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

Abstract

One of the characteristic features of human embryonic stem cells (hESCs) is the competence for self-renewal and pluripotency. To date, little is known about cell cycle regulation in these cells and how the cell cycle machinery influences hESCs properties. A common feature of human, murine and primate ESCs is the presence of a short G1 phase, which has been viewed as a time window during which stem cells are exposed to differentiation signals. We used the hESCs differentiation model and comparisons to human embryonic carcinoma (EC) cells to study the key regulators of G1 to S transition in hESCs. Our studies show that hESCs express all G1-specific CYCLINs (D1, D2, D3 and E) and cyclin-dependent kinases (CDK) (CDK2, CDK4 and CDK6) at variable levels. In contrast to murine ESCs, most of the cell cycle regulators in hESCs show cell cycle-dependent expression, thus revealing important differences in the expression of cell cycle regulatory components between these two embryonic cell types. Knockdown of CDK2 using RNA interference resulted in hESCs arrest at G1 phase of the cell cycle and differentiation to extraembryonic lineages. This suggests an important role for CDK2 in cell cycle regulation in hESCs that are likely to bear significant impacts on the maintenance of their pluripotent phenotype.

Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline

“…Role of CYE-1/CDK-2 in promoting the proliferative fate Our finding that CYE-1/CDK-2 regulates cell fate adds to a growing body of evidence supporting this role, including previous examples in Drosophila (Berger et al, 2005;Wang and Kalderon, 2009), C. elegans (Fujita et al, 2007) and mouse embryonic stem cells (Neganova et al, 2009). In the Drosophila ovary, similar to what we observe in the C. elegans germline, decreased cyclin E causes premature meiotic entry during cystocyte transit amplification (Lilly and Spradling, 1996).…”

Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

“…In contrast, several lines of evidence published by four independent laboratories and unpublished data from our laboratory indicate that hESCs can modify CDK activity to promote and/or inhibit cell cycle progression. These include the following: (a) cyclin quantities and CDK activities fluctuate during the cell cycle of hESCs [25,26, our unpublished data]; (b) CDK2 activity is controlled by Cdc25A [14]; (c) both hypo-and hyperphosphorylated forms of RB protein are detectable in hESCs [27, our unpublished data]; (d) miR92b regulates passage through the G1/S checkpoint by targeting the CDK inhibitor p57 [28]; and (e) regulation of the Sphase-associated CDK target, transcriptional coactivator The level of p21 protein in hESCs UVC-irradiated in G1 phase. The level of p21 was determined by western blot analysis 6 hours after irradiation (see Fig.…”

Human Embryonic Stem Cells Exhibit Increased Propensity to Differentiate During the G1 Phase Prior to Phosphorylation of Retinoblastoma Protein

“…Constitutive activity of CDK2 and CyclinE [7,11,12] in ESCs is important for their shortened G1 phase. Several studies in mouse and human ESCs showed that inhibition of CDK2 leads to cell cycle arrest in G1 and subsequent differentiation to extraembryonic lineages [11][12][13]. This differentiation, however, does not necessarily reflect increased propensity to differentiate in G1.…”

“…Functional studies addressing the interaction between cell cycle phase and differentiation in ESCs have been based primarily on manipulations of genes associated with cell cycle regulation, particularly CDK2 [10][11][12], p21 [13,14], p27 [15], and hTERT [16] (reviewed in [17]). Constitutive activity of CDK2 and CyclinE [7,11,12] in ESCs is important for their shortened G1 phase.…”