Cell synchronisation

Iron chelators of the hydroxypyridin-4-one class may well find application in treatment of tumour cell types which are relatively sensitive to cell synchronisation, an aspect which is under current investigation [86]. 

The mechanism of control of PFK-1 is mainly allosteric inhibition by ATP, citrate, long chain fatty acids and activation by AMP and F2,6-bisP. With the exception ofF2,6-bisP, the named regulators all indicate the fuel or energy status of the cell. Control by citrate helps to synchronise the rates of glycolysis and the TCA cycle. 

Hennequin C, Giocanti N, Favaudon V. S-phase specificity of cell killing by docetaxel (Taxotere) in synchronised HeLa cells. BrJ Cancer 1995 71(6) 1194—1198. 

Contrary to the previous case, using strategies with low MOTs, cell infection will not be synchronised and the cell population will be distributed over different cell status and different specific infection times (T]) [102], leading to a non-obvious definition of the optimal harvest time, extension of cell lysis and the increase in the proteolytic activity is not easy to predict and account. Although this could be considered a drawback in low MOI strategies, it is possible to control the extension of proteolysis [33,83] by the addition of protease inhibitors. 

If cells are synchronised at the G1 /S boundary and then released, the rate of DNA synthesis is initially slow but accelerates to reach a maximum at about 3h and then decelerates until S-phase is essentially complete in 6-7 h (Stubblefield and Mueller, 1962 Adams, 1969b). As replication occurs different numbers of replicons are active at any one time, and so it is not surprising that more careful labelling reveals bursts of tritiated thymidine incorporation throughout S-phase rather than a steady even progression (Klevecz, 1969 Lett and Sun, 1970 Klevecz et al., 1974). 

Several different methods of synchronising CHO cells have been compared by Grdina et al. (1987). 

More frequently a cell culture is allowed to leave the exponential growth phase and enter a stationary phase before it is subcultured (see 4.2). When this happens the subsequent round of DNA synthesis and cell division is partially synchronised. 

The mechanism of action of isoleucine starvation is not clear but it is not simply a general deficiency of amino acids as e.g. leucine starvation causes a far more drastic inhibition of cell growth without any synchronising action (Everhart, 1972 Tobey, 1973). Rather it may indicate some subdivision of G1-phase, and the presence of isoleucine appears to be important for the action of IGF ( 11.6) (Scher et al., 1977). 

This unbalanced growth rapidly leads to cell death if prolonged for more than a generation time (Ruekert and Mueller, 1960). Unbalanced growth will occur in any cells committed to division ( 10.4) yet blocked in one function including those maintained in colcemid for more than a few hours. Moreover, selective blocking of DNA synthesis may have effects which are not apparent until the synchronised cells are released and proceed to the next G1-phase (Firket and Mahieu, 1966 Cress and Gerner, 1977). Schindler et al. 

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). 

Myoblasts may differentiate and fuse without undergoing DNA synthesis (Nadel-Ginard, 1978), but fusion does not occur in calcium-free medium. Thus, if chick embryo myoblasts are set up in calcium-free (< 20/xM Ca2+) DMEM containing 5% heat inactivated FBS and 2% chick embryo extract (Appendix 1) the fusion which normally starts at around 24 h fails to occur. Addition of 1.4 mM CaCl 2 after 50 h will now produce synchronised fusion of cells (Wakelam and Pette, 1982). 

---