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8 h (25
m
M), 14 h (12.5
m
M), or even longer - despite concentrations of the test
compound being more than two orders of magnitude above the EC
50
value.
Although BYK54826 had only a slow effect on trypanosome viability, it
appeared to rapidly halt cell division and growth, indicating that PDE inhibition
leads to cell cycle aberrations. Figure
5
shows that, after 8 h of incubation with
1
m
M BYK54826, trypanosomes accumulate in the G2/M phase of the cell cycle,
with a greatly increased proportion of cells containing double the normal amount of
DNA (i.e., two nuclei). Longer incubations demonstrate the appearance of a further
peak, representing four nuclei (not shown).
The cell cycle of trypanosomes is relatively complex, with kinetoplasts dividing
first, before the nuclei, in a process that is linked to the generation of the new
flagellum (McKean
2003
). Fluorescence microscopy after DAPI staining showed a
statistically significant (
P
0.001) increase in trypanosomes with more than two
nuclei and kinetoplasts after 12 h of incubation with 1
m
M BYK54826. It thus
appears that the inhibition of TbrPDEB1/B2 does not stop DNA synthesis or indeed
the separation into new kinetoplasts and nuclei, but does prevent the completion of
cytokinesis into independent daughter cells, leading to the formation of nonviable,
multinucleated cells. Elevation of cAMP levels does not disrupt the coordination
between the kinetoplast and nuclear division cycles: we saw no evidence of cells
with more nuclei than kinetoplasts, for instance, and division of kinetoplasts
continued to precede nuclear division. Cells that reached the four nuclei stage
disintegrated within a few hours, leading to the clearance of the parasite population.
<
a
200
b
200
160
160
120
120
2C
80
80
4C
2C
4C
40
40
0
0
Fluorescence (AU)
Fluorescence (AU)
Fig. 5 BYK54826 causes cell cycle arrest in
T. brucei
. Flow cytometric analysis of the DNA
content of bloodstream forms of strain 427 trypanosomes, (a) untreated and (b) treated with 1
m
M
BYK54826, incubated under normal culturing conditions for 8 h. The histograms show the number
of cells with a particular fluorescence intensity that correlates with the amount of DNA in the cell.
The
peaks
labeled 2C represent the number of cells in the sample with a complete diploid
complement of DNA, having two copies of the genome; the
peaks
labeled 4C represent the cells
that have replicated their DNA but not yet undergone cytokinesis, therefore having four copies of
the genome
