Overview of Apoptosis

Cell death can occur via two processes–Accidental Cell Death (ACD) or Programmed Cell Death (PCD). Apoptosis is a type of PCD that is essential for maintaining the homeostasis of the cell and doubles as a defense mechanism in pathological conditions. Though first described by Carl Vogt in 1842, the term apoptosis was first used in 1972 by John Foxton Ross Kerr while distinguishing this specific mechanism from necrosis. While apoptosis is highly regulated, necrosis is cell death by injury and is not directly controlled by the cell. This article offers a brief overview of the major apoptotic mechanisms and key players along the various pathways.

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The hallmarks of apoptosis can be classified into changes pertaining to the nucleus, cell membrane and cytosolic components, and mitochondria function. Chromatin condensation (pyknosis), as well as nuclear and DNA fragmentation (karyorrhexis), are the significant nuclear changes associated with apoptosis. Following apoptosis initiation, the cellular junctions are lost, the cell membrane shrinks, and the cytosolic components are organized into apoptotic bodies. The presence of phosphatidylserine in the outer leaflet of the cell membrane marks the apoptotic bodies for phagocytic elimination.

In addition, cytoskeletal components and structural proteins like actin and spectrin are also deregulated, leading to a loss of cellular integrity. Apoptotic mechanisms can further trigger the release of pro-apoptotic factors from the mitochondria like cytochrome C, AIF, and SMAC/DIABLO.

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There are two major cellular signaling pathways that orchestrate the apoptotic processes—intrinsic and extrinsic.

Intrinsic Pathway

The apoptosis intrinsic pathway is triggered by stimuli causing DNA damage or metabolic stress. This results in the activation of pro-apoptotic BAX and BAK belonging to the Bcl2 family of proteins. BAX/BAK activation leads to mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome C, CYCS, SMAC/DIABLO, and HTRA2 into the cytoplasm. Cytochrome-C associates with cytoplasmic APAF1, leading to the formation of the apoptosome complex, which activates caspase-9. Active caspase-9, in turn, cleaves and activates the executioner caspases (caspases-3, 6, 7) that help in DNA fragmentation and the ensuing morphological and biochemical changes in apoptosis. Below are western blot and immunofluorescent data examples looking at two intrinsic targets: cytochrome C and DIABLO.

The apoptosis extrinsic pathway is triggered by diverse stimuli or the withdrawal of stimuli (e.g. growth factors). Apoptosis triggered by the extrinsic pathway involves transmembrane receptor interactions with respective ligands. These ‘death receptors’ belonging to the TNF gene superfamily consist of cysteine-rich extracellular domains and conserved intracellular ‘death domains’. The best studied ligand-receptor pairs are FasL/FasR, TNFα/TNFR1, Apo3L/DR3, Apo2L/DR4/DR5, and TRAIL/TRAILR1. Ligand binding to FasR and TNFR1 induces receptor clustering and recruitment of cytoplasmic adaptors FADD and TRADD, respectively. This results in the formation of DISC (death-inducing signaling complex) that further cleaves pro-caspase-8 to generate active caspase 8. Caspase 8 induces cleavage and myristoylation of cytoplasmic BID, a pro-apoptotic Bcl-2 family protein, and induces its translocation into mitochondria while it also activates the executioner caspases directly. Thus, BID acts as a common player between intrinsic and extrinsic apoptotic pathways. Below are western blot and immunofluorescent data examples looking at two extrinsic pathway targets: DR3 and BID.

Apoptosis pathways have multiple steps and multiple targets that need to be available for the process to proceed as outlined above. Here are some key targets to consider when researching apoptosis.

The studies on the CED-3 gene in nematode C. elegans led to the discovery of a homologous family of proteins in mammals, called caspases. These are cysteine aspartic-specific proteases that orchestrate the apoptotic processes. They are expressed as inactive zymogens in the cell and are activated via cleavage of pro-domains and inter-subunit linkers. The 14 different caspases in mammals are broadly classified into 3 groups based on sequence similarities and biological functions:

• Initiators (Caspases 2, 8, 9, 10) possess long N-terminal caspase recruitment domains (CARD). Of these, Caspase-2 is instrumental in apoptosis and is triggered by DNA damage, metabolic deregulations, and endoplasmic reticulum stress
• Executioners (Caspases 3, 6, 7, 14) are characterized by short pro-domains called death effector domains.
• Inflammatory (Caspases 1, 4, 5, 11, 12, 13) caspases possess long caspase recruitment domains (CARD) like initiator caspases, but prefer aromatic or hydrophobic residues in the P4 position of substrates while initiator caspases prefer leucine or valine residues.
   

Bcl-2 family proteins are classified based on their pro- or anti-apoptotic action and the Bcl-2 Homology (BH) domains. Anti-apoptotic/pro-survival Bcl-2-like proteins (e.g. Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1/Bfl-1) and pro-apoptotic/anti-survival BAX-like proteins (e.g. BAX, BAK, and BOK/Mtd) display 4 BH domains. In contrast, the pro-apoptotic BH3-only proteins (e.g. BID, Bim/Bod, BAD, Bmf, BIK/Nbk, BLK, NOXA, PUMA/Bbc3, and HRK/DP5) have only a short BH3 domain. The BH3 only proteins are activated upon various stress signals and in turn inactivate the pro-survival Bcl-2-like proteins. Subsequently, the pro-apoptotic proteins like BAX and BAK initiate the mitochondrial pathway leading to apoptosis. Below is a western blot analysis that confirms antibody specificity of a Bcl-2 antibody that would be suitable for apoptosis research and a flow cytometry analysis of Mcl-1 in human peripheral blood cells.

Figure 6. (A) Bcl-2 antibody in western blot. Western blot analysis was performed on whole cell extracts (30 µg lysate) of MCF-7 (Lane 1), Raji (Lane 2), T-47D (Lane 3), MDA-MB-231 (Lane 4), HeLa (Lane 5), and Jurkat (Lane 6). The blot was probed with Bcl-2 Monoclonal Antibody (124) (Cat. No. MA1-26233, 1:500 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A28177, 0.25 µg/mL, 1:4,000 dilution). A 27 kDa band corresponding to Bcl-2 was observed across the cell lines tested. (B) Knockdown of Bcl-2 was achieved by transfecting HeLa cells with Bcl-2 specific validated siRNAs. Western blot analysis (A) was performed using whole cell extracts from the Bcl-2 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with the same Bcl-2 Monoclonal Antibody at a 1:500 dilution) and the Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, HRP at a 1:4,000 dilution. Densitometric analysis of this western blot is shown in histogram (B). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Bcl-2.

The inhibitor of apoptosis proteins (IAPs) were first identified in insect baculoviruses. Cellular and viral IAPs are characterized by the presence of baculoviral repeat domain (BIR) repeats. The most widely studied mammalian IAPs are survivin, cellular IAP1 (cIAP1), cIAP2, and X-linked IAP (XIAP). Among the IAPs, only XIAP can directly bind and inhibit caspases (caspases-3 and -9). Upon apoptotic stimuli, IAP inhibitors, including SMAC/DIABLO and HtrA2/Omi, are released from the mitochondria and bind to XIAP’s BIR domains, releasing active caspases into the cytosol. In contrast, cIAP1and cIAP2 bind to tumor necrosis factor (TNF) receptor-associated factors (TRAFs) and inhibit cell death induced by TNF receptor 1 (TNFR1) by triggering the pro-survival signaling pathways. Below is immunofluorescent analysis of surviving in the cell cycle stages of HeLa cells.

Figure 8. Survivin antibody in IF. Immunofluorescence analysis of survivin using Survivin Monoclonal Antibody (1H5) (Cat. No. MA5-17035) shows differential subcellular localization of Survivin at various cell cycle stages in HeLa cells- low levels at early prophase and an increase at late prophase. During metaphase survivin is localized in the centromeres, remains in the spindle midzone at anaphase and localizes in the midbody between two daughter cells at telophase.

The TNF superfamily consists of the death receptors (DRs) and their ligands such as TNF, TRAIL, and Fas ligand (FasL). The binding of these ligands to their respective receptors trigger extrinsic apoptosis pathways. CD95 (DR2/Fas/APO-1), TNF receptor 1 (DR1/TNFR1), TRAIL-R1 (DR4), and TRAIL-R2 (DR5) are the well characterized DRs that bind CD95 ligand (CD95L/FasL), TNFα, lymphotoxin-α (both of these bind to TNFRI), and TRAIL (binds to TRAIL-R1 and TRAIL-R2), respectively.

The interaction of Fas (CD95/APO-1/DR2) with FasL results in conformational changes in the death domain (DD) of the receptor, which recruits FADD through its DD. FADD, in turn, interacts with pro-caspase-8 and -10 and activates them via auto-cleavage, thus triggering the downstream apoptotic signaling pathways. The CD95/FADD/caspase-8/-10 complex is termed the ‘death-inducing signaling complex’ (DISC).

Similarly, TRAIL binds to its receptors, TRAIL receptor 1 (death receptor 4, DR4) and TRAIL receptor 2 (death receptor 5, DR5) and recruits the adaptor protein FADD along with inactive caspase-8 and -10 to form the DISC complex. The cleaved caspase-8 and -10 are then released into the cytosol to activate effector caspases.

TNF-mediated apoptosis is generally carried out by TNFR1 (DR1). Binding of TNF to TNFR1 triggers the recruitment of TRADD through its DD. TRADD interacts with FADD resulting in the recruitment of pro-caspase-8, which is proteolytically cleaved to active caspase-8. Caspase-8, in turn, activates caspase-3. 

Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is a master regulator that inhibits TNF-α, Fas-L and TRAIL-induced apoptosis. c-FLIP binds to FADD and/or caspase-8 or -10 and DR5 and forms an apoptosis inhibitory complex (AIC), which prevents DISC formation.

p53 is a transcription factor that binds to DNA in a sequence-specific manner. p53 can regulate both intrinsic and extrinsic pathways of apoptosis by inducing transcription of several proteins like CD95, TRAILR2, PUMA, BID and BAX. p53 also translocates to the mitochondria in response to apoptotic signals where it forms inhibitory complexes with Bcl-XL and Bcl-2 causing the permeabilization of the mitochondrial membrane and cytochrome c release. Moreover, cytosolic p53 might induce the activation of pro-apoptotic BAX via direct protein-protein interactions.
 

Various novel non-apoptotic forms of regulated cell death have also emerged and are increasingly being implicated in multiple human diseases. Necroptosis is one such mechanism that works against pathogen-mediated infections. It is characterized by cell swelling followed by plasma membrane rupture and chromatin condensation. Generally, necroptosis is initiated upon caspase-8 inhibition and is mediated by ligands including FasL, TNF, and LPS leading to activation of RIPK3 (RIP3). The formation of necrosomes with RIPK1/RIPK3 and mixed lineage kinase domain-like pseudo kinase (MLKL) further leads to MLKL activation and its trans-location to the plasma membrane to initiate cell death. The figure below shows the validation of RIP3 antibody using relative expression approach. Other emerging cell death pathways include pyroptosis, ferroptosis, parthanatos, autophagy, and NETotic cell death that can be triggered by oxidative stress.

• Apoptosis Information
• Apoptosis Assays
• Apoptosis Pathways
• Apoptosis Protocols
• Behind the Bench Blog: Let’s get specific about the TNFR pathway!

BioProbes articles

• A Ratiometric Approach to Apoptosis Detection by Flow Cytometry
• Journal Club: Measurement and Characterization of Apoptosis by Flow Cytometry
• Journal Club: Multiparameter Detection of Early Apoptosis Markers Without Compensation
   

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