Prostaglandin-induced programmed cell death in Trypanosoma brucei involves oxidative stress

Recently, we reported the induction of a programmed cell death (PCD) in bloodstream forms of Trypanosoma brucei by prostaglandin D(2) (PGD(2)). As this prostanoid is readily metabolized in the presence of albumin, we were prompted to investigate if PGD(2) metabolites rather than PGD(2) itself are responsible for the observed PCD. In fact, J series metabolites, especially PGJ(2) and Delta(12)PGJ(2), were able to induce PCD more efficiently than PGD(2). However, the stable PGD(2) analog 17phenyl-trinor-PGD(2) led to the same phenotype as the natural PGD(2), 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 PGJ(2) and Delta(12)PGJ(2) formation within the serum represents a mechanism to amplify PGD(2)-induced PCD in trypanosomes via ROS production.     

自体吞噬在细胞死亡中的角色

一直以来人们认为自主性细胞死亡就是凋亡。然而 ,最近的研究表明 ,自主性细胞死亡不仅包括凋亡 ,还包括自体吞噬。自体吞噬通过与凋亡不同的降解机制 (膜包被降解 )和分子机制 (特定基因的激活 ) ,使细胞在某些特殊环境如饥饿、发育、分化等条件下自主死亡。在自主性细胞死亡中 ,自体吞噬与凋亡是两个既相互独立又紧密相关的过程。对自体吞噬机制的了解 ,必将为自主性细胞死亡机制的阐明及其相关疾病的治疗提供新的思路。     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

Prostaglandin D2 induces programmed cell death in Trypanosoma brucei bloodstream form

African trypanosomes produce some prostanoids, especially PGD2, PGE2 and PGF2alpha (Kubata et al. 2000, J. Exp. Med. 192: 1327-1338), probably to interfere with the host's physiological response. However, addition of prostaglandin D2 (but not PGE2 or PGF2alpha) to cultured bloodstream form trypanosomes led also to a significant inhibition of cell growth. Based on morphological alterations and specific staining methods using vital dyes, necrosis and autophagy were excluded. Here, we report that in bloodstream form trypanosomes PGD2 induces an apoptosis-like programmed cell death, which includes maintenance of plasma membrane integrity, phosphatidylserine exposure, loss of mitochondrial membrane potential, nuclear chromatin condensation and DNA degradation. The use of caspase inhibitors cannot prevent the cell death, indicating that the process is caspase-independent. Based on these results, we suggest that PGD2-induced programmed cell death is part of the population density regulation as observed in infected animals.     

Programmed cell death in procyclic form Trypanosoma brucei rhodesiense --identification of differentially expressed genes during con A induced death

Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR) to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described). Evidence for an ancestral death machinery, 'proto-apoptosis' in single celled organisms is discussed.     

Involvement of TatD nuclease during programmed cell death in the protozoan parasite Trypanosoma brucei

In this report, we describe the involvement of TatD nuclease during programmed cell death (PCD) in the human protozoan parasite Trypanosoma brucei. T. brucei TatD nuclease showed intrinsic DNase activity, was localized in the cytoplasm and translocated to the nucleus when cells were treated with inducers previously demonstrated to cause PCD in T. brucei. Overexpression of TatD nuclease resulted in elevated PCD and conversely, loss of TatD expression by RNAi conferred significant resistance to the induction of PCD in T. brucei. Co‐immunoprecipitation studies revealed that TatD nuclease interacts with endonucleaseG suggesting that these two nucleases could form a DNA degradation complex in the nucleus. Together, biochemical activity, RNAi and subcellular localization results demonstrate the role of TatD nuclease activity in DNA degradation during PCD in these evolutionarily ancient eukaryotic organisms. Further, in conjunction with endonucleaseG, TatD may represent a critical nuclease in a caspase‐independent PCD pathway in trypanosomatid parasites since caspases have not been identified in these organisms.     

Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs

Procyclic Trypanosoma brucei rhodesiense have a cell death mechanism which can be activated by an external signal, the lectin ConA, in vitro. ConA has been shown to cause profound changes in cellular morphology and induce fragmentation of nuclear DNA in T.b. rhodesiense which are characteristic of apoptosis, a form of programmed cell death (PCD) in other eukaryotic cells. RNA analysis of trypanosomes induced to undergo PCD revealed that RNA remains intact up to 48 h into the process, a time when nuclear DNA fragmentation has already started. Using the randomly amplified differentially expressed sequences polymerase chain reaction method, ConA-induced cell death in T.b. rhodesiense is shown to be associated with differential expression of mRNAs, including up regulation of mRNAs late in the death process. The results demonstrate that trypanosomes actively participate in their own destruction through a PCD process and confirm that cell death in trypanosomes is associated with de novo gene expression.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: A protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: Inhibition by Organotins

Activity and kinetics of phospholipase A₂ (PLA₂) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca²⁺-independent, strongly stimulated by Triton X-100 with optimum activity at 37°C and pH 6.5–8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA₂. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (K_M) for PLA₂ from T. b. brucei is 63.87 and 30.90 μM while for T. b. gambiense it is 119.64 and 32.90 μM for the substrates l,2-bis-(1-pyrenebutanoyl-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dode-canoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC), respectively.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins

Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.     

Biosynthesis of trypanothione in Trypanosoma brucei brucei

Trypanothione [T(SH)2], the major redox mediator in pathogenic trypanosomatids, is synthetized stepwise by two distinct enzymes in Crithidia fasciculata, while in Trypanosoma cruzi a single enzyme catalyzes both steps. A full-length reading frame presumed to encode trypanothione synthetase (TryS) was obtained by PCR using DNA of T. brucei as template and primers based on fragments of putative TryS genes. The recombinant protein produced by E. coli Origami (DE3) was purified to homogeneity by chelate and ion exchange chromatography. The enzyme catalyzed both reactions of T(SH)2 biosynthesis. Thus, T(SH)2 synthesis appears to be similar in African (T. brucei) and New World (T. cruzi) trypanosomes but distinct from that of Crithidia.     

The role of immunoglobulins in immunity to Trypanosoma brucei gambiense

The effects of IgM and IgG antibody molecules were compared on a weight basis by both agglutination and in vitro protection tests. It was shown that IgM is a better agglutinating antibody and in the presence of complement is also a better neutralizing antibody. However, when IgM and IgG were tested for their ability to passively protect infected animals, it was noted that the IgG fraction appeared to give greater protection. Based on differences in diffusion coefficients (and size) of the molecules, it is hypothesized that IgM is the antibody class responsible for the relapse phenomena observed in the blood, while, it is the IgG antibody molecule which is more active in extravascular locations.     

Coordination of cell cycle and cytokinesis in Trypanosoma brucei

In common with all eukaryotic cells, trypanosomes must coordinate a complex series of morphogenetic events both temporally and spatially during the cell cycle. The structural and molecular cues that synchronise these events in trypanosomes have started to be elucidated, and intriguingly although similarities to cell cycle events in other eukaryotes can be identified, trypanosomes have also evolved novel solutions to the common challenges faced by dividing eukaryotic cells. Although cellular morphology is clearly pivotal for successful progression through the trypanosome cell cycle, most cytological studies to date have focused exclusively on procyclic form trypanosomes. These studies provide an excellent framework for understanding cell cycle events in trypanosomes, however recent data indicates that profound differences might exist between different life cycle stages in relation to the regulation of cell cycle and cytokinesis.     

The role of autophagy in mammalian development: cell makeover rather than cell death

Autophagy is important for the degradation of bulk cytoplasm, long-lived proteins, and entire organelles. In lower eukaryotes, autophagy functions as a cell death mechanism or as a stress response during development. However, autophagy's significance in vertebrate development, and the role (if any) of vertebrate-specific factors in its regulation, remains unexplained. Through careful analysis of the current autophagy gene mutant mouse models, we propose that in mammals, autophagy may be involved in specific cytosolic rearrangements needed for proliferation, death, and differentiation during embryogenesis and postnatal development. Thus, autophagy is a process of cytosolic "renovation," crucial in cell fate decisions.     

The selectivity of autophagy and its role in cell death and survival

Autophagy is a cellular process whose primary function is to degrade long-lived proteins and recycle cellular components. Beside macroautophagy, there are several forms of selective autophagy, including chaperone-mediated autophagy (CMA), cytoplasm to vacuole targeting (Cvt), pexophagy, and mitophagy. In this review, we summarize what is currently known about selective autophagy, and discuss its role in cell death and survival. We also discuss possible mechanisms underlying the selectivity of macroautophagy.