Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities

Abstract

Pluripotent cells of embryonic origin proliferate at unusually rapid rates and have a characteristic cell cycle structure with truncated gap phases. To define the molecular basis for this we have characterized the cell cycle control of murine embryonic stem cells and early primitive ectoderm-like cells. These cells display precocious Cdk2, cyclin A and cyclin E kinase activities that are conspicuously cell cycle independent. Suppression of Cdk2 activity significantly decreased cycling times of pluripotent cells, indicating it to be rate-limiting for rapid cell division, although this had no impact on cell cycle structure and the establishment of extended gap phases. Cdc2-cyclin B was the only Cdk activity that was identified to be cell cycle regulated in pluripotent cells. Cell cycle regulation of cyclin B levels and Y 15 regulation of Cdc2 contribute to the temporal changes in Cdc2-cyclin B activity. E2F target genes are constitutively active throughout the cell cycle, reflecting the low activity of pocket proteins such as p107 and pRb and constitutive activity of pRb-kinases. These results show that rapid cell division cycles in primitive cells of embryonic origin are driven by extreme levels of Cdk activity that lack normal cell cycle periodicity.

Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells

“…Interestingly, inhibition of Cdk2/Cyclin E activity in murine ESCs using a pharmacological inhibitor results in lengthening of the cell cycle over all phases consistent with the cell cycle-independent expression of Cdk2 and Cyclin E, without impact on expression of pluripotent markers such as Oct4 or Rex1 (Stead et al, 2002). CDK2 showed the highest kinase activity compared with other G1-specific CDKs (CDK4 and CDK6); we, therefore, hypothesised that CDK2 might play an important role in maintaining hESCs proliferation and pluripotency, and set out to investigate its function.…”

“…For collection of cells at the G1 stage of the cycle, cells were first treated by Nocodazole for 16 h, then the drug was removed by several washes with media and a fresh media supplemented with 10 mg/ml of aphidicholine (Sigma) was applied for the next 9-10 h as it was reported for mouse embryonic stem cells (Stead et al, 2002). To demonstrate that cells were properly released from blocking procedures, cell cycle progression was analysed after release from inhibitors (Supplementary Figure 2) at every 1-2 h. For example, cells that were blocked in G1 by application of nocodazole-aphidicholine block took 4-5 h to resume the normal cell cycle distribution (Supplementary Figure 2).…”

“…Our understanding of the impact of cell cycle structure on hESCs pluripotency is far from complete (Neganova and Lako, 2008) and is mostly derived from the extension of studies of mouse embryonic stem cells, which demonstrate substantial differences in cell cycle regulation between these and somatic cells (Stead et al, 2002;Faast et al, 2004;Savatier and Malashicheva, 2004;White et al, 2005;Becker et al, 2006;Neganova and Lako, 2008).…”

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment

“…As in the case of cyclin B1, examples of cyclin E1 expression outside of the normal cell cycle phase have been reported in pluripotent murine embryonic cells (Stead et al, 2002) and in cells undergoing apoptosis (Mazumder et al, 2000). The cyclin E1 complex was active at 16 h after treatment, which was earlier than cyclin A or cyclin B1 complexes.…”

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q

“…One of the explanations for the high aneuploidy rate in hESCs cells, compared with hiPSCs and controls, might lies in the possibility that ESCs guarantee rapid cell division by bypassing certain mechanisms of cell-cycle control and by that, enabling the survival of chromosomally aberrant cells. As it turns out, in early development, the strict requirements for cell growth are suspended to allow rapid cell proliferation 38,39 and is activated again when ESCs are induced to differentiate into embryoid bodies. 40,41 A recent study, conducted by Mayshar et al 34 , identified a substantial number of hiPS cell lines carrying full and partial chromosomal aberrations.…”