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

Most research on chemical induction of apoptosis is carried out with primary cultures of cell lines (e.g., neurons, thymocytes, carcinoma cells, leukemia cells, neuroblastoma, breast cancer cells, lymphoma); little has been published on the in vivo effects of chemicals on apoptotic cells in animal models and none in humans. Therefore, it was of interest to examine the effects of exposure to low levels of benzene, as well as through drinking water concentrations of up to 14 ppb on the apoptotic cell population, as well as to examine possible changes in the cell cycle progression.⁹

Evidence is sufficient for the carcinogenicity of benzene in humans; therefore, there is no safe level of exposure to this chemical or its metabolites. Published case reports, a case series, epidemiologic studies, and both cohort and case–control studies have shown statistically significant associations between leukemia and occupational exposure to benzene and benzene-containing solvents.^17,18

It has been indicated that possibly 800,000 persons are exposed to benzene from coke oven emissions at levels less than 0.1 ppm, and 5 million may be exposed to benzene from petroleum refinery emissions at levels of 0.1 to 1 ppm. Since then, numerous chemicals have been implicated in apoptosis (or programmed cell death), which arises from damage to DNA. One of the authors, Vojdani along with collaborators, hypothesized that in individuals with a certain genetic makeup, benzene or its metabolites act as haptens, which may induce programmed cell death. The study involved a group of 60 male and female subjects who were exposed to benzene-contaminated water (at concentrations up to 14 ppm for a period of 3 to 5 years).^18a For comparison, a control group consisting of 30 healthy males and females with a similar age distribution and without a history of exposure to benzene were recruited. Peripheral blood lymphocytes of both groups were tested for percentage of apoptotic cell population, using flow cytometry. When exposed individuals were compared with the control group, statistically significant differences between each mean group were detected (27.5 ± 2.4 and 10 ± 2.6, respectively), indicating an increased rate of apoptosis in 86.6% of exposed individuals (P <0.0001; Mann-Whitney U-test). Flow cytometry analysis of apoptosis in a healthy control and a patient with chronic fatigue syndrome is shown in Figure 9-4.

FIGURE 9-4 Enhanced apoptotic cell population in benzene-exposed individuals with chronic fatigue syndrome. Flow cytometry analysis of apoptotic cell population in negative control cells (HL-60 leukemic cell line), positive control cells (HL-60 leukemic cells treated with the Apogen camptothecin), control subjects, and benzene-exposed individuals. Peripheral blood leukocytes were isolated, cultured for 12 hours, fixed in paraformaldehyde, labeled with fluorescent-tagged deoxyuridine triphosphate nucleotides, and analyzed for apoptosis by flow cytometry.

It has been demonstrated that benzene induction of apoptosis is caused by a discrete block of the cell cycle progression.

There is a tendency for normal cells to commit “suicide” when deprived of usual growth factors or physical contact with their neighbors due to chemical exposure, which may represent a built-in defense against metastasis. Prompt activation of apoptosis in tumor cells that leave their native tissue presumably eliminates many metastatic cells before they have a chance to proliferate. In cancer, it is tumor cells that neglect to sacrifice themselves or forget to die. Researchers increasingly describe cancer as a disease involving both excessive proliferation of cells and abandonment of their ability to die. The dysregulation of apoptosis in malignant cells underlies both the initiation and progression of cancer.

Cancer develops after a cell accumulates mutations in several genes that control cell growth and survival. When a mutation seems irreparable, the affected cell usually kills itself rather than risk becoming deranged and potentially dangerous. However, if the cell does not die, it or its progeny may live long enough to accumulate mutations that enable it to divide uncontrollably and metastasize.

In many tumors, genetic damage apparently fails to induce apoptosis because the constituent cells have inactivated the gene that codes for the p53 protein. This protein can lead to activation of the cell’s apoptotic machinery when DNA is injured by environmental agents, such as benzene or its metabolites. Therefore, it is important to study cell suicide in health and diseases.

Apoptosis in Cancer

The failure of apoptosis in malignant cells in the context of irreparable DNA damage leads to tumor progression. Cancer therapies, namely, chemotherapy and radiation, control cancer by inflicting cell damage, which, in turn, triggers apoptosis. Unfortunately, >50% of all human cancers involve a mutation of p53, a central gene in apoptosis. p53 stimulates both the extrinsic death receptor pathway of apoptosis as well as the intrinsic mitochondrial pathway involving a decreased Bcl-2:Bax ratio. Thus, it is imperative to find therapies that promote apoptosis independent of p53. Promising therapies in this regard include curcumin,¹⁹ derived from Curcuma longa, and resveratrol,²⁰ both of which are under investigative study for this application. Another promising cancer treatment involves the use of recombinant human apoptosis ligands to induce tumor necrosis factor-related apoptosis inducing ligand (TRAIL). These ligands induce apoptosis via TRAIL, a selective death receptor pathway in a broad range of cancer cell lines while sparing most normal cell types.²¹

The therapeutic potential for TRAIL inducing ligands is most promising in combination with cytotoxic chemotherapy agents. Advances in cancer therapy are likely to come in the arena of targeted therapies, the majority of which trigger specific receptor-driven pathways that culminate in apoptosis. The centrality of apoptosis induction in cancer cannot be overstated.

Apoptosis in Autoimmune Diseases

In cancer, it is the tumor cells that forget to die; in autoimmunity, immune cells fail to die when they are supposed to. Virtually all tissues harbor apoptotic cells at one time or another. Damaged cells usually commit suicide for the greater good of the body; when this does not occur, disease may develop. Autoimmunity occurs when the antigen receptors on immune cells recognize specific antigens on healthy cells and cause the cells bearing those particular substances to die. Autoimmune disease results from perpetuated immune-mediated tissue destruction, and can involve immune cells that are resistant to apoptosis. Under normal conditions, the body allows a certain number of self-reactive lymphocytes to circulate. These cells normally do little harm, but they can become overactive through several processes. For instance, if these reactive lymphocytes recognize some foreign antigen such as microbes on food and haptenic chemicals, then exposure to that antigen causes them to become excited. If, due to molecular mimicry, these antigens are similar to normal tissues, the activated cells may expand their numbers and attack the healthy tissue, thus causing an autoimmune disease.^1,22,23

Autoimmune reactions usually are self-limited—they disappear when the antigens that originally set them off are cleared away. In some instances, however, the autoreactive lymphocytes survive longer than they should and continue to induce apoptosis in normal cells. Some evidence in animals and humans has indicated that extended survival of autoreactive cells is implicated in at least two chronic autoimmune syndromes—systemic lupus erythematosus and rheumatoid arthritis. In other words, the lymphocytes undergo too little apoptosis, with the result that normal cells undergo too much.^24,25

Apoptosis During Viral Infection

Disturbance in the regulation of apoptosis is a component in various diseases. Viral illnesses are among the diseases caused by apoptosis dysregulation. After entering a cell, viruses attempt to shut down the cell’s ability to make any proteins except those needed to produce more virus. This act of stalling host protein synthesis is enough to induce many kinds of cells to undergo apoptosis. If the host cell dies, the virus is also eliminated. Therefore, certain viruses have evolved ways to inhibit apoptosis in the cells they infect.

Epstein-Barr virus, which causes mononucleosis and has been linked to lymphomas in humans, uses a mechanism that has been seen in other viruses. Epstein-Barr virus produces substances that inhibit apoptosis. Papillomavirus, a major cause of cervical cancer, inactivates p53, a central mediator of apoptosis. Cowpox virus, a relative of which is used as the smallpox vaccine, is another virus that inhibits caspase activation and attendant apoptosis. Investigators interested in antiviral therapy are now exploring ways to block the activity of the antiapoptotic molecules manufactured by viruses.²⁴

Apoptosis in Acquired Immunodeficiency Syndrome

Induction of apoptosis by viruses in healthy cells is believed to contribute to the immune deficiency found in patients with acquired immunodeficiency syndrome (AIDS). In these patients, infection with human immunodeficiency virus (HIV) causes T-helper cells to die. As T-helper cells gradually disappear, cytotoxic cells, such as natural killer cells, perish as well through apoptosis, because they cannot survive without the growth signals produced by T-helper cells. When the number of T cells dwindles, so does the body’s ability to fight infections, especially viral and parasitic infections. Researchers have shown that many more helper cells succumb in addition to those that are infected with HIV. It is also highly probable that a large number of the cells die through apoptosis. Apparently, Fas plays a crucial role in this process.

Normally, T cells make functional Fas only after they have been active for a few days and are ready to die. However, helper cells from AIDS patients may display high amounts of functional Fas even before the cells have encountered an antigen. This display of Fas would be expected to cause the cells to undergo apoptosis prematurely whenever they encounter Fas ligand on other cells (such as on T cells already activated against HIV or other microbes). In addition, if the primed cells encounter the antigen recognized by their receptors, they may trigger their own death.

It is also possible that oxygen-free radicals trigger the suicide of virus-free T cells. These highly reactive substances are produced by inflammatory cells drawn to infected lymph nodes in HIV patients. Free radicals can damage DNA and membranes in cells. They will cause necrosis if they do extensive damage, but they can induce apoptosis if the damage is more subtle. In support of the free-radical theory, researchers have found that molecules capable of neutralizing free radicals prevent apoptosis in T cells obtained from AIDS patients.^24,25

Therapies with antiapoptotic medication, such as Trolox, a water-soluble analog of vitamin E that prevents oxidative stress, and pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor-κB, are now the focus of AIDS and autoimmune disease studies.^26,27

Additionally, protease inhibitors, which are the mainstay of HIV therapy, inhibit apoptosis in immune cells.²⁸

The mechanism underlying the apoptosis inhibition is as of yet unknown, but interestingly, supratherapeutic doses of protease inhibitors have an opposite, pro-apoptotic, effect.

Apoptosis in the Heart and Brain

In contrast to cancer, where cells forget to die and insufficient apoptosis occurs, excessive apoptosis accounts for much of the cell death that follows heart attacks and strokes. In the heart, vessel blockage decimates cells that were fully dependent on the vessel. Those cells die by necrosis, partly because they are catastrophically starved of the oxygen and glucose they need to maintain themselves and partly because calcium ions, which are normally pumped out of the cell, rise to toxic levels.

Over the course of a few days, cells surrounding the dead zone, which initially survive because they continue to receive nourishment from other blood vessels, can die as well. Later, however, many cells die by necrosis after being overwhelmed by the destructive free radicals that are released when inflammatory cells swarm into the dead zone to remove necrotic tissue. The less injured cells commit suicide by apoptosis.

If the patient is treated by restoring blood flow, still more cells may die by necrosis or apoptosis because reperfusion leads to a transient increase in the production of free radicals. Similarly, in strokes due to inflammation, release of such neurotransmitters as glutamate lead to necrosis and apoptosis. Understanding of the factors that lead to the tissue death accompanying heart attack, stroke, and reperfusion has led to new ideas for treatment. Notably, cell death might be limited by drugs and other agents that block free-radical production or inhibit proteases.

Apoptosis also accounts for much of the pathology seen in such diseases as Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis (Lou Gehrig’s disease), which are marked by the loss of brain neurons. Elevated apoptosis in these neurologic diseases seems to be related to lack of production of the nerve growth factor and to free radical damage. It seems likely that a combination of such factors could cause many cells to destroy themselves. Manipulation of this process of cell killing may help in treating these neurologic diseases. Studies in animal models imply that long-term delivery of nerve growth factors could protect against programmed cell death in these conditions. Therefore, a greater understanding of the mechanisms involved in cell death should greatly enhance those important steps.^22,26,29