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    • Because cathepsins are located within lysosomes

    2018-10-22

    Because cathepsins are located within lysosomes and can be targeted by BA1, and because they participate in many cell death pathways, they are attractive candidates as effectors of charontosis in mESCs (Broker et al., 2004; Droga-Mazovec et al., 2008; Hsu et al., 2009; Kagedal et al., 2001; Zuzarte-Luis et al., 2007). We have shown that a broad spectrum cathepsin inhibitor, which targets cathepsins B, L, S, and H, produces a significant reduction or delay in ETO-induced PCD. These specific cathepsins, however, do not contribute to Parp-1 cleavage (Supplemental Fig. 9). Although many other cathepsins were elevated after treatment with ETO at the RNA level, their functional involvement in Parp-1 cleavage and charontosis is only inferential. Since cathepsins exist as zymogens that are not fully active until they are cleaved, RNA expression data cannot be considered sufficient to assign functional activity. Thus, the cathepsin(s) responsible for Parp-1 cleavage, and other cathepsins that promote charontosis has not yet been identified. Cathepsins other than B, L, S, and H can also be involved in PCD signaling. For example, the involvement of cathepsin D in PCD has been described in response to a variety of stimuli (Johnson, 2000). Cathepsin D is also released from lysosomes into the cytosol in U937 lymphoma aldehyde dehydrogenase and HeLa cells in response to several DNA damaging agents, including ETO (Emert-Sedlak et al., 2005). Furthermore, siRNAs that target cathepsin D in these cells can partially alleviate PCD (Emert-Sedlak et al., 2005). We have also observed cathepsin D activation following ETO exposure in mESCs (Fig. 3D), however, the lack of a specific pharmacological inhibitor to cathepsin D has made assaying its role in ETO-induced PCD difficult. In MEFs deficient for Bak and Bax, cathepsin Q plays a role in ETO-induced PCD signaling that requires p53 transcriptional activity (Tu et al., 2009). Thus, the possibility that additional cathepsins collaborate in ETO-induced charontosis remains a viable one, especially since the protective effect that zFA exerts on ETO-treated mESCs is not sufficient to completely alleviate the high level of PCD. In hESCs, p53 transactivation of target genes is important for ETO-induced PCD (Grandela et al., 2007). Inhibition of p53 by pifithrin μ also significantly reduced levels of cell death in these cells. Consistent with these data, we show that pifithrin μ protects mESCs from cell death, suggesting that p53 plays a role in mitochondrial-dependent PCD. Additionally, treatment of mESCs with pifithrin α was also protective against ETO-induced PCD, although not to the same extent as pifithrin μ. The ability of pifithrin α to prevent p53 transactivation is somewhat controversial, particularly at doses greater than 30μM (Walton et al., 2005). However, several other studies show that pifithrin α does indeed prevent the expression of p53 target genes, particularly at doses of 30μM or lower (Bragado et al., 2007; Jiang et al., 2006; Menendez et al., 2011; Schneider-Stock et al., 2005). At a high dose (30μM), pifithrin α had no effect on ETO-induced PCD levels (Supplemental Fig. 6). At lower doses, mESCs displayed slight but statistically significant reductions in ETO-induced PCD, indicating that p53 transactivation is partially responsible for ETO-induced PCD (Fig. 4D). A comparison of ETO-induced death levels in p53+/+ and p53−/− mESCs has shown that p53 is unequivocally involved in charontosis. However, since complete protection from death was not observed in p53−/− cells, then p53-independent pathways must also be involved. We cannot rule out the contribution of the p53 family members p63 and p73, particularly since data describing the functions of these proteins in mESCs are severely lacking. Both of these proteins have been reported to induce apoptosis in a variety of cell types, dependent and independent of p53 activity in PCD signaling (Alonso et al., 2009; Codelia et al., 2010; Dohn et al., 2001; Flores et al., 2002; Melino et al., 2004; Petre-Lazar et al., 2007; Pyati et al., 2011; Rana et al., 2010).