Atypical cell cycle profile of mouse embryonic stem cell is regulated by classic oncogenic and tumor suppressive genes in vitro

Abstract

Embryonic stem cells (ESCs) exhibit an unusual cell cycle profile containing a short G1 phase. Whether this feature is required to maintain pluripotency is a matter of debate. Here, we report that the short G1 phase is a consequence of MEK1/2 kinase-mediated promotion of G1/S transition, but not necessarily coupled with pluripotency maintenance. We find that compared to primed ESCs, naïve ESCs exhibit a significantly longer G1 phase due to the inhibition of MEK1/2 kinases. MEK1/2 inhibition increases intracellular level of reactive oxygen species (ROS), leading to the stabilization of p53 protein. The genetic ablation of p53 largely converts the cell cycle profile of naïve ESCs to that of primed ESCs. These results demonstrate that pluripotency and proliferation are separable cellular events, and the short G1 phase of primed ESCs is a manifestation of the intricate interplay between classical oncogenes MEK1/2 and tumor suppressor gene TP53 to promote G1/S transition.

Keywords: Cell cycle; Embryonic stem cell; P53; ROS.

Figure 1

ESCs grown in 2i condition exhibit a longer G1 phase. (A) Microscopic morphology of R1/E ESCs grown in serum (Serum) and 2i condition (N+2i). Scale bar, 200 μm. (B) Proliferative rate of ESCs in serum and 2i conditions. Equal number of cells were seeded at day 0, and cell numbers were counted every day for 4 days (n = 3). ∗, P < 0.05; ∗∗∗, P < 0.001 (unpaired t test). (C) Cell cycle profiling by fluorescence-activated cell sorting (FACS) analysis of ESCs grown in serum and 2i. Representative result was shown. (D) Quantification of FACS analysis as in (C) (n = 3). The bar plot represents mean ± SD. ∗∗∗, P < 0.001 (unpaired t test). (E) Cell cycle profiling by FACS analysis of additional ESC cell lines, Oct4-GFP and ES-E14TG2a (E14). (F) Quantification of FACS analysis as in (E) (n = 3). The bar plot represents mean ± SD. ∗∗∗, P < 0.001 (unpaired t test).(G) Hallmark analysis of differentially expressed genes between serum and 2i.

Figure 2

2i condition activates p53 to prolong G1 phase (A) Protein blot analysis of indicated proteins.(B) Knockdown of Tp53 expression by shRNA. Scrambled (scr) shRNA was used as control.(C) FACS analysis of ESCs with or without Tp53 knockdown in serum or 2i conditions.(D) Quantification of FACS analysis as in (C) (n = 3). The bar plot represents mean ± SD. ∗, P < 0.05; ∗∗∗, P < 0.001 (unpaired t test).(E) Quantification of FACS analysis and protein blot analysis of ESCs with Tp53 knockdown in high efficiency and in low efficiency. The bar plot represents mean ± SD.(F) Diagram of two independent p53-knockout cell lines.(G) Confirmation of knockout efficiency by MG132 or UV treatment. WT, wild-type.(H) FACS analysis of ESCs with or without Tp53 knockout in serum or 2i conditions.(I) Quantification of FACS analysis as in (G) (n = 3). The bar plot represents mean ± SD. ∗∗∗, P < 0.001 (unpaired t test). (J) Quantification of FACS analysis of another ESC cell line (Oct4-GFP) with or without Tp53 knockout. The bar plot represents mean ± SD. ∗∗, P < 0.01 (unpaired t test).

Figure 3

Tp53 is dispensable for pluripotency maintenance in vitro. (A) Alkaline phosphatase (AP) staining of ESCs with or without Tp53 knockout, grown in serum or 2i condition. Colonies with uniform AP staining, mixed positive and negative staining, or negative AP staining were assigned as pluripotent, mixed and differentiated ESC colonies, respectively (n = 3). (B) Microscopic examination of ESCs with or without Tp53 knockout, grown in serum or 2i condition. Scale bar, 200 μm.(C) Protein blot analysis of indicated proteins.(D) Immunofluorescence analysis of Oct4 and Nanog. Scale bar, 200μm. (E) qPCR analysis of linage-specific transcripts during the course of ESCs differentiation with or without Tp53 knockout. The bar plot represents mean ± SD. (F) Cluster analysis of expression levels of 140 genes in the category of “signaling pathways regulating pluripotency of stem cells - Mus musculus (mouse)” (KEGG pathway: mmu04550).(G) Transcriptomic differences between ESCs grown in serum and 2i, and their dependency on p53.

Figure 4

Inhibition of MEK pathway leads to p53 activation. (A) Effect of small-molecule inhibitors of MEK and GSK (MEKi and GSKi) on ESC cell cycle profile. 2i denotes MEKi plus GSKi. (B) Quantification of FACS analysis as in (A) (n = 3). The bar plot represents mean ± SD. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 (unpaired t test).(C) Effects of MEKi, GSKi or 2i on p53 protein.

Figure 5

Reactive oxygen species is induced by MEKi to promote p53 activity. (A) Effects of small-molecule inhibitors of ATM and ATR (ATMi and ATRi) on 2i- or hydroxyurea (HU)-induced p53 protein expression.(B) FACS analysis and MFI quantification of cellular reactive oxygen species (ROS) level in ESCs grown in indicated conditions.(C) Effect of antioxidant, N-acetylcysteine (NAC), on 2i-induced ROS.(D) FACS analysis and MFI quantification of ROS levels in ESCs with or without Tp53 knockout, grown in serum or 2i condition.(E) Effect of NAC on 2i-induced increase in the G1 duration. The bar plot represents mean ± SD. ∗, P < 0.05 (unpaired t test).(F) Effect of NAC on 2i-induced p53 protein expression.

Figure 6

2i promotes mitochondria-independent production of reactive oxygen species. (A) FACS analysis of mitochondrial ROS level in wild-type (WT) and p53 knockout cells (p53^−/−) in serum (S) or 2i condition. (B) FACS analysis of mitochondrial membrane potential in wild-type (WT) and p53 knockout cells (p53^−/−) in serum (S) or 2i condition. (C) Heatmap representation of differentially expressed genes, coding mitochondrial respiratory chain complex constituents and assembly factors, in serum and 2i conditions. (D) The effect of 2i-induced p53 on the expression of mitochondrial respiratory chain complex constituents and assembly factors as in (C). (E) Heatmap representation of differentially expressed genes, coding cellular oxidases capable of generation ROS, in serum and 2i conditions. (F) The effect of 2i-induced p53 on the expression of cellular oxidases as in (E). (G) Cellular localization of differentially expressed oxidases as in (E).