Apoptotic Cell Death


Introduction

Apoptosis is a form of programmed cell death characterized by a series of highly regulated biochemical events that lead to changes in cellular morphology and ultimately cell death. Apoptosis plays a central role in several physiological processes including development, cellular homeostasis, and aging. Moreover, it has been implicated in the pathogenesis of various human diseases including neurodegenerative disorders, autoimmune diseases, cancers, and viral infections. Not surprisingly, targeting the apoptotic pathway has emerged as a common therapeutic strategy in the treatment of such diseases.

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Apoptotic pathways

Apoptosis proceeds through one of two distinct signaling pathways known as the intrinsic and extrinsic pathways, both of which ultimately lead to the activation of proteolytic enzymes known as caspases. The intrinsic apoptotic pathway is activated in response to severe cellular damage or stress caused by various factors including DNA damage, hypoxia, and oncogenes resulting in mitochondrial outer membrane permeabilization (MOMP) and the consequent release of apoptogenic factors from the mitochondria. Initiation of the extrinsic apoptotic pathway relies on the activation of cell surface death receptors of the tumor necrosis factor (TNF) family of receptors by the binding of their corresponding ligands.

apoptotic pathway

 

 

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Immunohistochemistry of human prostate cancer tissue using FAS antibody from Proteintech
IHC of paraffin-embedded human prostate cancer tissue using FAS antibody (13098-1-AP) at a dilution of 1:50 (40x objective).

 

Immunofluorescent (IF) analysis of (4% PFA) fixed human liver cancer tissue using CoraLite® Plus 488 TNFR1 antibody (CL488-60192) at dilution of 1:200.
IF analysis of (4% PFA) fixed human liver cancer tissue using CoraLite® Plus 488 TNFR1 antibody (CL488-60192, Clone: 2A6E3 ) at dilution of 1:200.

 

The Bcl-2 protein family

The Bcl-2 protein family consists of a group of structurally related proteins that are critical for the regulation of the mitochondria-mediated intrinsic apoptotic pathway. Members of the Bcl-2 protein family are characterized by the presence of one or more conserved regions of sequence homology (Bcl-2 homology domains) and can be classified into anti-apoptotic or pro-apoptotic depending on their ability to promote or inhibit apoptosis. The pro-apoptotic Bcl-2 members can be further classified into BH3-only proteins and pore-forming proteins. BH3-only proteins function by either binding anti-apoptotic Bcl-2 members to inactivate them or binding pore-forming proteins to activate them. Once activated, pore-forming proteins undergo a series of conformational changes leading to their homo-oligomerization followed by MOMP and the subsequent release of mitochondrial apoptogenic factors. Anti-apoptotic Bcl-2 members, on the other hand, inhibit apoptosis by binding and sequestering both the BH3-only proteins as well as the pore-forming proteins.

 

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Immunohistochemical (IHC) analysis of paraffin-embedded human lymphoma tissue slide using 10435-1-AP (BAD Antibody) at dilution of 1:200 (under 10x lens).
IHC analysis of paraffin-embedded human lymphoma tissue slide using BAD Antibody (10435-1-AP) at dilution of 1:200 (under 10x lens).

 

Intracellular flow cytometry analysis of Hela cells using CoraLite®488 BAX antibody (CL488-60267, Clone:4G5E8) (red), or Mouse IgG2b Isotype Control (CL488-66360-3, Clone: K11B8C4B5) (blue).
Intracellular flow cytometry analysis of Hela cells using CoraLite®488 BAX antibody (CL488-60267, Clone:4G5E8) (red), or Mouse IgG2b Isotype Control (CL488-66360-3, Clone: K11B8C4B5) (blue).

 

Caspases

Caspases are a group of serine proteases that play a central role in the initiation and execution of the proteolytic cascade during apoptosis. Depending on their role during apoptosis, caspases can be divided into initiator caspases including caspases 2, 8, 9, and 10, and effector caspases including caspases 3, 6, and 7. While initiator caspases cleave and activate the effector caspases, the effector caspases cleave other cellular protein targets to complete the apoptotic program. Some caspases are also known to regulate inflammation, including caspases 1, 4, 5, 11, and 12. Caspase-14 is not involved in apoptosis or inflammation but plays an important role in skin cell development.

 

 

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Western blot (WB) analysis of untreated or staurosporine-treated Jurkat cells using Caspase 3 antibody (19677-1-AP) at a dilution of 1:500.
WB analysis of untreated or staurosporine-treated Jurkat cells using Caspase 3 antibody (19677-1-AP) at a dilution of 1:500.

 

IHC of paraffin-embedded human colon tissue using Caspase 8 antibody (13423-1-AP) at a dilution of 1:50 (40x objective).
IHC of paraffin-embedded human colon tissue using Caspase 8 antibody (13423-1-AP) at a dilution of 1:50 (40x objective).

 

Role of mitochondria in apoptosis

Mitochondria play a critical role in the regulation of apoptosis by initiating MOMP in response to the activation of the Bcl-2 family members, BAX and BAK. MOMP is followed by the release of soluble proteins such as cytochrome C from the mitochondrial intermembrane space into the cytoplasm leading to the activation of the apoptotic caspase cascade. During MOMP and cytochrome C release, mitochondria undergo massive fragmentation also known as mitochondrial fission. While healthy cells maintain a balance between mitochondrial fission and fusion, excessive mitochondrial fission is an integral part of apoptotic cell death. Therefore, not surprisingly, the mitochondrial fission machinery has been demonstrated to be actively involved in the induction of apoptosis.

 

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IF analysis of mouse eye tissue using Cytochrome c antibody (10993-1-AP) at a dilution of 1:200 and CoraLite®488-Conjugated AffiniPure Goat Anti-Rabbit IgG(H+L).
IF analysis of mouse eye tissue using Cytochrome c antibody (10993-1-AP) at a dilution of 1:200 and CoraLite®488-Conjugated AffiniPure Goat Anti-Rabbit IgG(H+L).

 

IF analysis of HepG2 cells using COXIV (11242-1-AP) antibody at a dilution of 1:50 and Alexa Fluor 488-Conjugated AffiniPure Goat Anti-Rabbit IgG(H+L).
IF analysis of HepG2 cells using COXIV antibody (11242-1-AP) at a dilution of 1:50 and Alexa Fluor 488-Conjugated AffiniPure Goat Anti-Rabbit IgG(H+L).

 

Measuring apoptosis

While detecting activated caspases is a common approach for studying apoptosis, several other assays can be used for quantifying apoptosis. The TUNEL method, for example, is a widely used apoptotic assay that detects fragmented DNA in apoptotic cells by incorporating fluorophore-labeled dUTP at the 3’-OH ends of broken DNA. The fluorophore-labeled DNA can then be quantified using a flow cytometer. The primary advantage of the TUNEL method is that it can selectively detect apoptotic cells, but not necrotic cells or cells with DNA strand breaks caused by irradiation and drug treatment.

Annexin V/propidium iodide double labeling is another widely used method for studying apoptosis that can also be used for differentiating apoptotic cells from necrotic cells. Annexin V detects early apoptotic cells by binding to phosphatidylserine (PS), which is flipped to the extracellular membrane surface during early apoptosis. PI on the other hand is a DNA-binding dye that can stain the nucleus of necrotic cells or cells that lose cell membrane integrity during the late stages of apoptosis.

The cell population was analyzed in THE SSC/FSC diagram

Flow cytometry analysis of Jurkat cells treated with staurosporine and stained using the CoraLite®488-Annexin V and PI Apoptosis Kit (PF00005). CL488-AnnexinV and PI are represented by the X and the Y axis of the scatterplot, respectively. Positively stained cells in the various regions of the scatterplot can be interpreted as follows:

  • Q1-UL (CL488-AnnexinV)-/PI+, cells in this region are primarily necrotic.
  • Q1-UR :(CL488-Annexinv)+/PI+, cells in this region are late apoptotic cells.
  • Q1-LR :(CL488-Annexinv)+/PI-, cells in this region are early apoptotic cells.
  • Q1-LL :(CL488-Annexinv)-/PI-, cells in this region are living cells.

 

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