**6. Transient bi-potentiality of CSC for senescence and self-renewal displays the population features of "noisy" expression and activated transposable elements**

One of the interesting facets of this dual expression of self-renewal and senescence regulators in the PA1-ETO model was the high heterogeneity in response, with individual cells expressing wildly differing levels of OCT4 and p21 (**Figure 4A**). This explorative chaos continued for 4–6 days and culminated with massive cell death selecting a small proportion (<1%) of resistant survivors. Earlier studies on ESC observing the extensive heterogeneity and fluctuations of gene expression in individual stem cells led the authors to suggest that "noise "may be the central driving force behind multipotency [32, 50, 52].

Therefore, notably, a similar long 'stochastic' phase of choice between senescence and selfrenewal (initiated by activating DNA repair and mesenchymal to epithelial transition), with

cellular senescence accompanying DNA damage or DNA damage as such favors cell reprogramming in vivo models. It should be noted however, that the frequency (chance) of survival in our PA1-ETO model was not high. It stresses the importance of another possible player in

**Figure 2.** Response of PA -1 cells to ETO treatment. PA -1 cells were treated with 8 μM ETO for 20 h, then washed and assessed at the indicated time point. (**A**) Cells were cytospun, fixed and stained for DNA image cytometry as previously described [46]. DNA content was determined for at least 200 cells in each condition and is represented as a percentage. Profound G2 arrest on day 2 was observed followed by the simultaneous emergence of a polyploid (>4C) and 4nG1 fraction on day 5 before the recovery of the normal cell cycle profile by day 7. (**B**) The proportion of pCHK2-positive cells was examined in the context of DNA content with cells sub-divided into small or large cells. In the NT control sample, all cells were pCHK2-negative with an expected nuclei size distribution (2C 80%; ≥ 4C 20%). On day 2, all cells were pCHK2-positive and the vast majority of nuclei were large (≥4C). By day 5, cells with small nuclei appeared, all of which were pCHK2-negative. Data are representative of>three independent experiments; (**C**) Immunoblot analysis of TP53, OCT4A and P21CIP1 in PA-1 cells after ETO treatment. PA-1 cells were treated with non-target (ntg) siRNA (−) or siRNA-TP53 (+) for 24 h before treatment with 8 μM ETO, washing after 20 h and cell lysates made and assessed by immunoblotting for p53, P21CIP1, OCT4A or GAPDH as a loading control at the indicated time-points (day 3 and 5). p53 was upregulated in response to ETO treatment and suppressed by siRNA-*TP53*. P21CIP1 and OCT4A were also upregulated by ETO treatment, and the upregulation was restricted by treatment with siRNA-*TP53*. Data are representative of three independent experiments. (**A**–**C**) republished from Ref. [45]. (**D**) OCT4A suppresses p21CIP1 and induces senescence. PA-1 cells were treated with ntg-siRNA or OCT4-siRNA for 24 h before treatment with 8 μM ETO for 20 h and replacement with fresh media. Protein expression was assessed at the indicated time points by immunoblotting. Cell lysates were made and assessed by immunoblotting for pCHK2, RAD51, p53, p21CIP1, OCT4A and GAPDH as a

this "undecided" stage between senescence and self-renewal, of transcriptional noise.

loading control. Republished from Ref. [46].

**Figure 4.** Population heterogeneity in expression of OCT4A and p21CIP1 in response of PA1 cells to ETO treatment. (**A**) Population heterogeneity in expression of OCT4A and p21CIP1 is extended for days 2–6 post ETO treatment, before recovery from day 7, when it reduces; (**B**) Population heterogeneity is accompanied by a degree of polyploidy as represented by DNA histograms on day 4, with underreplication of the cells in late S-phase of diploid and polyploid cell cohorts; this phenomenon is enhanced after impediment of autophagic flux. Republished from Ref. [46].

heterogenous activation of pluripotency genes, preceding the period of further determination of self-renewal circuitry has also been reported during the induction of pluripotent stem cells [53].

Our study of DNA histograms in ETO-treated PA1 cells revealed in addition to G2M arrest, a strong under-replication in late S-phase (**Figure 4B**), the time of constitutive heterochromatin replication [46]. Similarly, arrest in late S- and G2M phase was reported after Doxorubicin treatment in p53 mutant cancer cells [54]. This feature may therefore be equally required for any senescence causing release from silencing and subsequent activation of TE nested in constitutive heterochromatin as retrotransposition was found in replicative cell senescence [55, 56]. In particular, under-replication may cause de-repression of TE genes and result in the epigenetic activation of the developmental genes in the poised chromatin regions [30] enabling the reprogramming by senescence. Indeed, we observed the activation and clustering of *ALU* elements in the ETO treated PA1 cells [57]. The initial de-repression of transposons could provide the necessary "noisy" background of transcription allowing chaotic fluctuations of gene expression, which enable stochastic choice of the appropriate attractors for cell fate change and escape from terminal senescence.
