Mechanisms of Cell Death: Apoptosis, Autophagy, and Necrosis Flashcards by Joel Glotfelty

Programmed cell death

-i.e. death by suicide

Apoptosis

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Catabolic process involving the degradation of a cell’s own components through the lisosomal machinery

-i.e. Death by self-cannibalism

Autophagy

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Defined as the premature death of cells by external factors

-i.e. Death by accident or murder

Necrosis

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Most of what we know about apoptosis was discovered in

C. elegans

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An integral part of both plan and animal tissue development

Apoptosis

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In C. elegans, out of 1090 somatic cells, 131 of them underwent apoptosis. The different cells died at different times in

-remarkably accurate

Development

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In normal development, apoptosis is required for

Tissue sculpting

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Apoptosis is important in the immune system after infection for for eliminating used

T and B cells

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A balance between proliferation and cell death is required to maintain

Tissue homeostasis

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Crucial for eliminating cells that have been damaged by UV, radiation, chemical toxins, or viral infection

-i.e. it can prevent cancer

Apoptosis

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An example of apoptosis function during tissue development is the development of the

Fingers and toes

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Problems in apoptosis during finger and toe development results in

-2 or more fingers/toes are joined together

Syndactily

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Lack of apoptosis during embryo development also disrupts

Normal brain development

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Humans who are born with tails have deficiencies in

Apoptosis

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What are five morphological markers of apoptosis?

) Electron dense nucleus
) Nuclear fragmentation
) Large, clear vacuoles
) Blebs at the surface
) Loss of cell adhesion

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What is one very common biochemical marker of apoptosis?

Phosphatidylserine flipping from inner leaflet to outer leaflet

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Phosphatidylserine flipping from inner leaflet to outer leaflet enables

Apoptotic cells to enter cell

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Enables us to track nicks in DNA

Terminal Transferase dUTP Nick-End Labeling (TUNEL)

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Used to add labeled dUTP to 3’ terminal end of the DNA fragments

Terminal Transferase

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Highly sensitive and can detect fewer than 100 cells

-Fast: can be completed in 3 hours

TUNEL Assay

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Has high reproducibility with good precision

TUNEL Assay

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One of the disadvantages of TUNEL Assays are that we don’t know the minimum number of strand breaks necessary for detection. Thus we may miss the

Early stages of apoptosis

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Designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis

-Based on the ability of dUPT to label blunt ends of double-stranded DNA breaks independent of a template

Terminal Transferase dUTP Nick-End Labeling (TUNEL Assay)

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Can generate false positives in a TUNEL assay

Necrotic cells

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Another disadvantage of a TUNEL assay is that the detergent used to permeabilize cells can make them

Frgile

A distinctive feature of DNA degraded by caspase-activated DNAse (CAD)

DNA laddering

Cleaves genomic DNA at internucleosomal linker regions, resulting in DNA fragments that are multiples of 180-185 bps in length

Caspase-activated DNAse (CAD)

Must be cleaved to become active

Caspases

What are the two major apoptotic pathways?

1. ) Cell-extrinsic pathway | 2. ) Cell-intrinsic pathway

The key enzymes that give rise to all of the morphological and biochemical changes arising from apoptosis

Caspases

What are the two types of apoptotic caspases?

1. ) Initiator (apical) caspases (#'s 2, 8, 9, and 10) | 2. ) Effector (executioner) caspases (#'s 3, 6, and 7)

Regulated at a post-translational level, ensuring that they can be rapidly activated

Caspases

What are the steps of the caspase cascade?

Pro-apoptotic stimulus ---> initiator caspases ---> effector caspases ---> apoptosis

Initiator caspase is activated by

2 proteolytic cleavages

Initiator caspase then cleaves and activates

Effector caspase

Cleaves nuclear lamins, inhibitors of DNAases, and Actin

Effector Caspases

The intrinsic apoptosis pathway is also known as the

Mitochondrial mediated pathway

The extrinsic apoptosis pathway is also known as the

Death receptor mediated pathway

Family of proteins that regulates the integrity of the mitochondrial membrane

BCL2

What are the two pro-apoptotic BCL2 proteins?

BAX and BAK

What are the three anti-apoptotic BCL2 proteins?

BCL2, BCL-XL, and MCL1

The regulators/sensors of the intrinsic pathway are "BH3-only proteins" and include

BAD, BID, and PUMA

Characterized by permeabilisation of the mitochondria and release of cytochrome c into the cytoplasm

Intrinsic apoptotic pathway

Forms a multi-protein complex known as the ‘apoptosome’ and initiates activation of the caspase cascade through caspase 9

Cytochrome C

Cytochrome C activates -activates caspase 9

Apoptotic protease activating factor 1 (APAF1)

Antagonism of BCL2 causes the activation of the BAX/BAK channel which causes the mitochondria to release

Cytochrome C

Responsive to DNA damage by UV radiation -one of the most studied genes of all time

p53

Can activate DNA repair proteins, arrest cell at G1/S checkpoint, and initiate apoptosis

p53

p53 is a transcription factor that regulates

Cell cycle and apoptosis

Acts as a ubiquitin ligase and covalently attaches ubiquitin to p53, thus marking it for degredation

Mdm2

Also transports p53 from nucleus to cytosol

Mdm2

The critical event leading to the activation of p53 is

Phorphorylation of its N-terminus

The protein kinases known to target this transcriptional activation domain of p53 can be broken down into what two groups?

1. ) Members of MAPK family | 2. ) Checkpoint kinases

Oncogenes also stimulate p53 activation, mediated by the protein

p14ARF

Contains many pro-apoptotic proteins such as cytochrome C

Mitochondria

Can be release from the mitochondria in response to pro-apoptotic signals

Cytochrome C

Translocate to the mitochondrial membrane and stimulate the formation of pores allowing Cytochrome c to leak out -normally found in the cytosol

Pro-apoptotic molecules (i.e. Bad, Bax, Bid)

Interacts with APAF-1 to form the apoptosome

Cytochrome C

Interaction between cytochrome C and APAF-1 activate -promotes caspase cascade

Caspase 9

Promote apoptosis by tying up Bcl2 and freeing Bax/Bak

Bid and Bad

Binds Bax/Bak and prevents pore formation -anti-apoptotic

Bcl-2

Promote pore formation, release of cytochrome C and APAF1, and initiation of apoptosis

Bax and Bak

Results from a chromosomal translocation between the 14th and 18th chromosomes, which places the BCL-2 gene next to the IgG heavy chain locus

Follicular Lymphoma

The fusion gene of BCL-2 and IgG heavy chain leads to

High expression of BCL-2

High expression of BCL-2 then results in a decreased propensity of cells for undergoing

Apoptosis

Occurs in several cancers and is linked to poor disease outcome

Increased Bcl-2 protein expression

Triggered by the p53 tumor-suppressor in | response to DNA damage and other types of severe cell stress

Intrinsic pathway

Conventional anticancer therapies, such as chemotherapy, activate the intrinsic pathway via

p53

p53 activates the intrinsic pathway through transcriptional upregulation of pro-apoptotic members of the BCL2 family of proteins such as

PUMA and Bax

The extrinsic apoptotic pathway is apoptosis via the pro-apoptotic receptors

DR4 and DR5 (Fas receptors)

Activate the pro-apoptotic receptors DR4 and DR5

Endogenous Apo2L/TRAIL

The activated pro-apoptotic receptors D4 and D5 then recruit

Fas-associated death domain (FADD)

Recruits initiator caspase 8 and/or 10 to the death-inducing signaling complex (DISC)

FADD

The DISC activates caspases 8 and 10 and releases them into the cytoplasm, where they activate

Caspases 3, 6, an 7

Triggers apoptosis in response to the activation of pro-apoptotic receptors, such as DR4 and DR5, by specific pro-apoptotic ligands, such as Apo2L/TRAIL

Extrinsic apoptotic pathway

The extrinsic apoptotic pathway stimulates apoptosis independently of

p53

Ligand-induced activation of DR4 and DR5 leads to the rapid assembly of the death-inducing signaling complex (DISC) and the recruitment of initiator caspases 8 and 10 through the adaptor

Fas-associate death domain (FADD)

The extrinsic and intrinsic apoptosis pathways converge on

Caspases 3, 6, and 7

Also, caspases 8 and 10 (extrinsic) activate Bid,which activates

Bax and Bak (intrinsic)

Responsible for degrading cellular proteins and organelles and recycling them -A survival pathway

Autophagy

Autophagy is actually a response to

Cellular starvation

Occurs in many disease states, including cancer, inflammatory bowel disease, and neurodegenerative disorders

Disregulation of Autophagy

The sequestration of cellular organelles into cytoplasmic autophagic vacuoles (autophagosomes) that fuse with lysosomes and digest the enclosed material

Autophagy

What are the four stages of autophagy?

1. ) Induction 2. ) Autophagosome formation 3. ) Autophagosome-lysosome fusion 4. ) Autophagosome breakdown

A key regulator of autophagic induction is -has an inhibitory affect

mTOR (mammalian target of rapamycin)

A serine-theronine kinase

mTOR

Many of the genes that are upregulated in response to mTOR deactivation participate in

Autophagosome formation

Involves formation of a membrane around a targeted portion of the cell

Autophagosome formation

In autophagosome-lysosome fusion, the autophagosome fuses w/ the lysosome releasing its contents into the lysosome for

Degredation by proteases

Inevitably breaks down the autophagosome

Lysosome

Has contrasting roles during cancer development, progression, and treatment

Autophagy

Typically caused by factors external to the cell or tissue, such as infection, toxins, or trauma

Necrosis

Unlike apoptosis and autophagy, is almost always detrimental and can be fatal

Necrosis

What are the five types of necrosis?

1. ) Coagulation necrosis 2. ) Liquefactive necrosis 3. ) Enzymatic fat necrosis 4. ) Caseous necrosis 5. ) Gangrenous necrosis

Cell destruction leading to escape of hydrolases

Liquefactive necrosis

Typically begins with cell swelling, chromatin digestion, and disruption of the plasma and organelle membranes

Necrosis

Charactrized by extensive DNA hydrolysis, vacuolation of the endoplasmic reticulum, organelle breakdown, and cell lysis

Late necrosis

What is the cause of inflammation in necrosis?

Release of intracellular contents after plasma membrane rupture