Apoptosis is a controlled process of cell death where a sequential event leads to discarding
harmful cells ensuring they don’t release toxic substances into neighboring cells and spaces. By
removing worn out, unused and sickly cells, apoptosis has a very important function of
development and maintenance of the general body health. If any factor or condition interrupt or
prevent apoptosis from happening, harmful cells that are supposed to be eliminated by apoptosis
may persist and refuse to die like in cancers (Lowe & Lin, 2000). Apoptosis is a process that is
highly regulated because once it began it cannot be stopped. Two activation mechanisms regulate
the initiation of the apoptosis process, these activation mechanisms are the intrinsic pathway and
the extrinsic pathway.
In the intrinsic pathway, apoptotic proteins target the mitochondrial which is vital in
multicellular life. By targeting the mitochondria, they affect it in different ways, by forming
membrane pores they may result in mitochondria swelling, they may also affect the permeability
of the membranes of mitochondria and these can cause apoptotic effectors to ooze out (Nagata,
2018). Through the intrinsic pathway, tumors develop easily due to sensitivity caused than in the
extrinsic pathway. Extrinsic is governed by two direct initiation theories, the tumor necrosis
factor (TNF-induced) and Fas-Fas ligand-mediated models. TNF-alpha is a cytokine produced by
macrophages activation and it is the main extrinsic mediator of apoptosis (Elmore, 2007). The
first apoptosis signal (the fas receptor) is in its form transmembrane proteins that belong to the
TNF family and it holds the Fas-ligand (FasL) together. Fas and FasL interaction results in the
growth of death-inducing signaling complex (DISC) that contains caspase-8, caspase-10, and
FADD.
Characteristics of apoptotic cells
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DNA content: Apoptotic cells have been found to contain reduced DNA stainability. This after
staining apoptotic cells with different fluorochromes. The cells that appear to have low DNA
stainability that is lower than G1 cells that are treated with drugs are considered the marker for
cell death through apoptosis (Gorczyca et al., 1992). The reduced DNA stainability results from
partial loss of DNA due to endogenous endonuclease activation and diffusion of products of
DNA with low molecular weight before cell measurement.
Plasma membranes integrity: For cells undergoing apoptosis, the integrity of the plasma
membrane don’t change. Thus, apoptotic cells exclude assay viability dyes which include trypan
blue and PI.
This contradicts to cell necrosis because in necrosis loss of membrane functions and their
structural integrity are the earliest changes observed (Gorczyca et al., 1992). Apoptotic cells can
be classified erroneously as viable cells by flow cytometry due to their ability PI exclusion.
Mitochondrial transmembrane potential: In apoptotic cells, there exist no energy metabolism
changes. Since apoptosis is a process that requires energy, denovo protein synthesis is required to
ensure the process takes place (Darzynkiewicz et al,. 2009). Also in apoptotic cells, the
morphology of their mitochondria does not change. The necrosis contradicts this observation
since mitochondria swelling represent the earliest changes in necrosis.
The lysosomal proton pump: Apoptotic cells ability to accumulate AO in lysosomes does not
change. This simply indicates that, ATP-dependent proton pump is not too much affected during
apoptosis (Gorczyca et al., 1992). But in necrotic cells, they lose some of their ability to take AO
in lysosomes which results in less AO red luminescence.
Protein and RNA content: the apoptotic cells have been found to have low DNA content and
reduced protein content. This has been shown by differently staining DNA and also protein with
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DAPI and Sulforhadamine (Martin et al., 2009). The necrotic cells have leaky plasma membrane;
these have resulted in reduced protein content. But their DNA content remains unaltered
compared to live cells, therefore, the ratio of DNA/protein ratio is higher in necrotic cells than in
apoptotic cells or in live cells.
Necrosis though considered as an alternative to apoptosis is considered a harmful process with
the cell being a victim and it results in the death of the cell in a process that does not involve
energy. In some cases necrosis in considered inappropriate in description of cell death
mechanism since it involves degradative processes that happens after the death of a cell
(Nicotera et al., 1999). Therefore oncosis process brings about necrosis with cell swelling or
karyolysis but apoptosis cause death of cells with pyknosis, karyorrhexis, and shrinkage of cells
(Brown & Attardi, 2005). Whether the death of a cell is caused by apoptosis of necrosis is
dependent on the tissue type, signal of cell death, tissue development stage, and physiologic
milieu.
Apoptosis assays
Apoptosis happens through a complicated signaling cascade which regulated via many points;
therefore, many chances are there to investigate the activities of the protein involved. To counter
the tendency of features of necrosis overlapping with those of apoptosis, two or more assays are
employed to ascertain that apoptosis is the cause of cell death (Buzgariu,. 2009). One assay is
used to detect the early apoptotic events initiation while the other is used to target execution
events in a later moment. On deciding the appropriate method to use in detecting apoptosis in
cells, organs, and tissues, and the pros and cons in every assay are crucial to take into
consideration.
Cytomorphological Alterations assay
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This assay utilizes the ability of eosin-stained tissue parts and hematoxylin to allow visualization
of apoptotic cells under light microscopy (Kioussi et al., 2012). Just one apoptotic cell is
evaluated using this method and so the necessity to confirm with other methods is there.
Additionally, cells involved in early apoptosis phase might not be detected since this method
detects only late events of apoptosis. Semi-ultrathin extracted from epoxy-resin-embedded block
is stained with methylene blue or toluidine blue and it is evaluated using standard light
microscopy to show intensely strained apoptotic cells. This method depends on cytoplasmic and
nuclear condensation that happens during apoptosis. This technique preserves cellular and tissue
details and survey of substantial tissue regions; this is one of the advantages of this method.
However, minute apoptotic tissues might not be sensed and dense intracellular granules are
mistaken to be apoptotic cells.
Confirming apoptosis from this method can be achieved using transmission electron microscopy
(TEM). Transmission electron microscopy is known to the best method to ascertain apoptosis
since apoptotic cell categorization is irrefutable when the cells have ultrastructural
morphological characteristics. The following are those morphological characteristics: Electron-
dense nucleus, nuclear fragmentation, scattered cytoplasmic organelles, intact cell membrane,
clear vacuoles, and blebs found on the surface of the cell. Time usage, cost, and capability to
assay a little region at one time are the major disadvantage of TEM.
Apoptosis detection in assay of the whole mount
Dyes that includes Nile blue sulfate (NBS), neutral red (NR) and acridine orange (AO) are used
to show apoptosis in whole mounts of tissues and embryos. The outcome of this method will
need to be confirmed using other assays because dyes do not differentiate degrading of
lysosomes, apoptotic debris and debris degradation like microorganisms (Burchardt et al., 2017).
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The strength of these dyes is that they are inexpensive and fast. But they have demerits. AO is
mutagenic and toxic and it quenches under normal conditions. Nile blue sulfate and NR cannot
enter thick tissues and are easily diminished in preparation for doing sections. Lyso-Tracker Red
can be used as an alternative since it acts like other dyes but it can only be utilized under laser
confocal microscopy to show 3-dimensional images of apoptotic cells. Additionally this type dye
has quenching resistance, can penetrate thick tissues, and during processing it is stable. Besides,
this dye is utilized for cell culture and whole mounts of tissues, organs, and embryos.
Mitochondrial Assays
In mitochondrial assay the release of cytochrome c shows variations in the early stages of the
intrinsic pathway. Status of mitochondrial redox, mitochondrial permeability transition (MPT),
reactive oxygen, and calcium fluxes can be monitored using this method (Zamzami et al., 2007).
The gradient of the electrochemical on mitochondrial outer membrane (MOM) reduces during
apoptosis, these results in detection with a fluorescent cationic dye. In measuring metabolic
activity or redox potential of the cell’s mitochondria we can use other mitochondrial dyes.
However, some dyes fail to show cell death mechanism and they are too used together with other
methods.
Apoptosis defined as a strictly regulated process that as a dependency in energy is distinguished
by certain biochemical and morphological features and caspase activation holds a key role. Many
apoptotic proteins that are either activated or inactivated along the apoptotic pathway have been
discovered. But the molecular mechanisms and activation process of these proteins are still not
clear and they form a basis of continued research.
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References
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Traganos, F. (1992). Features of apoptotic cells measured by flow cytometry. Cytometry,
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Lowe, S. W., & Lin, A. W. (2000). Apoptosis in cancer. Carcinogenesis, 21(3), 485-495.
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Nicotera, P., Leist, M., & Ferrando-May, E. (1999, September). Apoptosis and necrosis: different
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Kyrylkova, K., Kyryachenko, S., Leid, M., & Kioussi, C. (2012). Detection of apoptosis by
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