Generation of active oxygen forms in rat thymocytes under action of hydrogen peroxide and fullerene C60

The dynamics of active oxygen forms (AOF) generation in rat thymocytes 50 min after treatment with 0.1 and 0.5 mM H2O2 was estimated with the use of fluorescent probe DCFDA. Both enhanced AOF generation, which was dependent on H2O2 concentration, and glutathione peroxidase and superoxide dismutase activation, followed by a decrease of thymocytes viability were demonstrated. Preincubation of cells with 10(-5) M fullerene C60 was shown not only to prevent H2O2--induced AOF generation but to increase viability of H2O2-treated thymocytes at more prolonged time period. The data obtained indicate to fullerene C60 ability to prevent oxidative stress in thymocytes.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells: role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway

Staphylococcus aureus infections can result in septic and toxic shock with depletion of immune cells and massive cytokine production. Recently, we showed that, in S. aureus-infected Jurkat T cells, alpha-toxin is the major mediator of caspase activation and apoptosis. Here, we investigated the mechanisms of cell death induced by alpha-toxin in peripheral blood mononuclear cells (MNC). We show that alpha-toxin is required and sufficient for S. aureus-induced cell death not only in transformed Jurkat T cells but also in MNC. Low alpha-toxin doses (3-30 ng ml-1) dose- and time-dependently induced apoptosis in both cell types, which was completely blocked by the caspase inhibitor zVAD-fmk. In Jurkat T cells and MNC, alpha-toxin induced the breakdown of the mitochondrial membrane potential and the intrinsic activation of caspase-3, -8 and -9. Interestingly, unlike in Jurkat T cells, apoptosis in MNC was additionally mediated by a caspase-9-independent component. MNC, but not Jurkat T cells, produced tumour necrosis factor (TNF)-alpha upon alpha-toxin stimulation. Blocking endogenous TNF-alpha with a TNF-alpha receptor antagonist partially decreased apoptosis in MNC. Our data therefore suggest that, whereas in Jurkat T cells apoptosis is solely mediated by the mitochondrial pathway, in MNC endogenous TNF-alpha and a death receptor-dependent pathway are also involved, which may contribute to depletion of immune cells during S. aureus infection.     

Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.     

The antioxidant 4b,5,9b,10-Tetrahydroindeno[1,2-b]indole inhibits apoptosis by preventing caspase activation following mitochondrial depolarization.

Oxidative stress appears to have a central role in the induction of apoptosis following the exposure of cells to a range of cytotoxic insults. The modulation of apoptosis by a diverse range of antioxidants has been reported in many systems. We demonstrate, for the first time, the anti-apoptotic properties of the antioxidant, 4b, 5,9b,10-tetrahydroindeno[1,2-b]indole (THII), in Jurkat T cells subjected to a number of cytotoxic insults. THII was found to inhibit the morphological features of apoptosis in cells treated with the cytotoxic agents camptothecin, actinomycin D and ultraviolet (UV) irradiation. However, THII was unable to inhibit apoptosis induced by anti-Fas IgM. Peroxide and superoxide anion production following UV treatment was monitored, and THII was found to only partially inhibit superoxide anion production. THII was unable to inhibit mitochondrial depolarization in UV, Camptothecin or anti-Fas-treated cells. Further downstream, THII exibited strong inhibition of caspase-3 activation in UV, but not in anti-Fas-treated cells. These results suggest that THII may exert its effects downstream of mitochondrial depolarization, but upstream of caspase-3 activation.     

Different roles of spermine in glucocorticoid- and Fas-induced apoptosis

Two experimental systems representative of the mitochondrial and death receptor apoptotic pathways are the dexamethasone-induced programmed cell death in mouse thymocytes and the antibody-mediated cross-ligation of the Fas receptor in the human leukemic T-cell line Jurkat, respectively. In both cell systems, caspase-9, -8, and -3 were activated upon induction of apoptosis and a sub-G(1) peak appeared as a sign of ongoing DNA fragmentation. Addition of 1 mM spermine together with dexamethasone inhibited caspase activation and the appearance of the sub-G(1) peak in mouse thymocytes. In contrast, Fas-induced cell death was totally unaffected by spermine addition. Spermine addition significantly elevated the spermine concentration in both thymocytes and Jurkat cells. Thus, spermine per se did not inhibit the caspases but rather their activation. The fact that spermine inhibited caspase activation only in the thymocytes implies that spermine inhibited dexamethasone-induced apoptosis upstream of caspase-9 activation.     

Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC₅₀ = ～80 μM), along with apoptotic sub-G₁ cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δψ_m) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G₁ cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δψ_m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c, causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.     

5-Fluorouracil enhances apoptosis sensitivity of T lymphocytes mediated by CD3 epsilon

Previous studies by our laboratory have reported that the T cell receptor (TCR) TCR/CD3 complex could mediate activation as well as apoptosis of T lymphocytes. Two tyrosine residues in the ITAM (immuno-receptor tyrosine-based activation motifs) of CD3 epsilon were required for apoptosis signalling of Jurkat T lymphocytes. Stable cell lines TJK and T3JK produced from CD8(-) Jurkat T lymphocytes by transfection with wild-type and mutant CD8 epsilon (fusion of the extracellular and transmembrane domains of human CD8 alpha to the intracellular domain of mouse CD3 epsilon), were used with CD8(-) Jurkat T lymphocytes for studying the role of single intact CD3 epsilon. 5-Fluorouracil (5-FU), a chemotherapeutic drug can induce cell death of many tumour cell lines. In the present experiments, we examined the expression of caspase-3, p53 and Bid in the three cell lines induced by 5-FU and/or anti-CD8 antibody. We found high expression of p53 during activation-induced cell death of TJK cells mediated by anti-CD8 antibody and apoptosis of TJK and T3JK induced by 5-FU, implicating p53 involvement in apoptosis of leukemia cells induced by anti-CD8 antibody and 5-FU. We also detected the active form of caspase-3 and Bid in apoptotic leukemia cells after treatment with 5-FU and/or anti-CD8 antibody, indicating that the drug and antibody induced cell death through caspase-3 and the signal pathway may involve the Bcl-2 protein family. Our results showed that combined treatment with 5-FU and anti-CD8 antibody could enhance the rate of apoptosis induced by 5-FU or anti-CD8 antibody through increased expression of p53 and by promoting activation of caspase-3 and Bid. This suggests that the combination of 5-FU and anti-CD8 antibody may play an important role in inducing apoptosis of leukemia cells.     

Hydroxylation at C4' or C6 is essential for apoptosis-inducing activity of flavanone through activation of the caspase-3 cascade and production of reactive oxygen species

Previous studies demonstrated that hydroxyl groups play important roles in the antioxidative activities of flavonoids; however, the importance of structurally related hydroxylation in their apoptosis-inducing activities is still undefined. In the present study, flavanone with hydroxylation at C4' and C6 had a significant cytotoxic effect in human leukemia HL-60 cells accompanied by the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, characteristics of apoptosis. The replacement of a hydroxyl group (OH) by a methoxyl (OCH3) group at C4' or C6 attenuated the apoptotic effect in cells, and there was no significant cytotocity of flavanone or flavanone with OH or OCH3 in C7-treated HL-60 cells. Induction of enzyme activity of caspase-3 and -9, but not caspase-1 and -8, accompanied by release of cytocrome C from mitochondria to cytosol and the appearance of cleaved of PARP (85 kDa), D4-GDI (23 kDa), and caspase-3 (p17/p15) fragments, was identified in 4'-OH- or 6-OH- flavanone-treated HL-60 cells. Caspase-3 and -9 inhibitors Ac-DEVD-FMK and Ac-LEHD-FMK, but not caspase-1 and -8 inhibitors Ac-YVAD-FMK and Ac-LETD-FMK, attenuated 4'-OH- or 6-OH-flavanone-induced cell death. And, inhibition of capsase-9 activity by Ac-LEHD-FMK suppresses caspase-3 protein procession induced by 4'-OH- and 6-OH-flavanone, indicative of caspase-9 activation locating upstream of caspase-3. A decrease in the antiapoptotic protein Mcl-1 and increases in the pro-apoptotic proteins Bax and Bad were found in 4'-OH- or 6-OH-flavanone-treated HL-60 cells. Induction of endogenous ROS production was detected in 4'-OH- or 6-OH-flavanone-treated HL-60 cells by the DCHF-DA assay. Antioxidants such as N-acetylcysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and allopurinol (ALL), but not pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited 4'-OH- or 6-OH-flavanone-induced ROS production, with blocking of the apoptosis induced by 4'-OH- or 6-OH-flavanone. The apoptosis-inducing activity of 4'-OH- or 6-OH-flavanone was also observed in another leukemia cell line (Jurkat), but was not found in mature monocytic cells (THP-1) and normal human polymorphonuclear neutrophils (PMNs). This suggests that hydroxylation at C4' or C6 is important to the apoptosis-inducing activities of flavanone through ROS production, and that activation of the caspase-3 cascade, downstream of caspase-9 activation, is involved.     

A Novel Apoptosis-like Pathway, Independent of Mitochondria and Caspases, Induced by Curcumin in Human Lymphoblastoid T (Jurkat) Cells

We have shown previously [E. Sikora, A. Bielak-Zmijewska, K. Piwocka, J. Skierski, and E. Radziszewska (1997) Biochem. Pharmacol. 54, 899-907] that curcumin prevents formation of oligonucleosomal DNA fragmentation in rat thymocytes and human leukemic T lymphocytes (Jurkat cells) induced to undergo apoptosis. In this paper we show that 50 microM curcumin by itself induces cell death in Jurkat cells, but its symptoms differ from those observed after a short ultraviolet (uv) irradiation. Ultraviolet-irradiated Jurkat cells displayed typical symptoms of apoptosis: morphological changes, internucleosomal and high-molecular-weight DNA fragmentation, formation of sub-G1 fractions in DNA content frequency histograms, and dissipation of the mitochondrial transmembrane electric potential (Delta psi). In contrast, curcumin-treated Jurkat cells exhibited DNA splitting into high-, but not low-, molecular-weight fragments. These cells retained their high mitochondrial Delta psi, and the content of Ca2+ in endoplasmic reticulum stores remained at the level typical for untreated cells. The frequency of opening of the mitochondrial permeability transition pores in curcumin-treated cells was decreased compared to the controls, whereas uv irradiation made these pores completely open. Curcumin did not produce any change in the activity of caspase-3, whereas uv irradiation considerably activated this protease. The morphology of curcumin-treated cells displayed chromatin condensation, which was insensitive to the caspase inhibitor z-VAD-fmk, but no formation of typical apoptotic bodies, as was the case after uv irradiation. In contrast to uv-irradiated cells, curcumin-treated Jurkat cells considerably increased the level of Bcl-2. It is concluded that the programmed cell death induced by curcumin in Jurkat cells differs from "classical" by the lack of mitochondrial depolarization and of the involvement of caspases.     

Inhibition of Nitric-Oxide-Mediated Apoptosis in Jurkat Leukemia Cells despite Cytochrome c Release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

Pramanicin induces apoptosis in Jurkat leukemia cells: A role for JNK,p38 and caspase activation

Pramanicin is a novel anti-fungal drug with a wide range of potential application against human diseases. It has been previously shown that pramanicin induces cell death and increases calcium levels in vascular endothelial cells. In the present study, we showed that pramanicin induced apoptosis in Jurkat T leukemia cells in a dose- and time-dependent manner. Our data reveal that pramanicin induced the release of cytochrome c and caspase-9 and caspase-3 activation, as evidenced by detection of active caspase fragments and fluorometric caspase assays. Pramanicin also activated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK 1/2) with different time and dose kinetics. Treatment of cells with specific MAP kinase and caspase inhibitors further confirmed the mechanistic involvement of these signalling cascades in pramanicin-induced apoptosis. JNK and p38 pathways acted as pro-apoptotic signalling pathways in pramanicin-induced apoptosis, in which they regulated release of cytochrome c and caspase activation. In contrast the ERK 1/2 pathway exerted a protective effect through inhibition of cytochrome c leakage from mitochondria and caspase activation, which were only observed when lower concentrations of pramanicin were used as apoptosis-inducing agent and which were masked by the intense apoptosis induction by higher concentrations of pramanicin. These results suggest pramanicin as a potential apoptosis-inducing small molecule, which acts through a well-defined JNK- and p38-dependent apoptosis signalling pathway in Jurkat T leukemia cells.     

Glucocorticoids engage different signal transduction pathways to induce apoptosis in thymocytes and mature T cells

Glucocorticoids (GC) induce apoptosis in a variety of cells, but their exact mode of action is controversial. Although initiation relies on the GC receptor (GR) and de novo gene expression, the effector phase differs among cell types. Proteasomal degradation as well as caspase-3, - 8, and -9 activity are essential for GC-induced apoptosis in murine thymocytes, but the same enzymes are dispensable in splenic T cells. Live imaging by confocal microscopy revealed that lysosomal cathepsin B, an unrecognized component of this pathway to date, becomes rapidly activated in thymocytes after GC exposure. This is followed by leakage of cathepsin B into the cytosol, nuclear condensation, and processing of caspase-8 and -3. According to our model, activation of caspase-3 by caspase-9 in thymocytes occurs both directly as well as indirectly via a lysosomal amplification loop. Interestingly, acute T lymphoblastic leukemia cells depend on caspase activity to undergo GC-induced cell death similar to thymocytes. Collectively, the apoptotic program induced by GCs comprises cell type-specific as well as common features.     

Pre-clinical antitumour evaluation of Biphosphinic Palladacycle Complex in human leukaemia cells

Previous studies reported by our group have introduced a new antitumoural drug called Biphosphinic Palladacycle Complex (BPC). In this paper we show that BPC causes apoptosis in leukaemia cells (HL60 and Jurkat), but not in normal human lymphocytes. IC₅₀ values obtained for both cell lines using the MTT and trypan blue exclusion assays 5 h after BPC treatment were lower than 8.0 μM. Using metachromatic fluorophore, acridine orange, we observed that BPC elicited lysosomal rupture of leukaemic cells. Furthermore, BPC triggered caspase-3 and caspase-6 activation and apoptosis in cell lines, inducing chromatin condensation, apoptotic bodies, and DNA fragmentation. Interestingly, the lysosomal cathepsin B inhibitor CA074 markedly decreased BPC-induced caspase-3 and caspase-6 activation as well as cell death. Lysosomal BPC-induced membrane destabilisation was not dependent on reactive oxygen species generation, which was consistent with the absence of cellular HL60 and Jurkat membrane lipid peroxidation. We conclude that, following BPC treatment, lysosomal membrane rupture precedes cell death and the apoptotic signalling pathway is initiated by the release of cathepsin B in the cytoplasm of leukaemia cells. As no toxic effects for human lymphocytes were observed, we suggest that BPC is more selective for transformed cells, mainly due to their exacerbated lysosome expression.     

Apoptosis induced by protein phosphatase 2A (PP2A) inhibition in T leukemia cells is negatively regulated by PP2A-associated p38 mitogen-activated protein kinase

Serine/threonine phosphatase regulation of phosphorylation-mediated intracellular signaling controls a number of important processes in mammalian cells. In this study, we show that constitutively active protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase, is essential for T leukemia cell survival. Jurkat and CCRF-CEM T leukemia cells treated with the PP2A-selective inhibitor okadaic acid (OA) showed a dose- and time-dependent induction of apoptosis, as indicated by loss of mitochondrial transmembrane potential (delta psi(m)), cleavage-induced activation of caspase-3, -8, and -9, and DNA fragmentation. In addition, caspase-8 or caspase-9 inhibition with z-IETD-fmk or z-LEHD-fmk, respectively, largely prevented OA-induced apoptosis. Although OA treatment did not affect constitutive Bcl-2 expression, overexpression of Bcl-2 prevented both OA-induced DNA fragmentation and dissipation of delta psi(m). Furthermore, inhibition of caspase-3, -8, or -9 partially protected against OA-induced loss of delta psi(m). In addition, caspase-9 and caspase-3 inhibition largely prevented procaspase-3 and procaspase-8 cleavage, respectively, while caspase-8 inhibition partially interfered with procaspase-9 cleavage in OA-treated T leukemia cells. Thus, PP2A inhibition triggered the intrinsic pathway of apoptosis, which was enhanced by a mitochondrial feedback amplification loop. PP2A has also been implicated in the regulation of p38 mitogen-activated protein kinase (MAPK). Co-immunoprecipitation analysis revealed a physical association between the catalytic subunit of PP2A and p38 MAPK in T leukemia cells. Moreover, OA treatment caused p38 MAPK to be phosphorylated in a dose- and time-dependent fashion, indicating that PP2A prevented p38 MAPK activation. Although p38 MAPK activation usually promotes apoptosis, pharmacologic inhibition of p38 MAPK exacerbated OA-induced DNA fragmentation and loss of delta psi(m) in T leukemia cells, suggesting that, in this instance, the p38 MAPK signaling pathway promoted cell survival. Collectively, these findings indicate that PP2A and p38 MAPK have coordinate effects on signaling pathways that regulate the survival of T leukemia cells.     

Calpain-dependent calpastatin cleavage regulates caspase-3 activation during apoptosis of Jurkat T cells induced by Entamoeba histolytica

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.     

A novel 2,6-diisopropylphenyl-docosahexaenoamide conjugate induces apoptosis in T cell acute lymphoblastic leukemia cell lines

We have previously characterized the effects of 2,6-diisopropylphenyl–docosahexaenoamide (DIP–DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP–DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP–DHA resulted in significantly greater inhibition of proliferation and induction of apoptosis than that of parent compounds, 2,6-diisopropylphenol (DIP) or docosahexaenoate (DHA). Treatment of the cells with DIP–DHA resulted in increased activation of caspase-3, and caspase-7. Furthermore, induction of apoptosis in both cell lines was reversed in the presence of a caspase family inhibitor. Treatment with DIP–DHA reduced mitochondrial membrane potential. These observations suggest that the effects are driven by intrinsic apoptotic pathways. DIP–DHA treatment also downregulated surface CXCR4 expression, an important chemokine receptor involved in cancer metastasis that is highly expressed in both CEM and Jurkat cells. In conclusion, our data suggest that the DIP–DHA conjugate exhibits significantly more potent effects on CEM and Jurkat cells than that of DIP or DHA alone. These conjugates have potential use for treatment of patients with T cell acute lymphoblastic leukemia.     

Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis

The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death-inducing stimulus. We demonstrate that the activation of caspase-3, in Jurkat cells stimulated to undergo apoptosis by a Fas-independent pathway, is catalyzed by caspase-6. Caspase-6 was found to co-purify with caspase-3 as part of a multiprotein activation complex from extracts of camptothecin-treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase-3 could be demonstrated by co-immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase-3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP-dependent 'foldase' activity of Hsp60 improves the vulnerability of pro-caspase-3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.