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

Abstract

Embryonic stem cells progress very rapidly through the cell cycle, allowing limited time for cell cycle regulatory circuits that typically function in somatic cells. Mechanisms that inhibit cell cycle progression upon DNA damage are of particular importance, as their malfunction may contribute to the genetic instability observed in human embryonic stem cells (hESCs). In this study, we exposed undifferentiated hESCs to DNA-damaging ultraviolet radiation-C range (UVC) light and examined their progression through the G1/S transition. We show that hESCs irradiated in G1 phase undergo cell cycle arrest before DNA synthesis and exhibit decreased cyclin-dependent kinase two (CDK2) activity. We also show that the phosphatase Cdc25A, which directly activates CDK2, is downregulated in irradiated hESCs through the action of the checkpoint kinases Chk1 and/or Chk2. Importantly, the classical effector of the p53-mediated pathway, protein p21, is not a regulator of G1/S progression in hESCs. Taken together, our data demonstrate that cultured undifferentiated hESCs are capable of preventing entry into Sphase by activating the G1/S checkpoint upon damage to their genetic complement.

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

“…While early work in mouse and human ESCs questioned the existence of functional G1-S restriction point [7,28], more recent data provided indirect evidence for activation of G1-S checkpoint in hESCs [9,10]. The distinction that we found between S-phase committed and non-committed G1 populations provides a strong support for this evidence.…”

“…Initial work in embryonic stem cells (ESCs) hypothesized that this may not be applicable to ESCs because of their abbreviated G1 phase, which was thought to reflect lack of sensitivity to environmental cues [7,8]. This hypothesis, however, was later questioned by studies of human ESC (hESC) response to UV irradiation, which suggested the existence of a functional checkpoint [9]. The existence of G1 checkpoint in hESCs was further supported by expression of the hypophosphorylated form of the retinoblastoma protein (pRB) [10].…”

Human Embryonic Stem Cells Fail to Activate CHK1 and Commit to Apoptosis in Response to DNA Replication Stress

“…While a number of studies have addressed the status of G1/S and G2/ M checkpoints responding to DNA damaging agents [14][15][16], the status of S-phase checkpoints in human ESCs is poorly understood. We show that, unlike somatic cells, human ESCs and EC cells fail to activate key S-phase checkpoint pathways that respond to replication stress and commit to apoptosis instead.…”

We heart cultured hearts. A comparative review of methodologies for targeted differentiation and maintenance of cardiomyocytes derived from pluripotent and multipotent stem cells

“…Again, the strictness of the checkpoint is fully restored after the cells had begun differentiation. Thus, cells in the mouse embryo that have sustained DNA damage are rerouted to the differentiation pathway, ensuring the survival of the embryo at the expense of introduction of mutations, whereas human embryonic cells still must check for DNA damage in G1 phase and repair it within the time limits of the abbreviated G1 phase in order to be allowed to replicate their DNA [154][155][156]. Embryonic cells that have been temporarily trapped in G1 phase must either embark on the differentiation pathway, or else revert to apoptosis.…”