Apoptosis

Causes

Current research suggests that apoptosis is one of the predominant cell death mechanisms, summarized below:

• Necroptosis: Occurs following activation of tumor necrosis factor-alpha (TNF-α), triggering several cell death receptors.[5]
• Pyroptosis: Mainly affects cell membrane integrity, engaging inflammasomes to activate caspases.[5][6]
• Apoptosis: Differentiated by the release of cytochrome c from the mitochondria, it is immunologically silent and non-lytic.[5][6]
• Ferroptosis: Characterized by iron-dependent phospholipid peroxides that accumulate in cell membranes, leading to non-apoptotic death.[7][8]

Given common mid-stream mediators, some researchers do not differentiate necroptosis from apoptosis as a separate mechanism. Instead, the simultaneous process is called PANoptosis, where pyroptosis, apoptosis, and necroptosis occur as programmed cell death.[9] In addition, autophagy refers to the process of digesting organelles or other parts of cells through lysosomal machinery, which can also lead to cell death.[10] Some parts of apoptosis are considered reversible, a phenomenon known as anastasis, particularly observed in cancer cell lines.[11]

Anatomical Pathology

Apoptosis is distinct due to the cascade of programmed cell death. Dying cells undergo shrinkage due to the disruption of the cell cytoskeleton, mainly caused by caspases. The cells become deeply eosinophilic, shrinking and distancing from their neighbors with the loss of cell-to-cell contact. The nucleus of the dying cell turns deeply basophilic. The hallmark of apoptosis is pyknosis, in which nuclear chromatin condenses to form 1 or more dark-staining masses against the nuclear envelope. Dissolution of the nuclear membrane occurs, and endonuclease slices the DNA into short, regularly spaced fragments (karyorrhexis).[3]

Subsequently, the condensed cytoplasm and nucleus break into fragments called apoptotic bodies that bud off from the cell. Macrophages then remove these apoptotic bodies in a process called efferocytosis. The cell membrane remains intact without inflammation, unlike necrosis, pyroptosis, or ferroptosis, where cell swelling and inflammation are common. Macrophages remove apoptotic cells quickly, with little or no inflammation occurring in the surrounding tissues. As such, the mechanism is considered immunologically silent.[12]

Clinical Pathology

Detecting apoptosis effectively requires the use of multiple assays, as its multi-stage complexity cannot be fully captured by a single method. Below is an overview of key assays organized by their specific detection targets.

Cytomorphological Changes

Apoptotic cells display distinct features such as chromatin condensation and membrane blebbing. Chromatin changes can be observed using DNA-binding dyes such as 4',6-diamidino-2-phenylindole (DAPI) or Hoechst, which emit brighter fluorescence in condensed nuclei under microscopic examination. Membrane blebbing, visible through phase-contrast microscopy in live cells, is linked to caspase-driven cleavage of proteins such as gelsolin and ROCK-1 kinase. Although fixed cells can be analyzed using caspase substrate markers, this approach may lead to false positives.[3]

DNA Fragmentation

A hallmark of late-stage apoptosis is the breakdown of DNA into 180 to 200 bp fragments. The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay commonly labels 3'-OH DNA ends with fluorescent markers, allowing detection through microscopy or flow cytometry. Alternatively, DNA laddering through agarose gel electrophoresis offers a more affordable but less sensitive option.[13]

Caspase Activity, Cleaved Substrates, and Regulatory Proteins

Caspase activation, particularly caspases-3, -8, and -9, can be measured using fluorogenic substrates or fluorescent inhibitors. Mid-stage apoptosis is often confirmed by detecting poly ADP-ribose polymerase cleavage through Western blot analysis. In addition, the balance between pro- and anti-apoptotic proteins such as Bcl-2 and Bax can be assessed using immunoassays to evaluate regulatory mechanisms.[14]

Membrane Changes

Early apoptosis is characterized by phosphatidylserine externalization, which is detected using Annexin V staining. This staining is often combined with propidium iodide to distinguish apoptotic cells from necrotic ones. In later stages, loss of membrane integrity is monitored using dye exclusion assays.[15]

Whole-Mount Detection

Apoptosis in embryos or tissues can be detected using acidophilic dyes such as acridine orange, Nile blue sulfate, and neutral red. However, these dyes require validation due to nonspecific debris staining and have limitations such as toxicity and poor penetration. LysoTracker Red provides stable, three-dimensional imaging in thick tissues through confocal microscopy.[3]

Mitochondrial Assays

Mitochondrial assays for apoptosis detection focus on key changes, including mitochondrial membrane potential loss, which is detected using dyes such as JC-1, tetramethylrhodamine methyl ester, or tetramethylrhodamine ethyl ester.[3] Permeability transition pore opening is monitored with calcein fluorescence quenching. In addition, cytochrome c release is tracked through immunofluorescence or subcellular fractionation. These assays are often combined with caspase activity measurements for improved specificity.[16]

Mechanisms

Cell proliferation and cell death are balanced in all normal tissues of multicellular organisms. This normal cell death, vital for cell development and health, is called apoptosis and involves the following pathways. All the pathways involve the activation of caspases as the final step.

Intrinsic Pathway

The intrinsic pathway is activated when the cell experiences internal stress due to various factors such as DNA damage from x-ray or UV light exposure, chemotherapeutic agents, hypoxia, or the accumulation of misfolded proteins inside the cell, as observed in conditions such as Alzheimer's disease, Parkinson's disease, or Huntington disease. When the cell undergoes stress, cytochrome c leaks from the intermembrane space of mitochondria into the cytosol, which leads to the activation of caspase-9. The regulation of this pathway is governed by the Bcl-2 and TP53 genes.[17][18] 

The Bcl-2 protein family includes both anti-apoptotic and pro-apoptotic members. Pro-apoptotic proteins detect death signals and trigger cell death, whereas anti-apoptotic proteins block this process. These evolutionarily conserved proteins may operate through varied mechanisms across species. Diverse cellular signals can alter their activity and location, creating a complex network that balances cell survival and death. Due to their key role in managing apoptosis from various triggers, Bcl-2 family proteins are vital for embryonic development and adult homeostasis.[19][20]

Extrinsic Pathway

The extrinsic pathway is triggered when the cell receives death signals from other cells. This pathway is receptor-linked, and the ligands from the other cells bind to these death receptors on the cell surface, activating apoptosis. This process involves the following cell surface receptors and their corresponding ligands, ultimately activating caspase-8, a key regulator in the cascade.

• TNF-α: TNF-α is a cytokine produced by macrophages and is the major extrinsic mediator of apoptosis. TNF-α binds to the receptor TNFR1, thereby activating caspases.

• Fas: T cells generate a surface receptor called Fas, which increases production during an infection. After a few days, the activated T lymphocytes release Fas ligands. When Fas binds to these ligands on the same or different cells, apoptosis is triggered through caspase activation. Fas receptor, a transmembrane protein of the TNF family, interacts with FasL to activate caspases. Apoptosis aids in the removal of the activated T lymphocytes when the infection has been cleared.

• Bcl-2 genes: These anti-apoptotic genes are located on chromosome 18 and produce the protein Bcl-2. Bcl-2 binds to and inhibits APAF-1, preventing the release of cytochrome c from the mitochondria. Cytochrome c is present between the inner and outer mitochondrial membranes. Cytochrome c release leads to its binding with APAF-1, activating procaspase-9.

• TP53 suppressor gene: This gene encodes a protein that regulates the cell cycle and promotes tumor suppression. Suppose DNA is damaged by ionizing radiation, chemotherapeutic agents, or hypoxia. In that case, TP53 arrests the cell in the G1 phase of the cell cycle, preventing the proliferation of cells with damaged DNA and facilitating DNA repair. Severe DNA damage prompts apoptosis by activating BAX apoptosis genes. BAX gene products inactivate the Bcl-2 anti-apoptosis gene.[21] The balance between pro-apoptotic and anti-apoptotic genes regulates the process (see Image. DNA Repair and Apoptosis).

• Cytotoxic CD8+ T-cell pathway: CD8+ T cells secrete perforins, creating holes in the target cells. Subsequently, CD8+ T cells secrete granzymes that enter the target cells through these holes and activate caspases.

• Caspases: Caspases are a group of protease-like enzymes. These enzymes exist in the cell in an inactive form and require proteolytic cleavage to become active. As described above, these enzymes are the primary effectors of apoptotic responses, activated by several regulators.[22]
  • Initiator caspases include caspases 2, 8, 9, and 10. When activated, the initiator caspases activate the effector caspases.
  • Effector caspases encompass caspases 3, 6, and 7. Active effector caspases cleave several proteins in the cell, leading to cell death and, ultimately, phagocytosis and removal of cellular debris.
  • Of all the caspases, caspase-3 is the most frequently activated, which catalyzes the cleavage of major cellular proteins and condensation of chromatin. Caspase also activates DNAse enzymes, which causes DNA fragmentation followed by internucleosomal fragmentation.[23][24]

Execution Pathway

The execution phase, the final step of apoptosis, dismantles the cell through effector caspases, such as caspases 3, 6, and 7, which are activated by upstream caspases from either the extrinsic or intrinsic pathways. These enzymes degrade the nuclear envelope, block DNA repair, and fragment DNA into a distinct pattern through an activated nuclease, marking irreversible breakdown. The cell shrinks, alters its membrane to expose phagocytic signals, and forms debris-filled vesicles, which are cleared by phagocytes to prevent inflammation. Pro-apoptotic and anti-apoptotic proteins tightly regulate this orderly process.[25]

Other Players

Following initial cell death, several danger-associated molecular patterns and pathogen-associated molecular patterns are released from the eliminated cells, signaling additional inflammatory mediators depending on the type of cell death and if other mechanisms are involved. Consequently, whether apoptosis is completely immunologically silent is still debated.[5] Apoptosis proteins are believed to be inhibited in several pathological conditions, particularly cancer, where apoptosis is typically suppressed. These modulators are a family of anti-apoptotic proteins called inhibitors of apoptosis proteins.[26] Cathepsin D is believed to trigger apoptosis, especially during tissue remodeling.[27]

Clinicopathologic Correlations

Embryogenesis

During embryogenesis in the fetus, the formation of the digits involves the apoptosis of interdigital tissues. Similarly, several body cavities undergo apoptosis in utero.[28] For example, a male fetus loses Müllerian structures due to a Müllerian inhibitory factor synthesized by Sertoli cells.[29]

Menstrual Cycle

The sloughing of the inner lining of the uterus (the endometrium) after the withdrawal of estrogen and progesterone in the menstrual cycle is a physiological process of apoptosis.[29]

Immunological Regulation

• Virus-infected cells: Cytotoxic T cells kill the virus-infected cells by apoptosis.
• Cells with DNA damage: Cells whose DNA is damaged by radiation exposure or chemotherapeutic agents are arrested in the G1 phase of the cell cycle for repair by activating p53. P53 is a tumor suppressor gene. A p53 mutation inhibits apoptosis, leading to the survival of abnormal cells and the development of carcinomas.
• Autoreactive T cells: Autoreactive T cells in the thymus are eliminated through apoptosis.[30]

Apoptosis is required for the development and maintenance of a healthy immune system. When B and T lymphocytes are initially produced, they are tested to see whether they react against any of the body's self-components. Cells that react are killed by apoptosis. If these cells are not removed, self-reactive cells may be released into the body, which can attack tissues and cause autoimmune conditions. Apoptosis is required to turn off the immune system after the offending pathogen is cleared from the body, such as removing acute inflammatory cells, including neutrophils, from healing sites. Furthermore, the destruction of B and T lymphocytes by corticosteroids occurs through apoptosis.[31]

Removal of Misfolded Proteins

Apoptosis removes misfolded proteins, such as amyloids and proteins in prion-related diseases. As a result, several formations that may lead to neurodegenerative diseases are eliminated.

Clinical Significance

Tumorigenesis

A decrease in apoptosis results in higher cell survival rates, leading to the development of cancers. In follicular lymphoma, a translocation event relocates the Bcl-2 gene from chromosome 18 to chromosome 14, leading to excessive transcription and increased levels of Bcl-2. Elevated Bcl-2 gene levels inhibit APAF-1, consequently inactivating caspases and intrinsic apoptosis, leading to follicular lymphoma. Mutations or deletions of the p53 gene increase the risk of tumor formation by enabling cells with damaged DNA to divide uncontrollably.[31]

Factors such as exposure to chemicals, radiation, and viruses can damage the p53 gene. Individuals with Li-Fraumeni syndrome have only 1 functional copy of p53; therefore, they are more likely to develop a tumor in early adulthood. When DNA repair mechanisms fail to remove the damaged, translocated, or deleted DNA, cells begin to evade cell cycle checkpoints that lead to apoptosis (see Image. The Mechanism of Apoptosis).[32][33]

Autoimmune Diseases

A decrease in the apoptosis of self-reactive immune cells can lead to the development of autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and autoimmune lymphoproliferative syndrome.[34] Recently, the role of mitochondria in regulating cell death has been linked to the development of diabetes due to the destruction of β-cells.[35]

Neurodegenerative Diseases

Cell death has also been implicated in many neurodegenerative disorders. Necrosis and apoptosis occur in neurologic diseases such as acute ischemic syndrome. In chronic neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, neuronal cell death predominantly occurs through apoptosis and has been implicated.[36]

Cardiovascular Diseases

Necrosis was long considered the sole cause of myocardial infarction. However, recent studies have shown that apoptosis also occurs mainly during the reperfusion phase after the acute infarction, leading to further myocardial damage. Staging atherosclerotic plaques and rupture is correlated to apoptosis, specifically the death of macrophages.[10][37]

Therapeutic Implications

Given the correlation of physiological and pathological processes, the identified players in intrinsic and extrinsic apoptosis are targets for immunotherapy.[37] Several cancer therapies inhibit Bcl-2, which is the most notorious example.[38] Given the multilevel effects of apoptotic pathways, no one specific therapeutic innovation has set the standard. Recently, a TUNEL assay was used to quantify apoptotic cell death, which is pertinent when staging pathological tissue samples.[11] Similarly, the applicability of antibody-specific inhibitors to malignant tumor antigens is perhaps the next level of treatment specificity.[9]