APOPTOSIS: INTRODUCTION, MORPHOLOGIC CHANGES AND MECHANISM
Author: Sanketh DS, MDS
INTRODUCTION AND ETIOLOGY
Apoptosis, also called “programmed cell death” is the process where the cell regulates its own death through the production of certain enzymes. These enzymes cause degradation of nuclear and cytoplasmic DNA and the cell breaks into fragments called apoptotic bodies. Apoptotic bodies are then gobbled up by phagocytes.
Apoptosis could be triggered by factors such as infections, especially viral infections, misfolding of proteins due to mutations and DNA damage due to mutation, radiation, hypoxia and free radicals.
Apart from pathologic factors inducing programmed cell death, apoptosis is also a homeostatic mechanism, where cells that are not needed are killed, thereby maintaining a steady state population of cells.
MORPHOLOGIC CHANGES DURING APOPTOSIS
During apoptosis, the cell undergoes certain morphologic changes that can be seen in light and electron microscopy.
The cell shrinks and becomes smaller in size. The cytoplasm and organelles become tightly packed. The nuclear chromatin shrinks and becomes condensed (usually peripherally – best seen in electron microscopy), a process called pyknosis. Following this, chromatin material undergoes karyorrhexis, i.e it disintegrates and becomes fragmented. Under the microscope, a cell undergoing apoptosis would appear shrunken, with a dense eosinophilic cytoplasm and small clumps of hematoxophilic chromatin material.
Further, the cell starts to form blebs on its surface and starts to break off into small fragments called apoptotic bodies. These apoptotic bodies have portions of cytoplasm, organelles and nuclear fragments of the cell. It is important to understand that apoptosis does not elicit inflammation, unlike another form of cell death called necrosis. This is because; apoptotic bodies have an intact plasma membrane and prevent any content from leaking out into the interstitial space. Also, apoptotic bodies are quickly recognised by phagocytes and removed from the environment.
MECHANISMS OF APOPTOSIS
A cell can undergo apoptosis due to several reasons and depending on the etiologic factors may have 3 different pathways of initiating cell death. Apoptosis could be initiated by signals from the intrinsic pathway, the extrinsic pathway and the perforin/granzyme pathway.
In the intrinsic pathway, mitochondria become leaky and ooze out proteins called cytochrome C, which initiate apoptosis. Usually the cytoplasm and mitochondrial membrane harbour proteins called Bcl-2 and Bcl-x which are anti-apoptotic and preserve the integrity of the mitochondrial membrane, preventing apoptotic proteins from leaking into the cytoplasm. However, in the absence of a growth signal, or insults due to radiation or protein misfolding, stress proteins called “BH3 only” proteins are stimulated. “BH3 only” proteins comprising of Bim, Bid and Bad proteins block the function of Bcl-2 and Bcl-x . These proteins further activate two pro-apoptotic effectors called Bax and Bak, which create channels in the mitochondrial membrane, allowing intra-mitochondrial proteins like cytochrome C to leak!
Cytochrome C, in the cytoplasm, binds with a protein called Apaf – 1 (Apoptosis activating factor -1) to form a complex called “apoptosome”. Apoptosome binds with Caspase 9 and begins to cleave and activate adjacent caspase-9 molecules. Caspase 9 is an initiator caspase and activated Caspase 9 molecules activate executioner Caspases 3 and 6 leading to apoptosis of the cell.
Extrinsic pathway and Granzyme/Perforin pathway
Cytotoxic T lymphocytes or CD8 T cells cause apoptosis of infected cells or tumor cells by FasL and Fas/death receptor interaction. Many cell types express a receptor called the Fas receptor and cytotoxic T cells express FasL. Cytotoxic T cells thus bind to the Fas receptor on tumor cells or infected cells through their FasL. This interaction may produce apoptotic signals through two pathways – the extrinsic or the Granzyme/Perforin pathway.
The extrinsic pathway involves binding of an adapter protein called Fas-associated death domain (FADD) to the cytoplasmic end of at least 3-4 Fas ligands. This then binds with caspase 8, an initiator caspase, which gets cleaved to become active. The active caspase 8 further activates other caspase 8 molecules. Caspase 8 moleules activate executioner Caspases 3 and 6 leading to apoptosis of the cell.
At times the Granzyme/Perforin pathway is initiated on FasL/Fas receptor interaction. T cells release perforins, which form trans-membrane pores, through which granzymes, another protein secreted by T-cells, enter. Granzymes, could either directly activate executioner caspase molecules 3 or cause DNA cleaving, leading to apoptosis.
Executioner caspases 3 and 6 cause degradation of chromosomal DNA and also degradation of cytoskeletal proteins, which cause the morphological changes such as nuclear fragmentation and cellular shrinkage respectively. However, it is not yet known what causes changes like cellular blebs and apoptotic bodies.
Apoptotic bodies are coated with a phospholipid called phosphotidylserine, which is recognised by phagocyte receptors. Also, apoptotic bodies may be coated with opsonins like antibody IgG or complement proteins like C3b which are recognised by phagocytes thus facilitating rapid phagocytosis of apoptotic bodies.
Robbins and Cotran, Pathologic Basis of Disease. Kumar, Abbas, Fausto, Aster. 8th Edition.
Elmore S. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol. 2007; 35(4): 495–516.
Apoptosis and Signal Transduction. Available at https://www.wehi.edu.au/wehi-tv/apoptosis-and-signal-transduction.
Apoptosis. Available at http://www.onkoview.com/das-buch/kapitel-7/die-apoptose.html.
OTHER (HACKDENTISTRY) PRACTICE/STUDY RESOURCES