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    • The current study has investigated the cell death pathway

    2018-11-12

    The current study has investigated the cell death pathway(s) utilized by mESCs in response to etoposide (ETO), a topoisomerase II poison that indirectly induces DNA double strand breaks (DSBs) (Baldwin and Osheroff, 2005). The data show that ETO not only induces cell death with hallmarks of both PCD and necrosis, but also that this cell death is predominantly caspase- and RIP-independent. We also demonstrate a role for lysosomal cathepsins as well as p53 in this PCD pathway. Finally, we show that the EndoG nuclease contributes to DNA fragmentation in ETO-induced PCD in mESCs.
    Materials and methods
    Results
    Discussion Although ETO induces PCD in several cell types (Fujino et al., 2002; Mayorga et al., 2004; Mizumoto et al., 1994; Tsujimoto, 1997; Yoo et al., 2012), the pathway(s) involved is not completely understood. We now show that ETO induces a PCD pathway in mESCs, which we now coin charontosis, that appears independent of caspase activity. Although there is a single report which argues that exposure to ETO induces caspase 3 activation (Grandela et al., 2007), it is based on hESCs, which may differ in behavior from mESCs. A separate account supports our findings that caspase 3 is not activated in mESCs (Mantel et al., 2007), arguing that Regorafenib cost of the same type but of different species origin may differ in their response to ETO exposure. In addition to their roles in apoptosis, the activity of caspases, particularly that of caspase 3, have been implicated in retinoic acid-induced differentiation of hESCs, and in the differentiation of both murine-derived myoblasts and neural stem cells (Fernando et al., 2005; Fujita et al., 2008; Larsen et al., 2010). Similarly, nucleoside analogs can trigger caspase-7-dependent cleavage of Oct4 to induce differentiation of embryonic carcinoma cells, which have many similarities to ESCs, suggesting that caspase activity is important during the differentiation process (Musch et al., 2010). We observe caspase 3 cleavage during retinoic acid-induced mESC differentiation (Supplemental Fig. 7), suggesting that caspase activation is stimulus-specific in mESCs. Although the data are not sufficient to argue unequivocally that caspases directly contribute to charontosis in mESCs, their participation remains a viable possibility. Survivin and XIAP, members of the inhibitor of apoptosis protein (IAP) family, can bind to and inhibit caspases, and are abundant in unchallenged mESCs (Supplemental Fig. 8A). Survivin is regulated by the Oct4 pluripotency transcription factor (Guo et al., 2008), which may explain why it is expressed at such high levels and why caspase activity appears inhibited in mESCs, at least after ETO treatment. Also, both Survivin and c-IAP2 proteins are induced following ETO treatment in mESCs (Supplemental Fig. 8B). Thus, the low caspase activity may be explained in part by the level of IAP expression in mESCs. After a four hour exposure to ETO and 44h of recovery, RIP1 expression was elevated, indicative of necroptosis. This observation is similar to what is seen in cell lines treated with ETO, and is consistent with the suggestion that an increase in RIP1 may contribute to necroptosis in ETO-mediated PCD (Tenev et al., 2011). A functional role for increased RIP1 expression in ETO-induced necroptosis in mESCs, however was not seen, either by siRNA inhibition of the RIP kinases, or by necrostatin-1 inhibition of RIP1. Interestingly, necroptosis, specifically the formation of the ripoptosome, can also be inhibited by c-IAP1 and c-IAP2 (Feoktistova et al., 2011). Expression of c-IAP2 was increased following ETO treatment (Supplemental Fig. 8B), suggesting that elevated IAPs might impair necroptosis in ESCs treated with ETO. Autophagy is frequently activated during PCD in many cell types, irrespective of how cell death is induced (Goehe et al., 2012; Hughson et al., 2012; Munoz-Gamez et al., 2009; Orlotti et al., 2012). Exposure of human cervical cancer cells to therapeutics such as ETO promotes autophagy, while inhibition of autophagy by 3-methyladenine reduces PCD (Lee et al., 2007). Similarly, ETO treatment of Bax/Bak-deficient MEFs induces cell death with autophagic features (Shimizu et al., 2004). Conflicting evidence, however, suggests that reducing autophagy in HepG2 cells with siRNA to Beclin 1 or with 3-methyladenine increases ETO-induced cell death (Xie et al., 2011). Similar results in other cancer model systems (Amaravadi et al., 2007; Apel et al., 2008; Guo et al., 2012; Han et al., 2011), have led to clinical trials in which autophagy is targeted (Amaravadi et al., 2011). Whether autophagy is a direct activator of cell death or a passive bystander activated during the process of PCD remains controversial. There are conflicting data from different cell models and a lack of specific pharmacological inhibitors of autophagy (Kroemer and Levine, 2008; Levine and Yuan, 2005). The resolution to this issue may not be simple, since autophagy may be a prerequisite to some PCD pathways (Chen et al., 2011), and inhibition of autophagy may induce compensatory pathways of PCD activation. The timing at which autophagy is inhibited and the impact that this timing may have on PCD is yet another confounding factor. Inhibition of early signaling within the autophagy pathway after treatment of glioma cells with imatinib promotes cell survival, while the inhibition of autophagy at a later stage results in loss of the protective effect and increased cell death (Shingu et al., 2009). Inhibition of autophagy in mESCs at several different stages of the autophagic process using siRNAs revealed no significant reduction of PCD following ETO exposure. In fact, knockdown of the autophagy mediators Atg5 or Beclin 1 tended to produce a higher level of cell death. Although treatment of mESCs with BA1, an inhibitor of late stage autophagy, significantly reduced PCD, an unequivocal role for autophagy in PCD was not established. Indeed, the data indicated that autophagy in mESCs has a pro-survival function in mESCs after ETO treatment. This finding suggested that other pathways, including those involving the lysosome, might be active in mESC PCD.