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    • DNA fragmentation is an essential component of PCD

    2018-11-06

    DNA fragmentation is an essential component of PCD. Of several nucleases that were tested for their involvement in charontosis, EndoG emerged as a likely candidate, although a role for CAD cannot be discounted due to the degradation of ICAD observed 44 h after ETO treatment. EndoG is activated in alpha adrenergic blockers undergoing oxidative stress and translocates to the nucleus to fragment DNA (Higgins et al., 2009; Ishihara and Shimamoto, 2006). ETO induces oxidative stress and reactive oxygen species (ROS) in a variety of cell types (England et al., 2004; Hirano et al., 2004; Rojas et al., 2009), an observation we have observed in mESCs after ETO treatment (Supplemental Fig. 10), adding credence to the involvement of EndoG in this process. Finally, since pifithrin μ inhibits p53 association with mitochondria and results in decreased PCD in ETO-treated mESCs, and since EndoG is released from mitochondria to promote DNA fragmentation in response to ETO treatment, p53 may contribute to EndoG release. In summary, we have shown that ETO induces massive DNA DSBs, an accumulation of cells in the G2 phase of the cell cycle, and extensive PCD in mESCs (Fig. 1; Supplemental Fig. 11). This PCD appears to be independent of caspase activity and of RIP kinases, which are active in necroptosis. Knockdown of proteins that are integral to autophagy did not reduce PCD, whereas chemical inhibitors of autophagy did significantly decrease the high levels of cell death after treatment with ETO. The possibility that autophagy itself promotes PCD was excluded, since the activation of autophagy during ETO treatment promoted cell survival and not cell death. When mESCs were exposed to a broad spectrum inhibitor of cathepsins, the level of cell death was significantly reduced suggesting that these lysosomal proteases were involved in PCD. The involvement of p53 was also queried by the use of inhibitors that prevent p53 transactivation or mitochondrial translocation. Inhibitors of both p53 activities significantly protected ESCs from ETO-induced PCD, albeit at varying degrees. Lastly, we have identified a novel role for EndoG in the ETO-induced PCD pathway in mESCs, a finding that has not been described in ETO-induced PCD in any other cell type. We have coined the term charontosis, after Charon, the ferryman on the river Styx in Greek mythology, to describe this PCD pathway, which is summarized in Fig. 6. The following are the supplementary data related to this article.
    Conflict of interest
    Acknowledgments We thank P Hexley and G Babcock for assistance with flow cytometry, performed at Shriners Hospitals for Children — Cincinnati, supported by a grant from the Shriners of North AmericaSSF 84070. We would also like to thank N White and the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children\'s Hospital Medical Center, supported in part by NIHAR-47363, NIH DK78392 and NIH DK90971, for assistance with Imagestream flow cytometry data acquisition and analysis. We also thank S. Mylavarapu and H. Ma for assistance with data acquisition. This work was supported in part by grants R01 ES012695 and R01 ES12695-4S1 to PJS from the National Institutes of Health and the Center for Environmental Genetics and grant P30 ES006096 from NIEHS. EDT was supported by a NIH training grant T32 ES007250.
    Introduction Definitive erythropoiesis occurs in the bone marrow, where a set of complex interactions encourages red blood cell (RBC) production. The central macrophage in the erythropoietic niches called erythroblastic islands plays a critical role in controlling the maturation, differentiation, and enucleation of erythroid cells (Bessis, 1958; Chasis and Mohandas, 2008). It is known that the main route of signal transport between macrophages and erythroid cells is physical contact via several ligands and receptors (Rhodes et al., 2008; Soni et al., 2006; Spring and Parsons, 2000; Telen, 2000). Various adhesion molecules responsible for maintaining this cell-to-cell contact within the erythroid islands have been identified, mainly through the relationship between macrophages and erythroid cells. However, not every erythroid cell is attached to a macrophage (Rhodes et al., 2008), and some erythroid cells contact only other erythroid cells. Erythroid cells seem to be able to autonomously regulate erythropoiesis, albeit with reduced efficiency (Chasis and Mohandas, 2008). Based on these observations, we speculated that there must be factors regulating erythroid cells that are not based on direct contact with macrophages.