Related Experiment Video
Updated: Sep 21, 2026
Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis
Published on: February 16, 2015
Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early
N Zamzami1, P Marchetti, M Castedo
1Centre National de la Recherche Scientifique, Unité Propre de Recherche 420, Villejuif, France.
Abstract:
Programmed cell death (PCD) is a physiological process commonly defined by alterations in nuclear morphology (apoptosis) and/or characteristic stepwise degradation of chromosomal DNA occurring before cytolysis. However, determined characteristics of PCD such as loss in mitochondrial reductase activity or cytolysis can be induced in enucleated cells, indicating cytoplasmic PCD control. Here we report a sequential disregulation of mitochondrial function that precedes cell shrinkage and nuclear fragmentation. A first cyclosporin A-inhibitable step of ongoing PCD is characterized by a reduction of mitochondrial transmembrane potential, as determined by specific fluorochromes (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine++ + iodide; 3,3'dihexyloxacarbocyanine iodide). Cytofluorometrically purified cells with reduced mitochondrial transmembrane potential are initially incapable of oxidizing hydroethidine (HE) into ethidium. Upon short-term in vitro culture, such cells acquire the capacity of HE oxidation, thus revealing a second step of PCD marked by mitochondrial generation of reactive oxygen species (ROS). This step can be selectively inhibited by rotenone and ruthenium red yet is not affected by cyclosporin A. Finally, cells reduce their volume, a step that is delayed by radical scavengers, indicating the implication of ROS in the apoptotic process. This sequence of alterations accompanying early PCD is found in very different models of apoptosis induction: glucocorticoid-induced death of lymphocytes, activation-induced PCD of T cell hybridomas, and tumor necrosis factor-induced death of U937 cells. Transfection with the antiapoptotic protooncogene Bcl-2 simultaneously inhibits mitochondrial alterations and apoptotic cell death triggered by steroids or ceramide. In vivo injection of fluorochromes such as 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide; 3,3'dihexyloxacarbocyanine iodide; or HE allows for the detection of cells that are programmed for death but still lack nuclear DNA fragmentation. In particular, assessment of mitochondrial ROS generation provides an accurate picture of PCD-mediated lymphocyte depletion. In conclusion, alterations of mitochondrial function constitute an important feature of early PCD.
Insights
Programmed cell death involves early mitochondrial dysfunction, including reduced membrane potential and reactive oxygen species (ROS) generation, preceding nuclear changes. This mitochondrial pathway is crucial for cell death and can be detected in vivo.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Programmed cell death (PCD) is traditionally defined by nuclear morphology changes (apoptosis) and DNA fragmentation.
- However, cytoplasmic factors can control PCD, as evidenced by enucleated cell death.
- Mitochondrial involvement in PCD has been suggested but not fully elucidated sequentially.
Purpose of the Study:
- To investigate the sequential events of mitochondrial dysfunction during programmed cell death (PCD).
- To identify specific mitochondrial alterations that precede nuclear fragmentation in PCD.
- To explore the potential of targeting mitochondrial pathways for detecting and understanding PCD.
Main Methods:
- Utilized fluorochromes (e.g., JC-1, DiOC6) to assess mitochondrial transmembrane potential.
- Employed hydroethidine (HE) oxidation assay to detect reactive oxygen species (ROS) generation.
- Investigated effects of cyclosporin A, rotenone, ruthenium red, and radical scavengers on PCD.
- Examined PCD in lymphocytes, T cell hybridomas, and U937 cells induced by various stimuli.
- Assessed the impact of Bcl-2 transfection on mitochondrial alterations and PCD.
Main Results:
- Identified a cyclosporin A-sensitive reduction in mitochondrial transmembrane potential as an early PCD event.
- Demonstrated a subsequent, rotenone/ruthenium red-sensitive increase in mitochondrial ROS generation.
- Observed that cell shrinkage is delayed by radical scavengers, implicating ROS in the apoptotic process.
- Confirmed this mitochondrial sequence across diverse PCD models (glucocorticoid, T cell activation, TNF-induced).
- Showed that Bcl-2 inhibits both mitochondrial changes and PCD.
Conclusions:
- Mitochondrial dysfunction, including loss of membrane potential and ROS production, is a critical early event in PCD.
- These mitochondrial alterations precede nuclear fragmentation and cytolysis.
- Mitochondrial fluorochromes can detect cells undergoing PCD before nuclear DNA fragmentation, offering diagnostic potential.
Related Concept Videos
01:46Electron Transport Chain: Complex I and II
ROS generation is regulated and maintained at moderate levels necessary...
01:30Overview of Cell Death
Cell death was observed in the early 19th century, but there was no experimental evidence to prove it. In 1842, Carl Vogt first discovered cell death in a metamorphic toad; however, it was not termed ‘cell death.’ Scientists discovered different cell death pathways only in the 20th century...
01:30Apoptosis
01:00Cellular Injury I: Introduction
01:16Cellular Injury IV: Necrosis
01:22Cellular Injury V: Apoptosis and Autophagy