Histology, Cell Death

Issues of Concern

When a cell is exposed to an insult, whether external or internal, it can respond differently depending on the circumstances. Cell response can lead to adaptation, apoptosis, or necrosis. Adaptation successfully maintains homeostasis by reversible responses that can alter cell function and structure. A physiological example of adaptation is seen in pregnancy, where uterine cells increase in size (hypertrophy) and number (hyperplasia). Adaptations may also result in atrophy, a decrease in cell size and activity, which is classically seen with the disuse of certain muscle groups. Metaplasia is an adaptation that results in a change of the cell type. Pathologically, this is seen in diseases such as Barrett esophagitis, where increased exposure of esophageal cells to gastric acid results in a phenotypic change from columnar to squamous.

Function

If a cell is unable to adapt to increased stress, it results in injury. Cell injury is reversible until a threshold is reached, after which it progresses to cell death. Historically, cell death has been designated into 2 classes: necrosis and apoptosis. Necrosis is often coined as accidental death, as it is generally seen as not controlled by the cell. Apoptosis, on the other hand, is typically viewed as programmed cell death, regulated and controlled. The 2 differ in presentation, morphology, and mechanism, though recognized overlap is discussed at the end of this review.

Necrosis typically occurs when extensive damage to the cell membrane and internal structures pushes the cell past the point of reversible injury. It is usually the result of acute external factors, such as trauma, ischemia, toxins, or other non-physiologic mechanisms. Necrotic cell death results in a specific set of features, including plasma membrane damage, cellular swelling, and unregulated nuclear degradation. 

Ischemia, for example, typically ends in necrotic cell death. With reduced oxygen delivery to the cell, oxidative phosphorylation in mitochondria becomes compromised. This results in a decreased amount of ATP available in the cytoplasm. In cells with a Na+/K+ (sodium-potassium) pump, a lack of sufficient ATP disables the transporter. Sodium begins to increase within the cell as it can no longer be pumped out. This results in cellular swelling as the increased intracellular sodium pulls water into the cell. Cellular swelling, blebbing, and loss of microvilli are typical presentations of necrotic cell death. If ATP is restored in time, these presentations are reversible. If ATP is not restored before further cell damage, the loss of ATP progresses to have extensive effects on the cell.

Without ATP restoration, the Ca+ pump ceases to function, leading to an influx of calcium ions. Free calcium levels are usually kept low inside the cytoplasm as calcium activates many cellular processes. With the increased free calcium now in the cell, it begins to interact with cellular enzymes, causing further membrane and nuclear damage.[2] Phospholipase, protease, endonuclease, and ATPase are all activated by increased cytoplasmic calcium. Phospholipase damages the plasma membrane by hydrolyzing phospholipids, resulting in whorled patterns called myelin figures. Proteases catalyze proteolysis, breaking down proteins within the cell and disrupting normal cellular activity. Endonuclease cleaves the phosphodiester bond in a polynucleotide chain, collapsing nuclear chromatin into clumps. ATPase breaks down any remaining ATP, accelerating energy loss and causing further damage.

Free cytoplasmic calcium increases mitochondrial permeability by opening a transitional pore. Exposure of mitochondrial contents artificially releases cytochrome C (Cyt C), an important factor in the electron transport chain and in apoptosis and cell death. Cyt C initiates a cascade of cell injury by directly activating caspases. This is a way that necrosis and apoptosis begin to overlap, as caspase activation is a primary mechanism of apoptotic death. In necrotic cell death, the release of caspases is coincidental to cellular injury. In apoptosis, caspase activation is purposeful and initiated through a series of regulatory factors.

The cellular characteristics of necrotic cell death do not always follow the pattern seen in ischemic injury, but it serves as a good example. Necrosis results in cell lysis and exposure of cytoplasmic contents in an uncontrolled setting as the plasma membrane is damaged, regardless of the type of damage that led it to this stage. The release of cellular contents into the surrounding area generally elicits an inflammatory response, unlike apoptosis.

Clinically, cell lysis and enzymatic leakage are used to assess a potential pathology in patients. When a cardiac cell is damaged, it undergoes lysis, and cell-specific enzymes are released into the extracellular space. The presence of these cell-specific enzymes, troponin, and CK-MB (creatine kinase-muscle/brain), can be used to diagnose myocardial infarctions.

If necrotic cell death is considered accidental cell death, then apoptosis would be referred to as a type of programmed cell death. Recently, a form of controlled necrosis called necroptosis was identified.[3][4] Apoptosis may be viewed as a form of self-sacrifice by the cell to prevent collateral damage to the surrounding tissue. Although the mechanisms that lead to necrosis are always pathological, apoptosis does not share the same distinction. Apoptotic mechanisms are used during embryology to shape fingers, selectively resorbing the webbing between digits.[5] Immunologically, apoptotic mechanisms are used during T-cell development to destroy cells that attack self-antigens. Apoptosis is an essential topic in cancer research and DNA damage. When cell cycle regulators detect irreparable DNA damage, signals are released to trigger cell death. Cancer cells often employ mechanisms to evade these signals, leading to immortal cell lines with mutated DNA that is no longer constrained by cell cycle checkpoints.

Characteristics of apoptosis include cell shrinkage, an intact membrane, chromatin condensation (pyknosis), apoptotic bodies, and a lack of an inflammatory response. Potentially dangerous cytoplasmic enzymes are carefully packaged and blebbed off the membrane in vacuoles called apoptotic bodies. Lymphocytes later phagocytose these bodies without causing local damage. The key to controlled apoptosis is caspases.

Caspase is a portmanteau for cysteine-aspartic proteases, named for the active site cysteine and its cleaving after aspartic acid residues. Caspases exist as inactive enzymes (zymogens) until apoptotic signals activate them.[6] These pro-caspases are the targets of Cyt C and FADD.

If a cell recognizes it is infected, it may signal its destruction by altering its membrane, flipping the inner leaflet out to expose phospholipids (specifically, phosphatidylserine) to the outer leaflet, and ultimately triggering a response from patrolling lymphocytes. Upon noticing the altered membrane, lymphocytes initiate the extrinsic apoptosis pathway in that cell.

An outside signal initiates the extrinsic pathway via death receptors (DRs). In T-cell-mediated death, the plasma membrane-associated death receptor (FasR) is engaged by the T-cell death ligand (FasL). The FasR/FasL interaction, also known as the death receptor pathway, recruits inner membrane Fas-associated death domain (FADD) proteins. FADD (or the death effector domain), newly formed, can combine with pro-caspase 8 (zymogen of caspase 8).[7] This fusion creates the death-inducing signaling complex (DISC). DISC can then self-cleave, releasing active caspase 8 from its zymogen form.

Caspases are broken into 2 groups: activators and executioners. Appropriately named, activator caspases 2, 8, 9, and 10 switch on the executioners, caspases 3, 6, and 7. The executioner caspases regulate DNA fragmentation, membrane alterations, chromatin condensation, and many other factors that modulate the cellular milieu for apoptosis. Caspase 9, released from the extrinsic pathway, extinguishes the cell unless inhibited by the survival factor XIAP (X-linked inhibitor of apoptosis protein). Because of its ability to escape the death cycle of a cell, XIAP has been connected to several types of cancer.[8]

The intrinsic apoptotic pathway (also called the mitochondrial pathway) responds to triggers within the cell, such as irreparable DNA damage.[9] MOMP (mitochondrial outer membrane permeabilization) is the climactic deciding factor in the intrinsic pathway.[10][11] As in ischemic cell death, mitochondrial contents, such as Cyt C, initiate terminal events by activating caspases. Upstream from MOMP is the controller (and often main target of cancer mutations) of the intrinsic pathway, BCL-2 (B-cell lymphoma 2).

BCL-2 is part of the BCL family, which has anti-apoptotic properties (along with BCL-X and MCL-1).[12] It is located on the outer mitochondrial membrane (OMM) and blocks pro-apoptotic, channel-forming proteins, Bax (bcl-2 associated X protein) and Bak (BCL-2 antagonist or killer), from causing MOMP and releasing inner mitochondrial membrane (IMM) content. Bax/Bak are transmembrane proteins that can oligomerize and create a pore allowing proapoptotic intermembrane space (IMS) proteins to leak out in the cytoplasm. One of these IMS proteins, known as Cyt C, initiates the caspase cascade when lost from the IMM. Loss of Cyt C from the IMM destroys the cell's energy-creating capacity via the electron transport chain. For these 2 reasons, MOMP is often seen as an “all or nothing” step, since without functioning mitochondria, the cell is unlikely to survive.

BCL-2 is inactivated by pro-apoptotic BH3 proteins Bim, Bad, or Bid (subsets of the BCL-2 family that contain only a single BH3 domain). Inactivation of BCL-2 allows Bax/Bak to combine and form a pore, letting IMS proteins leak into the cytoplasm. Newly released Cyt C begins the formation of apoptosomes by combining with adaptor proteins (Apaf-1) in the cytoplasm. Apoptosomes activate the zymogen pro-caspase 9, as DISC activates pro-caspase 8.

Clinical Significance

Cells that harbor a mutation in BCL-2, where it increases quantity or remains to inhibit Bak/Bax despite death signals, can become cancerous. For example, follicular lymphoma harbors a quantity mutation that leads to excessively high BCL-2 levels, decreasing the propensity of these cells to release Bak/Bax and enter apoptosis. If there is a lot of BCL-2 protecting the cell, there would need to be a greater increase in Bim/Bad/Bid to counteract it, which does not occur in this cancerous state.

p53 is a tumor suppressor gene that can function through the same Bax/Bak pathway. P53 functions as a regulator within the cell cycle at the G1/S checkpoint. It can arrest the cell cycle at this stage to allow time for DNA repair if P53 senses any mutation. P53 is upregulated when the cell experiences DNA damage or cell cycle abnormalities. The apoptotic pathway of P53 is activated when mutations are beyond repair, leading to Bax activation.[13] As a prominent tumor suppressor, P53 mutations are associated with a wide range of cancers. Endometrial carcinoma commonly harbors a P53 mutation that inactivates the protein’s ability to pause growth or trigger cell death. Pathology reports may show heavy P53 staining in these biopsies, suggesting upregulation due to mutations, but the increased P53 is nonfunctional.

Understanding cell death and the players involved is a subject of ongoing research. The better one understands the mechanism of cell death, the more likely it is that knowledge can be integrated into clinical medicine. It is possible to use these pathways to one's advantage, simulating apoptosis by inhibiting BCL-2 in tumor cells, as in the chemotherapy drug oblimersen used for chronic lymphocytic leukemia. Chemotherapy treatments with radiation can manipulate these pathways more directly by causing DNA damage that drives the cell to apoptosis.[14] Understanding the basics of cell death helps clarify how tumor cells may evade death and counter-evade clinically.