F1000Research 2017, 6:43 Last updated: 20 AUG 2021 RESEARCH ARTICLE Molecular signature of anastasis for reversal of apoptosis [version 2; peer review: 3 approved] Ho Man Tang1, C. Conover Talbot Jr1, Ming Chiu Fung2, Ho Lam Tang 3 1Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, Baltimore, USA 2School of Life Sciences, Chinese University of Hong Kong, Shatin, Hong Kong 3W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, USA v2 First published: 13 Jan 2017, 6:43 Open Peer Review https://doi.org/10.12688/f1000research.10568.1 Latest published: 09 Feb 2017, 6:43 https://doi.org/10.12688/f1000research.10568.2 Reviewer Status Invited Reviewers Abstract Anastasis (Greek for "rising to life") is a cell recovery phenomenon that 1 2 3 rescues dying cells from the brink of cell death. We recently discovered anastasis to occur after the execution-stage of apoptosis in version 2 vitro and in vivo. Promoting anastasis could in principle preserve (revision) report report injured cells that are difficult to replace, such as cardiomyocytes and 09 Feb 2017 neurons. Conversely, arresting anastasis in dying cancer cells after cancer therapies could improve treatment efficacy. To develop new version 1 therapies that promote or inhibit anastasis, it is essential to identify 13 Jan 2017 report report the key regulators and mediators of anastasis – the therapeutic targets. Therefore, we performed time-course microarray analysis to explore the molecular mechanisms of anastasis during reversal of 1. Takafumi Miyamoto, University of Tokyo, ethanol-induced apoptosis in mouse primary liver cells. We found Tokyo, Japan striking changes in transcription of genes involved in multiple pathways, including early activation of pro-cell survival, anti-oxidation, 2. Sanzhen Liu, Kansas State University, cell cycle arrest, histone modification, DNA-damage and stress- Manhattan, USA inducible responses, and at delayed times, angiogenesis and cell migration. Validation with RT-PCR confirmed similar changes in the 3. Leonard K. Kaczmarek, Yale School of human liver cancer cell line, HepG2, during anastasis. Here, we Medicine, New Haven, USA present the time-course whole-genome gene expression dataset revealing gene expression profiles during the reversal of apoptosis. Any reports and responses or comments on the This dataset provides important insights into the physiological, article can be found at the end of the article. pathological, and therapeutic implications of anastasis. Keywords Anastasis, apoptosis, Cell Death, Cell Survival, Gene Expression, Recovery, Repair, Reversal of Apoptosis Page 1 of 26 F1000Research 2017, 6:43 Last updated: 20 AUG 2021 Corresponding authors: Ho Man Tang ([email protected]), Ming Chiu Fung ([email protected]), Ho Lam Tang ( [email protected]) Competing interests: No competing interests were disclosed. Grant information: This work was supported by the Shurl and Kay Curci Foundation of the Life Sciences Research Foundation fellowship (H.L.T.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2017 Tang HM et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). How to cite this article: Tang HM, Talbot Jr CC, Fung MC and Tang HL. Molecular signature of anastasis for reversal of apoptosis [version 2; peer review: 3 approved] F1000Research 2017, 6:43 https://doi.org/10.12688/f1000research.10568.2 First published: 13 Jan 2017, 6:43 https://doi.org/10.12688/f1000research.10568.1 Page 2 of 26 F1000Research 2017, 6:43 Last updated: 20 AUG 2021 The physiological, pathological and therapeutic importance of REVISE D Amendments from Version 1 anastasis is not yet known. We proposed that anastasis could In the revised manuscript, we have added new data that be an unexpected tactic that cancer cells use to escape cancer support our conclusions. Specifically, our RT-PCR reveals that therapy29–31. Many tumours undergo dramatic initial responses a human liver cancer cell line displays similar gene expression to cell death-inducing radiation or chemotherapy36–39; however, profile during anastasis as observed in mouse primary liver these cells relapse, and metastasis often occurs in most types of cells. Additional microarray statistical analysis is included as 36–39 supplementary data. We have also discussed the potential cancer . Therefore, the ability of cells to recover from transient molecular mechanisms, physiological and pathological induction of cell death may allow tumour cells to escape treatment, consequences, and therapeutic potentials of anastasis. and survive and proliferate, resulting in relapse29–31. Furthermore, See referee reports cells may acquire new oncogenic mutations and transformation phenotypes during anastasis30,31, such as DNA damage caused by apoptotic nucleases. Therefore, anastasis could be one of the mechanisms underlying the observation that repeated tissue injury increases the risk of cancer in a variety of tissues40, such as Introduction liver damage due to alcoholism41, chronic thermal injury Apoptosis (Greek for “falling to death”) is essential for normal in the oesophagus induced by the consumption of very hot development and homeostasis of multicellular organisms by beverages42–44, evolution of drug resistance in recurrent eliminating unwanted, injured, or dangerous cells1–3. This cell cancers36–39,45, and development of a second cancer during sub- suicide process was generally assumed to be irreversible because sequent therapy46–49. Anastasis can also occur in primary cardiac it involves rapid and massive cell destruction4–9. During apopto- cells and neuronal cell lines30,31, and potentially in cardiomyo- sis, intrinsic and extrinsic pro-apoptotic signals can converge at cytes in vivo following transient ischemia50. These findings sug- mitochondria, leading to mitochondrial outer membrane permea- gest anastasis as an unexpected cellular protective mechanism. bilization (MOMP), which releases cell execution factors, such as Therefore, uncovering the mechanisms of anastasis may provide cytochrome c to trigger activation of apoptotic proteases including new insights into the regulation of cell death and survival, and caspase-3 and -710,11, small mitochondria-derived activator of cas- harnessing this mechanism via suppression or promotion of pases (Smac)/direct IAP binding protein with low pI (DIABLO) anastasis would aid treatment of intractable diseases including to eliminate inhibitor of apoptosis protein (IAP) which suppresses cancer, heart failure and neurodegeneration. caspase activation12,13, and apoptosis-inducing factor (AIF) and endonuclease G to destroy DNA14–17. Activated caspases commit Our previous study demonstrated reversibility of ethanol-induced cells to destruction by cleaving hundreds of functional and struc- apoptosis at late stages in mouse primary liver cells, and revealed tural cellular substrates4,18. Crosstalk between signalling path- that new transcription is important to reverse apoptosis30,31. ways amplifies the caspase cascade to mediate cell demolition via During recovery, we found up-regulation of genes involved in pro- nucleases (DNA fragmentation factor [DFF]/caspase-activated survival pathways and DNA damage responses during anastasis DNase [CAD]) to further destroy the genome19–21, and alter lipid (Bag3, Mcl1, Dnajb1, Dnajb9, Hsp90aa1, Hspa1b, and Hspb1, modifying enzymes to cause membrane blebbing and apoptotic Mdm2)30. Interestingly, inhibiting some of those genes by cor- body formation22,23. Therefore, cell death is considered to occur responding specific chemical inhibitors significantly suppresses after caspase activation within a few minutes24–26. anastasis30. However, the molecular mechanism of anastasis remains to be elucidated. To study the cellular processes of anasta- However, we and other groups have demonstrated reversal of early sis, we performed time-course RNA microarray analysis to deter- stage apoptosis, such as externalization of phosphatidylserine mine the gene expression profiles of the cultured mouse primary (PS) in cultured primary cells and cancer cell lines27–30. We have liver cells undergoing anastasis following transient exposure to further demonstrated that dying cells can reverse apoptosis even ethanol that triggers apoptosis, and identified unique gene expres- after reaching the generally assumed “point of no return”29–31, sion patterns during reversal of apoptosis. We also performed such as MOMP-mediated cytochrome c release, caspase-3 acti- reverse transcription polymerase chain reaction (RT-PCR) to vation, DNA damage, nuclear fragmentation, and apoptotic body validate the gene expression patterns in the human liver cancer formation4–9. Our observation of apoptosis reversal at late stages is cell line, HepG2, during anastasis. Here, we present our time- further supported by an independent study, which shows recovery course microarray data, which reveals the molecular signature of of cells after MOMP32. To detect reversal of apoptosis in live ani- anastasis. mals, we have further developed