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Effects of aphidicolin and hydroxyurea on the cell cycle and differentiation of Trypanosoma brucei bloodstream forms

https://doi.org/10.1016/0166-6851(96)02675-8Get rights and content

Abstract

The effects of aphidicolin (APH) and hydroxyurea (HU) on the cell cycle and differentiation of Trypanosoma brucei bloodstream forms were studied. APH (0.1 μg ml−1) inhibited cell division, but did not inhibit DNA synthesis. Most of the cells were arrested in the G2 phase of the cell cycle, with each cell containing two kinetoplasts, but only one nucleus. Recovery of the arrested cells showed a 24-h lag period compared to controls. Higher concentrations of APH (1 and 10 μg ml−1) were required to inhibit DNA synthesis, but the cells failed to resume growth after removal of the drug. Incubation of cells with HU (7.5 μg ml−1) did not inhibit DNA synthesis, but arrested cells after duplicating both the kinetoplast and the nucleus. Recovery from drug arrest also showed a 24-h lag period. We therefore conclude that neither APH nor HU arrests T. brucei at the G1S phase boundary as anticipated. The mechanisms of cell cycle arrest by APH and HU are not through inhibition of DNA synthesis, but rather through unidentified pathways, leading to growth arrest prior to nuclear division and cytokinesis respectively. Since the arrested cells do not resume normal development immediately following drug removal, APH and HU should be regarded as unsuitable agents for synchronizing T. brucei bloodstream forms. T. brucei bloodstream forms arrested with either APH or HU differentiated normally into procyclic forms in vitro, indicating that a cycle of cell division is not required for initiation of differentiation, and that the process can be initiated and completed when cells are arrested at the G2M and MG1 phase boundaries.

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Cited by (38)

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    In addition, HU induces double strand breaks in cells starting mitosis. It is interesting that T. brucei, rather than being arrested at G1 phase (Mutomba and Wang, 1996), could be induced to synchrony in S phase using low concentrations of HU (Chowdhury et al., 2008; Forsythe et al., 2009), suggesting that different check points operate in both organisms. In eukaryotes, some complexes formed by cyclins-CDKs need to be deactivated if DNA damage occur, in order to stop the progression of the cell cycle until the DNA is repaired (Gillis et al., 2009; Guo et al., 2000; Pontano and Diehl, 2009).

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    Therefore, we propose that in Tetrahymena low doses of APH induce only partial inhibition of polymerase α and perhaps polymerase δ⋅ The inhibition is strong enough to cause cell division arrest, but it does not prevent macronuclear DNA over-replication, even if this replication is slowed down. This explanation is consistent with the following data (i) increase in DNA content did not occur in cells treated with high concentration of APH, (ii) Luciani et al. (2004) showed that low concentrations of APH induced only partial inhibition of DNA strand elongation in early replication forks, and arrested firing of the late forks in mammalian nuclei, (iii) continuation of DNA replication and induction of polyploidy in the presence of low concentration of APH, and HU occurred in Trypanosoma cruzi (Mutomba and Wang 1996). It is relevant for this discussion that in Tetrahymena mutants of telomerase templates (Kirk et al. 1997, 2008; Petcherskaia et al. 2003; Yu et al. 1990) and in knockout mutants of the Pot-1 gene (Jacob et al. 2007) cell division arrest is correlated with an increase of macronuclear DNA content and cell size.

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