Abstract
Currently the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. We have previously shown that intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3+ ve/RIP3− ve), necroptosis (RIP3Hi + ve/Caspase-3− ve) and RIP1-dependent apoptosis (Caspase-3+ ve/RIP3+ ve) in a single Jurkat cell population. The addition of more intracellular markers allows the determination of the incidence of parthanatos (PARP), DNA Damage Response (DDR, H2AX), H2AX hyper-activation of PARP (H2AX/PARP) autophagy (LC3B) and ER stress (PERK), thus allowing the identification of 124 sub-populations both within live and dead cell populations. Shikonin simultaneously induced Jurkat cell apoptosis and necroptosis the degree of which can be shown flow cytometrically together with the effects of blockade of these forms of cell death by zVAD and necrostatin-1 have on specific RCD populations including necroptosis, early and late apoptosis and RIP1-dependent apoptosis phenotypes in live and dead cells. Necrostatin-1 and zVAD was shown to modulate levels of shikonin induced DDR, hyper-action of PARP and parthanatos in the four forms of RCD processes analysed. LC3B was up-regulated by combined treatment of zVAD with chloroquine which also revealed that DNA damage was reduced in live cells but enhanced in dead cells indicating the role of autophagy in maintaining cell health. This approach to RCD research should be a great advance to understanding the mechanisms of drugs and their effects upon RCD populations.
Highlights
The evolution of the cell death classification from simple macroscopic morphological characteristics termed Type I–III modes of cell death, including apoptosis, autophagy and necrosis, respectively to include biochemical and functional parameters has refined and reclassified the many forms of Regulated Cell Death (RCD) [1,2,3,4]. These include intrinsic and extrinsic apoptosis, immunogenic cell death (ICD) or viral-infection induced translocation of calreticulin (CALR) from the ER to the outer plasma membrane leaflet [5], autophagy and lysosome dependent cell death [4], necroptosis [6], MPT or mitochondrial “permeability transition pore complex”—driven necrosis (Oxidative stress and calcium overload) [4], parthanatos (hyper-activation of Poly (ADP-ribose) polymerase or PARP) caused by excessive DNA damage [7,8,9], pyroptosis [10], ferroptosis which is ROS and iron dependent lipid peroxidation and mitochondrial dysfunction controlled by GPX4 or GSH-dependent enzyme glutathione peroxidase [11], NETotic cell death and entotic cell death or loss of integrin signalling in epithelial cells [4]. This classification currently leaves out RIP1-dependent apoptosis RIP3/Caspase-3 [6, 12,13,14], the DNA Damage Response (DDR) [15] and ER stress, which causes autophagy and apoptotic cell death [4, 16], with cell senescence [17] and mitotic catastrophe being classed as a non-lethal process [18]
For example the drug shikonin a naturally occurring naphthoquinone which [19, 23,24,25,26] is known to induce apoptosis as well as necroptosis in Jurkat cells, so we investigated the modulation of the incidence of these and other types of RCD including RIP1dependent apoptosis, DDR, hyper-action of PARP and parthanatos by pan-caspase and RIP protein blocker zVAD and necrostatin-1 respectively [26, 27]
All live treated populations showed increased hyper-activation of PARP (DP, H2AX+ ve/ PARP+ ve) and parthanatos (H2AX− ve/PARP+ ve) with a decreased incidence of the quadruple negative population for all live cell phenotypes compared to untreated cells (QN, Fig. 4a, b, Fig. 1S i–l)
Summary
The evolution of the cell death classification from simple macroscopic morphological characteristics termed Type I–III modes of cell death, including apoptosis, autophagy and necrosis, respectively to include biochemical and functional parameters has refined and reclassified the many forms of Regulated Cell Death (RCD) [1,2,3,4] These include intrinsic and extrinsic apoptosis (often expressing active caspase-3), immunogenic cell death (ICD) or viral-infection induced translocation of calreticulin (CALR) from the ER to the outer plasma membrane leaflet [5], autophagy (up-regulation of LC3B) and lysosome dependent cell death (caused by cathepsins) [4], necroptosis (up-regulation of RIP3) [6], MPT or mitochondrial “permeability transition pore complex”—driven necrosis (Oxidative stress and calcium overload) [4], parthanatos (hyper-activation of Poly (ADP-ribose) polymerase or PARP) caused by excessive DNA damage (e.g. by H2AX) [7,8,9], pyroptosis (up-regulation of Caspase-1 and plasma membrane pre-formation by gasdermin) [10], ferroptosis which is ROS and iron dependent lipid peroxidation and mitochondrial dysfunction controlled by GPX4 or GSH-dependent enzyme glutathione peroxidase [11], NETotic cell death (granulocyte H2AX) and entotic cell death or loss of integrin signalling in epithelial cells [4]. The recent [19] development of a three colour flow cytometry assay in this laboratory targeting Caspase-3, RIP3 and cell viability has allowed the detection and quantification of multiple forms of RCD including, necroptosis, early and late apoptosis and RIP1-dependent apoptosis simultaneously in a single cell population.
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