Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line

This study delineates the molecular mechanism underlying psychosine-induced oligodendroglial cell death. An immortalized human oligodendroglial cell line, MO3.13, was treated with exogenous psychosine (beta-galactosylsphingosine), a toxic metabolite that accumulates in the tissues of patients with Krabbe's disease. The mode of cell death induced by psychosine was found to be apoptotic, as revealed by different apoptotic markers viz., TUNEL, DNA fragmentation and caspase cleavage/activation. The action of psychosine was redox sensitive, as measured by changes in mitochondrial membrane potential (psidelta), and this effect of psychosine could be reversed by pre-treatment with the antioxidant molecules N-acetyl-l-cysteine or pro-cysteine. Psychosine directly affects the mitochondria as revealed by the activation of caspase 9 but not caspase 8. Up-regulation of the c-jun/c-jun N-terminal kinase pathway by psychosine leads to the induction of AP-1 and, at the same time, psychosine also down-regulates the lipopolysaccharide-induced NF-kappaB transactivation. These observations indicate that the mechanism of action of psychosine is, through the up-regulation of AP-1, a pro-apoptotic pathway as well as, through the down-regulation of the NF-kappaB pathway, an antiapoptotic pathway.     

Micronucleus induction and reproductive death in a human cell line exposed to low-energy argon beam

Little is known about the action of charged particles of very high linear energy transfer (LET) on human cells and, in particular, the relationship between DNA damage and reproductive death. The aim of this study was to measure the biological efficiency of a low-energy argon beam (E=7.1 MeV/nucleon, LET= 1590 keV/µm) produced at GSI, Darmstadt, on a human melanoma cell line (CAL4) established in our Institute. Two different methods were used: the micronucleus (MN) test and the colonyforming assay. The MN test, using the cytochalasin-block method, is a measure of genotoxic damage. MN are scored in binucleate cells (BNC) and are formed from acentric fragments or whole chromosomes that have not been incorporated into daughter nuclei at mitosis. The colonyforming assay quantifies reproductive death. Parallel experiments were run with cobalt gamma-rays for comparison. After Co irradiation, the MN-free BNC dose-response curve coincided with that of the loss of colony-forming ability, suggesting the potential of the former as a predictive test of cell killing. After Ar irradiation, there was a dissociation between the two effects, especially at high doses: cell death was greater than the frequency of BNC with MN. The inactivation cross-section was 74 µm²; it was 39 µm² for MN yield. Therefore, the relative biological effectiveness (RBE) was higher for cell killing than for MN yield (0.8 and 0.5, respectively, at a Co dose of 3 Gy). The total MN count in BNC followed the same pattern of response as the fraction of BNC with MN. However, multiple (>2) MN in BNC were more frequently observed after low-dose Ar irradiation than after gamma-ray exposure (RBE>1). Moreover, the frequency of multiple MN induction exceeded that expected from a Poisson distribution attribution at all dose levels of Ar irradiation. These results indicate that (1) cell killing after 7.1-MeV Ar beam irradiation is less effective than after Co irradiation at an equivalent average energy deposition; (2) unlike gamma-rays, Ar particles are more efficient at cell killing than in producing MN; (3) the frequent scoring of multiple MN suggests the production of multiple damage sites, even at low fluences of Ar particles.     

Vaticanol C-induced cell death is associated with inhibition of pro-survival signaling in HL60 human leukemia cell line

Recently, we found that vaticanol C (a resveratrol tetramer), which was isolated from stem bark of Dipterocarpaceae, exhibited growth suppression and induction of apoptosis via the loss of mitochondrial membrane potential and consequent caspases activation. The detailed mechanisms are not clearly understood. We decided to attempt to gain further insight into the mechanisms underlying vaticanol C-induced apoptosis in HL-60 cells. Treatment of HL-60 cells with vaticanol C was found to cause a marked decrease in the level of phosphorylated extracellular signal-regulated kinase (ERK) concurrent with inhibited phosphorylation of its upstream kinase mitogen-activates protein kinase kinase (MEK). Moreover, exposure to vaticanol C led to a significant reduction in the level of phosphorylated Akt. Thus, vaticanol C induced inhibition of both ERK and Akt phosphorylation, resulting in reduced phosphorylation of Bad. These results suggest that vaticanol C might induce apoptosis via a mechanism involving activation of Bad through disruption of pro-survival signaling pathways.     

Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 M induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 M of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (_m) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC₆(3), a cationic lipophilic dye into mitochondria indicating the disruption of _m. This drop in _m was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation excess ROS production GSH depletion oxidative stress disruption of _m release of cytochrome C and other apoptosis related proteins to cytosol apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.     

A promising action of riboflavin as a mediator of leukaemia cell death

Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at μM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous disease.     

Aeromonas hydrophila vacuolating activity in the Caco-2 human enterocyte cell line as a putative virulence factor

A non-enterotoxigenic strain of Aeromonas hydrophila isolated from diarrheic stools of an 8-month-old child was found to cause vacuolation in Caco-2 cells. The vacuoles became prominent 60 min after addition of the bacterial culture to the cell monolayers and, after 120 min, a complete disruption of the monolayers was observed. Cell vacuolation was not detected when Caco-2 monolayers were tested with sterile filtrates of overnight cultures of the A. hydrophila vacuolating strain AH14846. This strain produced a diffuse adherence pattern in Caco-2 cell monolayers, but did not produce detectable cytotonic enterotoxin in the suckling mouse test and only produced small quantities of aerolysin. By demonstrating the ability to induce vacuolation in mammalian cells of enterocytic lineage, the current study raises the possibility that such activity might contribute to gastrointestinal symptoms in infections involving Aeromonas strains which do not express well-established enterotoxins.     

2'-Deoxyadenosine causes apoptotic cell death in a human colon carcinoma cell line

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.     

A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line

Background

Although chronic myeloid leukemia (CML) treatment has improved since the introduction of imatinib mesylate (IM), cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC) family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR) cells.

Results

The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of leucine-rich PPR motif-containing (LRPPRC) protein, minichromosome maintenance complex component 7 (MCM7) and ATP-binding cassette sub-family B (MDR/TAP) member 1 (ABCB1) were capable of defining samples from CML patients as responsive or resistant to therapy.

Conclusions

Through the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.     

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 microM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching. From these identifications a CHO 2-D reference map and annotated database have been established. Metabolic labelling and subsequent autoradiography showed the induction of a number of cellular proteins in response to the media additives butyrate and zinc sulphate. These were identified as GRP75, enolase and thioredoxin. The chaperone proteins GRP78, HSP90, GRP94 and HSP70 were not up-regulated under these conditions.     

Ca-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca^2 + across the OMM and because Ca^2 + has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca^2 + levels ([Ca^2 +]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca^2 + and induce VDAC1 over-expression. Direct elevation of [Ca^2 +]i by the Ca^2 +-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca^2 +]i using the cell-permeable Ca^2 +-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca^2 +]i levels and apoptosis induction, as induced by H₂O₂ or As₂O₃, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca^2 +-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca^2 +]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca^2 + promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau     

Ouabain activates signaling pathways associated with cell death in human neuroblastoma

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na⁺,K⁺-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 μM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca²⁺] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 μM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).     

Ouabain activates signaling pathways associated with cell death in human neuroblastoma

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na(+),K(+)-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 microM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca(2+)] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 microM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).     

Anti-IgM antibody-induced cell death in a human B lymphoma cell line, B104, represents a novel programmed cell death.

We investigated the mechanisms of anti-IgM antibody-induced cell death in a recently established human surface IgM+ IgD+ B lymphoma cell line, B104, the growth of which is irreversibly inhibited by anti-IgM antibody but not by anti-IgD antibody, and compared it with the cell death of T cells via TCR/CD3 complex and with the cell death of a murine anti-IgM antibody-sensitive B lymphoma cell line, WEHI-231. The rapid time course of B104 cell death and its requirements for de novo macromolecular synthesis and Ca2+ influx suggest that anti-IgM antibody-induced B104 cell death is an active Ca(2+)-dependent programmed cell death. Moreover, cyclosporin A rescued B104 cells from this lethal signal, via surface IgM, suggesting that the intracellular mechanisms involved are quite similar to those of T cell death. DNA fragmentation, which has been reported in TCR/CD3 complex-mediated T cell death, apoptosis, was not involved in the B104 cell death process, but the possible involvement of DNA single-strand breaks was suggested. Observations under light microscopy and transmission electron microscopy indicated that the morphologic features of dying B104 cells resembled necrosis rather than apoptosis. B104 cell death was shown to be quite distinct from that of WEHI-231 in cell death kinetics, the mode of cell death, and the response to cyclosporin A. These data collectively indicate that the death of B104 cells resulting from surface IgM cross-linking represents a hitherto undefined mode of programmed cell death.     

The induction of aneuploidy by nitrogen mustard in a human lymphoblastoid cell line

We report that the presence of an extra Y chromosome can be used as a marker for the induction of aneuploidy (mitotic non-disjunction) in a human lymphoblastoid cell line. This endpoint is easily visualized in metaphase chromosome preparations after staining with quinacrine mustard. The induction of cells with two Y chromosomes by nitrogen mustard (NM) was examined. Exposure to 150 ng/ml nitrogen mustard induced a 6-fold increase in aneuploid frequency relative to untreated control levels; maximal induction of aneuploidy was observed 2 days after treatment. Lower concentrations of nitrogen mustard (36 and 75 ng/ml) induced smaller increases in aneuploid frequency, with maximal induction observed 1 day after treatment. This system has the potential to be used as an assay for the induction of aneuploidy in cultured human cells.