**5. Ovarian germline cells challenged by genotoxic stress display the dual p53-dependent expression of p21CIP1 and OCT4A**

As a model for CSC we chose the ovarian germline cancer cells PA1. These cells are wt *TP53*, possessing features of embryonal carcinoma [44]. They were treated with etoposide (ETO), the inhibitor of Topoisomerase II, to induce DSB through the impairment of S-phase [45]. Cells entered massive, prolonged, G2-arrest, acquired flat morphology, and signaled persistent DDR for about 4–5 days in nearly 100% of cell nuclei (positive for γ-H2AX and CHK2 foci) with some cells overcoming the tetraploid barrier (**Figure 2A, B**). The most surprising fact was that regulators of the opposing processes of senescence (p21CIP1) and self-renewal OCT4A (POU5F1) were highly induced in the same G2-arrested cells (**Figures 2C** and **4A**). This was downstream of activated *TP53*, as both p21 and partly OCT4A became downregulated after *TP53* RNAi silencing (**Figure 2C**) [45]. Further study [46] revealed that p53-activated OCT4A down-regulated p21CIP1, moderating its expression and preventing cells from precipitating terminal senescence or apoptosis (**Figure 2D**), thus providing the opportunity for repair. Downregulation of p21 via OCT4 was previously shown in ESC [47].

Moreover, such stress-activated OCT4A was transiently disconnected from its self-renewal partners (SOX2 and NANOG), as those protein levels were low and/or not activated alongside Oct4A [46]. Therefore, the autoregulatory and feedforward loops seen in ESC [48] were not present, potentially due to the known down-regulation of the Nanog gene promoter by activated p53 [49] and/or the down-regulation of NANOG by high levels of OCT4A [50]. However, it is also possible that another, Cdk4-activating function of OCT4A [34] could be enhanced, while inhibiting action of p21on Cyclin D/cdk4,6 checkpoint function reduced forcing escape of the damaged cells from G1/S checkpoint leading to their accumulation in G2-arrest. **Figure 3** presents these hypothetical relationships between the cell cycle and pluripotency functions of stress-activated OCT4A induced by DNA damage through activated p53.

The bizarre duality of OCT4A with p21CIP1 was subdued and cell cycle in the PA1 clones returned to normal, on days 7–14 (**Figure 2A**), while silencing of stress-activated OCT4A prevented recovery [46]. This transient undecided state in G2 arrest, between true senescence and true self-renewal, was thus lasting as long as wt*TP53* was activated by the DNA DSB.

In accordance with our findings in PA1 cells after ETO treatment, a recent high-throughput RNAi screen revealed the intrinsic roles of S and G2, functionally establishing that pluripotency control is hardwired to the cell-cycle machinery and that the ATM/ATR-CHEK2-p53 axis enhances the TGF-b pathway to prevent premature cell death [36]. As well, [3] showed that

**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 loading control. Republished from Ref. [46].

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

Accelerated Senescence of Cancer Stem Cells: A Failure to Thrive or a Route to Survival? http://dx.doi.org/10.5772/intechopen.68582 51

**Figure 3.** Schematic of the response of wt *TP53* cancer stem cells to genotoxic damage. P53 is activated and simultaneously induces p21CIP1 and OCT4A. OCT4A moderates the expression of p21CIP1 preventing apoptosis or terminal senescence [45, 46]. Activated p53 also downregulates the promoter of Nanog gene [49]. Therefore, the multipotency circuit based on Nanog stimulation of OCT4A and SOX2 [48] (dashed lines) is interrupted. In addition, the overexpressed OCT4A also down-regulates NANOG [50]. Activated OCT4A also enhances the activity of cdk4 and favors cells exit from the G1/S restriction checkpoint [34] and accumulation in G2. Activated p53 also activates pAMPK, likely via OCT4A. pAMPK activates autophagy. Functional autophagy sequesters and digests p16INKA4 within the cytoplasm, preventing its transition into the cell nuclei and hence terminal senescence. With exhaustion of autophagic capacity (halting of autophagic flux) pAMPK activates a metabolic checkpoint [51], which precipitates cell death in the G1 and possibly also G2 cell compartments.
