Effect of phenols on natural killer (NK) cell-mediated death in the K562 human leukemic cell line

Cancer disease is a major cause of death in Western societies. Epidemiologically, antioxidant phenols have been associated with diminished incidence of cancer, while experimentally, they have cytotoxic effects on cancer cells. The aim of this study was to clarify whether natural antioxidant phenols render K562 human leukemic cells more susceptible to natural killer (NK) cell apoptosis and/or necrosis. K562 cells were pre-incubated with 7 different phenols (p-hydroxy benzoic acid, syringic acid, ferulic acid, p-coumaric acid, o-coumaric acid, gallic acid, and rutin) individually and afterwards targeted with NK cells at a ratio 1/5. Percentages of apoptotic and necrotic cells were assayed via flow cytometric analysis of annexin V and PI-stained cells. For the morphological assessment, cells were stained with acridine orange and ethidium bromide and were examined under a fluorescence microscope. Pre-treatment with gallic acid significantly rendered K562 cells more susceptible to NK cell-mediated necrosis, while pre-treatment with rutin significantly rendered K562 cells more susceptible to apoptosis. Gallic acid and rutin exert anticarcinogenic activity via the enhancement of K562 cell susceptibility to NK cell-mediated necrosis and apoptosis, respectively.     

Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes

Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 degrees C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV(+)), scarce propidium iodide stained cells (PI(+)) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 degrees C and rewarming at 37 degrees C. When DEM and BSO were added before cold storage, we observed a few AnXV(+) cells and an increase in PI(+) cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.     

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.     

Apoptosis in hematopoietic cells (FL5.12) caused by interleukin-3 withdrawal: relationship to caspase activity and the loss of glutathione

The mechanism of cell death caused by cytokine deprivation remains largely unknown. FL5.12 cells (a murine prolymphocytic cell line), following interleukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis. In the present study, the induction of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthionine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dependent increases in phosphatidylserine externalization, acridine orange and ethidium bromide staining, decreases in mitochondrial membrane potential, processing and activation of caspase-3 and proteolysis of the endogenous caspase substrate poly(adenosine diphosphate ribose)polymerase (PARP). Apoptosis induced by IL-3 deprivation but not BSO also caused lamin B1 cleavage, suggesting activation of caspase-6. Despite a more profound depletion of GSH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not after IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z-VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the cleavage and activation of caspase-3, the breakdown of lamin B1 and partially mitigated PARP degradation. However, the externalization of phosphatidylserine, the fall in mitochondrial membrane potential and subsequent apoptotic cell death still occurred. These results suggest that IL-3 withdrawal may mediate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH.     

Characteristics of the killing mechanism of human natural killer cells against hepatocellular carcinoma cell lines HepG2 and Hep3B

Purpose Unlike normal hepatocytes, most hepatocellular carcinomas (HCCs) are quite resistant to death receptor-mediated apoptosis when the cell surface death receptor is cross linked with either agonistic antibodies or soluble death ligand proteins in vitro. The resistance might play an essential role in the escape from the host immune surveillance; however, it has not been directly demonstrated that HCCs are actually resistant to natural killer (NK) cell-mediated death. Therefore, this study investigated the molecular mechanism of NK cell-mediated cytotoxicity against the HCCs, HepG2, and Hep3B, using two distinct cytotoxic assays: a 4-h ⁵¹Cr-release assay and a 2-h [³H] thymidine release assay which selectively measures the extent of necrotic and apoptotic target cell death, respectively.Methods Most of the target cells exhibited marked morphologic changes when they were co-incubated with the NK cells, and the NK cytotoxicity against these HCCs was comparable to that against K562, a NK-sensitive leukemia cell line, when the cytotoxicity was assessed by a 4-h ⁵¹Cr release assay.Results The NK cells also induced significant apoptotic cell death in the Hep3B targets, but not in the HepG2 targets, when the cytotoxicity was assessed by a 2-h [³H]-thymidine release assay. In agreement with these results, procaspase-3 was activated in the Hep3B targets, but not in the HepG2 targets. Interestingly, mildly fixed NK cells had no detectable activity in the 4-h ⁵¹Cr release assay against both HepG2 and Hep3B targets, while they were similarly effective as the untreated NK cells in the 2-h [³H]-thymidine release assay, suggesting that the level of apoptotic cell death of the Hep3B targets is granule independent and might be primarily mediated by the death ligands of the NK cells.Conclusion This study found that a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor interaction is involved in the NK cell-mediated apoptotic death of the Hep3B targets, but a Fas/Fas ligand (FasL) interaction is not.     

Osteoblastic MG-63 cell differentiation, contraction, and mRNA expression in stress-relaxed 3D collagen I gels

The present study was designed to evaluate the apoptotic efficacy of selenium (Se) under glutathione-deprived conditions. Testicular cells were used as a model to assess the above. For the study, cells were maintained for 4 h under various treatments; control (media only), selenium (0.5 microM and 1.5 microM), BSO (20 nM), selenium + BSO (0.5 microM Se + 20 nM BSO and 1.5 microM Se + 20 nM BSO). The treated cells were harvested for various estimations viz. viability, GSH, GSSG, redox ratio, ROS generation and integrity of DNA. mRNA was extracted for RT-PCR analysis of JNK, p38, caspase 3 and Bcl-2. It was observed that the cell viability decreased concomitant with the decrease in GSH levels, increase in GSSG levels and increase in the generation of ROS in the combined treatment group in comparison to control and individual treatments. Also, there was an increase in the mRNA expression of JNK and p38 MAPK along with an increase in caspase 3 expression and decrease in Bcl-2 expression. The integrity of DNA was also found to be altered in the combined treatment. Thus, the results presented in this work agree with those earlier reports in a notion that sodium selenite causes apoptosis and the toxicity of selenite is mediated by increase of intracellular ROS. Also, reduction in endogenous GSH along with selenite treatment is associated with increased apoptosis, increased expression of p38 and JNK MAPK, decreased Bcl-2 expression, and increase in caspase-3 expression. Our data indicates that GSH participates in apoptosis in testicular cells and that depletion of this molecule may be critical in predisposing these cells to apoptotic cell death.     

Removal of N-glycans from cell surface proteins induces apoptosis by reducing intracellular glutathione levels in the rhabdomyosarcoma cell line S4MH

Expression of determined Asn-bound glycans (N-glycans) in cell surface glycoproteins regulates different processes in tumour cell biology. Specific patterns of N-glycosylation are displayed by highly metastatic cells and it has been shown that inhibition of N-glycan processing restrains cell proliferation and induces cell death via apoptosis. However, the mechanisms by which different N-glycosylation states may regulate cell viability and growth are not understood. Since malignant cells express high levels of intracellular glutathione (GSH) and a reduction of intracellular GSH induces cell death via apoptosis, we investigated whether GSH was involved in the induction of apoptosis by removal of cell surface N-glycans. We found that removal of N-glycans from cell surface proteins by treating the rhabdomyosarcoma cell line S4MH with tunicamycin or N-glycosidase resulted in a reduction in intracellular GSH content and cell death via apoptosis. Moreover, GSH depletion caused by the specific inhibitor of GSH synthesis BSO induced apoptosis in S4MH cells. This data indicates that adequate N-glycosylation of cell surface glycoproteins is required for maintenance of intracellular GSH levels that are necessary for cell survival and proliferation.     

Gamma-linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant α-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.     

Arsenic trioxide-induced apoptosis and its enhancement by buthionine sulfoximine in hepatocellular carcinoma cell lines

We treated four hepatocellular carcinoma cell lines, HLE, HLF, HuH7, and HepG2 with ATO and demonstrated that arsenic trioxide (ATO) at low doses (1--3 muM) induced a concentration-dependent suppression of cell growth in HLE, HLF, and HuH7. HLE cells underwent apoptosis at 2 microM ATO, which was executed by the activation of caspase-3 through the mitochondrial pathway mediated by caspase-8 activation and Bid truncation. When these cell lines were exposed to ATO in combination with l-S,R-buthionine sulfoximine (BSO) which inhibits GSH synthesis, a synergistic growth suppression was induced, even in HepG2 showing a lower sensitivity to ATO than other cell lines tested. The intracellular GSH levels after the treatment with ATO plus BSO were considerably decreased in HLE cells compared with those after the treatment with ATO or BSO alone. The production of reactive oxygen species (ROS) which was examined by 2' ,7' -dichlorodihydrofluorescein diacetate, increased significantly after the treatment with ATO plus BSO in HLE cells. These findings indicate that ATO at low concentrations induces growth inhibition and apoptosis, and furthermore that the ATO-BSO combination treatment enhances apoptosis through increased production of ROS in hepatocellular carcinoma cells.     

Neuroprotection against neuroblastoma cell death induced by depletion of mitochondrial glutathione

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Thymosin alpha1 suppresses proliferation and induces apoptosis in human leukemia cell lines

Thymosin alpha1 (Talpha1), a 28-amino acid peptide, is a well-known immune system enhancer for the treatment of various diseases. In the present investigation, the effects of Talpha1 on the proliferation and apoptosis of human leukemia cell lines (HL-60, K562 and K562/ADM) were studied. The proliferation was significantly depressed after 96 h of treatment with Talpha1, and obvious signs of apoptosis, i.e., cell morphology, nuclei condensation and Annexin V binding, were observed thereafter. Moreover, the up-regulation of Fas/Apol (CD95) and decrease in bcl-2 anti-apoptotic gene expression were observed in apoptotic cells. The expression and the function of P-glycoprotein (P-gp) can be slightly inhibited by Talpha1. It is noteworthy that K562 and K562/ADM were more sensitive than HL-60 cells when subjected to Talpha1. Furthermore, HepG-2, the human hepatoma cell line, displayed significant less sensitivity to Talpha1 than all the human leukemia cell lines. D-Tubocurarine (TUB), a nicotinic acetylcholine receptors (nAChRs) antagonist, significantly antagonized the inhibition effects induced by Talpha1, whereas atropine, a muscarinic acetylcholine receptor antagonist, did not exhibit such effects. All the results indicate that Talpha1 was able to significantly suppress proliferation and induce apoptosis in human leukemia cell lines.     

Induction of apoptosis in K562 cells by jolkinolide B

Jolkinolide B, a bioactive diterpene isolated from the roots of Euphorbia fischeriana Steud, has various biological and pharmacological properties. In this study, the cytotoxicity of highly purified jolkinolide B was tested in human chronic myeloid leukemia (K562) and 2 other cell lines (human esophageal carcinoma Eca-109 and human hepatoma HepG2). The results indicate a significant decrease in the proliferation of all the 3 cell lines when treated with jolkinolide B for 24 h; the IC50 value of cytotoxicity was 12.1 microg/mL (for K562 cells), >50.0 microg/mL (for HepG2 cells), and 23.7 microg/mL (for Eca-109 cells). Further study of K562 cells involving fluorescence and transmission electron microscopy revealed characteristic apoptotic features, such as cell shrinkage, membrane blebbing, loss of microvilli, and nuclear condensation. Agarose electrophoresis of genomic DNA showed a typical fragmentation pattern for apoptotic cells. A kinetic cell-cycle analysis demonstrated that the cell cycle was arrested in the G1 phase. All these results suggest that the anti-proliferation effect of jolkinolide B on K562 cells is achieved by arresting the cell cycle in the G1 phase and subsequently inducing apoptosis.     

Induction of apoptosis in arsenic trioxide-treated lung cancer A549 cells by buthionine sulfoximine

Arsenic trioxide (ATO) affects many biological processes such as cell proliferation, apoptosis, differentiation and angiogenesis. L-buthionine sulfoximine (BSO) is an inhibitor of GSH synthesis. We tested whether ATO reduced the viability of lung cancer A549 cells in vitro, and investigated the in vitro effect of the combination of ATO and BSO on cell viability in relation to apoptosis and the cell cycle. ATO caused a dose-dependant decrease of viability of A549 cells with an IC50 of more than 50 muM. Low doses of ATO or BSO (1~10 muM) alone did not induce cell death. However, combined treatment depleted GSH content and induced apoptosis, loss of mitochondrial transmembrane potential (DeltaPsi(m)) and cell cycle arrest in G2. Reactive oxygen species (ROS) increased or decreased depending on the concentration of ATO. In addition, BSO generally increased ROS in ATO-treated A549 cells. ROS levels were at least in part related to apoptosis in cells treated with ATO and/or BSO. In conclusion, we have demonstrated that A549 lung cells are very resistant to ATO, and that BSO synergizes with clinically achievable concentration of ATO. Our results suggest that combination treatment with ATO and BSO may be useful for treating lung cancer.     

A novel tubulin polymerization inhibitor,MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.     

Endogenous Interleukin 6 Conveys Resistance to cis-Diamminedichloroplatinum-Mediated Apoptosis of the K562 Human Leukemic Cell Line

Cisplatin is an effective chemotherapeutic agent that elicits its antineoplastic activity by binding to DNA and disrupting template functions. IL-6 is a cytokine which has been shown to play a central role in host immunological defense mechanisms. Although K562 leukemic cells have been shown to secrete IL-6, little is known of whether there exists a correlation between the expression of IL-6 and the resistance of these cells to anticancer chemotherapeutic agents. To determine the contribution of IL-6 to the regulation of cisplatin-induced apoptosis in K562 cells, we examined whether treatment of K562 cells and cisplatin-resistant K562 subclones with anti-IL-6 mAb enhances their sensitivity to cisplatin. The results show that cis-diamminedichloroplatinum (CDDP) resistance was overcome by treatment with nontoxic doses of CDDP in combination with anti-IL-6 mAb. When we tested if the synergistic effect of anti-IL-6 and cisplatin could restore the ability of K562 mutant cells to undergo apoptosis, we found the typical DNA laddering in these cells, even in the presence of a nontoxic dose of the drug. Treatment of cells with anti-IL-6 reduced the levels of glutathione. The current studies show that anti-IL-6 mAb sensitized CDDP-resistant K562 cells to CDDP by induction of apoptotic death and the reduction of glutathione levels might be implicated in the enhanced cytotoxicity observed.     

A critical role of glutathione in determining apoptosis sensitivity and resistance in leukemia cells

In chemosensitive leukemias and solid tumors, anticancer drugs have been shown to induce apoptosis. Deficiencies in the apoptotic pathways may lead to chemoresistance. Here we report that glutathione (GSH) plays a critical role in activation of apoptosis pathways by CD95 (APO-1/Fas) or anticancer drugs. Upon treatment with anticancer drugs or CD95 triggering, CD95-resistant or Bcl-x(L) overexpressing CEM cells were deficient in activation of apoptosis pathways. CD95-resistant and Bcl-x(L) overexpressing CEM cells exhibited higher intracellular GSH levels in comparison to parental cells. Downregulation of GSH by L-buthionine-(S,R)-sulfoxime (BSO), a specific inhibitor of glutathione synthesis, reversed deficiencies in activation of apoptosis pathways by anticancer drugs or CD95. Interestingly, downregulation of GSH by BSO increased CD95 DISC formation in type I cells. In hybrids of CD95-resistant cells with sensitive cells and hybrids of overexpressing Bcl-x(L) cells with sensitive cells, the phenotype of apoptosis resistance was dominant. Also, in these hybrids, downregulation of GSH reversed CD95- and chemoresistance. We conclude that dominant apoptosis resistance depends, at least in part, on intracellular GSH levels, which may affect apoptosis signaling at different compartments, for example, the death receptor or mitochondria.