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Diverse effects on mitochondrial and nuclear functions elicited by drugs and genetic knockdowns in bloodstream stage Trypanosoma brucei.

Worthen C, Jensen BC, Parsons M - PLoS Negl Trop Dis (2010)

Bottom Line: Reduction in ATP levels lagged behind decreases in live cell number.Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds.Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent proliferation may identify new means to activate death pathways.

View Article: PubMed Central - PubMed

Affiliation: Seattle Biomedical Research Institute, Seattle, Washington, USA.

ABSTRACT

Background: The options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. New drugs that kill the causative agents, subspecies of Trypanosoma brucei, are therefore urgently needed. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage.

Methodology/principal findings: We therefore conducted the first side by side comparison of the cellular effects of multiple death inducers that target different systems in bloodstream form parasites, including six drugs (pentamidine, prostaglandin D(2), quercetin, etoposide, camptothecin, and a tetrahydroquinoline) and six RNAi knockdowns that target distinct cellular functions. All compounds tested were static at low concentrations and killed at high concentrations. Dead parasites were rapidly quantified by forward and side scatter during flow cytometry, as confirmed by ethidium homodimer and esterase staining, making these assays convenient for quantitating parasite death. The various treatments yielded different combinations of defects in mitochondrial potential, reactive oxygen species, cell cycle, and genome segregation. No evidence was seen for phosphatidylserine exposure, a marker of apoptosis. Reduction in ATP levels lagged behind decreases in live cell number. Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds.

Conclusions/significance: Parasites showed different phenotypes depending on the treatment, but none of them were clear predictors of whether apparently live cells could go on to proliferate after drugs were removed. We therefore suggest that clonal proliferation assays may be a useful step in selecting anti-trypanosomal compounds for further development. Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent proliferation may identify new means to activate death pathways.

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Annexin V staining of populations with dying cells.A) Annexin V and PI staining of unfixed cells. Untreated cells show predominantly annexin V-negative, PI-negative staining (lower left quadrant). All drug treated cells showed additional populations with higher annexin staining coupled with low to high PI staining. B) Annexin-positive cells are dead. The pentamidine-treated population shown in A was analyzed according to light scattering properties. The low forward scatter, dead population (D) contained all annexin-stained cells, including those with low to high PI staining. The live population contained the annexin-negative, PI-negative cells.
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pntd-0000678-g004: Annexin V staining of populations with dying cells.A) Annexin V and PI staining of unfixed cells. Untreated cells show predominantly annexin V-negative, PI-negative staining (lower left quadrant). All drug treated cells showed additional populations with higher annexin staining coupled with low to high PI staining. B) Annexin-positive cells are dead. The pentamidine-treated population shown in A was analyzed according to light scattering properties. The low forward scatter, dead population (D) contained all annexin-stained cells, including those with low to high PI staining. The live population contained the annexin-negative, PI-negative cells.

Mentions: Two of the drugs included in these studies, PGD2 and quercetin, have been implicated as eliciting a possible PCD mechanism in T. brucei. One hallmark of the apoptotic mode of PCD is the depolarization of the plasma membrane, in which the normally cytoplasmically facing phosphatidylserine is flipped to external face of the intact membrane bilayer. Exposed phosphatidylserine can be detected with annexin-V, a cell-impermeant protein. To verify that the cells have not lost membrane integrity (which would allow annexin-V binding to internal phosphatidylserine), the assays used non-fixed cells and included the membrane-impermeable stain PI. A parasite undergoing canonical apoptotic PCD should stain positive for annexin-V, but negative for PI. Under the conditions used, we never observed a clear annexin-positive/PI-negative population. Untreated cultures showed few cells staining with PI or annexin. The treated cultures all had similar patterns of staining (Figure 4A shows results from four drugs). Populations with moderate to high annexin staining emerged, and all showed increased PI staining. When the samples were gated on scatter (Figure 4B), all of the annexin-positive populations showed the low forward scatter indicative of dead cells. The reasons for multiple annexin-positive, PI-positive populations are unclear, but cells with sub-G1 DNA content are clearly present (as seen in Figure 3) and DNA degradation could reduce staining with PI.


Diverse effects on mitochondrial and nuclear functions elicited by drugs and genetic knockdowns in bloodstream stage Trypanosoma brucei.

Worthen C, Jensen BC, Parsons M - PLoS Negl Trop Dis (2010)

Annexin V staining of populations with dying cells.A) Annexin V and PI staining of unfixed cells. Untreated cells show predominantly annexin V-negative, PI-negative staining (lower left quadrant). All drug treated cells showed additional populations with higher annexin staining coupled with low to high PI staining. B) Annexin-positive cells are dead. The pentamidine-treated population shown in A was analyzed according to light scattering properties. The low forward scatter, dead population (D) contained all annexin-stained cells, including those with low to high PI staining. The live population contained the annexin-negative, PI-negative cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864271&req=5

pntd-0000678-g004: Annexin V staining of populations with dying cells.A) Annexin V and PI staining of unfixed cells. Untreated cells show predominantly annexin V-negative, PI-negative staining (lower left quadrant). All drug treated cells showed additional populations with higher annexin staining coupled with low to high PI staining. B) Annexin-positive cells are dead. The pentamidine-treated population shown in A was analyzed according to light scattering properties. The low forward scatter, dead population (D) contained all annexin-stained cells, including those with low to high PI staining. The live population contained the annexin-negative, PI-negative cells.
Mentions: Two of the drugs included in these studies, PGD2 and quercetin, have been implicated as eliciting a possible PCD mechanism in T. brucei. One hallmark of the apoptotic mode of PCD is the depolarization of the plasma membrane, in which the normally cytoplasmically facing phosphatidylserine is flipped to external face of the intact membrane bilayer. Exposed phosphatidylserine can be detected with annexin-V, a cell-impermeant protein. To verify that the cells have not lost membrane integrity (which would allow annexin-V binding to internal phosphatidylserine), the assays used non-fixed cells and included the membrane-impermeable stain PI. A parasite undergoing canonical apoptotic PCD should stain positive for annexin-V, but negative for PI. Under the conditions used, we never observed a clear annexin-positive/PI-negative population. Untreated cultures showed few cells staining with PI or annexin. The treated cultures all had similar patterns of staining (Figure 4A shows results from four drugs). Populations with moderate to high annexin staining emerged, and all showed increased PI staining. When the samples were gated on scatter (Figure 4B), all of the annexin-positive populations showed the low forward scatter indicative of dead cells. The reasons for multiple annexin-positive, PI-positive populations are unclear, but cells with sub-G1 DNA content are clearly present (as seen in Figure 3) and DNA degradation could reduce staining with PI.

Bottom Line: Reduction in ATP levels lagged behind decreases in live cell number.Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds.Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent proliferation may identify new means to activate death pathways.

View Article: PubMed Central - PubMed

Affiliation: Seattle Biomedical Research Institute, Seattle, Washington, USA.

ABSTRACT

Background: The options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. New drugs that kill the causative agents, subspecies of Trypanosoma brucei, are therefore urgently needed. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage.

Methodology/principal findings: We therefore conducted the first side by side comparison of the cellular effects of multiple death inducers that target different systems in bloodstream form parasites, including six drugs (pentamidine, prostaglandin D(2), quercetin, etoposide, camptothecin, and a tetrahydroquinoline) and six RNAi knockdowns that target distinct cellular functions. All compounds tested were static at low concentrations and killed at high concentrations. Dead parasites were rapidly quantified by forward and side scatter during flow cytometry, as confirmed by ethidium homodimer and esterase staining, making these assays convenient for quantitating parasite death. The various treatments yielded different combinations of defects in mitochondrial potential, reactive oxygen species, cell cycle, and genome segregation. No evidence was seen for phosphatidylserine exposure, a marker of apoptosis. Reduction in ATP levels lagged behind decreases in live cell number. Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds.

Conclusions/significance: Parasites showed different phenotypes depending on the treatment, but none of them were clear predictors of whether apparently live cells could go on to proliferate after drugs were removed. We therefore suggest that clonal proliferation assays may be a useful step in selecting anti-trypanosomal compounds for further development. Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent proliferation may identify new means to activate death pathways.

Show MeSH
Related in: MedlinePlus