How Does Cyclin E Deregulation Mediate Chromosome Instability

Cell lines deregulated for cyclin E expression exhibit several cell cycle perturbations. Flow cytometric analysis reveals a shortened G1 phase, consistent with cyclin E driving cells into S phase prematurely, and lengthened S and G2/M phases, respectively. Analysis ofBrdU pulse-labeled cells by immunofluorescence microscopy demonstrated directly that cyclin E deregulation reduces the rate of DNA replication. A hypothesis that could potentially link cyclin E deregulation to impairment of DNA replication is that inappropriate cyclin E/Cdk2 activity at the M/G1 boundary could interfere with pre-replication complex assembly. It has been demonstrated in yeast and in Xenopus egg extracts that cyclin-dependent kinase activity reduction is a prerequisite for assembly of pre-replication complexes (25). In somatic mammalian cells, pre-replication complexes begin to assemble during telophase (26). Therefore cells ectopically expressing cyclin E and controls were synchronized to enrich for telophase cells, which were then analyzed for pre-replication complex status both biochemically and by immunofluorescne microscopy. Pre-replication complexes contain a multi-protein complex known as Orc, to which Cdc6, Cdt1 and a hetero-hexomer consisting of Mcm2-7 are loaded in a sequential fashion (Figure 2). For immunofluorescence analysis, detergent extracted cells were fixed and analyzed for chromatin bound Cdc6 and Mcm proteins. For biochemical analysis, cells were fractionated to obtain a chromatin pellet, which was then analyzed for bound proteins by SDS-PAGE and western blotting. Both analyses revealed that all proteins were loaded onto chromatin equivalently in cyclin E-deregulated and control cells except for Mcm4, which was underrepresented on the chromatin of cyclin E-deregulated cells. Treating cells with the Cdk inhibitor roscovitine during mitotic exit reversed this effect, confirming that impairment of Mcm4 loading is a consequence of cyclin E/Cdk2 activity. Therefore, it is likely that the inefficient S phase observed in response to cyclin E deregulation is a consequence of a reduced number of competent pre-replication complexes resulting from an inhibition of Mcm4 loading.

How might impaired DNA replication lead to chromosome instability? We speculate that occasional failure of checkpoints that monitor the completion of DNA replication will allow cells with incompletely replicated genomes to enter mitosis, leading to chromatid non-disjunction events (Figure 3). The resolution of these non-disjunction events in progeny cells is likely to result in chromosome gains and losses as well as other form of aneuploidy.

Figure 2. Sequential assembly of pre-replication complexes. The ORC complex first binds defining origins of replication. Cdc6 and Cdtl are then loaded in an ORC-dependent manner in telophase. This potentiates the loading of the Mcm heterohexamer.

Figure 3. Incomplete DNA replication can lead to chromatid non-disjunction evens and aneuploidy. Normally, replicated sister chromatids align at metaphase and then are segregated to opposite poles at anaphase. However, an unreplicated segment (indicated in red) will not allow chromatid separation and segregation. Instead non-disjunction will occur, with both of the incompletely replicated chromatids segregating to the same pole and going to the same daughter cell. This will lead to chromosome loss and other forms of aneuploidy.

Cyclin E deregulation also leads to an accumulation of G2 and/or M phase cells. Comparing the distribution of mitotic phases in cyclin E-deregulated and control cells, there is an excess of metaphases and fewer anaphases. At high levels of cyclin E expression, some cells appear to be blocked at metaphase. This is consistent with cyclin E deregulation interfering with the metaphase-anaphase transition. We speculate that the increase in polyploidy observed when cyclin E is deregulated is a consequence of cells failing at the metaphase-anaphase transition and eventually exiting mitosis without division.

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