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Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of “stemness.” This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence.


Introduction
The relationship between cancer cells, normal stem cells, and cancer stem cells represents a question of substantial current interest. 1 It has been proposed that transcription networks that confer stem cell properties such as self-renewal, plasticity, or an increased resistance to genotoxic stimuli in normal stem cells may perform a similar function in cancer cells. 2 This hypothesis is supported by the growing clinical evidence that expression of key embryonal stem cell (ESC) transcription factors POU1F5 (OCT4A), NANOG and SOX2, are associated with poorer prognosis through tumor resistance, recurrence and progression in a wide variety of cancers. [3][4][5][6][7][8][9] Furthermore, it has been demonstrated by several groups that ESC transcription factors can be upregulated in response to DNA damage where they likely play a role in regulating survival. [10][11][12] Conversely, accelerated cellular senescence is a phenomenon that has also been shown to be induced by genotoxic treatments of cancer cells. 13 Cellular senescence has traditionally been considered a terminal cell fate. 13,14 However, more recently it has been shown to be reversible at early stages, at least in tumor cells. [15][16][17][18] Furthermore, a direct link between senescence and "stemness," essential cytological characteristics of a stem cell that distinguishes it from ordinary somatic cells, emerged in experiments where pluripotency is induced in normal cells. 19,20 The molecular regulators of these processes in normal embryonal development, such as p21Cip1, are slowly becoming discerned. 21 One intriguing observation is that embryonal cellular senescence is associated with upregulation of the same pathways which govern the epithelial-mesenchymal transition (EMT). 22 This, apparently paradoxical, link between opposites in cell fate provides a challenge for scientific reasoning.
We have previously observed in IMR90 fibroblasts that a presenescent phenotype is associated with the appearance of selfrenewal and senescence markers coupled to DNA damage. 23 We also demonstrated co-incident p53-dependent upregulation of 2 opposing cell fate regulators, p21Cip1 and OCT4A in embryonal carcinoma PA-1 cells treated with Etoposide (ETO). 24 We hypothesized that this bi-potential state favors DNA damage repair (DDR) while preventing full commitment to either senescence or self-renewal. In this system, p53 silencing promoted terminal senescence and premature mitosis. Together these data support the presence of a pre-senescent cell state which can arise in response to both senescence and stemness programmes being coactivated in response to genotoxic damage.
In the present study, we asked how key regulators of stemness (OCT4A, SOX2 and NANOG) and senescence (p16inka4a) behave in individual PA-1 cells during the response of ETO-induced DNA damage. Using siRNA silencing approaches we addressed the effect of OCT4A and p21Cip1 expression on each other and subsequent cell fates, determining the role of autophagy and how OCT4A activation impacts on the energy and genomic stress sensor and master metabolic regulator and activator of autophagy AMPactivated protein kinase (AMPK).

Results
Etoposide-treatment elicits a senescence-like phenotype in PA-1 cells Following ETO treatment many PA-1 cells undergo gradual cell apoptosis and anoikis, while the remainder arrest in G2M and upregulate p53 and p21Cip. 24 G2M arrest and nuclear swelling were evident on day 3 after ETO treatment, with restoration of normal cell cycle and nuclear size 4 d later ( Fig. 1A and B). Concurrent nuclear area assessments and DNA content measurements demonstrate that the nuclei of ETO treated cells increased in size irrespective of the stage of the cell cycle, but was most evident in G2M and polyploid cells ( Fig. 1C and D). Increased nuclear area and DNA content were also accompanied by an increase in cellular granularity, as determined by an increase in side scatter detected by flow cytometry analysis (Fig. 1E), and autophagy (see below). The majority of cells displayed flattened morphology (Fig. 1F), but only a proportion displayed p16Ink4a nuclear positivity ( Fig. 1 G, H). Furthermore, p16Ink4a expression was largely confined to the cytoplasm ( Fig. 1 G). All of these features are indicative of senescence.

Heterogeneous expression of OCT4A and p21Cip1 following Etoposide treatment
We previously showed that the senescence marker p21Cip1 and the stemness marker OCT4A simultaneously increase and co-exist in the same cells following ETO treatment. 24 After confirming the pre-senescent phenotype of these cells, we next wished to explore how these critical factors for cell fate were dynamically regulated, integrated and diversified. As judged by flow cytometry~60% of the cells become positive for both markers at the peak of the response (day 5), thereafter the expression of both markers and the proportion of double positive cells gradually decreases ( Fig. 2A) as clonogenic, mitotic division ensues. To exclude the possible impact of the senescence-associated cytoplasmic and nuclear area changes on the flow cytometry measurements, semi-automatic image cytometry was undertaken, measuring the expression of both factors in the cell nucleus (apoptotic cells possessing very high p21Cip1 positivity lacking OCT4A were deliberately excluded from this analysis as distorting stoichiometry of measurements). Both the expression and variation of expression of OCT4A and p21Cip1 increased on day 3 after ETO treatment; and increased further on day 5 (Fig2B and C, Fig. S1) and a high number of double-positive cells confirmed (Fig. 2B).

OCT4A upregulation is not accompanied by changes in SOX2 or NANOG
In its function as a master regulator of pluripotency and selfrenewal, OCT4A cooperates with SOX2 and NANOG whose expression are therefore required for maintaining the embryonic stem cell phenotype. 25 We thus explored the expression of these transcription factors in the DNA damage response of PA-1 cells. Immunoblotting confirmed that p53 increased in response to ETO from day 1, resulting in the induction of p21Cip1, which reached its maximum on day 5 ( Fig 3A). As documented previously, OCT4A expression was present in control cells and increased following ETO treatment, coincident with p53 upregulation. 24 Weak expression of SOX2 was found in untreated cells but did not alter following ETO-treatment (Fig. 3A); a result confirmed by image cytometry measurements in individual cells (Fig3B, Fig. S1). NANOG was not detected in PA-1 cells by immunoblotting ( Fig 3A) although rare NANOG positive cells (less than 5%) were found in control and ETO-treated samples by 3 different antibodies ( Supplementary Fig. 1). Intriguingly, SOX2 failed to show unambiguous antagonism with p21Cip1, which has previously been reported as directly inhibiting SOX2 in neurodifferentiation. 26 Furthermore, SOX2 expression did not show significant correlation with its self-renewal partner OCT4A (data not shown). Together, these data suggest a dissociation of OCT4A from its self-renewal capacity when it is induced by p53 (in the presence of p21Cip1) in response to DNA damage.
OCT4A suppresses p21Cip1 and promotes an enhanced senescence phenotype Given its potential lack of self-renewal capacity, we next investigated the role of OCT4A in PA-1 cells following ETO  treatment using siRNA. Prior to this analysis, we first established which OCT4 isoforms were expressed in PA-1 cells. Using a panel of specific mAb, immunoblotting revealed that OCT4B was undetectable in these cells both before and after ETO treatment (Fig.  S2), consistent with published previously RT-PCR data. 24 Having established that OCT4B was not expressed in PA-1 cells, siRNA silencing was used to assess the role of OCT4A in their response to ETO treatment. Efficient (80-95%) knock-down of OCT4A was confirmed with 3 different siRNA, in addition to a 3 siRNA pool (Fig. S3) and the molecular response to ETO was then examined by immunoblotting ( Fig 4A) for key regulators of DNA damage, (pCHK2, p53, p21Cip1, and RAD51). These experiments revealed p53, RAD51 and pCHK2 were all upregulated from day 1 independently of OCT4A expression. p21Cip1 expression rose gradually from day 1 throughout the course of experiment as seen in previous experiments. Silencing of OCT4A resulted in a marked increase in p21Cip1 expression from day 1. These results imply that the presence of OCT4A induced by DNA damage suppresses p21Cip1 expression despite comparable levels of p53 expression and equivalent DNA damage as judged by RAD51 and pCHK2 expression.
When investigating the cell cycle response, loss of OCT4A caused an almost identical phenotype to that previously reported with p53 silencing 24 i.e. OCT4A-silenced cells underwent a profound G2M arrest with apoptosis being greatly reduced ( Fig. 4B and C). The cells displayed the large, flattened morphology and Sa-b-gal staining associated with senescence on day 5 (Fig. 4D), identical to that previously reported when silencing p53. Thus, the increase in G2M arrest and increased accelerated senescence previously observed with p53 silencing appears to be entirely due to the loss of the induction of OCT4A. Prolonged loss of OCT4A lead to the polyploidisation of these senescent cells and a reduction in clonal recovery (Fig. 5). The absence of off target effects was confirmed by repeating silencing and cell cycle analysis experiments with 3 separate siRNA molecules targeting different sequences of OCT4A mRNA ( Supplementary Fig. 4). p21Cip1 silencing does not affect OCT4A expression or cell cycle arrest Having established that OCT4A suppresses p21Cip1 and a senescent phenotype we sought to investigate the possibility of a reciprocal suppression of OCT4A by p21Cip1. To do this p21Cip1 was silenced using siRNA. Silencing of p21Cip1 was efficient as judged by immunoblotting but was not found to affect the expression of OCT4A in the presence or absence of ETO treatment (Fig. 6). Furthermore, we detected no difference in the cell cycle profiles of p21Cip1-silenced cells compared to control siRNA treated cells following ETO treatment.
In summary, we found that OCT4A tempers p21Cip1 upregulation, restricting entry into senescence, indicating that coexpression defines the pre-senescent state. While p21Cip1 did not regulate OCT4A expression or change the cell cycle, depletion of OCT4A profoundly impacted it. We also found that p53-activated OCT4A does not support the pluripotency network in pre-senescent cells (owing to the lack of SOX2/NANOG co-ordination), leaving its biological role unclear. We, therefore, interrogated its possible role as a stress responder where one of these stresses may be metabolic. AMPK is a key sensor of metabolism and energy status. 27 Therefore, we next explored the activation of AMPK by virtue of its phosphorylation at Threonine 172 (pAMPK Thr172 ), in relation to OCT4A expression.

AMPK and OCT4A are coordinately activated in response to ETO-treatment
The results of this comparative study are shown in Fig. 7. Microscopy of control, untreated, cells revealed heterogeneous pAMPK Thr172 activation with expression in some cell nuclei and also in centrosomes, central spindle and mid-bodies of mitotic cells as described previously, 28 and rarely ( <1% cells), in the cytoplasm (Fig. 7A, upper row). In ETO-treated cells the staining for AMPK increased and strikingly coincided with the localization and intensity of staining for OCT4A in most cells (Fig. 7A, second row) as also confirmed by IF measurements, where clear correlation was shown (Fig. 7B). However, while OCT4A is seen homogenously throughout the nucleus pAMPK Thr172 staining revealed a fine (euchromatic) nuclear pattern, which indicates binding to chromatin, unlike OCT4A.
Intense staining of pAMPK Thr172 was also found in apoptotic cell nuclei and bodies, while OCT4A was not (Fig. 7C). In arrested metaphases appearing after ETO on day 4, pAMPK Thr172 was localized to multiple centrosomes, while OCT4A was not (Fig. 7A, second row). This difference in staining was confirmed by co-staining for g-tubulin and AURORA B kinase (not shown). Together, these data indicate that OCT4A is co-ordinately regulated alongside pAMPK Thr172 in interphase cells but that in apoptotic cells their functions may differ. Moreover, the concordant expression of OCT4A and pAMPK Thr172 in interphase cells suggests that the p53-dependent OCT4A induction is a result of a stress response following ETO treatment.
As a part of its role as a central metabolic regulator, AMPK is directly involved in the regulation of autophagy. 29 In this regard, previous studies have shown that autophagy is implicated in the chemo-resistance to ETO and this effect is reversed by inhibition of AMPK, which led to cell death by apoptosis. 30 Conversely, cellular senescence is also associated with autophagy 30 representing another potential cell fate phenotype following ETO-treatment. 24 p16ink4a is believed to drive irreversible senescence. 31 Therefore, we next decided to explore the relationship of p16ink4a and macroautophagy.

p16ink4a is sequestered by autophagy in ETO-treated cells
Autophagy was verified using immunofluorescence for p62 as a marker of autophagy. Post ETO treatment, an increase in the number of p62 foci/cell was observed by immunofluorescence ( Fig. 8A and B) and mean number of p62 foci/cell in 3 independent experiments was shown to increase > 5-fold ( Fig 8C). Bafilomycin A (Baf) halted autophagic flux in ETO-treated cells (shown as an accumulation of LC3B in relation to LC3A), confirming functional autophagy and its suppression (Fig. 8D). Baf treatment also enhanced cell death preventing clonogenic recovery only in ETO-treated cells (Fig. S5), which indicates the importance of autophagy for cell survival after DNA damage. We then assessed immunostaining for p16ink4a and LAMP2 in the presence and absence of Baf post ETO treatment. LAMP2 is an important regulator of lysosomal biogenesis, required for the maturation of functional autophagosomes. In control, untreated cells, p16ink4a was observed in~5% of cells as small aggresomes, surrounded by LAMP2-positive foci (data not shown). In ETO-treated cells (day 4), the cytoplasm was highly enriched with LAMP2, while p16ink4a-positive aggresomes (larger and more numerous than in control) were sequestered in Figure 4. OCT4A suppresses p21, G2 arrest and induces senescence. PA-1 cells were treated with ntg-siRNA or OCT4-siRNA for 24 h before treatment with 8 mM ETO for 20 h and replacement with fresh media. (A) 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. (B) Cells were fixed, stained with PtdIns and analyzed by flow cytometry. Both ntg-siRNA treated and OCT4-siRNA treated cells underwent a G2M arrest followibg ETO treatment, which is more profound at day 3 and 5 in OCT4A-siRNA-treated cells. There is subsequently a much lower extent of cell death (sub G1) in OCT4A-siRNA treated cells. (C) The G2M arrest was calculated from the DNA histograms shown in (B) and demonstrate significantly higher levels in OCT4A-silenced cells (*p < 0.05, n D 3). (D) PA-1 cells were treated with ETO as above and assessed for Sa-b-gal activity after 5 d Senescence, as monitored by Sa-b-gal activity, was increased by OCT4A-silencing. Both ntg-siRNA and OCT4A-siRNA treated cells became large following ETO treatment but non-silenced cells lacked Sab-gal activity.
autophago-lysosomes ( Fig. 9 A and B). Approximately 30% of cells were seen to accumulate large amounts of p16ink4a in multiple aggresomes, as measure by >3 aggresomes and/or diffusely detected within cytoplasm, or in large vacuoles ( Fig. 9A-C), and often showed DNA in cytoplasm (Fig. 9C). The proportion of such cells, with high levels of p16ink4a, increased fold2-(29 § 6 .5 and 58 § 3 .5, C/¡ Baf, respectively) as measured on day 4. These observations indicate the important role of autophagy in sequestering and degrading p16ink4a in ETO-treated PA-1 cells, thus preventing senescence. They also show that a considerable proportion of ETO-treated cells suffer from exhaustion of autophagy.
Failure of autophagy to sequester p16ink4a in the cytoplasm is accompanied by drastic nuclear changes and loss of DNA In ETO-treated samples counterstained with DAPI, we noted that the failure of macroautophagy to control and sequester p16ink4a in the cytoplasm coincided with prominent changes in cell nuclei, particularly following Baf treatment. Nuclei became thinner, less intensely DAPI-stained (e.g. Fig. 9C), or, more frequently, displaced to one of the cell poles by large autophagic vacuoles (Fig. 9B). Twisting, thinning, and disintegrating cell nuclei were the main phenotypes of ETOCBaf-treated cells. As detailed above, in their extreme distortions, the cell nuclei were displaced and squeezed by large autophagic vacuoles and attained a sickle-like shape (Fig. 9D). It remains possible that the loss of sequestration of p16ink4a, distortion of cell nuclei and loss of DNA are all linked, belonging to a related series of processes associated with exhausted autophagy, which is further exaggerated by Baf where full disintegration of the cell nucleus and alveolar LC3-positive cytoplasm are evident (Fig. 10 A).
Trying to understand how the penetrability of the cell nuclei was related to DNA damage we stained with an antibody (PL2-6) which detects a unique epitope involving a ternary complex of histones H2A and H2B and DNA at the site of the chromatin attachment to lamin B1. 32 We applied it to ETO-treated cell nuclei (day 4) combining it with a label for DNA damage (pCHK2). The result shows (Fig. 10B) bright nuclear staining with PL2-6 in cell nuclei without DNA damage and its reduction in cell nuclei containing DNA damage, possibly suggesting that binding to lamin B receptors in compromised.
In view of this data, we decided to use detailed stoichiometric DNA image cytometry following ETO treatment in the presence or absence of Baf. Typical histograms from one representative experiment are shown in Fig. 10C, while in Fig. S6 comparative DNA histograms of selected normal-and sickle-shaped cell nuclei from ETOCBaf treatment are presented. Measurements from ETO-treated cells revealed a peak of delay in late S-phase, which was augmented after Baf treatment showing apparent redistribution from G2 (4C) into <4C, which was exaggerated in selected sickle-type nuclei of ETOCBaf-treated cells (Fig. S6). These data support our microscopic observations that DNA is preferentially lost from the sickle-like shape nuclei in the cells as a result of failed autophagic processing with the proportion of sickle-like nuclei cells with sickle-like nuclei in ETO-treated samples increased more than 2-fold (2.38 § 0.2) following Baf treatment (n D 3).

Discussion
In this study, we expanded our investigation of the response of PA-1 embryonal carcinoma cells to DNA damaging chemotherapy. We previously observed, 24 a concurrent p53-dependent increase in OCT4A and p21Cip1 protein expression after ETOtreatment, with the resulting cells transiting through an apparently bi-potential state with hallmarks of both premature multicentriolar mitoses and senescence. Herein, we extended our analysis and highlight a hitherto unobserved DNA damage response (DDR) function of OCT4A following ETO treatment, which is different from its established pluripotency and self-renewal functions. This p53-dependant DDR-induced OCT4A is not coordinated with SOX2 or NANOG activity, but does serve to suppress p21Cip1. This finding is in accord with the previously shown observation that OCT4A can directly repress p21Cip1 expression by binding its promoter in the absence of SOX2 and NANOG. 33 Although not yet confirmed in other cell types or following different stimuli, this phenomenon likely relates to properties evidenced during embryonal 'stemness', as similar nuclear accumulation of OCT4A was similarly recently reported following laser beam damage of live ESC cells. 34 Our measurements of OCT4A and p21Cip1 expression in individual cells revealed that cell fate, in response to extensive DNA damage, occurs with high levels of heterogeneity. Both factors increase coordinately with huge variations in expression, covering (at day 5) a wide spectrum of p21Cip1 and OCT4A expression. 35 These observations demonstrate a typical characteristic of self-organizing, adapting systems, using heterogeneity over time to provide bifurcations of cell fate choice. 36,37 The increased variance in both factors -from day 3 until day 7 -corresponds to the period with most prominent cell death (99% cells will die and only <1% has the chance to survive, likely representing the degree of challenge that they must overcome). 24 When OCT4A is silenced the level of p21Cip1 increases, more cells enter senescence and mitotic recovery is reduced, abrogating fragile balance in bifurcation point and removing the possibility for rare cells to choose survival as their fate. It is worth noting that this response was phenotypically identical to that seen when silencing p53. 24 We hypothesize that the loss of p53 or OCT4A in silencing experiments causes the loss of the embryonal G2M checkpoint leading to increased mitotic slippage, ultimately ending in terminal senescence rather than executing the G2M checkpoint function via apoptosis. Continued silencing of OCT4A inhibited clonogenic recovery post ETO treatment highlighting the importance of the pre-senescent G2M arrest for clonogenic recovery of the small percentage of cells that are able to repair their DNA damage. Silencing of OCT4A also leads to endoreplication in senescent cells further eluding to the presence of mitotic slippage. 38 p21Cip1 silencing experiments demonstrated that the observed G2M arrest was independent of p21Cip1 expression.
Mitotic survival of the ETO-treated cells ultimately originates from metastable pre-senescence. Theoretically, this means that the self-renewal function of OCT4A is finally restored. The attractor of self-renewal fate in individual cells is high NANOG, 39 released from direct suppression by p53 40 and activated by OCT4A-SOX2 heterodimer. Although NANOG is only found in a very small number of PA-1 cells, its role requires further exploration.
This moderator function of DDR-induced OCT4A occurs alongside activation of AMPK. AMPK is an evolutionary conserved low-energy checkpoint that operates as a master regulator of cellular metabolism and activator of autophagy. AMPK also acts as a genomic stress sensor that is required for the adaptation of metabolism switches and the DDR for efficient repair of DNA. 41 We previously observed a bidirectional relationship between AMPK and ATM/CHK2 pathways that integrates both the metabolic and DNA damage stress signaling. 42 AMPK activation was previously shown as necessary for maximal stressinduced transcription of p53-dependent genes that promote cell  survival. 43 Interestingly, AMPK has also been shown to activate and stabilize p53. 44,45 Thus, the energetic stress-response of AMPK is tightly linked to the DDR of p53. The role of AMPK in the autophagy and cell death response after ETO has previously been reported. 30 We previously revealed the p53-dependant DDR-induced elevation in OCT4A in PA-1 cells following ETO treatment. Therefore, it was not unexpected that DDR-induced OCT4A and AMPK activation should be coordinately regulated by ETO as we observed here. We found that while autophagy apparently remains functional, cells retaining both AMPK and DDRinduced OCT4A in cell nuclei are capable of preventing terminal senescence. This data are in accordance with the report by Chitikova and colleagues, 46 who demonstrated that silencing mTOR (the antagonist of AMPK suppressing autophagy) in irradiated, apoptosis-resistant cells caused a reversal of senescence. Thus OCT4A moderating p21Cip1 in collaboration with pAMPK Thr172 , supporting cells with metabolites and energy through autophagy and other means, channel cells through disadaptation chaos caused by high DNA damage toward a survival phenotype. ETO-treated PA-1 cells struggle against terminal senescence in particular by autophagic sequestration and removal of its regulator p16ink4a.
There is much controversy regarding markers of terminal senescence and even what the process really is. It has become recently apparent that terminal senescence is not only growth arrest, 47 moreover, it is intrinsically associated with the origin of EMT 22 and predicts poor clinical outcome after cancer treatment. 48 In our previous and current article we have partly unravelled a mechanism showing that in response to DNA damage p53 induces 2 regulators of opposite cell fates. The setting is interesting: although they both are positively regulated by the same up-stream inducer, OCT4A suppresses p21Cip1, while the latter does not directly suppress OCT4A. Nevertheless, the scattering of the cells in expression of both through the phase space ending up in one of 2 cell fates presumes their reciprocity. The observation of cell-to-cell variability can also be a result from biological oscillations in time. 49 The best documented example of this is the p53-mdm2 oscillating system in which p53 oscillations are, indeed, crucial for cell recovery after DNA damage. 50 It is, therefore, possible that heterogeneity in p53 due to oscillations results in downstream heterogeneity of both OCT4A and p21Cip1 in PA-1 cells.
Senescence is intrinsically associated with macroautophagy induction (regulated by AMPK and persistent DNA damage). 51 Here, we found that persistent DNA damage was associated with destruction of the ternary complex of histones (H2A and H2B) and DNA in their attachment to nuclear lamin B1 using a unique antibody reagent. 32 This observation is in keeping with data of the thinning and disassembly of lamin B1 and leakage of DNA from cells as a feature of terminal senescence. 52 Our DNA cytometric observations revealed a delay in late Sphase after ETO, augmented by BafA1. The consequence of this delay would be under-replication of heterochromatin, 53 appearance of single strand DNA breaks, demethylation and activation of the transposable elements (TE) ALU/LINE 1, whose mobility after ETO has already been described. 54 As sequestering ALU mRNA and LINE 1 by autophagy in the cytoplasm has been reported as a mechanism for protecting against transposition, 55 it therefore remains possible, (although purely speculative) that functional autophagy prevents terminal senescence in this manner also. The model with the scenario of terminal senescence occurring due to activation of TE as discussed in several other studies. [56][57][58] It follows then that terminal senescence may in fact be mainly triggered by several mechanisms, which potentiate each other and result from failure of autophagy. Thus, autophagy should be a key target for preventing resistance of cancer stem cells to genotoxic therapies, a supposition supported by others. 59,60

Materials and Methods
Cells PA-1 is an ovarian teratocarcinoma (a germline tumor) cell-line, obtained from ATCC, with a stable near-diploid karyotype. 61 It possesses functional p53, in spite of possible acquisition of one mutated allele reported at passages over 407. 62 Importantly it retains the ability to differentiate into all 3 tissue types, thus possessing the features of embryonal carcinoma. 61 The NTera 2 (NT2) cell line, obtained from ATCC and used as a positive control for pluruipotency genes OCT4A, SOX2 and NANOG.

Antibodies
Primary antibodies and their uses are detailed in Table 1. Secondary antibodies and their uses are detailed in Table 2.
Cell culture and treatment PA-1 cells were cultured in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% foetal bovine serum (FBS) if not otherwise stated. NT2 cells were cultured in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% foetal bovine serum (FBS). Cells were grown without antibiotics in 5% CO 2 at 37 C. Exponentially growing PA-1 cells were incubated with 4-8 mM ETO for 20 h. Following ETO treatment, cells were maintained by replenishing culture medium every 48h. Bafilomycin A (Baf A, Cayman Chemicals) 50 nM was added to the culture medium for 24h between day 3 to 4 after 20h treatment with 8 mM ETO.

Flow cytometry
Cells were harvested at relevant time points, washed in cold PBS and fixed with 70% ethanol for 20 min at room temperature. After two washes in tris buffered saline (TBS), cells were permeabilised with TBS/4% bovine serum albumin (BSA)/0.1% Triton X-100 for 10 min at room temperature. Samples were then incubated with mouse monoclonal anti-OCT4 antibody solution (Santa Cruz) and rabbit polyclonal p21Cip1 (Pierce) in TBS/4% BSA/0.1% Triton X-100 for 1h at room temperature. Following two washes in TBS, cells were incubated with goat anti-rabbit-IgG-Alexa Fluor 488 (A31627, Invitrogen) and Chicken anti-mouse IgG-Alexa Fluor 647 (A21463, Invitrogen) (1:200) in TBS/4% BSA/0.1% Triton £ 100, for 30 min in the dark. DNA was counterstained with 10 mg/ml propidium iodide (PtdIns) in PBS, and assessed by flow cytometry using a FACS Aria (BD Biosciences) using Cell Quest Pro Software). Data was analyses using FlowJo analysis software.

Immunofluorescence
Cells were suspended in warm FBS and cytospun onto glass slides. Cytospins were fixed in methanol for 7 min at ¡20 C and dipped 10 times in ice cold acetone. Slides were then washed thrice in TBS 0.01% Tween 20 (TBST) for 5 min. Slides were subsequently blocked for 15 min in TBS, 0.05% Tween 20%, 1% BSA at room temperature. Samples were covered with TBS, 0.025% Tween 20%, 1% BSA containing primary antibody and incubated overnight at 4 C in a humidified chamber. Samples were then washed thrice in TBST and covered with TBST containing the appropriate secondary antibodies (Goat anti-mouse IgG Alexa Fluor 488 (A31619, Invitrogen) and Goat anti-rabbit-IgG Alexa Fluor 594 (A31631, Invitrogen)) and incubated for 40 min at room temperature in the dark. Slides were washed thrice for 5 min with TBST and once for 2 min in PBS. Samples were then counterstained with 0.25 mg/ml DAPI for 2 min, and finally embedded in Prolong Gold (Invitrogen). When staining for p-AMPKa1/2 (alone or in combination with the antibody for OCT4), fixation in 4% paraformaldehyde for 15 min was used, followed by washing thrice in PBS 0,1% glycine. Primary antibodies and their source are listed in Table 1.
Microscopy and immunofluorescence image cytometry Slides were evaluated using a Leitz Ergolux L03-10 microscope equipped with Sony DXC 390P color video camera, for microscopic observations; in addition to separate optical filters, a 3-band BRG (blue, red, green) optical filter (Leica) was used. Image cytometry was carried out by semi-automatic measuring fluorescence values for each cell nuclei in all 3 channels and analyzed using Image-Pro Plus 4.1 software (Media Cybernetics). Apoptotic cells, determined by nuclear morphology, were omitted from measurements.

DNA image cytometry
For DNA cytometry measurements, stoichiometric toluidine blue DNA staining was performed as previously. 63 In brief, cytospins were fixed in ethanol:acetone (1:1) for >30 min at 4 C and air dried. Slides were then treated with 5N HCl for 20 min at room temperature, washed in distilled water (5 £ 1 min) and stained for 10 min with 0.05% toluidine blue in  ). Apoptotic cells were omitted from measurements. The stoichiometry of DNA staining was verified using the values obtained for metaphases compared to anaphases and telophases (ratio 2.0); the summary error of the method and device was estimated to be less than 5%.
DNA flow cytometry Cells (including those in media) were harvested at indicated time points, washed in PBS and re-suspended in hypotonic fluorochrome solution [50 mg/ml propidium iodide (PI), 0.1% (w/ v) sodium citrate, 0.1% (v/v) Triton-X-100] and stored for at least 1 h in the dark at 4 C. Flow cytometry was performed using a FACScan (BD Biosciences) or Accuri TM C6 Cytometer (BD Biosciences). Data was analyses using FlowJo analysis software.

Detection of sa-b-galactosidase activity
The senescence b-galactosidase (sa-b-gal) staining kit (Cell Signaling Technology, UK) was used to detect sa-b-gal activity in cells according to the manufacturer's protocol.

Immunoblotting
For whole-cell lysates, cells were harvested, washed once with PBS and lysed using RIPA buffer containing protease inhibitor cocktail (Sigma P8340). Protein concentrations were determined by Bradford assay (Thermo Scientific), with 10-30 mg of total protein separated on 20% SDS polyacrylamide gels followed by electrophoretic transfer onto BA85 nitrocellulose membranes (Schleicher & Schuell GmbH) overnight. Equal protein loading in each lane was confirmed by Ponceau S staining. Blots were probed with appropriate antibodies primary and secondary detected using ECL Western Blotting Substrate (Pierce TM , 32106).
For comparison of OCT4 isoforms protein was transferred onto immobilon-FC transfer membrane and probed with primary antibodies. Blots were then probed with fluorescent secondary antibodies and the signal was visualized using a LICOR Odyssey imaging system.

Methods of statistical analysis
Statistical analysis was performed in GraphPad (GraphPad Software Ltd). Student t-test was used to calculate the statistical significance of difference of means where appropriate. Statistical significance was accepted when p < 0.05. Graphs were plotted in GraphPad Prism 5 and IBM SPSS statistics 22.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

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