| To maintain cell and genome integrity, cells need to exert tight control over cell cycle progression. Two cell cycle regulation mechanisms were examined in this thesis, namely (1) the centrosome reduplication block, and (2) the tetraploidy checkpoint. (1) In mammalian cells, the centrosome duplicates once in S phase. To determine whether there is a block to centrosome reduplication, a cell fusion assay was used to compare the duplication potential of unduplicated G1 centrosomes and recently duplicated G2 centrosomes. By fusing cells in different cell cycle stages, G2 centrosomes were found unable to reduplicate in a cellular environment that supports duplication. Furthermore, G2 cytoplasm did not inhibit centrosome duplication in fused cells, indicating that the block to reduplication is intrinsic to the centrosomes rather than the cytoplasm. To test the underlying mechanism, mononucleate G1 cells with two centrosomes were created by fusing cells with enucleated cytoplasts. Both centrosomes duplicated, indicating that the block is not controlled by centrosome:nucleus ratio. Human primary cells were also found to have tighter control over centrosome number during prolonged S phase arrest compared to transformed cells. This suggests a link between centrosome duplication control and genome stability maintenance. (2) Mammalian cells have been reported to have a tetraploidy checkpoint that blocks cell cycle progression in G1 in response to tetraploidy. However, other evidence argues against the existence of such a checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. The role of tetraploidy, aberrant centrosome number, and increased cell size was tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Furthermore, when cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed. Instead, various cell synchronization drug treatments were found to induce cellular damage, which was the likely cause of the arrest. Given that binucleate cells created with minimal manipulation can cycle, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used. |
