Flow Cytometry Reveals the Nature of Oncotic Cells

Abstract

The term necrosis is commonly applied to cells that have died via a non-specific pathway or mechanism but strictly is the description of the degradation processes involved once the plasma membrane of the cell has lost integrity. The signalling pathways potentially involved in accidental cell death (ACD) or oncosis are under-studied. In this study, the flow cytometric analysis of the intracellular antigens involved in regulated cell death (RCD) revealed the phenotypic nature of cells undergoing oncosis or necrosis. Sodium azide induced oncosis but also classic apoptosis, which was blocked by zVAD (z-Vla-Ala-Asp(OMe)-fluoromethylketone). Oncotic cells were found to be viability^+ve/caspase-3^-ve/RIP3^+ve/-ve (Receptor-interacting serine/threonine protein kinase 3). These two cell populations also displayed a DNA damage response (DDR) phenotype pH2AX^+ve/PARP^-ve, cleaved PARP induced caspase independent apoptosis H2AX^-ve/PARP^+ve and hyper-activation or parthanatos H2AX^+ve/PARP^+ve. Oncotic cells with phenotype cell viability^+ve/RIP3^-ve/caspase-3^-ve showed increased DDR and parthanatos. Necrostatin-1 down-regulated DDR in oncotic cells and increased sodium azide induced apoptosis. This flow cytometric approach to cell death research highlights the link between ACD and the RCD processes of programmed apoptosis and necrosis.

Keywords: DDR; accidental cell death; flow cytometry; oncosis; parthanatos.

Figure 1

Cell death and caspase-3 activation assay. Cells were (A) untreated; (B) treated with 0.25% sodium azide (NaN₃) for 24 h; (C) pre-treated with 20 µM zVAD for 2 h, then with 0.25% NaN₃; (D) pre-treated with 60 μM necrostatin-1 (Nec-1) for 2 h, then with 0.25% NaN₃; (E) pre-treated with 20 μM zVAD and 60 μM Nec-1 for 2 h, then with 0.25% NaN₃; (F) treated with 1 μM Etoposide (Etop) for 24 h; (G) pre-treated with 20 μM zVAD for 2 h, then with 1 μM Etop; (H) pre-treated with 60 μM Nec-1 for 2 h, then with 1 μM Etop; and (I) pre-treated with 20 μM zVAD and 60 μM Nec-1 for 2 h, then with 1 μM Etop. n = 3, % Mean ± % SEM; Student’s t-test: NS (not significant), * p < 0.05, ** p < 0.01**, *** p < 0.001; arrows indicate change compared with untreated cells.

Figure 2

RIP3 and caspase-3 activation analysis of oncosis. After gating on live and dead cells from a Zombie NIR vs. caspase3-BV650 dot-plot (A) untreated live and (B) dead Jurkat cells were analysed on a RIP3-PE vs. caspase-3-BV650 dot-plot with resting phenotype indicated by RIP3^+ve/caspase-3^–ve, apoptosis by RIP3^–ve/caspase-3^+ve, RIP1-dependent apoptosis RIP3^+ve/caspase-3^+ve, and double negative RIP3^–ve/caspase-3^–ve. Live and dead cells treated with (C,D) 0.25% NaN₃ for 24 h; (E,F) pre-treated with 20 μM zVAD for 2 h, then treated with 0.25% NaN₃; (G,H)pre-treated with 60 μM Nec-1 for 2 h, then treated with 0.25% NaN₃; and (I,J) pre-treated with 20 μM zVAD and 60 μM Nec-1 for 2 h, then treated with 0.25% NaN₃, respectively. n = 3, % Mean ± % SEM, Student’s t–test: NS (not significant), * p < 0.05, ** p < 0.01**, *** p < 0.001; arrows indicate change compared with untreated cells.

Figure 3

Parthanatos/hyper-activation of cleaved PARP, apoptosis via cleaved PARP, and DDR analysis of oncosis. Untreated Jurkat cells, treated with 0.25% NaN₃ for 24 h, or pre-treated with zVAD (20 μM) and/or Nec-1 (60 μM) for 2 h, then incubated with 0.25% NaN₃. Gating live and dead cells from a Zombie NIR vs. caspase-3-BV650 plot then both were analysed on a RIP3-PE vs. caspase-3-BV650 plot. Next, early and late apoptotic, necroptotic/resting, RIP1-dependent apoptotic, and double negative (DN) populations were analysed for pH2AX and cleaved PARP (Figures S2, S3). The incidence of (A) parthanatos/hyper-activation of cleaved PARP, (B) apoptosis via cleaved PARP, and (C) DDR were determined for all populations listed above. n = 3, % Mean, error bars % SEM, Student’s t-test; NS (not significant), * p < 0.05, ** p < 0.01**, *** p < 0.001 compared with untreated cells.

Figure 4

RIP3 and caspase-3 activation analysis of apoptosis. Gating on live and dead cells from a Zombie NIR vs. caspase-3-BV650 plot followed by analysis on a RIP3-PE vs. caspase-3-BV650 plot with resting phenotype indicated by RIP3^+ve/caspase-3^–ve, apoptosis by RIP3^–ve/caspase-3^+ve, RIP1-dependent apoptosis by RIP3^+ve/caspase-3^+ve and double negative by RIP3^–ve/caspase-3^–ve. (A,B) Treated with 1 μM Etop for 24 h; (C,D), pre-treated with 20 μM zVAD for 2 h, then treated with 1 μM Etop; (E,F) pre-treated with 60 μM Nec-1 for 2 h, then treated with 1 μM Etop; and (G,H) pre-treated with 20 μM zVAD and 60 μM Nec-1 for 2 h, then treated with 1 μM Etop. N = 3, % Mean ± % SEM, Student’s t–test: NS (not significant), * p < 0.05, ** p < 0.01**, *** p < 0.001; arrows indicate change compared with untreated cells.

Figure 5

Parthanatos/hyper-activation of cleaved PARP, apoptosis via cleaved PARP, and DDR analysis of apoptosis. Untreated Jurkat, treated with 1 μM Etop or pre-treated with zVAD (20 μM) and/or Nec-1 (60 μM) for 2 h, then incubated with 1 μM Etop for 24 h. Gating on live and dead cells from a Zombie NIR vs. caspase-3-BV650 plot then both were analysed on a RIP3-PE vs. caspase-3-BV650 plot. Early and late apoptotic, necroptotic/resting, RIP1-dependent apoptotic, and double negative (DN) populations were analysed for pH2AX and cleaved PARP (Figures S2,S4 for detailed information). The incidence of (A) parthanatos/hyper-activation of cleaved PARP, (B) apoptosis via cleaved PARP, and (C) DDR were determined for all populations listed above. Mean, error bars % SEM, Student’s t-test: NS (not significant), * p < 0.05, ** p < 0.01**, *** p < 0.001 compared with untreated cells.

Figure 6

ACD and RCD pathways. (A) Live cells can undergo either early apoptosis (EAPO) or oncosis after drug treatment, with early apoptotic cells moving to late apoptotic (LAPO), then later with cell degradation, to the oncotic or necrotic phenotype. (B) Live cells may express RIP3^+ve/caspase-3^–ve when resting, or be RIP3^high+ve/caspase-3^–ve when undergoing necroptosis, or be double negative (DN). EAPO cells lose RIP3 or, if retained, undergo RIP1-dependent apoptosis (RIP1-APO). EAPO cells can also become RIP1^+ve. (C) Loss of plasma membrane integrity or cell death results in cell phenotypes mirrored in (B), with degradation of cells resulting in the DN population. (B,C) Live and dead cell phenotypes can also express pH2AX (DDR) or cleaved PARP (apoptosis), both of which can ultimately express both proteins, (D,E) resulting in pH2AX hyper-activation of cleaved PARP in the presence of active caspase-3 or parthanatos in the absence of caspase-3. Arrows indicate movement of cell populations.