Fig 1.
RNA polymerase I (Pol I) transcription inhibitors selectively inhibit proliferation of bloodstream form Trypanosoma brucei.
An Alamar Blue in vitro cytotoxicity assay was used to determine sigmoidal dose response curves of T. brucei SM (A-D) or the MCF10A human breast epithelial cell line (E-H) incubated with the Pol I inhibitors quarfloxin (A, E), BMH-21 (B, F), CX-5461 (C, G) or the anti-trypanosomal agent suramin (D, H) [44]. The mean percentage of signal relative to the negative control (vehicle without drug) from three biological replicates is plotted with the standard deviation indicated with error bars.
Table 1.
Relative toxicity of Pol I inhibitors in T. brucei compared with human breast epithelial or fibroblast cells expressed as IC50 (μM ± SD)a.
The selectivity index for T. brucei is indicated in brackets.
Fig 2.
Pol I transcription inhibitors kill bloodstream form Trypanosoma brucei in a time and dose dependent manner.
Cell proliferation assays were performed in which T. brucei SM cells were exposed to a range of concentrations of Pol I inhibitors including quarfloxin (A), BMH-21 (B) or CX-5461 (C) using the anti-trypanosomal agent suramin (D) as a control. The graph shows the cell density of treated parasites in comparison with the untreated T. brucei SM line at 0, 16, 24, 40 and 48 hours. The mean of four biological replicate experiments for the Pol I inhibitors, or three replicates for the suramin control are shown with standard deviation indicated with error bars.
Fig 3.
Irreversible inhibition of T. brucei growth after incubation with quarfloxin or CX-5461 as determined using wash-out assays.
T. brucei SM cells were treated with various concentrations of quarfloxin (A), BMH-21 (B) or CX-5461 (C) for two hours, washed, diluted into fresh media and cell growth was monitored. Untreated cells, and parasites incubated with the appropriate drug vehicle (DMSO for quarfloxin and BMH-21) or NaH2PO4 (for CX-5461) were used as controls. The mean of three biological replicates is plotted with the standard deviation indicated with error bars.
Fig 4.
Rapid and specific inhibition of Pol I transcription by Pol I inhibitors in bloodstream form T. brucei.
(A) Schematic of the ten kilobase pair T. brucei ribosomal DNA (rDNA) transcription unit with the rDNA promoter indicated with a black flag, and the rRNA genes with black boxes. Different rRNA precursor transcripts are shown below according to [52], with the qPCR primers used indicated with letters. The first rRNA cleavage produces rRNA Precursor 1 (3.4 kb) and Precursor 2 (5.6 kb) transcripts. Subsequently, Precursor 2 is cleaved to generate Precursor 3 (5.0 kb) [52]. (B) Schematic of the sixty kilobase pair VSG221 expression site with the promoter indicated with a white flag. Various expression site associated genes are indicated with grey boxes, and the telomeric VSG pseudogene (ψ) and the VSG221 gene indicated with coloured boxes with relevant primers indicated below. These transcription units are not drawn according to scale. (C) Rapid and specific inhibition of Pol I transcription in the presence of quarfloxin. The T. brucei S16221PuroGFP cell line was incubated with 1 μM quarfloxin for 15 minutes. RNA precursor transcripts analysed were either from the Pol I transcribed rDNA (primer pairs a-d) or the active VSG221 ES. ES transcripts analysed corresponded to a VSG pseudogene (ψ) or a VSG221 precursor transcript (221). In comparison, levels of precursor transcript from the Pol II transcribed alpha-beta tubulin locus (tub) remained unaffected. RNA was normalised against actin mRNA, levels of which remained unchanged by the different Pol I inhibitors. Results shown are the mean of three biological replicates with standard deviation indicated with error bars. (D) As in (C), only cells were incubated in 1 μM BMH-21. (E) As in (C), only cells were incubated in 1 μM CX-5461. (F) As in (C), only cells were incubated with 800 nM suramin.
Fig 5.
Incubation of T. brucei with Pol I transcription inhibitors leads to rapid disappearance of the Pol I Expression Site Body (ESB) and fragmentation of the nucleolus.
Immunofluorescence analysis of T. brucei TY-YFP-RPA2 incubated with 3 μM quarfloxin, BMH-21, CX-5461 or 800nM suramin for three hours (h). The panels show representative T. brucei cells in the G1 cell cycle stage with DNA visualised with the DNA stain DAPI, and the nucleolus with the L1C6 nucleolar marker. The Pol I complex can be visualised using Yellow Fluorescence Protein (YFP), as one allele of the RPA2 gene (second largest Pol I subunit) is epitope tagged. Signal corresponding to the extra-nucleolar ESB is indicated with a white arrow head. Scale bar indicates 5 μm.
Fig 6.
Time dependent disappearance of the Pol I Expression Site Body (ESB) in T. brucei incubated with Pol I transcription inhibitors.
Quantitation of ESB presence in T. brucei cells in the G1 cell cycle stage incubated with various concentrations of quarfloxin (A), BMH-21 (B), CX-5461 (C) or 800 nM suramin (D). The percentage (%) of cells with an ESB after incubation for the time indicated in hours (h) is shown. A total of 100 cells (two biological replicates of ~50 cells) was analysed with the standard deviation indicated with error bars.
Fig 7.
Incubation of T. brucei with Pol I transcription inhibitors results in a dramatic decrease in VEX1 foci.
(A) Immunofluorescence analysis of T. brucei S16_221PurVEX1x12myc incubated with 3 μM quarfloxin, BMH-21, CX-5461 or 800 nM suramin for three hours (h). The panels show representative cells in the G1 cell cycle stage. DNA is stained with DAPI and the myc-epitope tagged VEX1 protein visualised with an anti-myc antibody and Alexa954 secondary. Scale bar indicates 5 μM. (B) Quantification of VEX1 foci present in nuclei of T. brucei in the G1 cell cycle stage (1K1N) at 0 hours or after a three hour incubation with 3 μM quarfloxin, BMH-21, CX-5461 or 800 nM suramin. The mean percentage (%) of G1 cells with either one or two VEX1 foci per nucleus (two biological replicates of 50 cells) is plotted with the standard deviation indicated with error bars.
Fig 8.
Incubation of T. brucei with Pol I transcription inhibitors leads to rapid disintegration of nucleoli.
The nucleolus can be visualised with the L1C6 nucleolar marker as one spot in T. brucei in the G1 cell cycle stage. Nucleoli rapidly disintegrate into multiple L1C6 positive spots after incubation of cells with 3 μM quarfloxin (A), BMH-21 (B), CX-5461 (C) or 800 nM suramin (D). Each dot indicates a cell, where the number of L1C6 positive spots is indicated. The mean indicated with a line, and standard deviation with error bars. A total of 100 cells was analysed (two biological replicates of ~50 cells).