Molecular & Biochemical Parasitology 193 (2014) 93–100

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Molecular & Biochemical Parasitology

How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor Aline Araujo Zuma a,b , Isabela Cecília Mendes c , Lissa Catherine Reignault a,b , Maria Carolina Elias d , Wanderley de Souza a,b,e , Carlos Renato Machado c,∗∗ , Maria Cristina M. Motta a,b,∗ a Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21491-590 Rio de Janeiro, RJ, Brazil b Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil c Laboratório de Genética Bioquímica, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil d Laboratório Especial de Ciclo Celular, Instituto Butantan, Center of Toxins, Immune Response and Cell Signaling, 05503-900 São Paulo, SP, Brazil e Instituto Nacional de Metrologia, Qualidade e Tecnologia-Inmetro, 20261-232 Duque de Caxias, RJ, Brazil

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Article history: Received 30 October 2013 Received in revised form 21 January 2014 Accepted 3 February 2014 Available online 11 February 2014 Keywords: Apoptosis Camptothecin Cell cycle Topoisomerase inhibitors Ultrastructure Trypanosoma cruzi

a b s t r a c t The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which affects approximately 8 million people in Latin America. This parasite contains a single nucleus and a kinetoplast, which harbors the mitochondrial DNA (kDNA). DNA topoisomerases act during replication, transcription and repair and modulate DNA topology by reverting supercoiling in the DNA double-strand. In this work, we evaluated the effects promoted by camptothecin, a topoisomerase I inhibitor that promotes protozoan proliferation impairment, cell cycle arrest, ultrastructure alterations and DNA lesions in epimastigotes of T. cruzi. The results showed that inhibition of cell proliferation was reversible only at the lowest drug concentration (1 ␮M) used. The unpacking of nuclear heterochromatin and mitochondrion swelling were the main ultrastructural modifications observed. Inhibition of parasite proliferation also led to cell cycle arrest, which was most likely caused by nuclear DNA lesions. Following camptothecin treatment, some of the cells restored their DNA, whereas others entered early apoptosis but did not progress to late apoptosis, indicating that the protozoa stay alive in a “senescence-like” state. This programmed cell death may be associated with a decrease in mitochondrial membrane potential and an increase in the production of reactive oxygen species. Taken together, these results indicate that the inhibition of T. cruzi proliferation is related to events capable of affecting cell cycle, DNA organization and mitochondrial activity. © 2014 Published by Elsevier B.V.

1. Introduction The parasite Trypanosoma cruzi is the etiological agent of Chagas disease, which is endemic to Latin America and affects 8 million people [1]. This protozoan has one nucleus, which presents condensed heterochromatin around the nucleolus and close to the nuclear envelope, and a single mitochondrion with an enlarged

∗ Corresponding author at: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21491-590 Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 6580; fax: +55 21 2260 2234. ∗∗ Corresponding author. Tel.: +55 31 3409 2628. E-mail addresses: [email protected] (A.A. Zuma), [email protected] (C.R. Machado), [email protected], [email protected] (M.C.M. Motta). http://dx.doi.org/10.1016/j.molbiopara.2014.02.001 0166-6851/© 2014 Published by Elsevier B.V.

portion (termed kinetoplast) that harbors the mitochondrial DNA (kDNA) [2–4]. The nucleus of trypanosomatids presents a similar ultrastructure as other eukaryotic organisms. However, in these protozoa, the nuclear envelope remains intact during the cell cycle, and there is no condensation of chromosomes. Chromatin distribution and condensation, as well as nucleolus morphology, vary during the T. cruzi cell cycle. During interphase, the nucleolus has a central position and is surrounded by heterochromatin, which is also found juxtaposed to the nuclear envelope. In mitosis, the nucleus becomes more elongated and more homogenous once the nucleolus and chromatin are dispersed in the nuclear space. As mitosis progresses, the chromatin condenses and migrates to the polar region of the nucleus, culminating in nuclear division during cytokinesis [5–7]. How the cell cycle is regulated in trypanosomatids remains to be elucidated. Such protozoa seem to use evolutionarily

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conserved mechanisms, as well as specific molecules, to control the transitions through different stages such as G1/S, G2/M and mitosis/cytokinesis. Ten members of the cyclin-dependent kinase (CDK) family (as cyclin related kinase – CRK) and ten orthologous cyclins (as CYCs) were identified in the T. cruzi genome [8]. TcCYC2 was able to rescue the cell cycle in G1-arrested yeast cells [9], and some evidence suggests that CRK3 has a role in cell cycle control [10]. DNA topoisomerases play an essential role in nuclear and kDNA organization and regulate the topological state of DNA by introducing or removing supercoiling in these molecules during replication, transcription and repair [11]. Topoisomerases are classified into two groups: type I topoisomerases (topo I) bind to one strand of the DNA double helix and break and rejoin this strand, whereas type II topoisomerases (topo II) bind, break, and rejoin double DNA strands [11]. Camptothecin is an alkaloid isolated from the Chinese plant Camptotheca acuminate that targets eukaryotic topoisomerase I. This compound acts as a non-competitive inhibitor by trapping the enzyme, which is bound to the DNA, thus forming a ternary complex that prevents the rejoining of the DNA strand. The collision between the camptothecin–topoisomerase–DNA complex and replication forks causes cell cycle arrest, which enables the repair of DNA damage or causes cell death by apoptosis [12–14]. T. cruzi contains several genes involved in different mechanisms of DNA repair that can mend the majority of lesions. Nonetheless, some damage cannot be repaired, leading to mutations or blockage of the cell cycle [15]. In the present work, we investigated the effects of camptothecin in T. cruzi. Blockage of parasite proliferation and cell cycle arrest induced changes in the nuclear ultrastructure and DNA breaks, which in turn induced apoptosis and reduced mitochondrial activity. Interestingly, protozoa did not enter late apoptosis but remained alive in a “senescence-like” state. 2. Materials and methods 2.1. Protozoa culture Epimastigote forms of T. cruzi Y were grown for 24 h at 28 ◦ C in liver infusion tryptose (LIT) medium [16] supplemented with 10% fetal calf serum. 2.2. Drug treatment Camptothecin was diluted in dimethyl sulphoxide (DMSO) to a concentration of 5 mM and concentrations used for treatment were determined according to previous report [17]: 1, 5, 10, and 50 ␮M. Cells were collected every 24 h for counting in a Neubauer chamber. To compare the control and the treated groups, paired t-tests were applied to the results using the 95% confidence interval (GraphPad Prism version 5.00 for windows; GraphPad Software Inc., San Diego, CA, USA). To evaluate the potential reversibility of cell proliferation, parasites were treated for 48 h, then were washed with LIT to remove the drug from the medium and incubated with LIT and fetal calf serum for 168 h. 2.3. Transmission electron microscopy The protozoa were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h and were washed in the same buffer. The cells were post-fixed in cacodylate buffer containing 1% OsO4 and 0.8% potassium ferricyanide for 1 h, were washed in the same buffer, dehydrated in a graded series of acetone, and embedded in Epon (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were stained with uranyl acetate and lead citrate and

observed using a Zeiss 900 transmission electron microscope (Zeiss, Oberkochen, Germany). 2.4. Cytochemical analysis with phosphotungstic acid (PTA) The cells were fixed in 2.5% glutaraldehyde in cacodylate buffer for 1 h, dehydrated in a graded series of ethanol, and post-fixed with OsO4 . The cells were incubated in 2% PTA in absolute ethanol for 2 h and embedded in Epon. 2.5. Cell cycle analysis by flow cytometry Parasites were treated for up to 72 h, washed with PBS (Phosphate Buffered Saline), pH 7.2, and fixed in 70% ethanol overnight. The cells were incubated with 10 ␮g/mL RNAse for 30 min at 37 ◦ C, and washed with PBS. Immediately before reading, the cells were incubated with 200 ␮g/mL propidium iodide. The analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson Bioscience BDB, San Jose, CA, USA), and the data analyzed using the WinMDI 2.9 software. 2.6. Analysis of DNA lesions using quantitative PCR assay (QPCR) Parasite cultures containing 1 × 107 cells mL−1 were harvested by centrifugation at 3000 × g for 10 min. The supernatant medium (conditioned medium) was saved for later use, and the cells were re-suspended in PBS. The cells were treated with 50 ␮M for up to 72 h. The DNA extraction, quantification, QPCR amplification, and analyses were conducted as reported previously [18]. The QPCR assay compared the amplification of the DNA from a treated sample with the amplification obtained with the undamaged control. Specific primers were used to amplify large and small fragments of the nuclear and mitochondrial DNA. The large nuclear fragment was amplified using the forward primer QPCRNuc2F (5 -GCACACGGCTGCGAGTGACCATTCAACTTT-3 ) the reverse primer QPCRNuc2R (5 and CCTCGCACATTTCTACCTTGTCCTTCAATGCCTGC-3 ). The small nuclear fragment was amplified with the internal primer (5 -TCGAGCAAGCTGACACTCGATGCAACCAAAGQPCRNuc2Int 3 ) and the reverse primer QPCRNuc2R. The large mitochondrial fragment was amplified using the forward primer QPCRMitF (5 -TTTTATTTGGGGGAGAACGGAGCG-3 ) and the reverse primer QPCRMitR (5 -TTGAAACTGCTTTCCCCAAACGCC-3 ). The small mitochondrial fragment was amplified with the internal primer QPCRMitInt (5 -CGCTCTGCCCCCATAAAAAACCTT-3 ). Because the probability of introducing a lesion in a short segment is very low, the small fragment (250 bp) was used to normalize the amplification results obtained with the large fragments (10 kb). This step eliminates the bias of changes in the proportion between the nuclear and the mitochondrial genomes. All of the primers sets were amplified in a specific manner, i.e., these produced only one band, as verified through gel electrophoresis (data not show). The normalized amplification of the treated samples was then compared with the controls, and the relative amplification was calculated. These values were used to estimate the average number of lesions per 10 kb of the genome through a Poisson distribution. The final results are the mean of two sets of PCR results for each target gene from at least two biological experiments. 2.7. Detection of apoptosis by flow cytometry Parasites were treated for up to 72 h and to evaluate the potential reversibility of phosphatidylserine exposure, the parasites were treated with 1 ␮M for 48 h, washed and incubated in LIT with fetal calf serum for 48 h. Then, 1 × 106 cells were washed, suspended in Annexin V binding buffer, and incubated for 15 min with Annexin

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V – Alexa 488 (Molecular Probes, Eugene, OR, USA) at the concentration indicated by the manufacturer. Then, 10 ␮g/mL propidium iodide (Molecular Probes, Eugene, OR, USA) was added to the samples. The data were collected in a BD FACSCalibur® and analyzed using the Summit v4.3® software (Dako, Fort Collins, CO, USA). 2.8. Measurement of mitochondrial membrane potential ( m) The parasites were added to a reaction medium (125 mM sucrose, 65 mM KCl, 10 mM HEPES/K+ (pH 7.2), 2 mM Pi, 1 mM MgCl2 , and 500 ␮M EGTA). Approximately 1 × 107 cells were incubated in 10 ␮g/mL JC-1 (a probe used to detect the loss of m), and readings were obtained every minute for a 30-min period using a microplate reader. As a positive control of the depolarization of m, 1 ␮M FCCP (an inhibitor of mitochondrial function) was used. At the end of the reading period, 2 ␮M of FCCP was added to the treated and the non-treated cells to decrease the m. 2.9. Measurement of the Production of reactive oxygen species (ROS) Approximately 2.5 × 107 cells were incubated in 10 ␮g/mL H2 DCFDA in PBS for 1 h at 28 ◦ C. H2 DCFDA is a non-fluorescent dye that becomes fluorescent in the presence of ROS. Oligomycin (10 ␮M) was used as a positive control for the production of ROS. The levels of ROS were analyzed with a microplate reader using an excitation wavelength of 507 nm and an emission wavelength of 530 nm. 2.10. Cell viability Parasites were analyzed through the MTS/PMS method [19]. The reagent PMS is reduced in viable cells and their electrons are transferred to MTS, which is converted by dehydrogenase enzymes into a water-soluble compound (formazan) in proportion to the number of viable cells. Parasites were incubated with the MTS/PMS solution for 4 h. Untreated parasites were fixed with 0.4% formaldehyde for 10 min and used as the negative control. The percentage of viable parasites was obtained through a spectrofluorometer (Molecular Devices Microplate Reader, SpectraMax M2/M2e , Molecular Devices) using a wavelength of 490 nm. 3. Results In this study, we confirmed, as previously reported, that T. cruzi epimastigote proliferation was strongly inhibited by camptothecin. One of the main effects of this compound was the appearance of a plateau in the growth curve, which indicates the arrest of cell proliferation as the treatment proceeded [17]. To verify whether the protozoa were able to recover their proliferation capability after camptothecin treatment, a reversibility test was performed. The results revealed that those parasites treated with 1 ␮M camptothecin, but not those treated with 5, 10, or 50 ␮M, recovered their cell proliferation capability (Fig. 1). Analysis of the T. cruzi ultrastructure showed that one of the most remarkable effects of replication inhibition occurred in the nucleus. The non-treated parasites presented condensed nuclear heterochromatin distributed around the nucleolus and close to the nuclear envelope (Fig. 2A). Parasites treated with 1 ␮M camptothecin for 24 h presented a notable unpacking of heterochromatin (Fig. 2B), and the same pattern was observed in cells treated with the highest drug concentration for 24 h (Fig. 2C) or for up to 72 h (data not shown). Treated parasites also presented mitochondrial swelling, although kinetoplast ultrastructure was not affected (Fig. 2B and C). Ultrastructural analysis of the cells subjected to the reversibility test was also performed, and the results showed that

Fig. 1. Cell reversibility after inhibition of T. cruzi replication. The asterisks indicate the addition of camptothecin to the culture medium, and the arrow indicates the time of drug removal. The data are the average of three independent experiments. DMSO, dimethyl sulfoxide.

the parasites treated with 1 ␮M camptothecin for 48 h and grown for further 96 h in the absence of the drug resumed a typical nuclear chromatin conformation similar to that observed in the control cells (Fig. 2D). However, at higher drug concentrations over the same treatment period, nuclear heterochromatin remained uncondensed after the drug was removed (Fig. 2E). Heterochromatin unpacking was better visualized after cytochemical analysis using phosphotungstic acid (PTA), which stains basic proteins such as histones. The results showed that in non-treated parasites, staining was associated with nuclear heterochromatin (Fig. 2F). In T. cruzi treated with 50 ␮M camptothecin for 72 h, nuclear labeling was less evident compared with the control cells (Fig. 2G), which reinforces the idea that DNA was less compact in the treated protozoa. Analysis of the growth curve [17] showed reduced cell proliferation and growth arrest, which suggests blockage of the T. cruzi cell cycle. To test this hypothesis, flow cytometry assays were performed (Fig. 3A–E). The results showed that 36% of the protozoa treated with 10 and 50 ␮M camptothecin for 24 h were in G1 and 50% were in the late S/G2/M phase, whereas 48% of the control cells were in G1 and 35% were in late S/G2/M. After treatment for 48 h, the effect of the drug on the T. cruzi cell cycle was similar to that observed after 24 h (Fig. 3A). After 72 h of treatment with 1 ␮M camptothecin, the number of cells in the G1 and late S/G2/M phases was very similar to the control group (Fig. 3B). However, after treatment with 5 ␮M camptothecin, the percentage of parasites in late S/G2/M was higher (43%) than the percentage in G1 (35%) (Fig. 3C). The percentage of cells in late S/G2/M increased to 58% and 66% after treatment with 10 and 50 ␮M camptothecin for 72 h, respectively, while the corresponding percentages of cells in the G1 phase were 27% and 18%, respectively (Fig. 3D and E). Taken together, these data clearly reveal that the number of trypanosomatids in the late S and G2/M phases increased in a dose-dependent manner, which indicates cell cycle arrest. To determine the occurrence of DNA lesions associated with cell cycle arrest, cells treated with camptothecin were analyzed by QPCR. The results showed that after treatment with 50 ␮M camptothecin for 24 h, few lesions were detected in the nuclear DNA, which may represent the main drug target. However, approximately 1 lesion per 10 kb of DNA was found in parasites treated for 48 h, and this ratio was maintained after 72 h of treatment (Fig. 4A). It is worth considering that we did not observe any lesions in kDNA, and coincidently, the kinetoplast ultrastructure was not affected (Fig. 4B).

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Fig. 2. Transmission electron microscopy analysis of T. cruzi epimastigotes treated with camptothecin. (A) Non-treated parasite showing the nucleus with condensed heterochromatin (ht) around the nucleolus (nu), the bar-shaped kinetoplast (k), the basal body (bb), the Golgi complex (gc) and the mitochondria (m). (B) and (C) T. cruzi treated with 1 and 50 ␮M camptothecin for 24 h, respectively, showing the unpacking of nuclear heterochromatin and mitochondrial swelling (asterisks). (D) T. cruzi treated with 1 ␮M camptothecin for 144 h in the reversibility test. (E) T. cruzi treated with 50 ␮M camptothecin for 144 h present unpacked chromatin. (F and G) Phosphotungstic acid (PTA) staining. (F) Non-treated parasite showing the distribution of basic proteins associated with nuclear heterochromatin (ht) and the kinetoplast periphery (k). (G) T. cruzi treated with 50 ␮M camptothecin for 72 h. Bars: A, F, and G = 2 ␮m; B, C, and E = 1 ␮m; D = 0.5 ␮m.

It has been reported that after cell cycle arrest for DNA break repair, cells can undergo apoptosis [20]. To determine whether parasites were apoptotic, we performed analyses by flow cytometry using labeled Annexin-V, which recognizes and binds to externalized phosphatidylserine (PS), and propidium iodide (PI), which is a cell viability marker. It has been reported that the plasma membrane is still intact during early apoptosis, which results in no PI labeling and Annexin-V binding only on PS that is exposed to the external leaflet of the membrane. During the late stage, the membrane loses its integrity, and it is possible to notice PI labeling and Annexin-V staining that result from binding to the PS located in the inner membrane leaflet [21]. Our results showed that after 24 h of treatment with 1 and 50 ␮M camptothecin, at

least 50% of the cells were in early apoptosis, and this number increased to approximately 95% PS-positive cells after 48 and 72 h of treatment (Fig. 5B–D). Notably, the percentage of cells in early apoptosis increased from 24 to 72 h of treatment; however, the number of protozoa in the late stage did not increase with increasing treatment time and remained between 2 and 3%. The reversibility assays, in which the protozoa were treated with 1 ␮M camptothecin, showed that the percentage of cells in early apoptosis at 48 h after drug removal decreased from 91% to 35% (Fig. 5E). Taking into account the mitochondrion ultrastructural alterations reported by transmission electron microscopy and the signaling for apoptosis detected by flow cytometry, mitochondrial

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Fig. 3. Cell cycle arrest analysis of T. cruzi epimastigotes treated with camptothecin by flow cytometry. (a) T. cruzi treated with different camptothecin concentrations for 24 and 48 h, (b) T. cruzi treated with 1 ␮M camptothecin, (c) T. cruzi treated with 5 ␮M camptothecin, (d) T. cruzi treated with 10 ␮M and (e) 50 ␮M camptothecin. The thin line represents the control, and the thicker line represents the treated parasites.

Fig. 4. Quantitative PCR-based measurement of camptothecin-induced DNA lesions after up to 72 h of treatment with 50 ␮M. Detection of lesions in nuclear DNA (a) and in mitochondrial DNA (b).

activity was also investigated. When T. cruzi was cultivated for 72 h with 1 and 5 ␮M camptothecin, we observed a decrease in the mitochondrial membrane potential equivalent to 30%. This effect became more pronounced at higher drug concentrations (10 and 50 ␮M; Fig. 6A); the membrane potential of the treated parasites was twofold lower compared with the control cells. Analysis of the generation of ROS showed that treatment with 10 and 50 ␮M camptothecin for 72 h promoted increases in ROS production of 12 and 22%, respectively (Fig. 6B). Cell viability, which was assessed based on dehydrogenase activity, was reduced by almost 10% and 60% after treatment with 1 and 50 ␮M camptothecin, respectively, for 72 h (Fig. 6C).

4. Discussion Camptothecin acts by binding to topoisomerase I and DNA to form a ternary complex. The collision of this complex with a replication fork leads to cell growth impairment and cell cycle arrest at the G2/M phase, resulting in DNA repair or apoptosis [20,22,23]. A previous study showed that the protozoan Leishmania donovani was very sensitive to this topoisomerase inhibitor because approximately 65% of its cell growth was inhibited after 4 h of treatment with 5 ␮M [13]. For T. cruzi, camptothecin presented an IC50 value of 2.08 ␮M after 72 h [17]. Furthermore, in this work, electron microscopy approaches revealed intense

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Fig. 5. Quantification of apoptotic parasites by flow cytometry after treatment with camptothecin. (a) Non-treated parasites, (b) T. cruzi treated with 1 and 50 ␮M camptothecin for 24 h, (c and d) T. cruzi treated with 1 and 50 ␮M camptothecin for 48 h and 72 h, respectively and (e) T. cruzi treated with 1 ␮M camptothecin for 48 h in the absence of the drug.

ultrastructural alterations, such as mitochondrial swelling and unpacking of nuclear heterochromatin, as were previously reported [17]. Interestingly, such effects have also been observed when T. cruzi is exposed to gamma radiation, which consequently activates DNA repair mechanisms [24]. Analysis of the effects promoted by T. cruzi proliferation inhibition using reversibility tests showed that only those parasites treated with 1 ␮M camptothecin resumed their cell growth capability and ultrastructure, i.e., the heterochromatin was redistributed around the nucleolus and close to the nuclear envelope. Such data suggest that the DNA repair mechanism of the treated cells was only efficient in the protozoa submitted to the lowest drug concentration (1 ␮M). We also found that the plateau presented in the growth curve indicates cell cycle arrest in the treated parasites. The flow cytometry data showed that 24 h of treatment with 10 and 50 ␮M camptothecin resulted in an increase in the percentage of cells in the late S and G2/M phases, and this percentage increased with time. These results indicate cell cycle arrest in late S/G2/M because the number of cells at this phase was approximately twofold higher in parasites treated for 72 h compared with the control group. The occurrence of apoptosis after replication arrest has been reported in tumor cells [20,23] and in L. donovani [13]. In protozoan parasites, the signs of apoptosis usually include chromatin condensation, DNA fragmentation, cell shrinkage, loss of mitochondrial membrane potential and exposure of phosphatidylserine from the

inner to the outer leaflet of the plasma membrane [25]. In accordance to these concepts, the flow cytometry analyses performed in the present work suggest the occurrence of signaling to initiate apoptosis in T. cruzi treated with camptothecin, however this effect did not occur in a dose-dependent manner. All of the doses tested demonstrated 50% of cells in early apoptosis after 24 h of treatment; this percentage increased to approximately 95% after 48 h and remained at 95% after 72 h. Interestingly, these protozoa did not progress to late apoptosis, and there is no indication of parasite death within 72 h of treatment according to the data obtained in the growth curve [17]. Thus, these data suggest that the parasites are refractory to the continuity of the apoptosis process and stay alive, but that their replication is blocked. The reversibility assays showed that the group treated with 1 ␮M presented a small number of cells with exposed PS compared with the parasites treated with the same concentration for longer periods, which is in agreement with the growth curve and ultrastructural data. These findings suggest that the signaling that induces cells to enter early apoptosis can be reverted. In T. cruzi, it has been shown that trypomastigotes, but not epimastigotes or amastigotes, can naturally expose PS on their surface. Thus, as was previously suggested for Leishmania spp. amastigotes [26], it was proposed that PS exposure on the trypomastigote surface would trigger a signaling cascade that would lead to the disappearance of iNOS and thus the subversion of the macrophage

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inhibition of topoisomerase I appears to be more deleterious to epimastigotes of T. cruzi than the break generated by gamma irradiation, which is spread in the genome [18]. This can be suggested because protozoa overexpressing the RAD51 repair protein seem to be unable to repair DNA breaks by homologous recombination when treated with camptothecin (Fig. S1). Consequently, the parasite cell cycle remains arrested and protozoa signal for apoptosis. It is worth mentioning that autophagy, another type of regulated cell death, can also be induced after DNA damage checkpoint activation and involves repair pathways as mismatch repair (MMR) and base excision repair (BER). Interestingly, during this process the DNA damage also induces chromatin unpacking and rDNA reorganization [30]. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molbiopara. 2014.02.001. In this work, our results indicate that after inhibition of T. cruzi proliferation using camptothecin, cells demonstrated chromatin unpacking, which led to cell cycle arrest. Consequently, some of the parasites repaired the DNA breaks and recovered proliferation; however, most of them entered apoptosis but was unable to continue this process, in contrast to Leishmania species [13]. Although apoptosis has been related as the major consequence in response to DNA damage [31], other types of regulated cell death may be also triggered, as necrosis and autophagy [30,32]. It has been demonstrated that trypanosomatids die after exposure to stress conditions, however part of the molecular machinery necessary to execute the programmed cell death was not convincingly identified in these protozoa. This includes caspases and enzymes involved in specific cell death signaling, as well as in the dissipation of mitochondrial membrane potential [33]. Taking together, our finding suggests that different trypanosomatid species respond to proliferation inhibition and to DNA damage in distinct manners [13] that are associated with some special features of their biology. Acknowledgements

Fig. 6. Analysis of mitochondrial activity in T. cruzi epimastigotes after inhibition of parasite proliferation with 1–50 ␮M camptothecin for 72 h. (a) Mitochondrial membrane potential, (b) ROS production and (c) cell viability. The data are the average of three independent experiments.

microbicidal system [27]. In the present study, the PS exposure in T. cruzi epimastigotes may be directly related to topoisomerase I inhibition and replication blockage because this developmental form does not naturally expose PS on its surface. A decrease in mitochondrial activity has been described in L. donovani after replication inhibition, resulting in mitochondrial membrane hyperpolarization just before apoptosis followed by depolarization [13]. In contrast, in T. cruzi, an increase in mitochondrial membrane potential was observed without a subsequent decrease. This effect is associated with an increase in the ROS levels in T. cruzi after treatment with 10 and 50 ␮M camptothecin for 72 h, as was also observed in L. donovani [13]. Mitochondrial alterations, such as loss of mitochondrial membrane potential and production of ROS, have been considered indicators of apoptosis in protozoan parasites [28]; however, in the present work, these were not sufficient to signal T. cruzi to enter late apoptosis. The inhibition of topoisomerase functions is expected to affect parasite proliferation and ultrastructure [13,17] and is closely related to cell cycle arrest and apoptosis. It has been reported that several topoisomerase inhibitors are able to induce apoptosis in trypanosomatids and promote DNA strand breaks and cell cycle blockage at the G2/M phase in tumor cells [20,22,29]. The double strand break provoked by replication fork arrest and caused by the

This work was supported by Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado od Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundac¸ão de Amparo à Pesquisa do estado de Minas Gerais (FAPEMIG) and Programa de Apoios a Núcleos de Excelência (Pronex). References [1] Rassi AJR, Rassi A, Resende MJ. American trypanosomiasis (Chagas disease). Infect Dis Clin North Am 2000;26:275–91. [2] Motta MCM, de Souza W, Thiry M. Immunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids. FEMS Microbiol Lett 2003;221:17–23. [3] Elias MC, da Cunha JP, Faria FP, Mortara RA, Freymuller E, Schenkman S. Morphological events during the Trypanosoma cruzi cell cycle. Protist 2007;158:147–57. [4] De Souza W. Structural organization of Trypanosoma cruzi. Mem Inst Oswaldo Cruz 2009;104:89–100. [5] Ogbadoyi E, Ersfeld K, Robinson D, Sherwin T, Gull K. Architecture of the Trypanosoma brucei nucleus during interphase and mitosis. Chromosoma 2000;108:501–13. [6] Elias MC, Faria M, Mortara RA, Motta MC, de Souza W, Thiry M, et al. Chromosome localization changes in the Trypanosoma cruzi nucleus. Eukaryot Cell 2002;1:944–53. [7] De Souza W. Basic cell biology of Trypanosoma cruzi. Curr Pharm Des 2002;8:269–85. [8] Naula C, Parsons M, Mottram JC. Protein kinases as drug targets in trypanosomes and Leishmania. Biochim Biophys Acta 2005;1754:151–9. ˜ [9] Potenza M, Schenkman S, Laverrière M, Tellez-Inón MT. Functional characterization of TcCYC2 cyclin from Trypanosoma cruzi. Exp Parasitol 2012;132:537–45. ˜ MT. Evidence for [10] Santori MI, Laría S, Gómez EB, Espinosa I, Galanti N, Téllez-Inón CRK3 participation in the cell division cycle of Trypanosoma cruzi. Mol Biochem Parasitol 2002;121:225–32.

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How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor.

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which affects approximately 8 million people in Latin America. This parasi...
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