Apoptosis

**What are the two major categories of cell death?**

Non-programmed cell death (necrosis) and programmed cell death.

**What is necrosis?**

Non-programmed cell death caused by cell injury.

**What structural changes occur in necrosis?**

Swelling of the cell, disruption of organelles, and cell breakage.

**Why does necrosis trigger an immune response?**

Cell breakage causes the release of intracellular contents, leading to inflammation.

**What is programmed cell death?**

A controlled process where a cell dismantles itself in response to molecular signals.

**What is apoptosis?**

A type of programmed cell death triggered by DNA damage or lack of survival signals.

**Where does the word "apoptosis" come from?**

It comes from a Greek word meaning "falling off," like leaves falling from a tree.

**How does apoptosis differ from necrosis?**

Apoptosis is a planned, controlled process that does not damage surrounding cells, while necrosis is uncontrolled and leads to inflammation.

**What are the key characteristics of apoptosis?**

Chromatin condensation, cell shrinkage, preservation of organelles/membranes, DNA fragmentation, and rapid engulfment by neighboring cells.

**How does apoptosis differ from necrosis in terms of cell structure?**

Apoptotic cells shrink and maintain organelle integrity, while necrotic cells swell and rupture.

**What happens to chromatin during apoptosis?**

Chromatin condenses and DNA is fragmented into precise 100 bp pieces.

**Why does apoptosis not trigger an immune response?**

Cellular components are neatly packaged into apoptotic bodies and engulfed by neighboring cells or macrophages, preventing inflammation.

**What natural processes require apoptosis?**

Separation of fingers during development, regression of the mammary gland during weaning, and removal of a tadpole's tail.

**What is membrane blebbing in apoptosis?**

The formation of small, bubble-like protrusions on the cell membrane as the cell breaks down.

**What happens to apoptotic bodies?**

They are engulfed by macrophages to prevent immune activation.

**What role do proteolytic enzymes play in apoptosis?**

They break down DNA and proteins to facilitate controlled cell disassembly.

**How do scientists detect apoptosis in the lab?**

By measuring the activity of proteolytic enzymes involved in DNA and protein breakdown.

**Why is apoptosis important in development?**

It allows for processes like finger separation, tadpole tail removal, and mammary gland regression during weaning.

**How does apoptosis help maintain healthy tissues?**

It removes seriously damaged or abnormal cells before they can cause harm.

**What happens when apoptosis fails in cancer?**

Cancer cells avoid apoptosis, allowing uncontrolled growth and tumor formation.

**How is apoptosis used in cancer treatment?**

Many therapies aim to trigger apoptosis in cancer cells to stop their proliferation.

**How does apoptosis relate to stroke and heart attacks?**

When blood flow is lost and then restored, cells can undergo apoptosis due to stress and damage.

**What diseases are associated with defects in apoptosis?**

Cancer, stroke, myocardial infarction, diabetes, and neurodegenerative diseases.

**How does necrosis differ from apoptosis in disease?**

Necrosis is caused by physical damage, trauma, or toxins, leading to uncontrolled cell death and inflammation.

**How does failure of apoptosis contribute to cancer progression?**

Cancer cells with DNA damage should undergo apoptosis, but mutations can allow them to escape, leading to uncontrolled growth.

**Why do cancer cells avoid apoptosis?**

Genetic mutations can disrupt apoptotic signaling, preventing the cell from receiving enough death signals.

**How can apoptosis help control cancer?**

If apoptosis functions properly, damaged or abnormal cells are eliminated before they can grow uncontrollably.

**How do some cancer treatments exploit apoptosis?**

Treatments like radiation cause massive DNA damage, triggering apoptosis in cancer cells.

**Why is radiation effective against tumors?**

It induces severe DNA damage, which activates the apoptotic pathway in cancer cells.

**What happens if the apoptosis pathway is compromised in cancer?**

The cancer cells survive and continue dividing, leading to tumor progression.

**What are the two main types of pathways that can activate apoptosis?**

Extrinsic and intrinsic pathways.

**How are extrinsic pathways activated?**

By external signals such as tumor necrosis factor (TNF) receptors and Fas receptors.

**What receptors are involved in the extrinsic pathway?**

TNFR (tumor necrosis factor receptor) and Fas receptor.

**What triggers intrinsic pathways?**

Internal signals like DNA damage or lack of survival signals.

**How does DNA damage trigger intrinsic apoptosis pathways?**

DNA damage activates p53, which then initiates the apoptosis pathway.

**What role does p53 play in apoptosis?**

p53 is activated by DNA damage or lack of survival signals and triggers the intrinsic apoptosis pathway.

**What is the common death mechanism used by both intrinsic and extrinsic apoptosis pathways?**

Caspases.

**What are caspases?**

Cysteine-dependent aspartate-specific proteases.

**Why are caspases called "executioners"?**

They are responsible for triggering the degradation of macromolecules during apoptosis.

**How many caspases have been identified in mammals?**

At least 14.

**How are caspases activated?**

They are synthesized as procaspases and are cleaved to remove the pro domain, activating them.

**What do activated caspases do?**

Trigger degradation pathways for various macromolecules in the cell.

**Where is the cysteine in caspases?**

Cysteine is in the active site of the caspase.

**Where is the "aspartate" in caspases?**

Caspases cleave after an aspartate residue in the proteins being targeted.

**How are caspases activated?**

Caspases are always present in the cell in an inactive form. The pro-domain is cleaved to activate them, which is a rapid way to regulate their activation.

**What happens after one caspase is activated?**

It triggers the activation of the next caspase in the apoptosis pathway.

**What are caspases?**

Cysteine-dependent, aspartate-specific proteases.

What happens once one caspase is active

It triggers the activation of the next one in the pathway

How does each caspase work

Each caspase can work on specific targets and activate the next caspase in the chain, amplifying the response at each step

What is the result of the caspase cascade

The response is amplified at each step, making it an efficient killing mechanism

What happens when a killer lymphocyte binds to a targeted cell

It interacts through the death receptor

What is the death receptor involved in the extrinsic pathway

The TNFR receptor, part of the TNF superfamily

What happens when the lymphocyte carrying TNF binds to the TNFR receptor

It activates caspase-8, causing it to be cleaved

What does caspase-8 activation trigger

It triggers the cleavage of other caspases, leading to cell death

What activates death receptors in the extrinsic pathway

extracellular ligands

What happens when death receptors are activated

They lead to the cleavage and activation of caspase 8

What does caspase 8 activate

It activates caspases 3 and 7

What is the result of caspase 3 and 7 activation

They lead to the execution phase of apoptosis

What regulates apoptosis through intrinsic pathways

the Bcl-2 family of proteins,

What family of surface proteins is involved in intrinsic apoptosis pathways

Bcl family

What are the anti-apoptotic proteins of the Bcl family

Bcl-2 and Bcl-XL

What are the pro-apoptotic proteins of the Bcl family

Bax and Bad

What does Bcl-2 prevent

Bcl-2 prevents the leakiness of cytochrome C from mitochondria

What does Bax allow

Bax allows cytochrome C escape from mitochondria

What happens when the balance shifts to more Bax proteins

Cytochrome C is released

What does the release of cytochrome C activate

The release of cytochrome C activates caspases

What regulates Bcl family members in intrinsic apoptosis

Intrinsic signals, such as lack of survival signals or DNA damage

What happens when there is a lack of survival signals or DNA damage

p53 expression is increased

What does increased p53 expression do to Bax and Bcl-2

It increases Bax and decreases Bcl-2

What does the increase in Bax and decrease in Bcl-2 lead to

Cytochrome C release

What is formed when cytochrome C is released

The apoptosome, composed of Apaf-1 bound to cytochrome C

What does the apoptosome activate

Caspase-9

What does the activation of caspase-9 trigger

The downstream caspase cascade

What happens when DNA damage or the lack of survival signals occurs

Increases p53 expression

What does increased p53 expression do to the Bax/Bad to Bcl-2 ratio

Increases the Bax/Bad ratio relative to Bcl-2

What happens if the Bax/Bad to Bcl-2 ratio favors more Bax/Bad

Cytochrome C is released from the mitochondria

What does cytochrome C bind to after release

Apaf-1

What happens when cytochrome C binds to Apaf-1

It causes the activation of caspase-9

What does caspase-9 activate

Caspases-3 and -7

What is the result of the caspase-9 activation

Activation of the downstream caspase cascade, leading to apoptosis

What can caspase-3 cleave in detection kits

Ac-DEVD-pNA

What happens when pNA is removed during caspase-3 detection

A yellow product is released

What type of substrate does caspase-3 cleave

Colorimetric substrate (pNA)

What is the role of pNA in the detection of caspase-3 activity

It is a yellow product that can be detected in a spectrophotometer

What do detection kits compare in caspase-3 detection

The relative color change compared to an untreated control

What chemotherapeutic agent is used as an example in caspase-3 detection

Doxorubicin

What is the function of doxorubicin

It is a topoisomerase II inhibitor that disrupts DNA replication

What happens to phosphatidylserine (PS) at the start of apoptosis

PS is translocated to the outer membrane

What does Annexin V bind to during apoptosis

Phosphatidylserine (PS)

How can Annexin V binding be measured

By flow cytometry or fluorescence microscopy

What additional dye can be used in flow cytometry to detect late apoptotic cells

Propidium iodide

Why is propidium iodide used in flow cytometry

To detect late apoptotic cells, as their membranes become more permeable to the dye

What is PARP (poly-ADP-ribose polymerase) typically involved in

Repair of single-strand DNA breaks

What happens to PARP during apoptosis

It is cleaved by caspases

Which caspases are involved in cleaving PARP

Caspase-3 and caspase-6

What is the normal size of PARP protein

116 kDa

What size product appears after cleavage of PARP

A smaller band around 85 kDa

How is cleavage of PARP typically detected

By western blotting

What happens to DNA during apoptosis

It is cleaved by nucleases into 180-200 bp fragments

What produces the "ladder-like" effect in DNA

Cleavage of DNA into 180-200 bp fragments