Embryonic Stem Cells Bypass Numerous Cell Cycle Checkpoints; Not Just G1.
Authors: Mantel Charlie , Broxmeyer Hal E. 
Published: April 2020

Abstract
It was recently demonstrated that human and mouse embryonic stem cells (ESC) have deficiencies in the mitotic spindle assembly checkpoint (SAC) and it’s uncoupling to apoptosis which leads to polyploidy (Mantel et.al. BLOOD10:4518; 2007), a source of genetic instability in ESC in-vitro. The G1 checkpoint is also absent in ESC, a fact already known. It was also shown that p53 phosphorylation is absent in SAC-bypassed murine ESC in contrast to somatic cells with intact checkpoints (Mantel, et.al. CELL CYCLE 7:484; 2008). This lack of p53 phosporylation likely contributes to apoptosis uncoupling and polyploidization in ESC after microtubule/spindle damage and SAC-bypass. Microtubule/spindle damage in somatic cells eventually causes M-phase slippage where cells enter a 4C-G1 state that has 4C DNA content, no cyclin B1, and highly phosphorylated Rb. 4C-G1 status has not been investigated in ESC. We have now begun studies to determine mechanisms of checkpoint-bypass and polyploidization in ESC using intracellular flow cytometric analysis and here we report on the phosphorylation status of Rb in polyploid ESC. Because histone acetylation has been linked to cell cycle checkpoint function and because chromatin structure is more “open” in ESC, we investigated the oscillatory acetylations of the four core nucleosomal histones during checkpoint-bypass in ESC. The effects of DNA strand breaks on cell cycle checkpoints in ESC were also investigated. Results demonstrated that Rb is highly phosphorylated at several sites when ESC are in a cell cycle phase consistent with that seen in somatic cells in 4C-G1 after microtubule damage. It is concluded that ESC polyploidization is accompanied by 4C-G1-exit without apoptosis, which contrasts to 4C-G1-exit in somatic cells that do initiate apoptosis. There were also pronounced differences in acetylation oscillations on histone H4 and histone H2B compared to histone H3 and histone H2A during checkpoint activation and bypass. Total histones increased linearly as DNA content increased, as expected. Bivalent histone acetylation/methylation site, histone H3K9, changed little during checkpoint-bypass. However, DNA strand breakage revealed that S, G2, and the following G1 DNA-damage checkpoints also appeared to be bypassed in ESC. Most unusual is the polyploidization after DNA strand breakage, which may be due to aborted G2/M phases, but not to SAC activation since DNA strand breakage is not known to activate the SAC. DNA damage caused polyploidy without accumulation of cells in 4C-G1, as noted by lack of Rb phosphorylation, lack of p53 phosphorylation (as previously determined), but with an increase in total p53 in all phases of the cell cycle including 8C/polyploid. We conclude that mouse ESC can bypass numerous cell cycle checkpoints and fail to couple them to apoptosis initiation. This could be related to differences in histone acetylation, Rb phosphorylation, and the absence of p53 phosphorylation when compared to results of similar studies of somatic cells. Bypass of numerous checkpoints is a likely source of genetic instability in ESC cultured in-vitro.