Cell line-specific differences in the control of cell cycle progression in the absence of mitosis.

Abstract

This paper reports that there are major fferences between mammalian cell lines in the propensity to progress into subsequent cell cycles when mitosis is inhibited with agents that disrupt the assembly of the mitotic spindle apparatus (Colcemid, nocodazole, and taxol). Human HeLa S3 cells, which represent one extreme, remain arrested in mitosis, with elevated levels of cyclin B and p34cdc2 kinase activity. InChinese hamster ovary cells, at the other extreme, the periodic rise and fall of cyclin B levels and p34cdc2 kinase activity is only transiently inhibited in the absence of mitosis. The cells progress into subsequent cell cycles, without dividing, resulting in serial doublings of cellular DNA content. In general, the propensity to progress into subsequent cell cycles in the absence of mitosis appears to be species related, such that human cell lines remain permanently blocked in a mitotic state, whereas rodent cell lines are only transiently inhibited when spindle assembly is disrupted. We interpret these results to indicate that in mammalian cell lines there exists a checkpoint which serves to couple cell cycle progression to the completion of certain karyokinetic events. Furthermore, either such a checkpoint exists in some cell lines but not in others or the stringency of the control mechanism varies among different cell lines.Current understanding of the cell cycle is based upon models in which phase transitions are regulated by complex networks of biochemical events, many of which are highly conserved in all eukaryotes. The activation of protein complexes containing the p34cdc2 kinase is central to the transitions from G1 to S and from interphase to mitosis (1-3). The proper temporal order of cell cycle processes is maintained by control mechanisms which ensure that the initiation oflater cell cycle events is dependent on the completion of earlier events (4).The activation of p34cdc2 to initiate the events of mitosis, for example, is modulated by several gene products that serve to couple mitosis to the completion of DNA synthesis and repair, as well as nutrient state and cell size (4, 5). It has been hypothesized that control mechanisms also exist that serve to couple the exit from mitosis with the correct assembly of the spindle apparatus (2).In this paper, we report that major differences exist between mammalian cell lines in the ability to proceed into the next cell cycle when mitosis is inhibited with agents that disrupt microtubule polymerization. These differences are correlated with differences in the modulation of p34cdc2 activity, as well as cellular concentrations of cyclin B, and indicate that mammalian cells differ significantly in the regulation of the transition from mitosis into subsequent cell cycles. MATERIALS AND METHODSCell Culture and Drug Treatment. HeLa S3 and Chinese hamster ovary cells strain AA8 (CHO-AA8) were maintained in minimal essential media a, supplemented with 10% fetal bovine serum, gentamicin at 50 ug/ml, and 2 mM glutamine, buffered with 10 mM Hepes at pH 7.4, ...

Inactivation of the ubiquitin conjugating enzyme UBE2Q2 causes a prophase arrest and enhanced apoptosis in response to microtubule inhibiting agents

“…Cells were harvested for analysis by flow cytometry at intervals thereafter. Control cells accumulate in mitosis after initiation of vincristine treatment, but escape from this arrest after twelve hours ( Figure 2d) and progress into G1 (Figure 2e), similar to what has been reported previously (Kung et al, 1990;Rieder and Palazzo, 1992). In contrast, DN-UBE2Q2-expressing cells continue to accumulate in mitosis ( Figure 2d) and fail to progress into the G1-phase of the cell cycle ( Figure 2e).…”

Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line

“…Cytometric analysis of DNA content indicated that MCF-7 and NIH-OVCAR-3 cells harbor G2/M DNA for 72 h after addition of paclitaxel. However, the measurement of the mitotic index showed that MCF-7 cells and NIH-OVCAR-3 cells exit mitosis 48 h and 72 h respectively, after exposure to paclitaxel, confirming that exit from mitotis occurs without cell division (Jordan et al, 1996;Long and Fairchild, 1994) and at di erent times in di erent cell types (Kung et al, 1990). Cell doubling times are 40 h and 44 h respectively for MCF-7 and NIH-OVCAR-3 cells (Figure 2a), a di erence of 4 h in their cell cycle time could not explain the di erence in the variation of mitotic index observed between these two cell lines.…”

“…It is generally thought that the mitotic block induced by low concentrations of paclitaxel is transient (Kung et al, 1990). Cells exit mitosis without completion of late mitotic events such as chromosome segregation and cytokinesis and progress to an interphase-like state (Andreassen and Margolis, 1994;Jordan et al, 1996).…”

p53 regulation by post-translational modification and nuclear retention in response to diverse stresses

“…In fact, high concentrations of nocodazole were shown to prevent rereplication in baby hamster kidney cells (Andreassen and Margolis, 1994). Moreover, several di erent cell lines varied in their propensity to rereplicate after colcemid treatment, suggesting that cell types di er in their response to microtubule inhibitors (Kung et al, 1990).…”

“…Persuasive data from several di erent experimental approaches demonstrate that extended exposure to microtubule destabilizing agents does not lead to a sustained mitotic arrest in mammalian cells. Instead, the cells undergo`mitotic slippage' whereby cells with a 4N DNA content bypass the mitotic block and maintain a long term arrest in a stage biochemically similar to G1 (Andreassen and Margolis, 1994;Kung et al, 1990;Rieder and Palazzo, 1992). This raises the possibility that p53 and pRb prevent reduplication after the cells have undergone mitotic slippage and enter a 4N`G1-like' state.…”