Measurable Features of Apoptosis

One of the most easily measured features of apoptotic cells is the breakup of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of DNA laddering when the DNA is analyzed by agarose gel electrophoresis. The DNA of nonapoptotic cells, which remains largely intact, does not display this laddering on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3’-hydroxyl termini of DNA ends. This property can also be used to identify apoptotic cells by labeling the DNA breaks with fluorescent-tagged deoxyuridine triphosphate nucleotides. The enzyme terminal deoxynucleotidyl transferase catalyzes a template-independent addition of deoxyribonucleotide triphosphates to the 3’-hydroxyl ends of double- or single-stranded DNA. A substantial number of these sites are available in apoptotic cells, providing the basis for the single-step fluorescent labeling and flow cytometric method. Nonapoptotic cells do not incorporate significant amounts of the fluorescent-tagged deoxyuridine triphosphate nucleotides due to the lack of exposed 3’-hydroxyl DNA ends.

Apoptosis can also be characterized by changes in cell membrane structure. During apoptosis, the cell membrane’s phospholipid asymmetry changes—phosphatidylserine is exposed on the outer membrane, whereas membrane integrity is maintained. Annexin V specifically binds phosphatidylserine, whereas propidium iodide is a DNA-binding fluorochrome. When a cell population is exposed to both reagents, apoptotic cells stain positive for annexin V and negative for propidium iodide; necrotic cells stain positive for both, and live cells stain negative for both.³

This process of apoptosis and its analysis by flow cytometry are shown in Figures 9-1 and 9-2.

FIGURE 9-1 Detection of apoptosis using damaged membrane or DNA single-strand break and flow cytometry.

FIGURE 9-2 Separation of cells by flow cytometry and detection of apoptotic population.

Another assessment of apoptosis involves ex vivo cell analysis. Specifically, the expression of active caspase-3 along with the Bcl-2/Bax ratio as markers of apoptosis can be measured. Immunohistochemical staining will reveal the expression of these apoptotic-related proteins, caspase-3 and cleaved caspase-3; the latter is indicative of apoptosis.⁷ Bcl-2 is anti-apoptotic gene product that exists in ratio to Bax and Bak, which are pro-apoptotic gene products. This ratio is indicative of the degree of apoptosis, with a decreased Bcl-2:Bax ratio indicative of apoptosis. Cells from Bax (−/−) and Bak(−/−) knockout animals do not respond to apoptosis inducers. In these cells, cytochrome C is not released from the mitochondrial membrane to initiate the caspase cascade.⁸ Thus, Bax and Bak are critical to apoptosis, and their expression in relation to Bcl-2 is highly correlative to apoptosis.

Different Stages of Apoptosis

The process of apoptosis is divided into three different stages:

These stages of apoptosis are depicted in Figure 9-3. Induction represents the initial events that signal a cell so that apoptosis may begin. This induction phase may be induced by various physical agents, such as toxic chemicals, hypoxia, radiation, chemotherapy agents, hormones, and CD95 or Fas ligation. It has been proposed that the induction stage of apoptosis is prevented by many antioxidants (vitamin C, β-carotene, and vitamin E) and also by various biological response modifiers, including lentinan, thymic hormones, viral antigens, and cytokines.

FIGURE 9-3 Various stages of “inside out” cell death or apoptosis.

The induction stage is followed by a decision on whether or not the cell will undergo apoptosis. The decision to die is under the control of a number of different pathways or cellular sensors that induce the apoptosis signal, which then triggers the central mechanisms. During this stage, enzymes such as interleukin1-β converting enzymes, serine protease, cysteine protease, granzymes, and cyclin-dependent kinases become activated. Once activated, these enzymes dismantle the cell and trigger the cell surface changes that cause direct cell recognition and engulfment of the dying cells by phagocytes. These central events are prevented by various antioxidants and biological response modifiers.

Apoptosis is Induced by Chemicals to Control Malignancy

Many chemicals have the capacity to bind to DNA, form DNA adducts, or cause DNA single-strand breaks, possibly leading to cancer. However, the body is equipped with many factors, enzymes, suppressor genes, and cellular sensors, all with the capacity to prevent the consequences of this DNA damage by activating apoptosis-inducing signals.

The role of apoptosis in regulating tissue growth is readily apparent in the simple equation in which the rate of growth is equal to the difference between the rates of cell proliferation and cell death. Thus, tissues expand if the rate of proliferation exceeds the rate of cell death. This is one of the reasons for suggesting that defects in apoptosis may contribute to the transformed state.

An important prediction of the relevance of apoptosis to malignancy is that the rate of apoptosis versus mitosis should influence the behavior of a tumor. Recently, the relationship between the apoptotic and mitotic indexes in a tumor was demonstrated as predictive of outcome: a higher ratio of apoptosis to mitosis within the tumor correlated with positive prognosis. Further, it was found that this was not simply a function of cell death per se. Tumors with a high incidence of necrosis rather than apoptosis were correlated with poor prognosis. It therefore follows that treatments or conditions that favor apoptosis should have desirable effects, and that defects in the pathways leading to apoptosis are likely to play important roles in the process of oncogenesis.^4,5

Many reactive chemicals and drugs such as acetaminophen, diquat, carbon tetrachloride, quinones, cyanide, polyhydroxyl polyether, methyl mercury, and organotin have been implicated in apoptosis (programmed cell death) and necrosis (toxic cell death).^9–16