Nocodazole Induced Suicidal Death of Human Erythrocytes

Abstract

Background: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis. Methods: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy. Results: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca²⁺ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin. Conclusions: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca²⁺ entry, oxidative stress and ceramide.

The Human Papillomavirus E6 PDZ Binding Motif Links DNA Damage Response Signaling to E6 Inhibition of p53 Transcriptional Activity

“…We therefore investigated whether the nocodazole-induced E6 phosphorylation patterns might be part of a stress response. Indeed, nocodazole has been implicated in induction of oxidative stress (38) and DNA damage (39,40) in the cell. One of the potential mechanisms is inhibition of MTH1 (41), which leads to the incorporation of oxidized nucleotides into the DNA and subsequent DNA damage (42).…”

Oxidative stress, eryptosis and anemia: a pivotal mechanistic nexus in systemic diseases

“…Eryptosis may be induced by oxidative stress , which not only plays a crucial role in eryptosis‐related pathophysiology in several diseases but also contributes to its stimulation by a wide spectrum of xenobiotics . Oxidative stress is defined as an augmented production and/or a diminished elimination of reactive oxygen species (ROS) affecting the major cellular components such as DNA, lipids and proteins.…”

Mesoscale Dynamics of Spectrin and Acto-Myosin shape Membrane Territories during Mechanoresponse

“…We next investigated the mechanisms regulating spectrin turnover by FRAP analysis upon drug treatment (LatA, Blebbistatin, Nocodazole, Diamide). Since maximal response to the drugs was observed after 5 minutes, a dual-FRAP assay on single cells expressing GFP-βII-spectrin was performed before and after 5 minutes of treatment to avoid secondary effects driven by long-term cytoskeletal perturbation (Mikulich, Kavaliauskiene and Juzenas, 2012; Signoretto et al ., 2016) (Figure 4 C-E). The impairment of actin filament turnover by latrunculin A did not affect significantly either the half-time recovery (Figure 4 D) or the mobile fraction of spectrin (Figure 4 E, Extended Table 2).…”