**8.** *TP53* **mutants with persistent DNA damage undergo mitotic slippage, ploidy cycles, and are capable of reversing senescence alongside polyploidy**

Some authors have reported that genotoxically treated cancer cells can paradoxically combine sa-β-gal-positivity (considered as a universal marker of senescence) with expression of Ki67, a hallmark signature of proliferation. This "swing phenotype" is apparently dependent on p21 and TERT [61]. Others have reported that sa-β-gal-positivity is also compatible with polylploid cells (induced by DNA chemotherapy) undergoing de-polyploidization and surviving [13, 16]. Overcoming the tetraploidy barrier in *TP53* mutants, boosting the self-renewal network [25, 26] – can likely convert tumor cells into CSCs or stabilize them. Moreover, paradoxically, genotoxically challenged *TP53* mutant tumor cells, which uncouple DNA replication from cell division and undergo mitotic slippage possessing both DNA DSBs and Ki67 expression (**Figure 6A**). As well the mitotic chromo-

**Figure 6.** Mutant *TP53* tumors have additional options for repair and sorting of DNA damage in ploidy cycles. The genotoxic damage in mutant TP53 cancer cell lines of various origins favors mitotic slippage with exaggerated: (**A**) H3ser10 activation by mitotic AURBK and (**B**) expression of proliferation marker Ki67 tolerating DNA damage detected by γ-H2AX foci. This is followed by (**C**) DNA damage sorting by micronuclei in the next mitosis of the polyploid cells and/or (**D**) by expelling and autophagic digestion of the whole subnuclei of multi-nucleated cells. (**A**) HeLa cells, 10 Gy irradiation, day 4; (**B**) MDA MB 231 breast cancer cells on day 4 after 100nM Doxorubicin treatment (in collaboration with A. Boiko); (**C**) SK-Mel-28 cell, 30Gy, day 2 (in collaboration with TR Jackson); (**D**) WI-L2-NS lymphoblastoma, 10Gy, day 6. Republished from Ref. [62]. Bars (A)=20 μm; (B–D)=10 μm.

**Figure 5.** Autophagic response of PA1 cells to ETO-mediated DNA damage: (**A**) Scatterplot of image cytometry of OCT4A and pAMPK in individual cells assessed on day 4 after ETO treatment. There is a clear correlation between enhanced expression of OCT4A and pAMPKThr172 of ETO-treated cells; (**B**) OCT4A and pAMPKThr172 immunofluorescence in PA-1 cells 4 days after ETO-treatment. (**C**) The relationship between autophagy and senescence. PA1-ETO cells were treated without or with Bafilomycin A1, prior to media being removed and replaced with fresh media; cells were harvested 48 h later (day 4). (i) imunofluorescent staining for p16ink4a, LAMP2 or DAPI as shown via the BRG optical filter, (ii) only LAMP2 demonstrating high level of functional autophagy sequestrating p16ink4a-containing aggresomes. (iii–iv) shows an example of autophagic failure, where sequestration of p16ink4a is partly lost, diffusing into the cytoplasm and nucleus, which is destroyed. Bars = 10 μm.

Republished from Ref. [46].

some passengers, such as catalytically active Aurora B kinase (**Figure 6B**) and Survivin are expressed during mitotic slippage and in resulting polyploidy interphase [19, 63, 64]. Notably, activated AMPK, responsible for the metabolic aspect of senescence-associated autophagy, also possesses these same chromosome passenger features [65]. All of these observations indicate that stress-induced "senescent" cancer cells retain their proliferation potential through induced polyploidy coupled to active autophagy. During this process or/and in the next tetraploid/octaploid cell cycle they can additionally repair DNA [42] and also sort the un-repaired DNA damage in micronuclei (**Figure 6C**), as first reported by Haaf et al. [66]. The autophagic nature of this sorting found by Rello-Varona et al. [67]; has been reviewed previously in Ref. [68]. This sorting of the DNA damage through micronucleation was observed by us in several tumor cell line models after different kinds of genotoxic treatments as exemplified in **Figure 6 (A–C)**. The autophagic elimination of large DNA portions or whole sub-nuclei with damaged DNA was also observed (**Figure 6D**) [62, 68, 69] as another intriguing feature of the late post-damage events of genotoxically treated *TP53* mutants.

All this indicates that *TP53* mutants have a strong capacity for surviving genotoxic damage and reversing cell senescence by reversible endopolyploidy through a pathway involving boosted stemness. This pathway is in fact far away from the regulations of the typical mammalian cell cycle. More likely, these tumor cells exploit the life-cycle-like regulations of the unicellular organisms recapitulated from evolutionary ploidy cycles as we have postulated previously in Refs. [70, 71] and showed recently by bioinformatics study of polyploidy [72]. It only remains to add that in general tumor cells cannot bear wild type *TP53* and inactivate it, if not by mutations, then in many other ways [73]. Perhaps the increased ability to access additional routes to cell survival by overcoming senescence and repairing DNA damage as detailed above, also help explain this inactivation of *TP53* function in tumors.
