Increase, Cell death

Cell death is part of normal development and maturation cycle, and is the component of many response patterns of living tissues to xenobiotic agents (i.e.. micro organisms and chemicals) and to endogenous modulations, such as inflammation and disturbed blood supply (Kanduc et al., 2002). Many physiological processes require cell death for their function (e.g.., embryonal development and immune selection of B and T cells) (Bertheloot et al., 2021). Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer (Crowley et al., 2016).

Cell death can be divided into: programmed cell death (cell death as a normal component of development) and non-programmed cell death (uncontrolled death of the cell). Although this simplistic view has blurred the intricate mechanisms separating these forms of cell death, studies have and will uncover new effectors, cell death pathways and reveal a more complex and intertwined landscape of processes involving cell death (Bertheloot et al., 2021).

Programmed cell death: is a form of cell death in which the dying cell plays an active part in its own demise (Cotter & Al-Rubeai, 1995).

Apoptosis At a morphological level, it is characterized by cell shrinkage rather than the swelling seen in necrotic cell death. It is characterized by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme‐dependent biochemical processes. The result of it being the clearance of cells from the body, with minimal damage to surrounding tissues. An essential feature of apoptosis is the release of cytochrome c from mitochondria, regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7). Apoptosis culminates in the breakdown of the nuclear membrane by caspase-6, the cleavage of many intracellular proteins (e.g., PARP and lamin), membrane blebbing, and the breakdown of genomic DNA into nucleosomal structures (Bertheloot et al., 2021). Mechanistically, two main pathways contribute to the caspase activation cascade downstream of mitochondrial cytochrome c release:

• Intrinsic pathway is triggered by dysregulation of or imbalance in intracellular homeostasis by toxic agents or DNA damage. It is characterized by mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome c into the cytosol.
• Extrinsic pathway is initiated by activation of cell surface death receptors. Proapoptotic death receptors include TNFR1/2, Fas and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5.

Other pathways of programmed cell death are called »non-apoptotic programmed cell-death« or »caspase-independent programmed cell-death« (Blank & Shiloh, 2007).

Necroptosis: This type of regulated cell death, occurs following the activation of the tumor necrosis receptor (TNFR1) by TNFα. Activation of other cellular receptors triggers necroptosis. These receptors include death receptors (i.e., Fas/FasL), Toll-like receptors (TLR4 and TLR3) and cytosolic nucleic acid sensors such as RIG-I and STING, which induce type I interferon (IFN-I) and TNFα production and thus promote necroptosis in an autocrine feedback loop. Most of these pathways trigger NFκB- dependent proinflammatory and prosurvival signals.

Pyroptosis is a type of cell death culminating in the loss of plasma membrane integrity and induced by activation of so-called inflammasome sensors. These include the Nod-like receptor (NLR) family, the DNA receptor Absent in Melanoma 2 (AIM2) and the Pyrin receptor.

Autophagy: is a process where cellular components such as macro proteins or even whole organelles are sequestered into lysosomes for degradation (Mizushima et al., 2008; Shintani & Klionsky, 2004). The lysosomes are then able to digest these substrates, the components of which can either be recycled to create new cellular structures and/or organelles or alternatively can be further processed and used as a source of energy (D’Arcy, 2019).

Anoikis is apoptosis induced by loss of attachment to substrate or to other cells (anoikis). Anoikis overlaps with apoptosis in molecular terms, but is classified as a separate entity because of its specific form od induction (Blank & Shiloh, 2007). Induction of anoikis occurs when cells lose attachment to ECM, or adhere to an inappropriate type of ECM, the latter being the more relevant in vivo (Gilmore, 2005).

Cornification: is programmed cell death of keratinocytes. Cell death in the context of cornification involves distinct enzyme classes such as transglutaminases, proteases, DNases and others (Eckhart et al., 2013).

Non-programmed cell death: occurs accidentally in an unplanned manner.

Necrosis is generally characterized to be the uncontrolled death of the cell, usually following a severe insult, resulting in spillage of the contents of the cell into surrounding tissues and subsequent damage thereof (D’Arcy, 2019).

Assays for Quantitating Cell Death:

• Cell death can be measured by staining a sample of cells with trypan blue, assay is described in protocol: Measuring Cell Death by Trypan Blue Uptake and Light Microscopy (Crowley, Marfell, Christensen, et al., 2015d). Or with propidium Iodide, assay is described in protocol: Measuring Cell Death by Propidium Iodide (PI) Uptake and Flow Cytometry (Crowley & Waterhouse, 2015a)
• TUNEL technique: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Uribe et al., 2013).

Assays for Quantitating Cell Survival          

Colony-forming assay can be used to define the number of cells in a population that are capable of proliferating and forming large groups of cells. Described in Protocol: Measuring Survival of Adherent Cells with the Colony-Forming Assay (Crowley, Christensen, & Waterhouse, 2015c); Measuring Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar (Crowley & Waterhouse, 2015b).

ASSAYS TO DISTINGUISH APOPTOSIS FROM NECROSIS AND OTHER DEATH MODALITIES

Detecting Nuclear Condensation: The nucleus is generally round in healthy cells but fragmented in apoptotic cells. Dyes such as Giemsa or hematoxylin, which are purple in color and therefore easily viewed using light microscopy, are commonly used to stain the nucleus. Other features of apoptosis and necrosis, such as plasma membrane blebbing or rupture, can be identified by staining the cytoplasm with eosin. Eosin is pinkish in color and can also be viewed using light microscopy. Hematoxylin and eosin are, therefore, commonly used together to stain cells. Assay is described in Protocol: Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining (Crowley, Marfell, & Waterhouse, 2015c); Analyzing Cell Death by Nuclear Staining with Hoechst 33342 (Crowley, Marfell, & Waterhouse, 2015a).

Detection of DNA Fragmentation: Apoptotic cells with fragmented DNA can be identified and distinguished from live cells by staining with Propidium Iodide (PI) and measuring DNA content by flow cytometry. This assay is described in Protocol: Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry (Crowley, Chojnowski, & Waterhouse, 2015a). TUNEL technique can also be used: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Crowley, Marfell, & Waterhouse, 2015b; Uribe et al., 2013).

Detecting Phosphatidylserine Exposure: Apoptosis is also characterized by exposure of phosphatidylserine (PS) on the outside of apoptotic cells, which acts as a signal that triggers removal of the dying cell by phagocytosis. Annexin V, can selectively bind to PS to label apoptotic cells in which PS is exposed. Purified annexin V can be conjugated to various fluorochromes, which can then be visualized by fluorescence microscopy or detected by flow cytometry. This assay is described in protocol: Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry (Crowley, Marfell, Scott, et al., 2015e).

Detecting Caspase Activity: antibodies that specifically recognize the cleaved fragments of caspases and their substrates can be used to specifically detect caspase activity in apoptotic cells by immunocytochemistry. Flow cytometry (using primary antibodies conjugated to fluorescent molecules, or by counter staining with fluorescently labeled antibodies against the primary antibody) can then be used to quantitate the number of apoptotic cells. This assay is described in protocol: Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry (Crowley & Waterhouse, 2015a).

Detecting Mitochondrial Damage: flow cytometry can be used to quantitate the number of cells that have reduced mitochondrial transmembrane potential, which is commonly associated with cytochrome c release during apoptosis. For this assay see protocol: Measuring Mitochondrial Transmembrane Potential by TMRE Staining (Crowley, Christensen, & Waterhouse, 2015b).

Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed. 

 Measures of apoptotic cytomorphological alterations: 

 Apoptotic cells exhibit electron dense nuclei, nuclear fragmentation, intact cell membrane up to the disintegration phase, disorganized cytoplasmic organelles, large clear vacuoles, blebs at cell surface, and apoptotic bodies, which can be visualized with various methods. (Elmore, 2007; Watanabe et al., 2002) 

DNA damage/fragmentation assays: 

Measurement of apoptotic markers through immunochemistry: 

Measures of altered caspase activity: 

Measures of altered mitochondrial physiology: 

Other measures: 

The process of cell death is highly conserved within multi‐cellular organisms. (Lockshin & Zakeri, 2004).

Taxonomic applicability: Increased cell death is applicable to all animals. This includes vertebrates such as humans, mice and rats (Alberts et al., 2002).  

Life stage applicability: There is insufficient data on life stage applicability of this KE. 

Sex applicability: This key event is not sex specific (Forger and de Vries, 2010; Ortona Matarrese, and Malorni, 2014).  

Evidence for perturbation by a stressor: Multiple studies show that cell death can be increased or disrupted by many types of stressors including ionizing radiation and altered gravity (Zhu et al., 2016).  

Alberts, B. et al. (2002), “Programmed Cell Death (Apoptosis)”, in Molecular Biology of the Cell. 4th edition, Garland Science, New York, https://www.ncbi.nlm.nih.gov/books/NBK26873/  

Bertheloot, D., Latz, E., & Franklin, B. S. (2021). Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cellular & Molecular Immunology, 18, 1106–1121. https://doi.org/10.1038/s41423-020-00630-3

Bever, M. M., & Fekete, D. M. (1999). Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears. Journal of Neurocytology, 28(10–11), 781–793. https://doi.org/10.1023/a:1007005702187

Blank, M., & Shiloh, Y. (2007). Cell Cycle Programs for Cell Death: Apoptosis is Only One Way to Go. Cell Cycle, 6(6), 686–695. https://doi.org/10.4161/cc.6.6.3990

Cotter, T. G., & Al-Rubeai, M. (1995). Cell death (apoptosis) in cell culture systems. Trends in Biotechnology, 13(4), 150–155. https://doi.org/10.1016/S0167-7799(00)88926-X

Crowley, L. C., Chojnowski, G., & Waterhouse, N. J. (2015a). Measuring the DNA content of cells in apoptosis and at different cell-cycle stages by propidium iodide staining and flow cytometry. Cold Spring Harbor Protocols, 10, 905–910. https://doi.org/10.1101/pdb.prot087247

Crowley, L. C., Christensen, M. E., & Waterhouse, N. J. (2015b). Measuring mitochondrial transmembrane potential by TMRE staining. Cold Spring Harbor Protocols, 12, 1092–1096. https://doi.org/10.1101/pdb.prot087361

Crowley, L. C., Christensen, M. E., & Waterhouse, N. J. (2015c). Measuring survival of adherent cells with the Colony-forming assay. Cold Spring Harbor Protocols, 8, 721–724. https://doi.org/10.1101/pdb.prot087171

Crowley, L. C., Marfell, B. J., Christensen, M. E., & Waterhouse, N. J. (2015d). Measuring cell death by trypan blue uptake and light microscopy. Cold Spring Harbor Protocols, 7, 643–646. https://doi.org/10.1101/pdb.prot087155

Crowley, L. C., Marfell, B. J., Scott, A. P., Boughaba, J. A., Chojnowski, G., Christensen, M. E., & Waterhouse, N. J. (2016). Dead cert: Measuring cell death. Cold Spring Harbor Protocols, 2016(12), 1064–1072. https://doi.org/10.1101/pdb.top070318

Crowley, L. C., Marfell, B. J., Scott, A. P., & Waterhouse, N. J. (2015e). Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harbor Protocols, 11, 953–957. https://doi.org/10.1101/pdb.prot087288

Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015a). Analyzing cell death by nuclear staining with Hoechst 33342. Cold Spring Harbor Protocols, 9, 778–781. https://doi.org/10.1101/pdb.prot087205

Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015b). Detection of DNA fragmentation in apoptotic cells by TUNEL. Cold Spring Harbor Protocols, 10, 900–905. https://doi.org/10.1101/pdb.prot087221

Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015c). Morphological analysis of cell death by cytospinning followed by rapid staining. Cold Spring Harbor Protocols, 9, 773–777. https://doi.org/10.1101/pdb.prot087197

Crowley, L. C., & Waterhouse, N. J. (2015a). Detecting cleaved caspase-3 in apoptotic cells by flow cytometry. Cold Spring Harbor Protocols, 11, 958–962. https://doi.org/10.1101/pdb.prot087312

Crowley, L. C., & Waterhouse, N. J. (2015b). Measuring survival of hematopoietic cancer cells with the Colony-forming assay in soft agar. Cold Spring Harbor Protocols, 8, 725. https://doi.org/10.1101/pdb.prot087189

D’Arcy, M. S. (2019). Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biology International, 43(6), 582–592. https://doi.org/10.1002/cbin.11137

Eckhart, L., Lippens, S., Tschachler, E., & Declercq, W. (2013). Cell death by cornification. Biochimica et Biophysica Acta - Molecular Cell Research, 1833(12), 3471–3480. https://doi.org/10.1016/j.bbamcr.2013.06.010

Elmore, S. (2007), “Apoptosis: A Review of Programmed Cell Death”, Toxical Pathology, Vol. 35/4, SAGE, https://doi.org/10.1080/01926230701320337. 

Forger, N. G. and G. J. de Vries (2010), “Cell death and sexual differentiation of behavior: worms, flies, and mammals”, Current opinion in neurobiology, Vol. 20/6, Elsevier, Amsterdam, https://doi.org/10.1016/j.conb.2010.09.006  

Gilmore, A. P. (2005). Anoikis. Cell Death and Differentiation, 12, 1473–1477. https://doi.org/10.1038/sj.cdd.4401723

Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A. A., Natale, C., Santacroce, R., Di Corcia, M. G., Lucchese, A., Dini, L., Pani, P., Santacroce, S., Simone, S., Bucci, R., & Farber, E. (2002). Cell death: apoptosis versus necrosis (review). International Journal of Oncology, 21(1), 165–170. https://doi.org/10.3892/ijo.21.1.165

Kressel, M. and P. Groscurth (1994), "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA", Cell and tissue research, Vol. 278/3, Nature, https://doi.org/10.1007/BF00331373. 

Lockshin, R. A., & Zakeri, Z. (2004). Apoptosis, autophagy, and more. International Journal of Biochemistry and Cell Biology, 36(12), 2405–2419. https://doi.org/10.1016/j.biocel.2004.04.011

Martinez, M. M., R. D. Reif, and D. Pappas (2010), “Detection of apoptosis: A review of conventional and novel techniques”, Analytical Methods, Vol. 2/8, Royal Society of Chemistry, https://doi.org/10.1039/C0AY00247J 

Mizushima, N., Levine, B., Cuervo, A. M., & Klionsky, D. J. (2008). Autophagy fights disease through cellular self-digestion. Nature, 451(7182), 1069–1075. https://doi.org/10.1038/nature06639

Nicoletti I. et al. (1991), “A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry”, Journal of Immunological Methods, Vol. 139/2, Elsevier, Amsterdam, https://doi.org/10.1016/0022-1759(91)90198-O

Ortona, E., P. Matarrese, and W. Malorni (2014), “Taking into account the gender issue in cell death studies”, Cell Death & Disease, Vol. 5, Nature, https://doi.org/10.1038/cddis.2014.73.  

Parajuli, K. R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", American journal of clinical and experimental urology, Vol. 2/4, pp. 300-312. 

Shintani, T., & Klionsky, D. J. (2004). Autophagy in health and disease: A double-edged sword. Science, 306(5698), 990–995. https://doi.org/10.1126/science.1099993

Sivandzade, F., A. Bhalerao and L. Cucullo (2019), “Analysis of the Mitochondrial Membrane Potential Using Cationic JC-1 Dye as a Sensitive Fluorescent Probe”, Bio Protocol, Vol. 9/1, https://doi.org/10.21769/BioProtoc.3128. 

Uribe, P. M., Sun, H., Wang, K., Asuncion, J. D., & Wang, Q. (2013). Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio). PLoS ONE, 8(3), 58755. https://doi.org/10.1371/journal.pone.0058755

Wade, M. G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biology of Reproduction, Vol. 78/5, Oxford University Press, Oxford, https://doi.org/10.1095/biolreprod.107.065151 

Watanabe, M., et al. (2002), “The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs”, Microscopy and microanalysis, Vol. 8/5, Cambridge University Press, Cambridge, https://doi.org/10.1017/S1431927602010346. 

Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One, Vol. 11/12, PLOS, San Francisco, https://doi.org/10.1371/journal.pone.0167052. 

Zhu, M., et al. (2021), “Immunogenic Cell Death Induction by Ionizing Radiation”, Frontiers in Immunology, Vol. 12, https://doi.org/10.3389/FIMMU.2021.705361.