterol accumulation in lysosomes induced by the well characterized cholesterol transport inhibitor, 3-β-[2-(diethyl-amino)ethoxy]androst-5-en-17-one (U18666A), inhibited Dp44mT-induced LMP and markedly and significantly (p<0.001) reduced the ability of Dp44mT to inhibit cancer cell proliferation (i.e., increased the IC50) by 140-fold. On the other hand, cholesterol extraction using methyl-β-cyclodextrin enhanced Dp44mT-induced LMP and significantly (p<0.01) increased its anti-proliferative activity. The protective effect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.

Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells.

PurposeCell-in-cell structures are created by one living cell entering another homotypic or heterotypic living cell, which usually leads to the death of the internalized cell, specifically through caspase-dependent cell death (emperitosis) or lysosome-dependent cell death (entosis). Although entosis has attracted great attention, its occurrence is controversial, because one cell line used in its study (MCF-7) is deficient in caspase-3.MethodsWe investigated this issue using MCF-7 and A431 cell lines, which often display cell-in-cell invasion, and have different levels of caspase-3 expression. Cell-in-cell death morphology, microstructures, and signaling pathways were compared in the two cell lines.ResultsOur results confirmed that MCF-7 cells are caspase-3 deficient with a partial deletion in the CASP-3 gene. These cells underwent cell death that lacked typical apoptotic properties after staurosporine treatment, whereas caspase-3-sufficient A431 cells displayed typical apoptosis. The presence of caspase-3 was related neither to the lysosome-dependent nor to the caspase-dependent cell-in-cell death pathway. However, the existence of caspase-3 was associated with a switch from lysosome-dependent cell-in-cell death to the apoptotic cell-in-cell death pathway during entosis. Moreover, cellular hypoxia, mitochondrial swelling, release of cytochrome C, and autophagy were observed in internalized cells during entosis.ConclusionThe occurrence of caspase-independent entosis is not a cell-specific process. In addition, entosis actually represents a cellular self-repair system, functioning through autophagy, to degrade damaged mitochondria resulting from cellular hypoxia in cell-in-cell structures. However, sustained autophagy-associated signal activation, without reduction in cellular hypoxia, eventually leads to lysosome-dependent intracellular cell death.

Activation of Lysosomal Function and Reactive Oxygen Species Play Crucial Roles in SAR650984-Induced Direct Killing of Human Multiple Myeloma Cells with Mutated p53, Which Is Further Augmented By Pomalidomide

The anti-CD38 monoclonal antibody (mAb) SAR650984 (SAR) is showing promising clinical activities in ongoing trials in relapsed/refractory multiple myeloma (MM), a setting in which p53 is often mutated. Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define additional molecular mechanisms of SAR-directed MM cell death, and further delineate enhanced anti-MM activity triggered by combining SAR with pomalidomide (Pom) or lenalidomide (Len). Since CD38 expression on MM cell lines is not as high as on primary patient MM cells, we first used lentivirus to overexpress CD38 in 3 MM cell lines with lower CD38 levels and p53 mutations. Without Fc-cross-linking agents or effector cells, SAR, but not other anti-CD38 mAb, induced homotypic aggregation (HA)-associated MM cell death in these cell lines, which was dependent on CD38 level, membrane lipid raft, and actin cytoskeleton. SAR and its F(ab)'2 fragments triggered comparable caspase 3/7 activation in CD38-highly expressing MM cells, indicating that SAR-induced caspase-dependent apoptosis is both CD38-specific and Fc-independent. Most importantly, SAR and its F(ab)'2 fragments induce lysosome-dependent cell death (LCD) in CD38 overexpressing MM cells even with p53 mutations, evidenced by enlarged lysosomes and increased lysosomal membrane permeabilization (LMP) along with leakage of cathepsin B and lysosome associated membrane protein 1 (LAMP1), in the presence or absence of IL-6 or bone marrow stromal cells. SAR significantly induced mobilization of cathepsin B and LAMP1 from lysosomes to the cytosol, particularly at the intercellular junctions, further confirming that lysosomes relocalize to sites of cell adhesion following SAR treatment. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR stimulated reactive oxygen species (ROS), whereas the antioxidant N-acetylcysteine (NAC) blocks SAR-induced ROS and MM cell death. Since NAC does not inhibit HA or lysosome enlargement or LMP, SAR-induced ROS occurs downstream of HA and activation of lysosomal function. SAR does not induce autophagy, indicating that autophagy is not required for this unique mode of cell death. Finally Pom, more potently than Len, enhances SAR-induced direct and indirect killing of MM cells, even resistant to Pom/Len. Moreover, combined treatment of MM cells with SAR and Pom augments caspase 3/7 activity induced by either agent alone. Taken together, these data show that SAR is the first naked therapeutic mAb with convincing direct killing of MM cell lines and patient MM cells, including those with p53 mutations and drug resistance, mainly via lysosome activation and ROS. Coupled with augmented both caspase 3/7-dependent and effector-mediated MM cytotoxicity when combined with Pom, our data further provide the framework for combination clinical trials. DisclosuresSong:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

SAR650984 directly induces multiple myeloma cell death via lysosomal-associated and apoptotic pathways, which is further enhanced by pomalidomide.

The anti-CD38 monoclonal antibody SAR650984 (SAR) is showing promising clinical activity in treatment of relapsed and refractory multiple myeloma (MM). Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define molecular mechanisms of SAR-directed MM cell death and enhanced anti-MM activity triggered by SAR with Pomalidomide (Pom). Without Fc-cross-linking agents or effector cells, SAR specifically induces homotypic aggregation (HA)-associated cell death in MM cells dependent on the level of cell surface CD38 expression, actin cytoskeleton and membrane lipid raft. SAR and its F(ab)'2 fragments trigger caspase 3/7-dependent apoptosis in MM cells highly expressing CD38, even with p53 mutation. Importantly, SAR specifically induces lysosome-dependent cell death (LCD) by enlarging lysosomes and increasing lysosomal membrane permeabilization associated with leakage of cathepsin B and LAMP-1, regardless of the presence of interleukin-6 or bone marrow stromal cells. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR further upregulates reactive oxygen species. Pom enhances SAR-induced direct and indirect killing even in MM cells resistant to Pom/Len. Taken together, SAR is the first therapeutic monoclonal antibody mediating direct cytotoxicity against MM cells via multiple mechanisms of action. Our data show that Pom augments both direct and effector cell-mediated MM cytotoxicity of SAR, providing the framework for combination clinical trials.

A Calcium-Dependent Pathway Determines Response to Lenalidomide in Del(5q) Myelodysplastic Syndromes

Deletions of the long arm of chromosome 5 (del(5q)) are common cytogenetic alterations in myelodysplastic syndrome (MDS), a disease characterized by refractory anemia, megakaryocyte dysplasia, and thrombocytosis. The thalidomide analogue lenalidomide (LEN) produces durable erythroid responses in ~60% of del(5q) MDS patients, including a majority of cytogenetic responses in which the del(5q) clone becomes undetectable in the bone marrow. Despite high response rates, clinical and cytogenetic relapse occur within 2-3 years. Mechanisms of clinical response, resistance and relapse with LEN therapy remain to be elucidated. The target of LEN has recently been identified as the cereblon (CRBN) component of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN). Upon LEN binding, the substrate-specificity of the CRL4-CRBN complex is altered, and LEN-regulated substrates are beginning to be identified.An RNA interference screen was performed to identify genes/pathways that mediate LEN sensitivity and resistance in del(5q) MDS. The LEN-sensitive del(5q) MDS patient-derived cell line MDSL was screened with a genome-wide shRNA library (SBI GeneNet Human 50K Library) in the presence and absence of LEN treatment (0 and 10 μM) for 7 days. Three independent shRNAs targeting the proton-sensing G protein-coupled receptor 1 (GPR68 or OGR1) were among the most enriched shRNAs in LEN-treated cells, suggesting that loss of GPR68 expression conferred resistance to LEN. This finding was validated in MDSL cells, using an independent set of shRNAs. Conversely, a GPR68 agonist (N-cyclopropoyl-5-[thiophen-2-yl]-isoxazole-3-carboxamid) enhanced LEN-induced cytotoxicity to MDSL cells. GPR68 is a proton-sensing G-protein coupled receptor that stimulates inositol phosphate production and/or intracellular calcium (Ca2+) mobilization. Curiously, CRBN was originally identified as a binding protein of calcium-activated potassium channels. These data led us to hypothesize that Ca2+ signaling may be responsible for LEN-mediated cytotoxic effect in MDS cells. Reducing intracellular Ca2+ level with chelators reversed LEN’s cytotoxic effects, while increasing intracellular Ca2+ level with ionomycin enhanced LEN’s cytotoxic effect, indicating that intracellular Ca2+ levels determine cellular responsiveness to LEN. Although LEN did not induce an instant burst of Ca2+ influx, a gradual increase of basal intracellular free Ca2+ was observed following LEN treatment in LEN-sensitive cell lines and primary MDS marrow cells, but not in LEN-resistant cells, suggesting that LEN cytotoxicity was dependent on the cell’s ability to release Ca2+ from intracellular stores. GPR68 and CRBN were both necessary for the LEN-induced increase in Ca2+, as knockdown of GPR68 or CRBN in LEN-sensitive cells prevented the Ca2+increase.To identify the Ca2+-dependent signaling pathway responsible for mediating the cytotoxic effect of LEN, a panel of seven inhibitors that blocked mitochondrial/caspase-, calpain-, autophagy-, or lysosomal-dependent cell death pathways was tested in combination with LEN on MDSL cells. Only the inhibitor of calpains (PD150606) prevented LEN-induced cytotoxic effects in MDSL cells, indicating that calpain activation was necessary for mediating cell death in LEN-treated cells. Calpains are Ca2+-dependent cysteine proteases that can induce apoptotic and necrotic cell death by proteolytic cleavage of protein substrates. Calpastatin, the only endogenous calpain inhibitor, is localized to 5q15 and its expression is haploinsufficient in del(5q) MDS as compared to normal karyotype MDS. Taken together, our results show that LEN increased intracellular Ca2+ levels by a CRBN- and GPR68-dependent mechanism, leading to calpain-mediated cytotoxicity in del(5q) MDS cells. We propose a model in which haploinsufficient expression of calpastatin in del(5q) MDS sensitizes cells to cytotoxic effects of LEN. Further studies are required to identify the direct LEN-modulated substrates of CRBN that mediate this effect. DisclosuresOliva:Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria. MacBeth:Celgene: Employment, Equity Ownership. Starczynowski:Celgene: Research Funding.

Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation

In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.

Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation

In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.

Regulation of apoptosis-associated lysosomal membrane permeabilization

Lysosomal membrane permeabilization (LMP) occurs in response to a large variety of cell death stimuli causing release of cathepsins from the lysosomal lumen into the cytosol where they participate in apoptosis signaling. In some settings, apoptosis induction is dependent on an early release of cathepsins, while under other circumstances LMP occurs late in the cell death process and contributes to amplification of the death signal. The mechanism underlying LMP is still incompletely understood; however, a growing body of evidence suggests that LMP may be governed by several distinct mechanisms that are likely engaged in a death stimulus- and cell-type-dependent fashion. In this review, factors contributing to permeabilization of the lysosomal membrane including reactive oxygen species, lysosomal membrane lipid composition, proteases, p53, and Bcl-2 family proteins, are described. Potential mechanisms to safeguard lysosomal integrity and confer resistance to lysosome-dependent cell death are also discussed.

Alternative pathways of programmed cell death are activated in cells with defective caspase-dependent apoptosis

Loss of programmed cell death pathways is one of the features of malignancy that complicate the response of cancer cells to a therapy. Activation of alternative cell death pathways offers a promising approach to enhance efficiency of cancer chemotherapy. We analysed programmed cell death pathways of v -myb-transformed BM2 monoblasts induced by arsenic trioxide, cycloheximide and camptothecin with U937 promonocytes as a reference cell line. We show that induced death of BM2 cells is not executed by caspases but rather by alternative cell death pathways. Camptothecin induces the lysosome-dependent cell death, arsenic trioxide induces autophagy, and most of cycloheximide-treated BM2 cells die by necrosis. The fact that alternative cell death pathways can be switched in cells with defects in activation and/or function of caspases suggests that understanding and targeting of these pathways could improve therapy of cancer cells suffering from defective apoptosis.

Chloroquine-induced neuronal cell death is p53 and Bcl-2 family-dependent but caspase-independent.

Chloroquine is a lysosomotropic agent that causes marked changes in intracellular protein processing and trafficking and extensive autophagic vacuole formation. Chloroquine may be cytotoxic and has been used as a model of lysosomal-dependent cell death. Recent studies indicate that autophagic cell death may involve Bcl-2 family members and share some features with caspase-dependent apoptotic death. To determine the molecular pathway of chloroquine-induced neuronal cell death, we examined the effects of chloroquine on primary telencephalic neuronal cultures derived from mice with targeted gene disruptions in p53, and various caspase and bcl-2 family members. In wild-type neurons, chloroquine produced concentration- and time-dependent accumulation of autophagosomes, caspase-3 activation, and cell death. Cell death was inhibited by 3-methyladenine, an inhibitor of autophagic vacuole formation, but not by Boc-Asp-FMK (BAF), a broad caspase inhibitor. Targeted gene disruptions of p53 and bax inhibited and bcl-x potentiated chloroquine-induced neuron death. Caspase-9- and caspase-3-deficient neurons were not protected from chloroquine cytotoxicity. These studies indicate that chloroquine activates a regulated cell death pathway that partially overlaps with the apoptotic cascade.