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

G. Warnes

doi:10.1007/s10495-020-01613-5

Abstract

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.

Highlights

The regulated necrotic cell death process (RCD) necroptosis is a caspase independent process which after TNFα binding to the TNF receptor becomes associated with TRADD (TNF receptor type 1-associated with death domain protein) and TRAF-2 (TNFR-associated factor 2 forming complex I [1,2,3,4,5,6])
The non-exclusion of dead cells induced by the cytotoxic drug in question leads to a massive under-reporting of the degree of MMP in the remaining live cells. This study investigates this discrepancy by using a cell viability probe to show changes in MMP and Reactive Oxygen Species (ROS) generation in live cells undergoing necroptosis as well as oncosis, early, late and RIP1-dependent apoptosis and relates this to parthanatos, cleaved PARP expression and the degree of DNA Damage during RCD and ACD (Accidental Cell Death) processes
The main aim of this flow cytometric based study was to show that shikonin induced Jurkat cell necroptosis and apoptosis resulted in measurable changes in mitochondrial function, MMP and cellular ROS in live, early, late apoptotic and oncotic cell populations

Summary

Introduction

The regulated necrotic cell death process (RCD) necroptosis is a caspase independent process which after TNFα binding to the TNF receptor becomes associated with TRADD (TNF receptor type 1-associated with death domain protein) and TRAF-2 (TNFR-associated factor 2 forming complex I [1,2,3,4,5,6]) This complex together with the RIP1 protein auto-phosphorylates after inhibition of caspase-8 (active caspase-8 cleaves RIP1), [7] which acts as the first ROS sensor in this form of regulated cell death [2,3,4,5,6, 8]. RIP1 is mainly located in the cytoplasm in the form of the necrosome as part of the necroptosis process, nuclear RIP1 has been shown to associate in a kinase independent manner and activate PARP1 inducing PARP1-dependent regulated necrosis or parthanatos Fig. 1 [15]

Methods

Results

Conclusion