Limits...
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.

Show MeSH

Related in: MedlinePlus

Representative growth curves for RNAi cell lines targeting TOPIBL, NOPP44/46, or TOPIImt.Tet was added at day 0 to initiate destruction of the targeted RNA. The RNAi-induced cells from the final day of the growth curve were used in the experiments shown in Figure 6. The percent of dead cells at that point is listed. NOPP44/46 protein levels were reduced 44% as shown by immunoblotting (unpublished results).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2864271&req=5

pntd-0000678-g005: Representative growth curves for RNAi cell lines targeting TOPIBL, NOPP44/46, or TOPIImt.Tet was added at day 0 to initiate destruction of the targeted RNA. The RNAi-induced cells from the final day of the growth curve were used in the experiments shown in Figure 6. The percent of dead cells at that point is listed. NOPP44/46 protein levels were reduced 44% as shown by immunoblotting (unpublished results).

Mentions: Three other RNAi or conditional knockout lines were chosen for analysis because they target disparate key functions of the cell. PEX19 is involved in targeting of proteins to the glycosome, which compartmentalizes the first steps of glycolysis and glycerol metabolism [21], [36]. Failure to properly localize glycosomal proteins leads to the accumulation of toxic levels of intermediates in these pathways [37] and cell death [38]. KREPA3 is a component of the mitochondrial RNA editosome, which prepares mitochondrial transcripts for translation by insertion or deletion of uridines [19]. The nucleolar protein NOPP44/46 is involved in biogenesis of the large ribosomal subunit [22]. For RNAi lines, addition of Tet to the media induces knockdown of the corresponding mRNA. For the conditional knockout of KREPA3, removal of Tet blocks the expression of the complementing gene. Although some of these genes had not previously been tested for essentiality in BF (TOPIBL and TOP1BS, TOPIImt, and NOPP44/46), all strains showed compromised growth after disruption of gene expression (Figure 5 and Figure S4). From initial growth curves, we determined when the growth of the strains was strongly affected, which varied depending on the gene. Within the period when population expansion was strongly impacted and before resistant populations emerged, samples were analyzed with selected assays.


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)

Representative growth curves for RNAi cell lines targeting TOPIBL, NOPP44/46, or TOPIImt.Tet was added at day 0 to initiate destruction of the targeted RNA. The RNAi-induced cells from the final day of the growth curve were used in the experiments shown in Figure 6. The percent of dead cells at that point is listed. NOPP44/46 protein levels were reduced 44% as shown by immunoblotting (unpublished results).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000678-g005: Representative growth curves for RNAi cell lines targeting TOPIBL, NOPP44/46, or TOPIImt.Tet was added at day 0 to initiate destruction of the targeted RNA. The RNAi-induced cells from the final day of the growth curve were used in the experiments shown in Figure 6. The percent of dead cells at that point is listed. NOPP44/46 protein levels were reduced 44% as shown by immunoblotting (unpublished results).
Mentions: Three other RNAi or conditional knockout lines were chosen for analysis because they target disparate key functions of the cell. PEX19 is involved in targeting of proteins to the glycosome, which compartmentalizes the first steps of glycolysis and glycerol metabolism [21], [36]. Failure to properly localize glycosomal proteins leads to the accumulation of toxic levels of intermediates in these pathways [37] and cell death [38]. KREPA3 is a component of the mitochondrial RNA editosome, which prepares mitochondrial transcripts for translation by insertion or deletion of uridines [19]. The nucleolar protein NOPP44/46 is involved in biogenesis of the large ribosomal subunit [22]. For RNAi lines, addition of Tet to the media induces knockdown of the corresponding mRNA. For the conditional knockout of KREPA3, removal of Tet blocks the expression of the complementing gene. Although some of these genes had not previously been tested for essentiality in BF (TOPIBL and TOP1BS, TOPIImt, and NOPP44/46), all strains showed compromised growth after disruption of gene expression (Figure 5 and Figure S4). From initial growth curves, we determined when the growth of the strains was strongly affected, which varied depending on the gene. Within the period when population expansion was strongly impacted and before resistant populations emerged, samples were analyzed with selected assays.

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