Form Of Regulated Cell Death Research Articles

Flow cytometric detection of hyper-polarized mitochondria in regulated and accidental cell death processes

Shikonin induced necroptosis in Jurkat cells were identified flow cytometrically by the up-regulation of RIP3 in live cells and that a proportion of these cells underwent other forms of regulated cell death (RCD) which included parthanatos (< 10%), or cleaved PARP (< 10%) and DNA Damage (> 30%). Live necroptotic cells also possessed functioning mitochondria with hyper-polarized mitochondria membrane potential and generated a fivefold increase in cellular reactive oxygen species (ROS) which was resistant to inhibition by zVAD and necrostatin-1 (Nec-1). After loss of plasma membrane integrity these dead necroptotic cells then showed a higher incidence of parthanatos (> 40%), or cleaved PARP (> 15%) but less DNA Damage (< 15%). Inhibition of shikonin induced apoptosis and necroptosis by zVAD and Nec-1 respectively resulted in live necroptotic cells with an increased incidence of cleaved PARP and reduced levels of DNA Damage respectively. Dead necroptotic cells then showed a reduced incidence of parthanatos and DNA Damage after inhibition by zVAD and Nec-1 respectively. A high proportion of these dead necroptotic cells (30%) which lacked plasma membrane integrity also displayed functioning hyper-polarized mitochondria with high levels of cellular ROS and thus had the capacity to influence the outcome of RCD processes rather than just been the end product of cell death, the necrotic cell. Flow cytometry can thus measure multiple forms of RCD and the level of cellular ROS and MMP which highlights the inter-connection between cell death processes and that a single cell may simultaneously display multiple forms of RCD.

ACTL6A to drive gastric cancer progression through induction of glutathione synthesis.

e16521 Background: Actin-like 6A (ACTL6A), an actin-like protein, is a member of ATP-dependent SWI/SNF like BAF chromatin remodeling complexes. Previous reports revealed that ACTL6A was involved in varying cellular processes including chromatin remodeling, transcriptional regulation, vesicular transport, and nuclear migration. Nevertheless, its role and mechanism in Gastric Cancer (GC), the second leading causes of cancer-related death worldwide, has not been reported. To explore it, we found that ACTL6A inhibits ferroptosis of GC cells via regulating glutathione (GSH) metabolism. Ferroptosis is a regulated form of cell death driven by accumulation of lipid-based reactive oxygen species (ROS). Methods: GC cell line snu638 was used for studies. Firstly, silencing ACTL6A in snu638 with shRNA, cell proliferation was measured by counting cell confluence in incucyte. Overlapping the Gene Set Enrichment Analysis (GSEA) results of RNA-sequencing and two databases, we found that ACTL6A is positively correlated with GSH metabolism, which can be validated by real time-PCR. Next, cells were treated with ferrostatin-1 (fer-1), a ferroptosis inhibitor, to check if ACTL6A can inhibit ferroptosis. Finally, 13C-glucose and 13C-glutamine were used for tracing the metabolites in snu638, which were measured by LC-MS system. Results: Suppression of ACTL6A significantly inhibits GC cell growth. In our RNA-sequence results, ACTL6A is positively correlated with GSH metabolism, which is also enrich in two GC databases. ROS level was increased after ACTL6A knockdown. Since ROS plays an important role in ferroptosis, we found that fer-1 can rescue cell death caused by suppression of ACTL6A. When tracing with 13C-glucose, total GSH and 13C incorporation from 13C-glucose in the m+2 and m+4 isotopomer of GSH is reduced when ACTL6A is depleted, while total contribution of U-13C glucose to serine, glycine and glutamate have no significant changes. When tracing with 13C-glutamine, m+5 fractional contributions of 13C-glutamine to γ-glutamyl-cysteine and GSH are decreased when silencing ACTL6A. These results reveal that ACTL6A promotes de novo GSH synthesis. Conclusions: ACTL6A acts as an oncogene in GC via regulating GSH metabolism, and ferroptosis is inhibited for increasing GSH by ACTL6A, which highlights the importance of ACTL6A in GC metabolism and tumorigenesis. Therefore, ACTL6A could be a potential diagnosis marker or target gene of chemotherapy for GC.

Identification of the hallmarks of necroptosis and ferroptosis by transmission electron microscopy

Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and ferroptosis. The molecular mechanisms underlying the execution of these processes have been intensively investigated, yet the hallmarks of their morphology are not fully understood. Here, we report that electron lucent cytoplasm was a common feature of both necroptosis and ferroptosis, which was consistent with cytoplasmic vacuolization due to a defect in the cytoplasmic membrane integrity. Notably, the perinuclear space was dilated in necroptosis, but such dilation did not occur in ferroptosis. Cells undergoing ferroptosis, but not necroptosis, exhibited an electron lucent nucleus. We previously reported that one of the nuclear danger-associated molecular patterns (DAMPs), high mobility group box (HMGB)1, is rapidly released from the nucleus to the extracellular spaces of cells undergoing necroptosis through the ruptured nuclear and cytoplasmic membrane. Via time-lapse imaging of cells stably expressing HMGB1 fused to a fluorescence protein, we found that HMGB1 was also released from the nucleus to the cytosol, and then eventually released into the extracellular spaces in cells undergoing ferroptosis. Thus, nuclear membrane damage was induced prior to cytoplasmic membrane rupture in ferroptosis. Thus, dilation of the perinuclear space and an electron lucent nucleus may be the hallmarks of necroptosis and ferroptosis, respectively.

SnapShot: Ferroptosis

Ferroptosis is a regulated form of cell death that occurs when phospholipids with polyunsaturated fatty acyl tails are oxidized in an iron-dependent manner. Research in recent years has uncovered complex cellular networks that induce and suppress lethal lipid peroxidation. This SnapShot provides an overview of ferroptosis-related pathways, including relevant biomolecules and small-molecule modulators regulating them.

Double agents of cell death: novel emerging functions of apoptotic regulators.

Apoptosis is a highly regulated form of cell death that is required for many homeostatic and pathological processes. Recently, alternative cell death pathways have emerged whose regulation is dependent on proteins with canonical functions in apoptosis. Dysregulation of apoptotic signaling frequently underlies the pathogenesis of many cancers, reinforcing the need to develop therapies that initiate alternative cell death processes. This review outlines the convergence points between apoptosis and other death pathways with the purpose of identifying novel strategies for the treatment of apoptosis-refractory cancers. Apoptosis proteins can play key roles in the initiation, regulation, and execution of nonapoptotic death processes that include necroptosis, autophagy, pyroptosis, mPTP-mediated necrosis, and ferroptosis. Notably, recent evidence illustrates that dying cells can exhibit biochemical and molecular characteristics of more than one different type of regulated cell death. Thus, this review highlights the amazing complexity and interconnectivity of cell death processes and also raises the idea that a top-to-bottom approach to describing cell death mechanisms may be inadequate for fully understanding the means by which cells die.

Ceruloplasmin suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma cells

Ferroptosis is a regulated form of cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Ceruloplasmin (CP) is a glycoprotein that plays an essential role in iron homeostasis. However, whether CP regulates ferroptosis has not been reported. Here, we show that CP suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma (HCC) cells. Depletion of CP promoted erastin- and RSL3-induced ferroptotic cell death and resulted in the accumulation of intracellular ferrous iron (Fe2+) and lipid reactive oxygen species (ROS). Moreover, overexpression of CP suppressed erastin- and RSL3-induced ferroptosis in HCC cells. In addition, a novel frameshift mutation (c.1192-1196del, p.leu398serfs) of CP gene newly identified in patients with iron accumulation and neurodegenerative diseases lost its ability to regulate iron homeostasis and thus failed to participate in the regulation of ferroptosis. Collectively, these data suggest that CP plays an indispensable role in ferroptosis by regulating iron metabolism and indicate a potential therapeutic approach for hepatocellular carcinoma.

Lipid Droplet Proteome Dynamics and Lipotoxicity

Lipid droplets are ER‐derived neutral lipid storage organelles that function as cellular hubs of lipid metabolism, providing an “on demand” source of fatty acids for energy and sequestering fatty acids to protect cells from lipotoxic damage. Lipid droplet functions are controlled by associated integral and peripheral proteins. To understand the composition of lipid droplet proteomes, we recently developed a proximity labeling strategy to define high confidence lipid droplet proteomes. This approach uses lipid droplet‐targeted ascorbate peroxidase (APEX2), which enables the biotinylation of neighboring proteins in a spatially and temporally defined manner. Employing this method, we defined high confidence lipid droplet proteomes in multiple cell lines and identified pathways that impact lipid droplet proteome composition, uncovering a role for a ubiquitin‐dependent ER‐associated degradation (ERAD) pathway in the clearance of select lipid droplet proteins. A key function of lipid droplets is to sequester fatty acids to prevent lipotoxicity. To further understand how the cell responds to lipotoxic insults, we leveraged whole‐genome CRISPR‐Cas9 screens to dissect pathways that regulate lipid damage. For example, exploiting a synthetic lethal screening strategy, we discovered a new pathway that promotes cancer cell resistance to a regulated form of cell death known as ferroptosis, which involves the iron‐dependent accumulation of oxidatively damaged lipids. This study uncovered an unexpected role for non‐mitochondrial CoQ as a lipophilic antioxidant that prevents membrane damage. Together, our findings highlight the integration of systems level discovery approaches and cell biology methods as a powerful strategy to understand the mechanisms that regulate lipid droplets and lipotoxicity.Support or Funding InformationThis research was supported by grants from the National Institutes of Health (R00DK095921and R01GM112948) and from the American Heart Association (16GRNT30870005).

Regenerating islet-derived protein (Reg)3β plays a crucial role in attenuation of ileitis and colitis in mice

Regenerating islet-derived protein (Reg)3β belongs to a member of the Reg family of proteins and has pleiotropic functions, including antimicrobial activity and tissue repair. However, whether Reg3β plays a protective role in the development of colitis and ileitis has not been fully investigated. We generated transgenic mice expressing a short form of cellular FLICE-inhibitory protein (cFLIPs) that promotes necroptosis, a regulated form of cell death. cFLIPs transgenic (CFLARs Tg) mice develop severe ileitis in utero. Although Reg3β is undetectable in the small intestine of wild-type embryos, its expression is aberrantly elevated in the small intestine of CFLARs Tg embryos. To test whether elevated Reg3β attenuates or exacerbates ileitis in CFLARs Tg mice, we generated a Reg3b−/− strain. Reg3b−/− mice grew to adulthood without apparent abnormalities. Deletion of Reg3b in CFLARs Tg mice exacerbated the embryonic lethality of CFLARs Tg mice. Dextran sulfate sodium-induced colitis, characterized by body weight loss and infiltration of neutrophils, was exacerbated in Reg3b−/− compared to wild-type mice. Moreover, the expression of Interleukin 6, an inflammatory cytokine and Chitinase-like 3, a marker for tissue repair macrophages was elevated in the colon of Reg3b−/− mice compared to wild-type mice after DSS treatment. Together, these results suggest that attenuation of colitis and ileitis is a result of Reg3β′s real function.

Ferroptosis in Cancer Cell Biology.

A major hallmark of cancer is successful evasion of regulated forms of cell death. Ferroptosis is a recently discovered type of regulated necrosis which, unlike apoptosis or necroptosis, is independent of caspase activity and receptor-interacting protein 1 (RIPK1) kinase activity. Instead, ferroptotic cells die following iron-dependent lipid peroxidation, a process which is antagonised by glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1). Importantly, tumour cells escaping other forms of cell death have been suggested to maintain or acquire sensitivity to ferroptosis. Therefore, therapeutic exploitation of ferroptosis in cancer has received increasing attention. Here, we systematically review current literature on ferroptosis signalling, cross-signalling to cellular metabolism in cancer and a potential role for ferroptosis in tumour suppression and tumour immunology. By summarising current findings on cell biology relevant to ferroptosis in cancer, we aim to point out new conceptual avenues for utilising ferroptosis in systemic treatment approaches for cancer.

Loss of Microglial Parkin Inhibits Necroptosis and Contributes to Neuroinflammation.

Parkin is an E3 ubiquitin ligase involved in Parkinson's disease (PD). Necroptosis is a regulated form of cell death that depends on receptor interacting protein 1 (RIP1) and 3 (RIP3). Importantly, parkin has been implicated in ubiquitination events that can alter inflammation and necroptosis. Here, we investigated how parkin influences microglial function. Incubation of BV-2 microglial cells with zVAD.fmk (zVAD) induced high levels of cell death and viability loss, while N9 microglial cells and primary microglia required further stimuli. Importantly, necrostatin-1 (Nec-1), an inhibitor of RIP1 kinase activity, abrogated cell death, thus implicating RIP1-dependent necroptosis in cell death. Cell death was characterized by necrosome assembly, as determined by sequestration of RIP1/RIP3 in insoluble fractions and by MLKL phosphorylation, which were all abolished by Nec-1. Also, necroptosis-inducing conditions led to TNF-α secretion, which may in turn contribute to autocrine necroptosis activation. Interestingly, parkin knockdown protected BV-2 cells from zVAD-induced necroptosis, which may depend on the higher RIP1 ubiquitination levels detected in siRNA-PARK2 transfected cells. This effect was independent of inflammation, since pro-inflammatory stimulation of BV-2 and primary microglia with silenced parkin resulted in stronger pro-inflammatory gene expression, an opposite observation from zVAD-exposed BV-2 cells. LPS-mediated inflammation was exacerbated by NF-κB/JNK over-activation. Finally, no alterations in mitochondrial ROS production were detected in any condition, thereby excluding the role of parkin in mitophagy. In conclusion, here, we reveal that parkin may have unsuspected roles in microglia by modulating ubiquitination. Parkin loss exacerbates inflammation and promotes survival of activated microglia, thus contributing to chronic neuroinflammation.

Abstract 2326: Time-resolved studies of necroptosis reveal distinct regulatory functions for NFκB

Abstract Resistance to regulated cell death (RCD) is an important hallmark of cancer and reason why chemotherapy fails. Efficient reprogramming of cancer cells requires a molecular and dynamic understanding of regulatory circuits. Research has uncovered several resistance mechanisms for apoptosis with nuclear factor κB (NFκB) as a key inhibitory transcription factor. Targeted induction of necroptosis, which was recently discovered as an alternative form of RCD, could be a promising strategy to overcome apoptosis resistance. However, whether NFκB has a regulatory role in necroptosis is less clear. Here, we develop a molecular and time-resolved understanding of how NFκB regulates necroptosis. We present a real-time experimental system to quantify necroptosis in single cancer cells to study the cytotoxic effect of substances while avoiding the bias of proliferation in endpoint protocols. Using live-cell microscopy, computational cell tracking and automated image analysis we quantitate necroptotic death rates in L929 cells, a murine fibrosarcoma cell line, treated with tumor necrosis factor (TNF). While it is known that L929 cells predominantly respond with necroptosis despite rapid activation of NFκB, our experiments reveal an unsuspected temporal phasing of necroptosis over a time course of 24 hours. By perturbing NFκB activation dynamics using targeted CRISPR/Cas9 of IκB and/or Rel proteins, we find that NFκB has at least two distinct roles regulating early and late phases of TNF-induced necroptosis. Further, we will present our molecular analysis that identified NFκB-responsive candidate genes and mapped them into a functional temporo-spatial context within the NFκB-necroptosis signaling network. Our combined approach may inform novel personalized anticancer strategies to exploit targeted necroptosis while avoiding NFκB-induced drug resistance. Citation Format: Marie Oliver Metzig, Simon Mitchell, Brooks Taylor, Alexander Hoffmann. Time-resolved studies of necroptosis reveal distinct regulatory functions for NFκB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2326.

MiR-137 regulates ferroptosis by targeting glutamine transporter SLC1A5 in melanoma

Ferroptosis is a regulated form of cell death driven by small molecules or conditions that induce lipid-based reactive oxygen species (ROS) accumulation. This form of iron-dependent cell death is morphologically and genetically distinct from apoptosis, necroptosis, and autophagy. miRNAs are known to play crucial roles in diverse fundamental biological processes. However, to date no study has reported miRNA-mediated regulation of ferroptosis. Here we show that miR-137 negatively regulates ferroptosis by directly targeting glutamine transporter SLC1A5 in melanoma cells. Ectopic expression of miR-137 suppressed SLC1A5, resulting in decreased glutamine uptake and malondialdehyde (MDA) accumulation. Meanwhile, antagomir-mediated inactivation of endogenous miR-137 increased the sensitivity of melanoma cells to erastin- and RSL3-induced ferroptosis. Importantly, knockdown of miR-137 increased the antitumor activity of erastin by enhancing ferroptosis both in vitro and in vivo. Collectively, these data indicate that miR-137 plays a novel and indispensable role in ferroptosis by inhibiting glutaminolysis and suggest a potential therapeutic approach for melanoma.

Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis.

Flow cytometry was been widely used to measure apoptosis for many decades but the researcher has no definitive way of determining other forms of cell death using this technology. The use of Western Blot technology has numerous drawbacks in that all the cells in the sample whether live, dead or maybe undergoing multiple discrete forms of cell death are analysed as one population. Flow cytometry given that it can analyse different sub-populations of cells within a sample would reveal the expression of cell death markers within these sub-populations rather than just give a single result from the entire population. Here we describe a flow cytometric assay fully realising that potential by the use of anti-RIP-3 (Receptor-interacting serine/threonine-protein kinase 3) and anti-active caspase-3 fluorescently tagged antibodies and a fixable live dead fluorescent dye. This allows the determination of the degree of necroptosis, apoptosis and RIP1-dependent apoptosis within live and dead populations. Necroptosis was identified by the up-regulation of RIP3, while RIP1-dependent apoptosis was described by double positive for RIP3/active Caspase-3 events in live and dead populations. Apoptotic cells were defined by an active-Caspase-3+ve/RIP3-ve phenotype. Pan-caspase blocker zVAD and RIP1 inhibitors GSK'481 or necrostatin-1 revealed interesting modulations of such sub-populations of Jurkat cells. This novel flow cytometric assay employing two antibodies and a fixable viability probe provides the researcher with in-depth analysis of various forms of regulated forms of cell death beyond what is currently available and is a major methodological advancement in this field.

Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.

Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis.

Abstract 2315: Overcoming treatment resistance in cisplatin-resistant ovarian carcinoma cells by tissue-specific miRNAs

Abstract Ovarian cancer is a common human cancer with a poor outcome and a high risk of death. Due to resistances to current standard therapies (carboplatin and paclitaxel for 3 to 6 cycles) additional therapeutic approaches are required and novel specific biomarkers for ovarian cancer patients are necessary. MicroRNAs (miRNA) are conserved, small noncoding RNAs regulating gene expression by binding to their target mRNA which have been shown to regulate biological processes including apoptosis. Apoptosis is a conserved, irreversible process that allows cells to undergo a highly regulated form of cell death. However, the final role of miRNAs in apoptotic signaling has not yet been fully determined. Modulating the expression of key molecular components of the cell death machinery is an attractive strategy for cancer therapy overcoming chemo drug resistance. To extend the pool of potential miRNA as anticancer agents and biomarkers, we transferred a high content miRNA screening for pro-apoptotic miRNAs to human cancer cell lines. Based on the high recovery rate of apoptosis inducing miRNAs in SKOV3 cells analyses were extended to multiple ovarian carcinoma cell lines with different genetic background. After transfection of miR-1912-5p the apoptosis rate increased strongly in cisplatin-resistant cells. In combination treatment with carboplatin the apoptotic effect increased after transfection of miR-1912-5p in the cisplatin-resistant A2780 cells. Furthermore, miR-1912-5p, miR-147b and miR-3073a showed significant apoptosis induction in the various ovarian cancer cell lines alone and in combined therapy with carboplatin. Western Blot analysis revealed an enhanced expression of the pro-apoptotic proteins Bak1 and Bax and a decrease inBcl2 and Bcl-xL. We characterized the miRNAs in regard to their endogenous expression and detected an enhanced expression after apoptosis induction by chemotherapeutic drugs. Finally, analysis of The Cancer Genome Atlas data showed an influence on the median survival of ovarian cancer patients (&amp;gt; 69 years). Our data indicate a potential benefit of tissue specific pro-apoptotic miRNAs in regard to cisplatin-resistant cells. Furthermore the study identified novel pro-apoptotic miRNAs with their potential use as novel therapeutic entities or prognostic biomarkers for ovarian cancer. Citation Format: Michael Kleemann, Jeremias Bereuther, Simon Fischer, Kim Marquart, Simon Hänle, Kristian Unger, Verena Jendrossek, Christian U. Riedel, René Handrick, Kerstin Otte. Overcoming treatment resistance in cisplatin-resistant ovarian carcinoma cells by tissue-specific miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2315. doi:10.1158/1538-7445.AM2017-2315

Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death

Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia–reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia–reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death.

Regulated cell death and adaptive stress responses.

Eukaryotic cells react to potentially dangerous perturbations of the intracellular or extracellular microenvironment by activating rapid (transcription-independent) mechanisms that attempt to restore homeostasis. If such perturbations persist, cells may still try to cope with stress by activating delayed and robust (transcription-dependent) adaptive systems, or they may actively engage in cellular suicide. This regulated form of cell death can manifest with various morphological, biochemical and immunological correlates, and constitutes an ultimate attempt of stressed cells to maintain organismal homeostasis. Here, we dissect the general organization of adaptive cellular responses to stress, their intimate connection with regulated cell death, and how the latter operates for the preservation of organismal homeostasis.

Cdk2 phosphorylation of Bcl-xL after stress converts it to a pro-apoptotic protein mimicking Bax/Bak.

Apoptosis is a regulated form of cell death that proceeds by defined biochemical pathways. Most apoptosis is controlled by interactions between pro-survival and pro-apoptotic Bcl-2 family proteins in which death is often the consequence of permeabilization of the mitochondrial outer membrane. Many drugs affect this equilibrium to favor apoptosis but this process is not completely understood. We show that the chemotherapeutic drug cisplatin initiates an apoptotic pathway by phosphorylation of a pro-survival Bcl-2 family member, Bcl-xL, by cyclin-dependent kinase 2. The phosphorylation occurred at a previously unreported site and its biologic significance was demonstrated by a phosphomimetic modification of Bcl-xL that was able to induce apoptosis without addition of cisplatin. The mechanism of cell death induction was similar to that initiated by pro-apoptotic Bcl-2 family proteins, that is, phosphorylated Bcl-xL translocated to the mitochondrial membrane, and formed pores in the membrane. This initiated cytochrome c release and caspase activation that resulted in cell death.

Ferroptosis: Death by Lipid Peroxidation.

Ferroptosis is a regulated form of cell death driven by loss of activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and subsequent accumulation of lipid-based reactive oxygen species (ROS), particularly lipid hydroperoxides. This form of iron-dependent cell death is genetically, biochemically, and morphologically distinct from other cell death modalities, including apoptosis, unregulated necrosis, and necroptosis. Ferroptosis is regulated by specific pathways and is involved in diverse biological contexts. Here we summarize the discovery of ferroptosis, the mechanism of ferroptosis regulation, and its increasingly appreciated relevance to both normal and pathological physiology.

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53.

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.