Abstract
Recently, we reported the induction of a programmed cell death (PCD) in bloodstream forms of Trypanosoma brucei by prostaglandin D2 (PGD2). As this prostanoid is readily metabolized in the presence of albumin, we were prompted to investigate if PGD2 metabolites rather than PGD2 itself are responsible for the observed PCD. In fact, J series metabolites, especially PGJ2 and Δ12PGJ2, were able to induce PCD more efficiently than PGD2. However, the stable PGD2 analog 17phenyl-trinor-PGD2 led to the same phenotype as the natural PGD2, indicating that the latter induces PCD as well. Interestingly, the intracellular reactive oxygen species (ROS) level increased significantly under J series metabolites treatment and, incubation with N-acetyl-L-cysteine or glutathione reduced ROS production and cell death significantly. We conclude that PGJ2 and Δ12PGJ2 formation within the serum represents a mechanism to amplify PGD2-induced PCD in trypanosomes via ROS production.
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Introduction
The protozoan hemoflagellate Trypanosoma brucei is the causative agent of sleeping sickness in humans and of nagana in domestic animals. This unicellular eucaryote has a complex life cycle involving several morphologically and biochemically distinguishable stages during its development in the mammalian host (bloodstream form) and the tsetse fly vector (procyclic form).1, 2 Trypanosomes evade the host immune system by a combination of antigenic variation3 and immune suppression.4, 5 The appearance of parasitic waves in blood during the course of infection is attributed to the action of specific antibodies. However, this behavior was also observed in vitro if culture medium was regularly replaced by fresh medium.6 Thus, this parasitemia is controlled by the immune system of the host and is also regulated by the parasite itself.7
Untreated trypanosome infections are lethal. Patients with advanced sleeping sickness show an elevated prostaglandin (PG) level in their cerebrospinal fluid.8 PGs are produced by many cell types in response to a variety of stimuli.9, 10 They are synthesized from arachidonic acid via an unstable endoperoxide intermediate by a cyclooxygenase and are eventually converted by specific synthases to one of several related products, such as PGD2, PGE2 and PGF2α. PGs have been shown to play a prominent role in inflammatory processes, regulation of vascular tone, infertility, pain, fever, sleep, apoptosis and others.11, 12, 13, 14, 15, 16 The PG production is not restricted to mammals but has also been described in parasites such as cestodes, trematodes, nematodes and protozoa.17, 18, 19, 20 In Schistosoma mansoni, the PG production has been associated with the transformation of cercaria into schistosomules,21 whereas in Amoeba proteus, its synthesis plays a role during phagocytosis, eliciting vacuole formation.22 In the bloodstream form of T. brucei, an apoptosis-like programmed cell death (PCD) is induced by prostaglandin D2 (PGD2).23 PGD2 is the major prostanoid produced and secreted by the stumpy bloodstream form of T. brucei,24 which was shown to be more sensitive to this compound than the slender bloodstream form.23 In this sense, the physiological function of PGD2 in trypanosomes can be related to the population density regulation by PCD induction. However, it has already been shown that PGD2 undergoes dehydration in vivo and in vitro to additional, biologically active PGs of the J series.25, 26 Metabolites of the J series have their own unique spectrum of biological activities, including inhibition of cell cycle progression, suppression of viral replication, induction of heat-shock proteins, stimulation of osteogenesis and induction of apoptosis in different cell types.26, 27, 28 In this study, we analyzed the PGD2 metabolism under in vitro conditions and evaluated the effect of the produced metabolites on the bloodstream forms of T. brucei.
Results
Separation and identification of PGD2 metabolites in cultures
PGD2 is known to be spontaneously converted in blood and albumin-containing media to different metabolites, especially to the J derivatives (Figure 1).25, 26, 29, 30 In order to identify the respective metabolites produced under our experimental conditions, PGs were analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS) in the negative-ion mode. PGD2 was metabolized to derivatives of the J series PGJ2, Δ12PGJ2 and 15deoxy-PGJ2 and derivatives of the D series 15keto-PGD2 and 15deoxy-PGD2, as detected by their comigration with authentic and commercially available standards and by their ionic masses (m/z). Figure 2a shows the extracted ion chromatograms (EICs) of the single charged molecular ion m/z 351.1, [M-H]− of the PGs. PGD2 could be detected by the formation of an intense peak eluting after 15.8 min in the 0 h sample, which strongly decreased after 20 h and was practically absent after 40 h. Furthermore, the formation of 15k-PGD2 could be observed by the detection of an additional peak in the m/z 351.1 ion trace (14.2 min) after 20 and 40 h (Figure 2a). PGJ2, Δ12PGJ2 and 15d-PGD2 were detected by EIC of the single charged molecular ion m/z 333.0, [M-H]− (Figure 2b). The formation of PGJ2 and 15d-PGD2 was detected after 20 h incubation, which further increased after 40 h incubation. In addition, the formation of Δ12PGJ2 (16.9 min) was observed only after 40 h incubation (Figure 2b). Finally, the PGD2 metabolite 15d-PGJ2 was detected as a peak eluting after 24 min in both the 20 and 40 h samples, as monitored by the EIC of the single charged molecular ion m/z 315.1 (Figure 2c).
Inhibition of cellular proliferation and induction of morphological changes by PGs of the J2 series
Following identification of PGD2 metabolites in culture media, we investigated their effects on cell growth of T. brucei. In addition, we used a stable PGD2 analog, 17phenyl-trinor-prostaglandin D2 (17phenyl-PGD2), in order to explore if PGD2 itself is able to induce PCD.23 Using a [3H]thymidine incorporation assay, we found that PGs of the J series, PGJ2 and Δ12PGJ2, strongly reduced cellular proliferation of T. brucei at concentrations significantly lower than those required for PGD2 (5 μM). 15d-PGJ2 showed similar effects as PGD2 in the inhibition of proliferation, whereas metabolites of the D series, 15k-PGD2 and 15d-PGD2, remained ineffective (Figure 3). The IC50 for each metabolite was determined after 24 h incubation of trypanosomes in culture media. As shown in Table 1, metabolites of the D series were cytotoxic only at concentrations higher than 30 μM, suggesting that they do not play an important role in the PGD2-induced cell death. In contrast, J series metabolites have similar or lower IC50 than that for PGD2 (Table 1). In addition, 17phenyl-PGD2 inhibited proliferation with an IC50 of 7.7 μM (i.e. at a concentration two-fold higher than that for PGD2), demonstrating that PGD2 itself is able to inhibit cellular growth. On the other hand, the results suggest that part of the PGD2 effects depend on the formation of J series metabolites.
Electron microscopic studies of the ultrastructure of J series metabolites-treated cells showed that these PGs induced typical morphological changes of PCD. After an incubation time of 20 h, control cells show nuclei with a prominent central nucleolus and equally distributed chromatin, a not dilated mitochondrion and characteristic organelles (Figure 4a–c). In contrast, most of the treated cells displayed a fragmented nucleus with condensed chromatin (Figure 4d–g), a swollen mitochondrion (Figure 4k, m and n) and an increase of lysosomal structures (Figure 4g–j). Additionally, we observed a dilated flagellar pocket and the appearance of two or more flagellae in the same flagellar pocket (Figure 4o and p).
Analysis of the mitochondrial membrane potential and the DNA content
PGD2-induced cell death of T. brucei includes a set of different cellular changes such as loss of mitochondrial membrane potential (Ψm), DNA degradation and phosphatidylserine exposure in the outer leaflet. To investigate these parameters individually, exponentially growing cells were treated for 24 h with either J series or D series metabolites before they were sorted according to size and fluorescent label using a FACScalibur® cell sorter (BD Biosciences). Using propidium iodide staining as a control for necrosis, we could never detect a significant staining (data not shown). As shown in Figure 5a, treated cells were generally smaller in size than control cells. In addition, cell death was associated with a loss of the Ψm, as reflected by a leftward shift of the histograms obtained using tetramethylrhodamine (TMRE) staining (Figure 5b). After 24 h, 24.5% of PGJ2-treated cells had a low Ψm, as compared with 6.1% of control cells. Cells treated with Δ12PGJ2 and 15d-PGJ2 showed 18.3 and 12.8% Ψm disruption, respectively. Treatment of cells with 17phenyl-PGD2 depolarized the mitochondrial membrane, which is consistent with the results obtained after PGD2 treatment.23 Control experiments using valinomycin completely abolished the Ψm (Figure 5b).
We also observed that cell death induced by J series metabolites was associated with a loss of DNA content. As shown in Figure 5c, a significant percentage of the nuclei of treated cells had a low DNA content: 47.1% for PGJ2- and 14.5% for Δ12PGJ2-treated cells versus 3.9% for control cells. Δ12PGJ2 treatment increased the percentage of nuclei with low DNA content to 36.9% after 32 h (data not shown). Interestingly, 15d-PGJ2 was not able to induce DNA degradation, even after 32 h treatment with concentrations up to 5 μM. However, when this experiment was carried out with cells of the stationary phase, which had been shown to be more sensitive to the action of PGs than logarithmically growing cells,23 DNA degradation reached 16.2 versus 2.5% for control cells after only 4 h of incubation with 15d-PGJ2.
In addition, cell cycle analysis of treated cells clearly indicated an increase of cells in the G1 phase, which was paralleled by a decrease of cells remaining in the G2 and S phases (Figure 5d). After a 32 h treatment, the level of cells in the G1 phase was about 20% increased as compared with control cells. The cell cycle arrest induced by 15d-PGJ2 was rather moderate, with only a 10% difference as compared with cells incubated under control conditions, which did not increase further with time.
Detection of DNA disruption following J series PG treatment
In another set of experiments, we examined the incorporation of deoxyuridinetriphosphate (dUTP) using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay to investigate if J series metabolites are able to induce DNA degradation. This assay was performed using cells from the exponential growing phase after 24 h of incubation with the respective PGs. Figure 6 shows the incorporation of dUTP into nuclei of control and treated cells. Only samples that were treated with PGJ2, Δ12PGJ2, or 17phenyl-PGD2 showed a nuclear staining, which was not seen in control cell and scarcely observed in cells treated with 15d-PGJ2 (data not shown). Quantification was visually monitored by counting of 100 labeled and unlabeled cells each in three independent experiments. After 24 h, PGJ2-, Δ12PGJ2- and 17phenyl-PGD2-treated cells showed 40, 21 and 39% positive staining, respectively (Figure 6). These results are consistent with those obtained by fluorescence-activated cell sorting (FACS) analysis.
Detection of phosphatidylserine exposure after treatment with PGs of the J series
One of the first phenotypic changes associated to PCD is the exposition of phosphatidylserine residues on the surface of the plasma membrane.31 In order to assay this parameter and, if observed, to confirm that it is an early event in T. brucei PCD that requires protein biosynthesis, we used cells from the stationary phase (i.e. stumpy cells) as they are more sensitive than dividing parasites.
As shown in Figure 7a, J series metabolites were able to induce phosphatidylserine exposure after only a few hours of treatment. This phenotype was prevented by treatment with cycloheximide before the incubation with PGs (Figure 7b). Likewise, cycloheximide delayed the cell death. The results indicate that phosphatidylserine exposure and the subsequent cell death caused by the administered PGs are processes that depend on protein biosynthesis.
Taken together, all these results indicate that the J series metabolites, mainly PGJ2, are able to induce cell death more rapidly and at lower concentrations than their precursor PGD2.
Measurement of reactive oxygen species after treatment with PGD2 and their active metabolites
Previous studies have demonstrated that PGs are potential inducers of intracellular stress via production of reactive oxygen species (ROS).32, 33 In order to investigate if this phenomenon is involved in the PCD induced by PGD2 metabolites in T. brucei, we used the sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). This non-fluorescent dye diffuses across cell membranes and is hydrolyzed to dichlorohydrofluorescein (DCFH) intracellularly. In the presence of ROS, DCFH is rapidly oxidized to highly fluorescent dichlorofluorescein. For this assay, we used cells from the stationary phase (after about 40 h), again because they are more sensitive to the apoptosis-like effects of PGs than the slender form of parasites.23 As shown in Figure 8, treatment of the bloodstream forms of trypanosomes with PGJ2 for 2 h evoked an abrupt ROS generation. This effect was also induced by Δ12PGJ2, 15d-PGJ2 and 17-phenyl-PGD2, although after a much longer incubation time. Furthermore, ROS production induced either by PGD2 or its metabolites was completely abolished by pretreatment with the thiol compounds NAC or glutathione. Likewise, NAC was also able to inhibit the PG effect on cellular growth, and occurrence of PCD phenotype markers such as morphological changes, phosphatidylserine exposure and DNA degradation (Figure 9), suggesting that the mechanism by which PGs mediate their effects involves alteration of the cellular redox status. Interestingly, untreated trypanosomes from the late stationary phase showed also an increased intracellular ROS level (25.1±3.5%) as compared with NAC-treated parasites from the same phase (7.9±1.0%). These parasites died more rapidly than those treated with NAC (data not shown).
On the other hand, ROS formation has been shown to require an upregulation of protein biosynthesis, in particular of enzymes involved in their formation.34, 35 Interestingly, pretreatment of trypanosomes with cycloheximide led to a significant inhibition of ROS production, suggesting that protein synthesis is a prerequisite for PCD (Figure 8).
Discussion
In a previous study, we reported the capacity of PGD2 to induce PCD in the bloodstream forms of trypanosomes, which appeared to be a caspase-independent apoptosis.23 However, the instability of this prostanoid in albumin-containing aqueous solutions and its subsequent conversion to different metabolites has been already demonstrated.25 In addition, it was reported earlier that growth inhibition induced in murine leukemia cells after exposition to PGD2 was in fact due to an active metabolite, Δ12PGJ2, and that PGD2 exerted no growth inhibition by itself.36 We thus analyzed the metabolic conversion of PGD2 under our experimental conditions in order to determine if the induced cell death was indeed due to the formation of more potent products. PGD2 metabolization was a quick event, as it was reduced to about 80% after 12 h of incubation (data not shown). Using LC-ESI/MS, we identified various metabolites of the D and J series. Addition of D series metabolites had no effects on trypanosomes. The IC50 values for these metabolites were 10- to 30-fold higher than those for PGD2. In contrast, addition of J series metabolites led to a significant inhibition of cellular proliferation. The IC50 values for PGJ2 and Δ12PGJ2 were significantly lower than those for PGD2, whereas 15d-PGJ2 inhibited cell growth at concentrations similar to those of PGD2. Treatment with these metabolites led to cell cycle arrest in the G1 phase, as evidenced by FACS analysis and morphological changes, suggesting that this phenomenon is the principal effect exerted by J series metabolites on the slender form. The PG concentrations used in the described experiments are in the low micromolar or nanomolar range and thus, comparable and often lower than those used in similar studies with different mammalian cell types.15, 16, 32 In addition, it is widely described in the literature that the effective PG concentration is only a small fraction of the total, as they bind readily to albumin.37 At our conditions, at least 80% of PGD2 was sequestered owing to albumin binding.23 On assuming that this is also the case for the J metabolites, the effective PG concentrations have been in the low nanomolar range. On the other hand, the PGs are local mediators, and it has been reported that PG concentrations increase to the micromolar range during inflammation.14 Local events may play an important role in the parasite's life, because bloodstream forms shuttle between blood and extravascular tissue. Indeed, it has been demonstrated that differentiation from slender to stumpy form increased owing to local effects.38 Therefore, the actual PG concentrations may well be of physiological significance.
Similar to PGD2, J series metabolites induced characteristic features of PCD in trypanosomes. Using electron microscopy, FACS analysis and TUNEL test, we observed an increase of vacuolization in the cytoplasm, nuclear segmentation, chromatin condensation, DNA degradation and phosphatidylserine exposition. In addition, the recorded loss of Ψm correlates with the microscopic observation of dilated mitochondria.
Using cells from the stationary phase, which represent stumpy-like cells39 and are more sensitive to PGs than the slender form of parasites,23 confirmed that phosphatidylserine exposure is an early event in T. brucei PCD. This phenotype depends on protein biosynthesis, because short-time pretreatment with cycloheximide inhibited the phosphatidylserine exposure and significantly delayed PCD.
On the other hand, 17phenyl-PGD2, which was only slowly metabolized under our experimental conditions (data not shown), led to the same phenotype as PGD2, indicating that this latter compound is able to induce PCD itself. However, the concentration required to obtain the same effects was about two-fold higher than that of PGD2. These results suggest that the induced phenotype is partially owing to the action of PGD2, and that J series metabolites may be the more physiological inducers of PCD in the bloodstream form of T. brucei.
In general, PCD induced by J series metabolites was highly dependent on the growth phase. These PGs trigger cell death more rapidly and efficiently in the stationary than in the exponential growth phase. Similar results were found during PGD2 treatment or pretreatment with a membrane-permeable cAMP analog, which induced differentiation from slender to stumpy form, and enhanced the parasite's sensitivity for PGD2.23 Therefore, our data indicate that these PGs primarily exert their effects on the stumpy form of parasites.
Despite the well-documented action of ROS as harmful molecules, they have also been shown to play an important role in signal transduction in innumerable physiological events.35, 40, 41, 42, 43, 44 In addition, ROS have also been described to participate in the signal cascades associated to PCD.28, 32, 33, 45, 46, 47, 48, 49 Likewise, PGs are potent inductors of oxidative stress in different cell types. This alteration of the redox state is known to be the basis of many of their biological effects and was recently discovered to be involved in the activation of PCD.28, 32, 33 15d-PGJ2 has been reported to cause apoptosis in human hepatic myofibroblasts and breast cancer cells via oxidative stress.28, 33 PGD2 and PGJ2 as well as their metabolites (but not PGE2 or PGF2α) induce apoptosis in human leukemia cells through ROS production and caspase 3 activation.49 Therefore, we investigated if PCD in T. brucei is also induced by PGs via ROS generation. FACS analyses using DCFH-DA showed that all three J metabolites assayed stimulated ROS production. PGJ2 was the most potent ROS inductor, followed by Δ12PGJ2 and 15d-PGJ2. Interestingly, the potency to induce ROS was always closely related with the capacity to cause PCD. In addition, PGD2 and its analog 17phenyl-PGD2 also generated high ROS levels. Interestingly, ROS appearance was always the first event observed after treatment of the parasites with prostanoids. As shown earlier, different biological effects induced by PGs via ROS production (even PCD in other cells) were prevented by treatment with antioxidants.28, 32 In trypanosomes, pretreatment with two ROS scavengers, NAC or glutathione, completely inhibited the ROS production and occurrence of the other PCD phenotype markers induced by PGs and led to a significant reduction of cell death. Taken together, we propose that ROS are implicated as intermediates in the PCD signaling pathway induced by J series PGs. These results are consistent with the data published by Ridgley et al.,50 who stated that the increase of ROS led to PCD-like phenomena in procyclic trypanosomes. In addition, Fang and Beattie51 showed that the trypanosome alternative oxidase (TAO) decreases the mitochondrial ROS production, and recently Tsuda et al.52 demonstrated that TAO overexpression prevents PCD-like phenomena in the bloodstream form of T. b. rhodesiense.
The sensibility of PCD to protein synthesis inhibitors is an important feature, which clearly links this phenomenon to gene expression. Similarly, other compounds involved in the ROS signal pathway could require an upregulation of protein biosynthesis. This was the rationale to examine if ROS generation by PGs depends on this process. Parasites pretreated with cycloheximide and exposed to the different prostanoids showed no elevated ROS levels or phosphatidylserine exposition and survived a much longer time. These results are consistent with those reported for different neuronal cell types, in which apoptosis is triggered by ROS; their formation was totally or partially blocked by transcription and translation inhibitors.53, 54 We thus conclude that PG-induced ROS formation is a prerequisite for PCD in T. brucei.
Oxidative stress is considered to be an important condition to promote cell death in response to a variety of signals. Our results provide evidences for the existence of an active process of PCD in T. brucei, which involves de novo protein synthesis and oxidative stress.
PCD in single-cell organisms, from bacteria to unicellular eucaryotes, is well documented.55, 56, 57, 58, 59 To date, the physiological role of PCD in single-cell organisms has not clearly been defined. However, PCD functions similar to those known for metazoa have been suggested for unicellular organisms too,59, 60 because protozoa behave as a population, that is, as a complex community with many cell–cell interactions, which in many ways resemble metazoa.59, 60 Evidence of this crosstalk among trypanosomes is well documented for the differentiation process from the proliferative slender to the non-dividing stumpy form. Differentiation depends on cell density, where local effects like cell-to-cell communication play an important role.38 Thus, differentiation has been suggested as an autoregulatory process, stopping significantly the cellular proliferation.38 Although it is known that wave parasitemia can be reproduced in vitro,6 mechanisms of the parasite to regulate the stumpy form density have not been described so far. We propose that PGD2 secretion by trypanosomes and production of their J series metabolites play a key role as regulators of cell density within their host by PCD induction in the stumpy form of parasites via cell cycle regulation. In this sense, an elevation of the PG levels could arrest or diminish the parasitemia by elimination of the non-dividing form, which represents a transitory stage unable to return to the proliferative slender form. This mechanism of regulation would not compromise the parasites population for two reasons: (1) it would not cause PCD in the proliferative slender form, guaranteeing a persisting parasitemia, and (2) it would be an autolimited process, as PGs involved in PCD are secreted mainly by the stumpy form.
Materials and Methods
Reagents
PGs were purchased from the Caiman Chemical Co. (Ann Arbor, MI, USA). Triethylamine and solvents used were obtained from Merck (Darmstadt, Germany). Methyl-[3H]thymidine was obtained from Hartmann Analytic (Braunschweig, Germany). The TUNEL kit and Annexin-V-Fluos were purchased from Roche Diagnostics GmbH (Mannheim, Germany). NAC and glutathione were obtained from Sigma-Aldrich (Deisenhofen, Germany). DCFH-DA was purchased from Molecular Probes (Karlsruhe, Germany).
PGs were reconstituted in ethanol and diluted to the respective concentrations using culture medium. The respective ethanol concentration was always applied to control cells as well.
Separation and identification of PGD2 metabolites by LC-ESI/MS
In order to analyze the PGD2 metabolism, this PG was incubated in medium at 37°C. After different time points (0, 20, 40 h), PGs in the medium were extracted with 4 ml of ethylacetate (1 : 2, v/v) three times. The upper organic phases were pooled and evaporated to dryness under a stream of nitrogen at room temperature. All extraction procedures were performed under conditions of minimal light. Before the LC-ESI/MS analysis, samples (resuspended in 50 μl of acetonitrile : methanol (65 : 35, v/v)) were diluted 1 : 10 (v/v) with 10 mM triethylamine and 10 μl was directly injected onto the column. LC was performed on a 0.5 × 50 mm Saphire 110 C18 (5 μm) capillary column (Grom, Herrenberg, Germany) attached to an HP1100 capillary HPLC system (Agilent Technologies, Waldbronn, Germany). Mobile phase A was 10 mM triethylamine in water and mobile phase B was 65 : 35 (v/v) acetonitrile : methanol. Samples were separated by a gradient program: 0–5 min 10% mobile phase B and 5–30 min 10–40% mobile phase B at a flow rate of 15 μl/min. The outlet of the column was directly interfaced with the electrospray ionization source of an Esquire 3000plus ion trap mass spectrometer (Bruker-Daltonics, Bremen, Germany). The mass spectrometer was operated in the negative-ion mode. Dry gas (6 l/min) temperature was set to 275°C, nebulizer was set to 20.0 psi and the electrospray voltage was set to +3000 V. Maximal accumulation time was set to 200 ms. Loading of the trap was controlled by the instrument (ICC 15000). Mass spectra were acquired from 150 to750 m/z, using four averages. All signals were detected as monoisotopic masses.
Cell culture
The monomorphic strain T. brucei MITat 1.2 (VSG-variant 221) was taken from frozen stabilates, seeded at a cell density of 2 × 105 cells/ml and grown at 37°C and 5% CO2 for about 16 h, until a cell density of 8 × 105 cells/ml was reached.6, 39 These precultures were then adjusted to 2 × 105 trypanosomes/ml with fresh medium and incubated again in a CO2 incubator with PGs at the given concentrations. Experiments were performed with exponentially growing parasites (slender form) to evaluate cellular proliferation, cell cycle and partially PCD. Parasites from the stationary phase, equivalent to stumpy forms, were used to evaluate PCD. For experiments using cycloheximide (an inhibitor of protein synthesis), cells were grown until they reached the stationary phase, preincubated for 1 h with this inhibitor and treated with the respective PG. All experiments were performed at least in triplicate.
Cytotoxicity assay
The IC50 values were determined according to Bodley et al.61 Exponentially growing parasites were diluted to 2 × 105 cells/ml, placed in 96-well microtiter plates and grown in the absence or presence of different PG concentrations. Plates were incubated for 24 h at 37°C in a CO2 incubator. Cell growth was stopped by the addition of lysis buffer containing p-nitrophenyl phosphate (20 mg/ml in 1 M sodium acetate, pH 5.5, containing 1% Triton X-100). Likewise, addition of this buffer started the acid phosphatase reaction, which was measured at 405 nm after 6 h incubation by using an ELISA reader (MRX II, Dynex Technologies, Middlesex, England).
Proliferation assay
The cellular proliferation was determined by measuring thymidine incorporation. Cultures were adjusted to 2 × 105 cells/ml and seeded into 96-well microtiter plates. After 24 h incubation at 37°C and 5% CO2, trypanosomes were pulsed for 4 h with methyl-[3H]thymidine (1 μCi/well), harvested immediately and washed twice in PBS. Cells were lysed in 5% SDS and counted in a liquid scintillation counter.
Transmission electron microscopy
Control or treated cells were harvested after 24 h incubation with PGs and prepared for TEM. The cells were fixed in 2% glutaraldehyde in 0.2 M sodium cacodylate buffer containing 0.12 M sucrose for 1 h at 4°C. After washing and storage overnight in sodium cacodylate buffer, cells were post-fixed in osmium tetroxide (1.5%, w/v) and stained in 0.5% uranyl acetate.62 Dehydration in ethanol, clearing in propylene oxide and embedding in Agar 100 (equivalent to Epon 812) were performed according to standard procedures.63 Sections were stained in 5% (w/v) uranyl acetate and 0.4% (w/v) lead citrate.
TUNEL assay
We used the in situ cell death detection kit obtained from Roche (Mannheim, Germany) in order to detect DNA double-strand ruptures. The assay procedure was performed as previously described.55 Briefly, trypanosomes were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in phosphate buffer for 1 h before they were placed on a slide for staining. The percentage of cells showing a clearly visible nucleus staining was determined in treated and untreated cells by counting three times 100 cells each.
FACS analysis
Mitochondrial membrane potential: This parameter was measured after incubation of PG-treated and untreated trypanosomes with 25 nM TMRE for 30 min at 37°C. Fluorescence analysis was performed in a FACSCalibur® apparatus (Becton Dickinson & Co., NJ, USA). The ionophore valinomycin (100 nM) was used as a control for a complete depolarization of the inner mitochondrial membrane.
DNA content: The assay was performed as previously described.23 Briefly, following a 30 min permeabilization of trypanosomes in 10 mM phosphate buffer containing 6 μM digitonin, nuclei were stained with a propidium iodide solution (10 μg/ml final concentration in 10 mM phosphate buffer) and kept on ice until measurement in the cell sorter.
Necrosis: In order to analyze disruption of the plasma membrane after PG treatment, nuclei were stained with propidium iodide (5 μg/ml) in the absence of a cell permeant and analyzed by flow cytometry.
Phosphatidylserine exposure: Exposed phosphatidylserine was detected on the outer membrane of cells using Annexin-V-Fluos® (Roche, Mannheim, Germany) according to the manufacturer's protocol. Fluorescence was measured using FACS analysis as described before.
Reactive oxygen species: DCFH-DA was employed to measure intracellular oxidative stress.34 Control and treated cells were incubated with 10 μM DCFH-DA for 1 h at 37°C. The cellular fluorescence intensity was measured using a flow cytometer as described above.
Abbreviations
- DCFH-DA:
-
2′,7′-dichlorodihydrofluorescein diacetate
- dUTP:
-
deoxyuridinetriphosphate
- EIC:
-
extracted ion chromatogram
- Ψm:
-
mitochondrial membrane potential
- FACS:
-
fluorescence-activated cell sorting
- LC-ESI/MS:
-
liquid chromatography electrospray ionization mass spectrometry
- NAC:
-
N-acetyl-L-cysteine
- PCD:
-
programmed cell death
- PG:
-
prostaglandin
- ROS:
-
reactive oxygen species
- TEM:
-
transmission electron microscopy
- TUNEL:
-
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
References
Mahan SM and Black SJ (1989) Differentiation, multiplication and control of bloodstream form Trypanosoma (Duttonella) vivax in mice. J. Protozool. 36: 424–428
Vickerman K (1985) Developmental cycles and biology of pathogenic trypanosomes. Br. Med. Bull. 41: 105–114
Borst P, Rudenko G, Taylor MC, Blundell PA, Van Leeuwen F, Bitter W, Cross M and McCulloch R (1996) Antigenic variation in trypanosomes. Arch. Med. Res. 27: 379–388
Sternberg MJ and Mabbott NA (1996) Nitric oxide-mediated suppression of T cell responses during Trypanosoma brucei infection: soluble trypanosome products and interferon-gamma are synergistic inducers of nitric oxide synthase. Eur. J. Immunol. 26: 539–543
Schleifer KW and Mansfield JM (1993) Suppressor macrophages in African trypanosomiasis inhibit T cell proliferative responses by nitric oxide and prostaglandins. J. Immunol. 151: 5492–5503
Hesse F, Selzer PM, Muhlstadt K and Duszenko M (1995) A novel cultivation technique for long-term maintenance of bloodstream form trypanosomes in vitro. Mol. Biochem. Parasitol. 70: 157–166
Tyler KM, Higgs PG, Matthews KR and Gull K (2001) Limitation of Trypanosoma brucei parasitaemia results from density-dependent parasite differentiation and parasite killing by the host immune response. Proc. R. Soc. London B 268: 2235–2243
Pentreath VW, Rees K, Owolabi OA, Philip KA and Doua F (1990) The somnogenic T lymphocyte suppressor prostaglandin D2 is selectively elevated in cerebrospinal fluid of advanced sleeping sickness patients. Trans. R. Soc. Trop. Med. Hyg. 84: 795–799
Smith SK (1989) Prostaglandins and growth factors in the endometrium. Baillieres Clin. Obstet. Gynaecol. 3: 249–270
Smith SK and Ahmed A (1992) Eicosanoid synthesis and Helicobacter pylori associated gastritis: increase in leukotriene C4 generation associated with H. pylori colonization. Prostaglandins 44: 457–470
Samuelsson B (1979) Prostaglandins, thromboxanes, and leukotrienes: formation and biological roles. Harvey Lect. 75: 1–40
Dubois R, Lapillonne A, Laine X, Claris O, Bouvier R, Chappuis JP and Tsujii M (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Pediatr. Surg. Int. 13: 346–349
Hayaishi O (2000) Molecular mechanisms of sleep-wake regulation: a role of prostaglandin D2. Philos. Trans. R. Soc. London B 355: 275–280
Nencioni A, Lauber K, Grunebach F, Van Parijs L, Denzlinger C, Wesselborg S and Brossart P (2003) Cyclopentenone prostaglandins induce lymphocyte apoptosis by activating the mitochondrial apoptosis pathway independent of external death receptor signaling. J. Immunol. 171: 5148–5156
Shibata T, Kondo M, Osawa T, Shibata N, Kobayashi M and Uchida K (2002) 15-Deoxy-delta 12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes. J. Biol. Chem. 277: 10459–10466
Ward C, Dransfield I, Murray J, Farrow SN, Haslett C and Rossi AG (2002) Prostaglandin D2 and its metabolites induce caspase-dependent granulocyte apoptosis that is mediated via inhibition of I kappa B alpha degradation using a peroxisome proliferator-activated receptor-gamma-independent mechanism. J. Immunol. 168: 6232–6243
Alam M (1986) Ionophore A23187 stimulates Entamoeba histolytica to release prostaglandin F2 alpha. Prostaglandins Leukotr. Med. 22: 259–264
Kubata BK, Eguchi N, Urade Y, Yamashita K, Mitamura T, Tai K, Hayaishi O and Horii T (1998) Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans. J. Exp. Med. 188: 1197–1202
Belley A and Chadee K (2000) Production of prostaglandin E(2) by Entamoeba histolytica via a novel cyclooxygenase. Arch. Med. Res. 31: 74–75
Kubata BK, Duszenko M, Kabututu Z, Rawer M, Szallies A, Fujimori K, Inui T, Nozaki T, Yamashita K, Horii T, Urade Y and Hayaishi O (2000) Identification of a novel prostaglandin f(2alpha) synthase in Trypanosoma brucei. J. Exp. Med. 192: 1327–1338
Fusco AC, Salafsky B and Delbrook K (1986) Schistosoma mansoni: production of cercarial eicosanoids as correlates of penetration and transformation. J. Parasitol. 72: 397–404
Prusch RD, Goette SM and Haberman P (1989) Prostaglandins may play a signal-coupling role during phagocytosis in Amoeba proteus. Cell Tissue Res. 255: 553–557
Figarella K, Rawer M, Uzcategui NL, Kubata BK, Lauber K, Madeo F, Wesselborg S and Duszenko M (2005) Prostaglandin D2 induces programmed cell death in Trypanosoma brucei bloodstream form. Cell Death Differ. 12: 335–346
Kubata BK, Duszenko M, Kabututu Z, Rawer M, Szallies A, Inui T, Urade Y and Hayaishi O (2002) Enzymatic formation of prostaglandin D2, E2, and F2a in the parasitic protozoan Trypanosoma brucei. Int. Congress Ser. 1233: 461–466
Fitzpatrick FA and Wynalda MA (1983) Albumin-catalyzed metabolism of prostaglandin D2. Identification of products formed in vitro. J. Biol. Chem. 258: 11713–11718
Fukushima M (1992) Biological activities and mechanisms of action of PGJ2 and related compounds: an update. Prostaglandins Leukotr. Essent. Fatty Acids 47: 1–12
Padilla J, Kaur K and Phipps RP (2000) Effects of 15-deoxy-delta 12,14-PGJ2 on B lineage cells. Ann. NY Acad. Sci. 905: 322–325
Li L, Tao J, Davaille J, Feral C, Mallat A, Rieusset J, Vidal H and Lotersztajn S (2001) 15-Deoxy-delta 12,14-prostaglandin j2 induces apoptosis of human hepatic myofibroblasts. A pathway involving oxidative stress independently of peroxisome-proliferator-activated receptors. J. Biol. Chem. 276: 38152–38158
Kikawa Y, Narumiya S, Fukushima M, Wakatsuka H and Hayaishi O (1984) 9-Deoxy-delta 9, delta 12-13,14 dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma. Proc. Natl. Acad. Sci. USA 81: 1317–1321
Haberl C, Hultner L, Flugel A, Falk M, Geuenich S, Wilmanns W and Denzlinger C (1998) Release of prostaglandin D2 by murine mast cells: importance of metabolite formation for antiproliferative activity. Mediators Inflamm. 7: 79–84
Bratton DL, Fadok VA, Richter DA, Kailey JM, Guthrie LA and Henson PM (1997) Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase. J. Biol. Chem. 272: 26159–26165
Kondo M, Oya-Ito T, Kumagai T, Osawa T and Uchida K (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress. J. Biol. Chem. 276: 12076–12083
Pignatelli M, Sanchez-Rodriguez J, Santos A and Perez-Castillo A (2005) 15-Deoxy-Delta-12,14-prostaglandin J2 induces programmed cell death of breast cancer cells by a pleiotropic mechanism. Carcinogenesis 26: 81–92
Herrera B, Murillo MM, Alvarez-Barrientos A, Beltran J, Fernandez M and Fabregat I (2004) Source of early reactive oxygen species in the apoptosis induced by transforming growth factor-beta in fetal rat hepatocytes. Free Radic. Biol. Med. 36: 16–26
Thannickal VJ and Fanburg BL (2000) Reactive oxygen species in cell signaling. Am. J. Physiol. Lung Cell. Mol. Physiol. 279: 1005–1028
Narumiya S and Fukushima M (1985) Delta 12-prostaglandin J2, an ultimate metabolite of prostaglandin D2 exerting cell growth inhibition. Biochem. Biophys. Res. Commun. 127: 739–745
Raz A (1972) Interaction of prostaglandins with blood plasma proteins. Comparative binding of prostaglandins A2, F2 and E2 to human plasma proteins. Biochem. J. 130: 631–636
Reuner B, Vassella E, Yutzy B and Boshart M (1997) Cell density triggers slender to stumpy differentiation of Trypanosoma brucei bloodstream forms in culture. Mol. Biochem. Parasitol. 90: 269–280
Hamm B, Schindler A, Mecke D and Duszenko M (1990) Differentiation of Trypanosoma brucei bloodstream trypomastigotes from long slender to short stumpy-like forms in axenic culture. Mol. Biochem. Parasitol. 40: 13–22
White AA, Crawford KM, Patt CS and Lad PJ (1976) Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide. J. Biol. Chem. 251: 7304–7312
Mittal CK and Murad F (1977) Activation of guanylate cyclase by superoxide dismutase and hydroxyl radical: a physiological regulator of guanosine 3′,5′-monophosphate formation. Proc. Natl. Acad. Sci. USA 74: 4360–4364
Roth S and Droege W (1987) Regulation of T cell activation and T cell growth factor (TCGF) production by hydrogen peroxide. Cell Immunol. 108: 417–424
Schreck R, Rieber P and Baeuerle PA (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-κB transcription factor and HIV-1. EMBO J. 10: 2247–2258
Droege W (2001) Free radicals in the physiological control of cell function. Physiol. Rev. 82: 47–95
Chaudhri G, Hunt NH, Clark JA and Ceredig R (1988) Antioxidants inhibit proliferation and cell surface expression of receptors for interleukin-2 and transferrin in T-lymphocytes stimulated with phorbol myristate acetate and ionomycin. Cell Immunol. 115: 204–213
Nakamura K, Hori T, Sato N, Sugie K, Kawakami T and Yodoi J (1993) Redox regulation of a src family protein tyrosine kinase p56lck in T cells. Oncogene 8: 3133–3139
Russo T, Zambrano N, Esposito F, Ammendola R, Cimino F, Fiscella M, Jackman J, O’Connor PM, Anderson CW and Appella E (1995) A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress. J. Biol. Chem. 270: 29386–29391
Esposito F, Cuccovillo F, Vanoni M, Cimino F, Anderson CW, Appella E and Russo T (1997) Redox-mediated regulation of p21waf/cip1 expression involves a post-transcriptional mechanism and activation of the mitogen-activated protein kinase pathway. Eur. J. Biochem. 245: 730–737
Chen YC, Shen SC and Tsai SH (2005) Prostaglandin D2 and J2 induce apoptosis in human leukemia cells via activation of caspase 3 cascade and production of reactive oxygen species. Biochem. Biophys. Acta 1743: 291–304
Ridgley EL, Xiong ZH and Ruben L (1999) Reactive oxygen species activate a Ca2+-dependent cell death pathway in the unicellular organism Trypanosoma brucei brucei. Biochem. J. 340: 33–40
Fang J and Beattie DS (2003) Alternative oxidase present in procyclic Trypanosoma brucei may act to lower the mitochondrial production of superoxide. Arch. Biochem. Biophys. 414: 294–302
Tsuda A, Witola WH, Ohashi K and Onuma M (2005) Expression of alternative oxidase inhibits programmed cell death-like phenomenon in bloodstream form of Trypanosoma brucei rhodesiense. Parasitol. Int. 54: 243–251
Schulz JB, Henshaw DR, Siwek D, Jenkins BG, Ferrante RJ, Cipolloni PB, Kowall NW, Rosen BR and Beal MF (1995) Involvement of free radicals in excitotoxicity in vivo. J. Neurochem. 64: 2239–2247
Tan S, Sagara Y, Liu Y, Maher P and Schubert D (1998) The regulation of reactive oxygen species production during programmed cell death. J. Cell Biol. 141: 1423–1432
Madeo F, Frohlich E and Frohlich KU (1997) A yeast mutant showing diagnostic markers of early and late apoptosis. J. Cell Biol. 139: 729–734
Arnoult D, Tatischeff I, Staquier J, Girard M, Sureau F, Tissier JP, Grodet A, Dellinger M, Traincard F, Kahn A, Ameisen JC and Petit PX (2001) On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death. Mol. Cell. Biol. 12: 3016–3030
Arnoult D, Akarid K, Grodet A, Petit PX, Estaquier J and Ameisen JC (2002) On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilisation. Cell Death Differ. 9: 65–81
Piacenza L, Peluffo G and Radi R (2001) -Arginine-dependent suppression of apoptosis in Trypanosoma cruzi: contribution of the nitric oxide and polyamine pathways. Proc. Natl. Acad. Sci. USA 98: 7301–7306
Lewis K (2000) Programmed death in bacteria. Microbiol. Mol. Biol. Rev. 64: 503–514
Tan K and Nasirudeen A (2005) Protozoan programmed cell death – insights from Blastocystis deathstyles. Trends Parasitol. 21: 547–550
Bodley AL, McGarry MW and Shapiro TA (1995) Drug cytotoxicity assay for African trypanosomes and Leishmania species. J. Infect. Dis. 172: 1157–1159
Hirsch JG and Fedorko ME (1968) Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and ‘postfixation’ in uranyl acetate. J. Cell Biol. 38: 615–627
Glauert AM, Butterworth AE, Sturrock RF and Houba V (1978) The mechansim of antibody-dependent, eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro: a study by phase-contrast and electron microscopy. J. Cell Sci. 34: 173–192
Acknowledgements
We are grateful for the valuable technical help from Alexandra Thor. This work was funded by the Deutsche Forschungsgemeinschaft (DFG); KF and NU are recipients of personal grants from DAAD (Germany) and CDCH (Venezuela), respectively.
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Figarella, K., Uzcategui, N., Beck, A. et al. Prostaglandin-induced programmed cell death in Trypanosoma brucei involves oxidative stress. Cell Death Differ 13, 1802–1814 (2006). https://doi.org/10.1038/sj.cdd.4401862
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DOI: https://doi.org/10.1038/sj.cdd.4401862
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