Camptothecin-induced Imbalance in Intracellular Cation Homeostasis Regulates Programmed Cell Death in Unicellular Hemoflagellate Leishmania donovani*

, a trypanosomatid protozoan parasite, causes a wide range of human diseases ranging from the localized self-healing cutaneous lesions to fatal visceral leishmaniasis. However, it undergoes a process of programmed cell death during treatment with the topoisomerase I poison camptothecin (CPT). The present study shows that CPT-induced formation of reactive oxygen species increases the level of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential ( (cid:1)(cid:2) m ), which is followed by a significant decrease in intracellular pH levels. CPT-induced oxidative stress also causes impairment of the Na (cid:3) -K (cid:3) -ATPase pump and subsequently decreases the intracellular K (cid:3) level in leishmanial cells. A decrease in both intracellular pH and K (cid:3) levels propa-gates the apoptotic process through activation of caspase 3-like proteases by rapid formation of cytochrome c -mediated apoptotic complex. In addition to caspase-like protease activation,

Leishmania, a unicellular trypanosomatid protozoan parasite, causes a wide range of human diseases ranging from the localized self-healing cutaneous lesions to fatal visceral leishmaniasis. However, it undergoes a process of programmed cell death during treatment with the topoisomerase I poison camptothecin (CPT). The present study shows that CPT-induced formation of reactive oxygen species increases the level of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (⌬⌿ m ), which is followed by a significant decrease in intracellular pH levels. CPT-induced oxidative stress also causes impairment of the Na ؉ -K ؉ -ATPase pump and subsequently decreases the intracellular K ؉ level in leishmanial cells. A decrease in both intracellular pH and K ؉ levels propagates the apoptotic process through activation of caspase 3-like proteases by rapid formation of cytochrome c-mediated apoptotic complex. In addition to caspase-like protease activation, a lower level of intracellular K ؉ also enhances the activation of apoptotic nucleases at the late stage of apoptosis. This suggests that the physiological level of pH and K ؉ are inhibitory for apoptotic DNA fragmentation and caspase-like protease activation in leishmanial cells. Moreover, unlike mammalian cells, the intracellular ATP level gradually decreases with an increase in the number of apoptotic cells after the loss of ⌬⌿ m . Taken together, the elucidation of biochemical events, which tightly regulate the process of growth arrest and death of Leishmania donovani promastigotes, allows us to define a more comprehensive view of cell death during treatment with CPT.
DNA topoisomerases are ubiquitous enzymes that catalyze the breakage and rejoining of DNA strands to permit topological changes in DNA (1). They are classified into two types. Type I topoisomerase breaks and rejoins one strand of duplex DNA, whereas type II topoisomerase breaks and rejoins both strands of DNA by using ATP as cofactor (2). These enzymes play a pivotal role in the maintenance of genome integrity and are essential for many chromosomal functions including DNA replication, recombinations, transcription, and chromosome segregation (3). Other than the orderly synthesis of nucleic acids, these enzymes also have been identified as the molecular targets for numerous clinically important antibacterial and antitumor agents like fluoroquinolone, etoposide, and camptothecin (CPT), 1 etc. (2,3).
CPT, an inhibitor of DNA topoisomerase I, has been widely used to induce apoptosis under experimental conditions and is in phase III clinical trials for colon cancer (4,5). Poisoning of topoisomerase I by CPT causes protein-linked single strand breaks, but the breaks are by themselves not sufficient for cell death. The collision between the DNA replication fork with CPT-stabilized topoisomerase I-DNA covalent complex is thought to be responsible for cell killing (6). The double strand breaks resulting from the fork arrest are repaired very slowly and lead to prolonged S phase or G 2 arrest of the cell cycle, followed by apoptosis (7).
Apoptosis, a physiological mode of cell death, results from the action of a genetically encoded suicide program that leads to series of characteristic morphological and biochemical changes (8). These changes include activation of caspases, cell shrinkage, chromatin condensation, and nucleosomal degradation (9). But the most significant event in apoptosis is mitochondrial dysfunction, which was shown to be involved in an early phase of apoptosis in a variety of cells upon induction of a number of stimuli including tumor necrosis factor (10), glucocorticoids (11), ceramides (12), and oxidative stress (13,14).
As compared with necrosis, apoptosis is an energy-dependent process requiring functional mitochondria. Without the supply of ATP, cell cannot transmit apoptotic death signals from the cytoplasm to the nucleus (15). Moreover, changes in the intracellular concentration of cations are responsible for alterations in cell volume, which is one of the most striking morphological changes during the process of apoptosis (16). But very little is known about the effects of these ionic changes on the activity of underlying apoptotic machineries, including caspases and nucleases.
Caspases are synthesized and maintained in the cytoplasm as proenzymes, which themselves must undergo a proteolytic activation, perhaps triggering apoptosis. The substrates cleaved by these enzymes are numerous, including structural proteins (17,18) and degradative enzymes (19). In addition to caspase activation, nucleases are activated, which destroy the genome to produce DNA fragments, recognized as the DNA ladder.
Leishmaniasis, one of the dreaded protozoal diseases threatening mankind, does not have enough combative measures. CPT has been shown to inhibit type I DNA topoisomerase of Leishmania donovani promastigotes and leads to apoptosis. To characterize the cellular events associated with apoptosis, we have found that CPT-induced oxidative stress causes depolarization of mitochondrial membrane potential. This is followed by the activation of caspase-like proteases inside leishmanial cells after the release of cytochrome c into the cytosol (20). But the molecular mechanisms connecting ion fluxes to the apoptotic machinery are still unknown, and these aspects remain to be investigated for the apoptotic cell death in unicellular parasites like L. donovani during treatment with CPT.
Here we have dissected the mechanism of action of CPT by analyzing the nuclear, mitochondrial, and cytosolic changes associated with apoptosis of leishmanial cells. In the present study we show that changes in the level of both cytosolic cations (calcium and potassium) and alterations in the level of ATP and pH regulate the apoptotic process by controlling the mitochondrial membrane potential and activity of caspase-like proteases and endonucleases. Taken together, our results provide the first insight into the mechanistic pathway of apoptosis in leishmanial cells where a decrease in cytosolic K ϩ ion concentrations and alterations in pH homeostasis by the topoisomerase I poison CPT appears to be an essential event responsible for the propagation of apoptosis.

EXPERIMENTAL PROCEDURES
Parasite Culture and Maintenance-Leishmania strain AG 83 promastigotes were grown at 22°C in M199 liquid media supplemented with 10% fetal calf serum.
Drug Solutions-CPT was dissolved in 100% Me 2 SO at 20 mM concentration and stored at Ϫ20°C. BHT, NAC, FFA, and verapamil were dissolved in 100% Me 2 SO at 50 mM concentration and stored at Ϫ20°C.
Measurement of ROS Level-Intracellular ROS level was measured in treated and untreated cells as described by Mukherjee et al. (21). Briefly, cells (2 ϫ 10 7 ) after different treatments were washed and resuspended in 500 l of medium 199 and were loaded with the cellpermeant probe 5-(and -6)-chloromethyl-2Ј,7Ј-dichlorodihydro-fluorescein diacetate acetyl ester. It is a nonpolar compound and is hydrolyzed within the cell to form a nonfluorescent derivative, which in the presence of a proper oxidant is converted to a fluorescent product. Fluorescence was measured through a spectrofluorometer by using 507 nm as excitation and 530 nm as emission wavelengths. For all measurements basal fluorescence was subtracted.
Intracellular Ca 2ϩ Measurement-Intracellular Ca 2ϩ concentration was measured with the fluorescent probe Fura 2AM as described by Sarkar and Bhaduri (22). Briefly, cells with differently treated groups were harvested and washed twice with wash buffer containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl 2 , 5.5 mM glucose, 1 mM CaCl 2 , and 50 mM MOPS, pH 7.4. Cells were then suspended in the same buffer containing 15% sucrose and were incubated with Fura 2AM (6 M) at 27°C for 1 h with mild shaking. Cells were pelleted down, and after two subsequent washing steps were suspended in the same wash buffer. Cells were visualized and quantified by TCS-SP Leica confocal microscope using a software, Z-profile measurement with statistics within the region of interest following the instruction manual (Leica Confocal Systems User Manual TCS SP2). Here this function was used to determine the maximum values of the intensity of Ca 2ϩ -bound Fura 2 within each parasite. It should be noted that 100 cells per group with comparable intensity were calculated for each condition. Another set of cells was used for spectrofluorometric analysis. Fluorescence measurements were performed with excitation at 340 nm and emission at 510 nm. To convert fluorescent values into absolute calcium concentration, calibration was performed at the end of the each experiment. Cytosolic Ca 2ϩ concentration was calculated using Equation 1, where K d is the dissociation constant of the calcium-bound Fura 2 complex, and F min represents the minimum fluorescence of the cells after adding 4 mM EGTA, 30 mM Tris-HCl, pH 7.4, and 0.1% Triton X-100 simultaneously to the sample. F max corresponds to the maximum fluorescence of the cells after adding 10 mM CaCl 2 to the above-treated cells.
Measurement of Intracellular K ϩ Level-Intracellular K ϩ level was measured in both treated and untreated cells using PBFI-AM as the cell-permeant probe (23). Briefly, cells (2 ϫ 10 7 ) after different treatments were harvested and were washed twice with wash buffer containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl 2 , 5.5 mM glucose, and 50 mM MOPS, pH 7.4. Cells were then suspended in the same buffer containing 15% sucrose and incubated with PBFI-AM (5 M) at 27°C for 1 h with mild shaking. Then cells were pelleted down and after two subsequent washing steps were suspended in the same wash buffer. Fluorescence was measured for the dye-treated and washed cells at 340 and 380 nm excitation and at 500 nm as emission wavelength. PBFI shows an excitation spectral shift upon binding and so was calibrated by using a ratio of the fluorescence intensities at two different wavelengths in varied K ϩ buffers in the presence of valinomycin (24). The value of intracellular K ϩ level in experimental samples was calculated from the calibration curve.
Assay for Na ϩ -K ϩ -ATPase Activity-The plasma membrane of L. donovani promastigotes has a characteristic structure that provides strength to the membrane against hypotonic shock. Unsealed ghosts devoid of flagella were prepared according to Mukherjee et al. (25). Briefly, 0.5 g of treated and untreated cells were collected at mid-log phase and suspended in 25 ml of 5 mM Tris-HCl, pH 7.4, containing 0.5 mM PMSF. Cell suspensions were kept at 4°C and were mixed by mild vortexing to detach flagella from the cell body. Formation of unsealed ghosts was confirmed by total leakage of the marker cytoplasmic enzymes (26).
Aliquots of each unsealed ghost (about 5 mg protein/ml) were incubated in a medium containing 3 mM ATP, 4 mM MgCl 2 , 1 mM DTT, 1 mM EGTA, 110 mM Tris-HCl, pH 7.4, 10 g/ml leupeptin, 10 g/ml soybean trypsin inhibitor, 15 g/ml bovine serum albumin, 120 mM NaCl, 30 mM KCl, and 75 nmol of [␥-32 P]ATP (0.15 Ci, specific activity 3000 Ci/ nmol) to a total volume of 150 l. After 45 min of incubation at 37°C, the reaction was stopped by adding trichloroacetic acid to a final concentration of 8%. A 150-l suspension of 50% activated charcoal in water and 10 l of 100 mM KH 2 PO 4 were added to the above suspension. After mild agitation for 10 min, charcoal was precipitated by centrifugation. The process was repeated two more times, and finally 100 l of the supernatant was transferred to a scintillation counter.
Measurement of Mitochondrial Membrane Potential-The fluorescence of the mitosenser reagent (BD Biosciences) is considered as an indicator of relative mitochondrial energy state. When mitochondrial membrane depolarizes, the dye remains as a monomer (emission 530 nm, green fluorescence), but during normal or higher membrane potential, the dye remains as an aggregate (red fluorescence, emission 590 nm) (20). Briefly, cells after different treatments were harvested and washed with 1ϫ PBS. Cells were then incubated at 37°C in a 5% CO 2 incubator for 1 h with a final concentration of mitosensor reagent (BD Biosciences) at 5 g/l (according to the manufacturer's protocol). Cells were then analyzed by flow cytometry as well as by fluorescence measurements. The ratio of the reading at 590 nm to the reading at 530 nm (590/530 ratio) was considered as the relative ⌬ m value.
Measurement of ATP Level-The ATP content was determined by the luciferin-luciferase method (27). The assay is based on the requirement of luciferase for ATP in producing light (emission maximum 560 nm at pH 7.8). Briefly, cells (2 ϫ 10 7 ) after different treatments were harvested and resuspended in 1ϫ PBS. An aliquot of this cell suspension was assayed for ATP using the Sigma chemical luciferase ATP assay kit. The amount of ATP in experimental samples was calculated from a standard curve prepared with ATP and expressed as nmol/10 7 cells.
Intracellular pH Measurements-Intracellular pH was measured with the fluorescent probe BCECF as described by Mukherjee et al. (25). Briefly, cells were harvested and washed two times with wash buffer containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl 2 , 5.5 mM glucose, and 50 mM MOPS, pH 7.4. Cells were then suspended in the same buffer containing 15% sucrose and incubated with BCECF-AM (10 M) at 27°C for 1 h with mild shaking. The cells were pelleted and after two subsequent washing steps were suspended in the same wash buffer. Fluorescence was measured for the dye-treated and washed cells in a Hitachi spectrofluorometer (model F4010) at 440 and 490 nm excitation and at 535 nm emission wavelengths. Calibration of the internal pH of the control promastigotes was done by incubating the BCECF-loaded cells in the varied pH buffers in the presence of nigericin (1 g/ml). The value of pH in the experimental samples was calculated from the calibration curve.
Preparation of Cytoplasmic Extract-Cytoplasmic extracts were prepared both in treated and untreated cells according to Das et al. (28). Briefly, cells (2 ϫ 10 7 ) after different treatments were harvested, suspended in cell extraction buffer (20 mM HEPES-KOH, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 200 M PMSF, 10 g/ml leupeptin and pepstatin), and lysed by a process of freeze-thaw using nitrogen cavitation and a 37°C water bath simultaneously. Then lysate was centrifuged at 10,000 ϫ g for 1 h, and supernatants were used as a source of cytoplasmic extract. Protein was estimated by the Bio-Rad protein determination kit.
Determination of Caspase Activity-Cytosolic extracts from untreated leishmanial cells were adjusted to different pH values by mixing various concentrations of NaOH or HCl solutions. To initiate caspaselike protease activation, 10-l aliquots of the extracts were incubated with cytochrome c (10 M) and/or 1 mM dATP at 30°C for 1 h. For caspase activity measurements, 4 l of the mixture (30 g of protein) was brought to 100 l in caspase buffer (final concentration 50 mM Tris-HCl, 50 mM KCl, 10% sucrose, 0.1% CHAPS, 10 mM DTT) together with 100 M DEVD-AFC. AFC release was measured fluorometrically at 505 nm (29).
Isolation of Nuclear Fraction from Leishmanial Cells-Nuclear fractions were prepared from CPT-treated and untreated cells according to Chakraborty et al. (30). Briefly, cells were suspended in a hypotonic buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mM EGTA, 1 mM PMSF, 1 mM benzamidine hydrochloride, and 5 mM DTT) and were homogenized. The homogenate was centrifuged for 20 min at 10,000 rpm in Sorvall RC5B centrifuge using an SS34 rotor. The pellet was washed with the same buffer and centrifuged as above. The pellet was suspended in Tris-HCl, pH 7.5, and was used as a source for nuclear fraction.
Preparation of Nuclear Extract from Leishmanial Cells-To prepare nuclear extract from isolated nuclear fractions of CPT-treated leishmanial cells, nuclei were suspended in 400 mM NaCl, 1 mM EDTA, and 20 mM Tris, pH 7.5, and were ultracentrifuged at 1,65,000 ϫ g at 4°C for 1 h. Supernatants were used as a source of nuclear extract like other mammalian cells (31,32).
HeLa Nuclei Assay-The nuclei from healthy growing HeLa cells were isolated according to Schwartzman and Cidlowski (31,32). Briefly, cells were harvested and lysed in ice-cold 10 mM MgCl 2 and 0.25% Nonidet P-40. Nuclei were pelleted down and suspended in 50 mM Tris-HCl, pH 7.5. For the DNA fragmentation assay, 2 ϫ 10 7 nuclei were suspended in CPT-treated nuclear extract of leishmanial cells and incubated at 37°C for 3 h in the presence of 2 mM MgCl 2 and 1 mM CaCl 2 . After incubation, 25 mM EDTA, 540 mM NaCl, and 0.5% SDS were added to these solutions. Then proteinase K was added to a final concentration of 0.5 mg/ml and was incubated at 55°C for 1 h. This was followed by phenol/chloroform extraction. Then DNAs were precipitated, dissolved in TE buffer, and electrophoresed in 1.5% agarose gel.
Double Staining with Hoechst 33342 and Propidium Iodide-Condensation of genomic DNA is one of the characteristic nuclear phenotypes of apoptotic cells. To distinguish between apoptotic, necrotic, and viable cells, these were stained with Hoechst 33342 and PI according to Das et al. (28). Briefly, after fixing with 2% paraformaldehyde, cells were incubated with 0.2% Triton X-100 for 5 min for permeabilization, washed with 1ϫ PBS, and incubated with Hoechst 33342 and PI for 10 min. Then cells were visualized with a TCS-SP Leica confocal microscope. Healthy viable cells were stained only with Hoechst and were identified by dull blue nuclei, but apoptotic cells were identified with bright blue nuclei. PI only penetrates necrotic cells and stained the nuclei red. Total cells versus only bright blue cells were calculated, and data are expressed as percentage of apoptotic cells. It should be noted that 100 cells per group with identical morphology were calculated for each condition.
Statistical Analysis-Data are expressed as mean Ϯ S.D. unless mentioned. Comparisons were made between different treatments using unpaired Student's t test.

RESULTS
CPT Induces Oxidative Stress in L. donovani Promastigotes-When L. donovani promastigotes were treated with CPT, ROS were generated inside cells (20), which can be measured fluorometrically by conversion of 5-(and -6)-chloromethyl-2Ј,7Јdichlorodihydro-fluorescein diacetate acetyl ester to highly fluorescent 2,7-dichlorofluorescein in the presence of a proper oxidant. The level of ROS in CPT-treated cells remains 4-fold higher compared with the levels of ROS of control cells throughout the experiment (Fig. 1). When cells were treated with NAC prior to the treatment with CPT, the level of ROS generation was reduced to the extent of 80% compared with that in Me 2 SO-treated control cells.
CPT-induced Oxidative Stress Inactivates Na ϩ -K ϩ -ATPase Pump and Subsequently Decreases Intracellular Potassium Level-Extrusion of K ϩ ions from cells has been suggested to mediate the loss of cell volume, which is one of the most significant events in apoptosis (23). Here we analyzed K ϩ levels in these parasites using the dye PBFI-AM that is specific for intracellular K ϩ . As shown in Fig. 2A, the PBFI-AM fluorescence decreased more than 65% in the CPTtreated leishmanial cells relative to the normal cells, demonstrating that lowered K ϩ levels are restricted to the apoptotic cells. The decrease in intracellular K ϩ ions is because of impairment of the Na ϩ -K ϩ -ATPase pump, which is a main mechanism for the regulation of K ϩ ions inside cells both in higher eukaryotes as well as in protozoan parasites (33,34). So we have measured the activity of the Na ϩ -K ϩ -ATPase pump during treatment with CPT. The activity of Na ϩ -K ϩ -ATPase was about 56 nmol/mg protein/min, which remained almost unchanged throughout the experiment. In the presence of CPT, the activity of Na ϩ -K ϩ -ATPase decreased to the extent of 48% after 1 h. The inhibition of the activity of this P-type ATPase further decreased to 67% after 2 h of treatment with CPT. Based on the data published elsewhere (20), it appears that the impairment of Na ϩ -K ϩ -ATPase was because of both formation of ROS inside the cell and an increase in lipid peroxidation. This was confirmed by recovery of the Na ϩ -K ϩ -ATPase activity to about 80% after treating cells with different antioxidants like NAC (20 mM) and BHT (20 mM) (a specific inhibitor of lipid peroxidation) prior to the treatment with CPT (Fig. 2B).

CPT-induced Oxidative Stress Causes Increase in Cytosolic
Ca 2ϩ Level-Many studies have shown that calcium flux is absolutely necessary not only for the activation of different proteases (35) but also for the appearance of phosphatidylserine on the outer leaflet of the plasma membrane (36) during apoptosis. We have shown by confocal microscopy and spectrofluorometric analysis that over the time course of drug treatment leishmanial cells showed a significant increase in intracellular calcium (Fig. 3). Control leishmanial cells maintained intracellular [Ca 2ϩ ] at 85 nM, and the concentration increases with the increase in ROS formation of CPT-treated cells at different times to the extent of 140 nM (Fig. 3A). This was confirmed by a decrease in elevation of cellular Ca 2ϩ level during treatment with different antioxidants like NAC and BHT separately prior to the treatment with CPT. To investigate possible sources for the elevation of intracellular calcium, we have treated cells with a selective calcium channel blocker like verapamil (100 nM) and a nonselective cation channel blocker like FFA separately prior to the treatment with CPT. Each can prevent the increase of intracellular calcium level significantly (Fig. 3B). Thapsigargin (TG) causes an increase in cytosolic calcium levels by inhibiting the specific sarcoplasmic reticulum Ca 2ϩ -ATPase pump (37). When TG (15 M) was added to CPT-treated cells, a further increase in Ca 2ϩ level does not occur, indicating that CPT induces the release of Ca 2ϩ from an intracellular pool (Fig. 3B). The result is also consistent with the changes in maximum intensity of Ca 2ϩ -bound Fura 2 dye, which were quantitated by confocal microscopy, and these were represented in separate histograms (Fig. 3C). When cells were treated with CPT, maximum intensity was increased, whereas the level of maximum intensity was decreased during treatment with NAC, BHT, FFA, and verapamil separately prior to the treatment with CPT. The maximum intensity of Ca 2ϩ -bound Fura 2 dye was not further increased when TG was added to CPT-treated cells. The above observation clearly confirmed that CPT-induced oxidative stress causes damage in both selective and nonselective cation channels and provides definite reasons for the increase in Ca 2ϩ in CPT-treated cells through influx of extracellular Ca 2ϩ and release of Ca 2ϩ from intracellular pools.
Increase in Cytosolic Calcium Results in Mitochondrial Depolarization-Dissipation of ⌬ m (mitochondrial transmembrane potential) is a characteristic feature of apoptosis (38). To determine the changes in the ⌬ m , we used the BD Biosciences mitosensor reagent, which is a cationic dye that aggregates in the mitochondria of healthy cells. These aggregates fluoresce red at higher potentials, but at a lower potential this reagent cannot accumulate in the mitochondria and remains as monomers in the cytoplasm that fluoresces green. To confirm the sensitivity of the reagent to the change in mitochondrial mem- brane potential, leishmanial cells were treated with the mitochondrial uncoupling agent CCCP (1 M). Within 1 h of treatment with CCCP, the entire cell population shifted to the right side of the FL-1 channel, indicating the depolarization of mitochondrial membrane potential. The sharp increase in mean green fluorescence intensity as determined by fluorescenceactivated cell sorter analysis is also consistent with a decrease in the ratio of 590/530 as measured by spectrofluorometeric reading. Endogenous toxic ROS is responsible for elevation in cytosolic Ca 2ϩ levels, and this is linked to the dissipation of mitochondrial membrane potential. It was confirmed that when cells were treated with the L-type voltage-gated channel inhibitor (verapamil) or with the nonspecific calcium channel inhibitor (FFA) before the treatment with CPT, loss of mitochondrial membrane potential was prevented to the extent of 68% (Fig. 4). This was also confirmed by the ratio of fluorescence intensity (590/530) as represented in Table I.

Decrease in Mitochondrial Membrane Potential Causes Depletion in Cytosolic ATP Level and a Decrease in Intracellular
pH-In CPT-treated leishmanial cells, disruption in the function of mitochondria caused reduced ATP generation. Here we have measured the ATP level in differently treated cells. As shown in Fig. 5A, there was a gradual fall in ATP level to the extent of 75% after 3.5 h of CPT treatment. Treatments with different antioxidants like NAC or BHT prior to the treatment with CPT prevent further a decrease in ATP level. Similar results were also obtained when cells were treated with selective Ca 2ϩ channel inhibitor (verapamil) or with nonselective Ca 2ϩ channel inhibitor (FFA) prior to treatment with CPT. This may be due to the fact that both an increase in ROS and intracellular Ca 2ϩ are responsible for the loss of ⌬⌿ m , which subsequently decreases cellular ATP level. Moreover, a tight link exists between the intracellular ATP level and the number of apoptotic cells during treatment with CPT. As shown in Fig.  5B, a decrease in ATP level is consistent with an increase in the number of apoptotic cells that were detected by their condensed nuclei, which exhibited a bright blue fluorescence in the presence of Hoechst 33342 dye, but no red fluorescence of PI was observed in these apoptotic cells.
A fluorescent method was adopted to measure intracellular pH levels in leishmanial cells treated with or without CPT by using the dye BCECF. Leishmanial cells were loaded with acetomethyl ester derivative of BCECF (BCECF-AM), a dye whose fluorescence emission is sensitive to pH variations. Cell samples were excited at the 440 and 490 nm wavelengths, and the emission was recorded at a single wavelength of 535 nm. The ratio of fluorescence excitation was used as a quantitative measure of the pH, independent of cell volume or dye concentration. The pH of untreated leishmanial cells was 7.4 Ϯ 0.03. A significant decrease in the level of pH was observed to the extent of 7.1 immediately after loss of ⌬ m upon treatment with CPT for 2 h. This was further decreased to 6.8 Ϯ 0.05 after 3.0 h of treatment with CPT (Fig. 5C). These experiments show that leishmanial cells treated with CPT become significantly more acidic than untreated cells. But treatment with NAC (20 mM), BHT (20 mM), and FFA (250 M) separately prior to CPT treatment can prevent the decrease of intracellular pH.
CPT-induced Decrease in Intracellular pH and K ϩ Enhances Cytochrome c-induced Caspase-like Protease Activation-Release of cytochrome c into cytosol is a common event in an apoptotic process irrespective of apoptotic stimuli both in mammalian cells as well as in protozoan parasites like L. donovani (20,39). This was followed by activation of caspases, which is a central event that occurs upstream of DNA fragmentation (40).
To determine the effect of K ϩ on the activity of mature caspase 3-like proteases in CPT-treated L. donovani promastigotes, we have prepared cytoplasmic extracts from CPTtreated leishmanial cells and incubated the extract at 30°C with different concentrations of KCl (0 -200 mM). The substrate DEVD-AFC was added to this extract, and the release of AFC was used as a measure of mature caspase 3-like protease activity in CPT-treated cells. It was observed that the activity of the mature protease was not altered significantly in leishmanial cells (Fig. 6A). Similarly, to assess the effects of K ϩ on the activation of the pro-enzyme, we incubated the extracts from untreated leishmanial cells with dATP and cytochrome c for 1 h in the presence of increasing concentrations of KCl at 30°C. Then DEVD-AFC was added to determine the activity of this protease. At a lower K ϩ ion concentration, caspase 3-like protease activity is induced, whereas at a higher concentration the activity decreases (Fig. 6B). Thus, the physiological concentration of K ϩ expected to be found in nonapoptotic leishmanial cells can directly inhibit the activity of pro-caspase 3-like enzymes, but it has no effect on mature caspase 3-like proteases.
To see the effects of a decrease in cytosolic pH on the activity  4. Flow cytometry analysis of mitochondrial membrane potential. Changes in mitochondrial membrane potential after treatment with CCCP, 0.2% Me 2 SO alone (control), and 5 M CPT for 2.5 and 5 h, respectively. This was also determined after treatment with FFA and verapamil separately prior to treatment with CPT. FL1 channel indicates green fluorescence intensity. of pro-caspase 3-like proteases in cytosol, we prepared cytosolic extracts from untreated leishmanial cells, buffered them to various pH values, and then added cytochrome c in combination with dATP and 50 mM KCl in the assay mixture. Caspase activation induced by the combination of cytochrome c and dATP exhibited pH dependence, optimal activation occurring at pH 6.3-7.0. At pH 6.8, which represents the approximate cytosolic pH observed in cells undergoing apoptosis by CPT (Fig. 6C), the efficiency of cytochrome c/dATP-stimulated caspase activation was 1.8-fold higher than at physiological pH 7.4 (Fig. 6C). Moreover, the progress curve obtained at pH 6.8, 6.3, and 7.0 exhibited roughly similar slopes, but with a pH- dependent delay in the time of onset of caspase activity following addition of cytochrome c and dATP. It indicates that lower pH accelerates the rate of formation of active apoptotic complex, as opposed to increasing the V max of cytochrome c and dATP-activated caspase 3-like proteases. So, the pH-dependent differences in cytochrome c/dATP-induced caspase activation were not due to faster substrate depletion as determined by evaluation of enzyme activities at different pH values (Fig. 6D).
CPT-induced Decrease in Intracellular K ϩ Level Facilitates Apoptotic DNA Fragmentation in Vitro-Apoptotic DNA fragmentation was observed in vitro by incubation of isolated nuclei from log phase culture of leishmanial cells with Ca 2ϩ and Mg 2ϩ at 37°C by a process known as autodigestion. The nuclease present within the nuclei of untreated leishmanial cells in an inactive form, perhaps as a proenzyme, becomes activated in the presence of Ca 2ϩ and Mg 2ϩ during incubation at 37°C. When KCl was included in this reaction, DNA fragmentation was suppressed in a dose-dependent manner (Fig. 7A). At a physiological concentration of K ϩ (150 mM), inhibition of DNA fragmentation was observed, which suggests that K ϩ efflux from apoptotic cells is an important regulator of chromatin degradation.
Autodigestion of leishmanial cells consists of essentially two molecular events, activation of a latent nuclease and degradation of the chromatin structure. Treatments with CPT cause the activation of latent nucleases, which were present in their nuclear extract. When CPT-treated nuclear extracts from leish-manial cells were added to nuclei from healthy growing HeLa cells (that do not have the capacity to autodigest their own nuclei) in the presence or absence of KCl (31,32,41), DNA fragmentation is inhibited in a dose-dependent manner as shown in the case of autodigestion of leishmanial cells. But nuclear extracts from CPT-treated cells, pre-treated with the CED-3/CPP32 group of protease inhibitor VAD-fmk, show no capacity to digest nuclei from HeLa cells even in presence of 50 mM KCl. This suggests that physiological concentration of K ϩ has a direct inhibitory effect on the active apoptotic nuclease, and a decrease in intracellular K ϩ concentration is a prerequisite for apoptotic DNA degradation. DISCUSSION Apoptosis or programmed cell death can be activated in variety of cells through diverse signaling pathways. However, all apoptotic stimuli result in a highly conserved series of morphological and biochemical changes both in mammalian cells as well as in protozoan parasites, suggesting a common pathway distal to cell-specific events. But very little is known about the importance of cytosolic cations in the process of apoptosis particularly in the case of protozoan parasites like L. donovani.
In earlier studies we have found that CPT-induced formation of ROS inside leishmanial cells causes an increase in the level of lipid peroxidation (20). In general, the overall effect of lipid peroxidation is to decrease membrane fluidity, as well as in- crease leakiness of the membrane leading to complete loss of membrane integrity (42). This in turn causes an increase in intracellular Ca 2ϩ level inside cells, which is a common feature of apoptosis. The elevation in cytosolic Ca 2ϩ causes cellular damage and death through disruption of the cytoskeletal networks and the action of Ca 2ϩ -stimulated catabolic enzymes, such as protease, phospholipases, and endonucleases, which are involved in the nuclear apoptosis in mammalian cells (35). Here in the case of leishmanial cells, CPT-induced formation of ROS causes an increase in cytosolic Ca 2ϩ due to opening of nonselective and L-type voltage-gated calcium channels and causes dysregulation of sarcoplasmic Ca 2ϩ -ATPase channels. The excessive free cytosolic Ca 2ϩ leads to uncoupling of mitochondrial oxidative phosphorylation and directs the cell to follow the executionary part of apoptosis.
Several studies (38) support the hypothesis that disruption of ⌬⌿ m is an irreversible commitment of the cell to death. One of the consequences of this is the release of apoptogenic factors including cytochrome c into the cytosol of mammalian cells as well as in leishmanial cells after treatment with CPT (20,43). But concerning the cellular bio-energy of leishmanial cells in apoptosis, it is observed that the ATP level gradually decreases as opposed to mammalian cells. This may be due to the fact that in higher eukaryotes not all mitochondria are responsible for cytochrome c release and that a portion maintain their transmembrane potential and supply ATP to continue the apoptosis. Thus it appears that healthy mitochondria ensure compensation for the injured ones in mammalian cells after induction of apoptosis. But for organisms with single mitochondria like L. donovani, there is no possibility for the compensation of injured mitochondria and survival depends on proper function-ing of a single organelle. In the absence of proper functional mitochondria, cells would cease to synthesize ATP from their mitochondrial source and cause a rapid decrease in cellular ATP levels to the extent of 75%. Our result is consistent with the study reported earlier (21) that the ATP level gradually decreases after the loss of ⌬⌿ m during treatment with H 2 O 2 . But ATP is a key molecule for chromatin condensation, nuclear fragmentation and regulation, and maintenance of ion homeostasis during apoptosis. So we can assume that the ATP levels generated before the loss of ⌬⌿ m and ATP supplied by glycolysis are sufficient to carry out these cellular activities and to propagate apoptosis in leishmanial cells. This was also confirmed when cells were treated with oligomycin, where about 82% leishmanial cells had undergone apoptosis (data not shown) unlike other mammalian cells (44).
On the other hand, immediately after the loss of ⌬⌿ m , protons are released into the cytosol from mitochondria, and this contributes to the intracellular acidification in leishmanial cells like other mammalian cells (45). The pH changes modulate the apoptotic responsiveness of the cell as well as amplify the apoptotic program by regulating the activity of caspase-like proteases during treatment with CPT in leishmanial cells. The pH-dependent differences in cytochrome c and dATP-induced caspase activation depend on the rate of formation of active complex in CPT-treated leishmanial cells. In addition, the activity of caspase 3-like proteases also depends on the concentration of K ϩ inside the cells. At a lower concentration of K ϩ , caspase 3-like protease activity was inhibited at the level of cleavage of pro-enzyme but not at the level of mature enzyme, suggesting that a physiological concentration of K ϩ is inhibitory for the activation of caspase 3-like proteases. Intracellular K ϩ is known to be one of the most important determinants for the maintenance of ionic balance, which is directly related to osmotic pressure inside cells (33). Most of the cells can achieve and maintain this osmotic balance through the continuous activity of an ATP-dependent Na ϩ -K ϩ -ATPase pump that exchanges 3Na ϩ for 2K ϩ against the electrochemical gradient (34). Here in case of leishmanial cells, CPT-induced oxidative stress and lipid peroxidation cause impairment of the Na ϩ -K ϩ -ATPase pump, which consequently decreases in intracellular K ϩ levels and facilitates apoptosis through the increase in the activity of these death enzymes. Collectively, it can be inferred that a decrease in intracellular pH level and [K ϩ ] are prerequisite events during apoptosis and are needed for the activation of caspase 3-like proteases in leishmanial cells during treatment with CPT.
Downstream of the caspase 3-like protease activation, nucleases become active and cause apoptotic DNA fragmentation in leishmanial cells during treatment with CPT. Evidence presented here also supports the fact that the nuclear extract from CPT-treated leishmanial cells causes degradation of HeLa nuclei but was suppressed at 150 mM concentration of K ϩ ions. So the importance of K ϩ ions to the apoptotic nuclease activity can be explained in such a way that it is normally present within a cell in inhibitory concentration and therefore is in a position to apply a tonic suppressive force on the nuclease activity. Changes in the level of K ϩ ions inside CPT-treated leishmanial cells may cause alteration in ionic strength, which can affect various aspects of structure and function of caspase 3-like proteases and nucleases. However, further studies are needed to determine the actual mechanism as to how intracellular K ϩ suppresses the enzymatic activity of these death enzymes during treatment with CPT in leishmanial cells.
In conclusion, our study demonstrates for the first time that apoptosis in leishmanial cells is orchestrated by the coordinated alterations in ion fluxes and subsequent activation of caspase 3-like proteases and endonucleases during treatment with CPT. Moreover, modification of pH by the topoisomerase I poison CPT likely represents an essential event responsible for the propagation of apoptosis and can act as a central regulator of the apoptotic machineries. So understanding the components and the steps involved in this intricate process provides the opportunities for discovering and evaluating molecular targets for drug designing, which now form a rational basis for development of improved therapy against leishmaniasis.