Mitotic selection

Figure 3. Scanning electron micrographs of synchronized KB cells at various stages of cell cycle. Cells pretreated for 20 hr with 2 mM thymidine, released for 8 hr, and mitotically selected by shaking, (a) Mitotic cells (X200) (b) early G, phase (X800) (c) late G,-early S phase (X1200) (d) S phase (X2000).

Figure 8. Effect of adding butyrate at different points in the cell cycle of mitotically selected KB cells on [3H]-thymidine incorporation.

Mitotically selected KB cells were plated into culture flasks and allowed 2 hr to attach to the substrate. At 2-hr intervals after attachment 2 mM sodium butyrate (B) was added to a set of flasks, and incorporation of [3H]-thymidine was measured every 2 hr after the addition of the drug by a 1-hr [3H]-thymidine pulse, (see Materials and Methods). C control cells, no butyrate added B2 B added 2 hr after plating B,t B added 4 hr after plating Be B added 6 hr after plating Bs B added 8 hr after plating B10 B added 10 hr... 

This method gives results very comparable to selection of mitotic cells in that almost 100% of the population can be obtained in Gl. It is much simpler than mitotic selection and is more readily applied to larger numbers of cells. 

Variations and other combinations of these methods are obviously possible, e.g. mitotic selection and thymidine block aminopterin block reversed with low thymidine followed by a high thymidine block reversed with deoxycytidine. 

The G2-phase of the cell cycle is perhaps the most difficult to study as it is the most difficult phase in which to obtain a synchronised cell population. This is because, if cells are synchronised by selection at mitosis or accumulation at the Gl/S boundary, by the time they reach G2 much of the synchrony has been lost. This is because of the dispersion forces arising from the different rates at which individual cells in a population traverse the cycle. G2 populations are always contaminated with cells in other phases of the cycle and the maximum fractions of Chinese hamster (CHO) cells obtainable in G2 are 0.7 by double thymidine block and 0.4 by mitotic selection (Enger et al., 1968). 

Among the many phosphorus compounds selected by Mitchell and his collaborators may be mentioned tetrasodium 2-methyl l 4-naphthohydroquinone diphosphate (VTH). A concentration of 4 x 10 6 M of (VIII) produced a 50 per cent mitotic inhibition using chick fibroblasts in tissue culture. It is thought that the inhibition of the entry of cells into mitosis depends on the blockage of cellular synthetic processes involving phosphoryla-... 

Aristarkhov, A., Eytan, E., Moghe, A., Admon, a., Hershko, A., and Ruderman, j. V. E2-C a cydin-selective ubiquitin carrier protein required for the destruction of mitotic cydins. Proc. Natl. Acad. Sd. USA 1996, 93, 4294-99. 

Crebelli R, Bellincampi D, Conti G, et al. 1986. A comparative study on selected chemical carcinogens for chromosome malsegregation, mitotic crossing-over and forward mutation induction in Aspergillus nidulans. Mutat Res 172 139-149. 

The group at Millennium Pharmaceuticals has claimed MLN-8054 (97) to be the first kinase inhibitor selective for Aurora-A over Aurora-B, which gives robust inhibition of human tumor xenografts [228-230]. Treatment of cultured human tumor cells with 97 resulted in the accumulation of mitotic cells with spindle abnormalities, a phenotype consistent with selective Aurora-A inhibition. In a pharmacodynamic model the time-dependent accumulation of... 

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