The role of cyclin E in cell cycle regulation and genomic instability

Abstract: Cyclin E, a positive regulatory subunit of Cdk2, normally accumulates periodically at the G1/S transition, where it promotes entry into S phase and other DNA replication-associated functions. However, in many types of human cancer, cyclin E is overexpressed and in some cases its expression becomes deregulated, a phenomenon generally associated with poor prognosis. Although it is not yet known how cyclin E deregulation promotes tumorigenesis, one possible mechanism may be through the generation of genomic instability, a common characteristic of tumor cells. The restriction point (R) is a checkpoint in mid G1 phase, defined as the point after which cells can complete a division cycle independently of mitogenic signals. Regulation at this checkpoint is often lost in cancer cells. Passage through R has been shown to depend on the accumulation of a labile protein, suggested to be cyclin E. We carried out single cell analysis of cyclin E expression to determine the exact timing of cyclin E protein accumulation in relation to previous mitosis, passage through the restriction point (R) and subsequent S phase. By time-lapse video microscopy and immunofluorescence staining, we showed however, that passage through R is independent of the accumulation of cyclin E and does not require Cdk2 activity. These results rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest that passage through R is a prerequisite for cyclin E accumulation. Deregulation of cyclin E with respect to the cell cycle has been linked to a lengthening of S phase. We therefore investigated the effect of deregulated cyclin E on pre-replication complex (pre-RC) assembly in telophase cells. By immunofluorescence and biochemical analysis of chromatin bound pre-RC components in telophase cells, we show that loading of Mcm2, Mcm4 and Mcm7, believed to function in a complex with other Mcm proteins as a replicative DNA helicase, is severely impaired in these cells. We also show that DNA replication is impaired in cells constitutively expressing cyclin E. These results suggest that cyclin E interferes with proper pre-RC assembly, which may explain the observed replication defects that these cells exhibit. Cyclin E-mediated impairment of DNA replication constitutes a potential mechanism for the chromosome instability that has been observed in cells constitutively expressing cyclin E and might thereby represent the link between deregulation of cyclin E and tumorigenesis. Recent studies demonstrate a link between cancer and mutations in hCdc4, the gene encoding the F-box protein that facilitates SCF-mediated degradation of cyclin E. However, in many of these tumors cyclin E is not overexpressed. We investigated whether deregulation of cyclin E expression rather than simple overexpression might be a more critical factor in tumorigenesis. We found a strong correlation between hCDC4 mutation and deregulation of cyclin E expression, suggesting that hCDC4 function is necessary for proper regulation of cyclin E expression. Thus, deregulation of cyclin E-Cdk2 activity and as a result, specific phosphorylation events, may be the critical factor in cyclin-E-mediated pathology.