Mechanisms of cell death Flashcards

1
Q

Pathways that trigger apoptosis culminate in widespread intracellular proteolysis. Which of the following proteases is a downstream executioner that directly participates in the breakdown of numerous cellular proteins?

A. caspase-8 (CASP8)
B. caspase-9 (CASP9)
C. caspase-3 (CASP3)
D. caspase-10 (CASP10)
E. XIAP (BIRC4)

A

C

Apoptotic signals trigger a series of proteolytic events known as the caspase cascade. There are at least 14 human caspases, which fall into two categories: the initiator caspases (caspases-2, -8, -9 and -10), which activate the downstream caspases, and the executioner caspases (caspases-3, -6 and -7), which cleave cellular substrates. The actions of the executioner caspases produce the cellular effects that distinguish apoptosis from other forms of cell death. XIAP is a protein that binds to and inhibits the action of caspases.

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2
Q

Which of the following statements regarding cell death following radiotherapy is TRUE?

A. The majority of solid epithelial tumors regress during treatment because of radiation-induced apoptosis
B. The intrinsic apoptotic pathway can be triggered either by radiation-induced DNA damage or by sphingomyelin-mediated damage to the outer plasma membrane
C. A novel drug that abolishes the G1 checkpoint would be expected to reduce the incidence of mitotic catastrophe in irradiated cells.
D. Cells that undergo replicative senescence following radiotherapy are characterized by increased membrane blebbing and DNA fragmentation
E. The presence of gamma-H2AX histone foci in irradiated cells is indicative of sphingomyelin activation

A

B

The intrinsic apoptotic pathway can be triggered either by damage to DNA or by damage to the plasma membrane. Radiation acts directly on the plasma membrane, activating acid sphingomyelinase, which generates ceramide by enzymatic hydrolysis of sphingomyelin. Ceramide then acts as a second messenger in initiating an apoptotic response via the mitochondrial system. Mitotic catastrophe, and not apoptosis, is the major mechanism of cell death in epithelial tumors. Inhibition of the G1 checkpoint in irradiated cells may increase the probability of mitotic catastrophe since cells are more likely to enter mitosis with damaged chromosomes. Radiation-induced senescent cells cease dividing and can remain metabolically active for extended periods before dying, but do not show membrane blebbing and DNA fragmentation, which are characteristic of apoptosis. gamma-H2AX foci noted in the nuclei of irradiated cells are indicative of the presence of DNA double-strand breaks.

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3
Q

Radiation-induced cellular senescence is often the result of:

A. Cellular nutrient deprivation
B. Oxidative stress secondary to mitochondrial dysfunction
C. p16-mediated cell cycle arrest
D. Telomere shortening
E. Mitotic catastrophe

A

C

The term “senescence” refers to the loss of cellular replicative potential leading to a reduced capability to repopulate a tissue after exposure to genotoxic agents, including ionizing radiation. Senescence is most often the result of a permanent arrest in G1, associated with elevated expression of the cell cycle inhibitors p16INK4A (CDKN2A) and p21 (CDKN1A, WAF1/CIP1). Importantly, senescence is not a type of cell death per se because cells remain morphologically intact and metabolically active when senescent. Depending on the level of tumor suppressor proteins and the oncogenic signal, senescence can be reversible in a small subset of cells though in most cells this process is irreversible.

A clinically relevant scenario for radiation-induced senescence is the loss of salivary gland function and xerostomia commonly seen in head and neck cancer patients undergoing radiotherapy. Another one is radiation induced premature senescence in fibroblasts that triggers proinflammatory and profibrotic senescence associated secretory phenotype (SASP) and ultimately drives fibrosis in the lung.

Mitochondrial dysfunction is a hallmark of apoptotic cell death, not senescence (Answer Choice B).

Telomere shortening occurs in most normal somatic cells as part of each cell cycle (“end replication problem”) and triggers senescence once a critical low threshold is reached, but telomere shortening tends not to be the cause for radiation-induced senescence which is driven by DNA damage and cell cycle arrest (Answer Choice D).

Nutrient deprivation can lead to autophagy, and ultimately autophagic death cell distinct from apoptosis (Answer Choice A).

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4
Q

The extrinsic pathway of apoptotic cell death requires:

A. Signals derived from changes in chromatin conformation
B. Activation of death receptors that translocate from the plasma membrane to the nucleus and degrade DNA
C. Engagement of death receptors located on the plasma membrane that lead to activation of the initiator caspase-8 (CASP8)
D. p53 (TP53) activation
E. The triggering of changes in mitochondrial membrane potential

A

C

There are two principal pathways that can lead to apoptotic death. One of these, the extrinsic pathway, involves extracellular signaling through death receptors located on the plasma membrane such as TRAILR-1 (TNFRSF10A), TRAILR-2 (TNFRSF10B) or FAS (CD95/APO-1). These death receptors are activated in response to ligand binding of TRAIL (TNFSF10) or FAS ligand (FASLG/CD95-L) and signal through a series of adapter molecules such as the adapter molecule Fas- associated death domain (FADD) within the death-inducing signaling complex (DISC). Upon recruitment and oligomerization FADD then binds pro-caspases-8 and -10, causing their homodimerization and activation.

The activation of procaspase-8 is thought to occur via an induced proximity model leading to its conversion to the active enzyme, caspase-8. Ionizing radiation can elicit activation of the extrinsic pathway leading to apoptosis. The other pathway by which ionizing radiation can elicit an apoptotic response is the intrinsic pathway. This can be stimulated by DNA damage leading to signaling to mitochondria, changes in mitochondrial membrane potential, release of cytochrome c, and activation of procaspase-9.

In most cases, activated caspase-8 induces apoptosis through activation of pro-caspase-3 at the DISC independently of mitochondria. However, in some cells, especially when only a low amount of active caspase-8 is generated (and hence not sufficient amounts of pro-caspase-3), caspase-8 cleaves the ‘Bcl-2 homology (BH) 3-only protein’ Bid, generating an active fragment (tBid) that activates the (intrinsic) mitochondrial death pathway. In this manner, the extrinsic death signal may be amplified through formation and activation of the apoptosome which contributes to effector caspase activation. In other words, the extrinsic pathway can feed into the intrinsic one and additionally change mitochondrial membrane potential.

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5
Q

One hallmark of the apoptotic process is the display of phosphatidylserine residues on the outer surface of the plasma membrane. This is an important event in terms of the tissue response to ionizing radiation because it:

A. Helps recruit death ligands expressed by neighboring cells to receptors on the cell surface
B. Stimulates an inflammatory response to remove dying cells from the tissue
C. Signals the recruitment of phagocytes that engulf the dying cells without causing an inflammatory response
D. Is required for DNA condensation and fragmentation
E. Leads to increased ceramide levels

A

C

Apoptosis helps maintain tissue homeostasis because cells that are undergoing an apoptotic response recruit phagocytes that clear the dying cells, also known as “apoptotic corpses”, from the tissue without stimulating an inflammatory response. In fact, uptake of apoptotic cells
by macrophages can actually lead to the release of anti-inflammatory mediators such as TGF-β and IL-10, and the attenuation of the RIG-I/IRF-3 pathway and the cGAS/STING pathway through proteolytically inactivating RIP kinase 1 or the degradation of cytoplasmic DNA. As a result, apoptosis can decrease the expression of interferons and other inflammatory factors.

Of note, the concept that apoptosis is entirely non-inflammatory isn’t always strictly true. An example is the induction of apoptosis in hepatocytes following FAS activation that causes a strong inflammatory response probably because they can’t get cleared fast enough by phagocytes.

The exposure of phosphatidylserines (phospholipids) on the exterior of the plasma membrane is the signal that initially recruits phagocytes. Ordinarily, phosphatidylserine is sequestered on the inner leaflet of the phospholipid bilayer and is not displayed on the cell’s surface. The process of necrosis, which involves rupture of the cell membrane and the leakage of cellular contents into the surrounding tissue, does elicit an inflammatory response.

While DNA condensation and fragmentation are important steps in the apoptotic process, they are not coordinated directly through the exposure of phosphatidylserine on the plasma membrane. A number of stimuli lead to increased ceramide levels, including TNF, FasL and ionizing radiation, but not phosphatidylserine.

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6
Q

Regarding the regulation of apoptosis, which of the following pairs of mammalian proteins and their apoptosis-related functions is FALSE?

A. p53 (TP53) — upregulation of PUMA
B. DIABLO — caspase activation
C. XIAP (BIRC4) — caspase inhibition
D. BAX — cytochrome c release
E. caspase-3 (CASP3) — initiator caspase

A

E

The characteristic changes associated with apoptosis are due to activation of a family of intracellular cysteine proteases, known as caspases. Initiator caspases are the first to be activated, and include caspases-2, -8, -9 and -10. Initiator caspases cleave and activate the effector/executioner caspases, including caspases-3, -6, and -7, which then cleave, degrade or activate other cellular proteins. Activation of caspases is regulated by members of the BCL2 family and by the inhibitors of apoptotic protein (IAP) family. BCL2 family members can be either pro- or anti-apoptotic. BAX is one of a series of pro-apoptotic members of the BCL2 family.
These pro-apoptotic BCL2 family members regulate the release of cytochrome c from mitochondria and elicit the subsequent activation of
caspases. Another important function of p53 is that it causes upregulation of pro-apoptotic PUMA. X-linked IAP (XIAP) inhibits the activity of caspases directly. DIABLO is a pro-apoptotic protein that prevents IAPs from inhibiting caspases. BAX and p53 are required for some forms of DNA damage-induced apoptosis.

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7
Q

Which ONE of the following is a morphological or biochemical feature of apoptosis?

A. Random cleavage of DNA
B. Cellular swelling
C. Lack of dependence on ATP as an energy source
D. Chromatin condensation
E. Rupture of the plasma membrane

A

D

During the apoptotic process, endonucleases cut the DNA at precise sites corresponding to the linker region between nucleosomes. This leads to the formation of fragments that are multiples of 80 bp units. There is no cell swelling, such as occurs in necrosis, but rather cell shrinkage after the apoptotic process begins followed by condensation of chromatin at the periphery of the nucleus. Apoptosis is an energy-dependent process requiring ATP. During the apoptotic process, the plasma membrane initially remains intact but later fragments and surrounds the apoptotic bodies.

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8
Q

The TUNEL assay used to identify apoptotic cells detects:

A. The action of BAX on the mitochondria
B. Membrane integrity
C. Mitochondrial release of cytochrome c
D. Binding of TNF alpha to its receptor
E. DNA fragmentation

A

E

The terminal deoxynucleotidyl transferase (TdT) mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) technique has been used to identify apoptotic cells. It is based upon the binding of TdT to the exposed 3’-OH terminal ends of DNA fragments generated during apoptosis and catalyzes the addition of modified deoxynucleotides, conjugated with biotin or fluorescein, to the DNA termini.

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9
Q

Which of the following best describes radiation-induced bystander effects?

A. Damage to unirradiated normal tissue noted after irradiation of a tumor
B. Cell killing resulting from irradiation of the cell’s cytoplasm in the absence of direct irradiation of the nucleus
C. Radiation-induced increase in cell membrane permeability that causes increased sensitivity to cytotoxic drugs
D. DNA and/or chromosomal damage that occurs in unirradiated cells that are nearby irradiated cells
E. Intercellular communication that modifies the shoulder region of the radiation survival curve

A

D

While damage to cellular DNA was long considered the major initiator of cellular responses to ionizing radiation, more recent evidence suggests the involvement of non-targeted pathways, including radiation-induced bystander effects. Bystander effects are defined as radiation-like effects observed in cells that are not themselves irradiated, but that are in communication with irradiated cells through their location near these cells or by stimuli transferred from the irradiated cells through the intracellular medium. Various endpoints have been measured as bystander effects, including enhanced cell killing, induction of apoptosis, presence of
chromosome aberrations and micronuclei, presence of DNA double-strand breaks, increased oxidative stress, genetic effects (including induction of mutations, and neoplastic transformation) and altered gene expression

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10
Q

Mitotic death in irradiated cells results primarily from:

A. The mis-rejoining of DNA single strand breaks.
B. DNA ladder formation.
C. Stimulation of the extrinsic death pathway.
D. Mis-assortment of genetic material into daughter cells.
E. An alteration in cell membrane permeability.

A

D

Mitotic death in most irradiated cells results primarily from mis-assortment of genetic material into daughter cells as a result of the formation of asymmetrical chromosome aberrations. This aberrant mitosis triggers mitotic catastrophe, which is characterized by cells exhibiting multiple tubulin spindles and centrosomes as well as the formation of multinucleated giant cells that contain uncondensed chromosomes. Mitotic death can be of any molecular mechanism, including apoptosis or necrosis.

Single strand breaks are repaired rapidly and do not appear to play an important role in cell lethality (Answer Choice A).

DNA ladder formation is characteristic of apoptosis (Answer Choice B).

An alteration in cell permeability occurs in cells undergoing necrosis (Answer Choice E).

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11
Q

Which of the following concerning autophagy is INCORRECT?

A. Autophagy is a reversible process that can contribute both to tumor cell death and survival
B. The U.S. Food and Drug Administration–approved anti-malarial drugs chloroquine and hydroxychloroquine are inhibitors of autophagy
C. Autophagy contributes to cellular metabolism by degradation of damaged protein aggregates and organelles
D. Mitophagy refers to autophagy in mitotic cells
E. Autophagy is controlled by the Atg family of proteins

A

D

Autophagy can be nonselective or selective. Nonselective, bulk degradation of cytoplasm and organelles by autophagy provides material to support metabolism during periods of cellular stress. For example, autophagy provides internal nutrients, when external ones are unavailable. Whether mechanisms exist to prevent bulk autophagy from consuming essential components, such as a cell’s final mitochondrion, remains unclear, and in some cases such consumption may lead to cell death. Selective autophagy of proteins and of organelles such as mitochondria (mitophagy), ribosomes (ribophagy), endoplasmic reticulum (reticulophagy), peroxisomes (pexophagy), and lipids (lipophagy) occurs in specific situations.

Autophagy (‘self-eating’) tends to refer to macroautophagy: the sequestration process of cytoplasmic material for degradation. (Microautophagy and chaperone-mediated autophagy are other types.) After initiation, an isolation membrane encloses a small portion of cytoplasmic material, including damaged organelles and unused proteins, to form a double-membraned structure called an “autophagosome” that subsequently fuses with lysosomes to become an “autolysosome”, in which the cytoplasmic material is degraded by lysosomal enzymes.

The whole process is tightly regulated through at least 30 Atg-autophagy related genes that orchestrate initiation, cargo recognition, packaging, vesicle nucleation expansion and fusion and breakdown. The initial steps center around the Atg1 complex (Atg1–Atg13–Atg17– Atg29–Atg31) that translocates to the ER, (thought to be the major membrane source for autophagy). This leads to recruitment of the autophagy-specific form of the class III PI(3)K complex, which includes Vps34, Vps15, Atg6/Beclin1 and Atg14, to the ER. To form an autophagosome, elongation and closure of the isolation membrane requires 2 protein conjugation systems,
the Atg12–Atg5–Atg16 complex and the Atg8/LC3–
phosphatidylethanolamine (PE) complex. Detection of autophagy relies on the redistribution of GFP-LC3 fusion proteins into vesicular structures (which can be autophagosomes or autolysosomes).

‘Autophagic cell death’ is the excessive version of autophagy, that occurs in the absence of chromatin condensation. In contrast to apoptotic cells, there is little or no association of autophagic cells with cells phagocytes. Although the expression ‘autophagic cell death’ is a linguistic invitation to believe that cell death is executed by autophagy, the term simply describes cell death with autophagy.

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12
Q

Which of the following is NOT a feature of apoptosis?

A. Chromatin condensation
B. Cell shrinkage
C. DNA fragmentation
D. Rapid engulfment by neighboring cells
E. Pro-inflammatory response

A

E

There is no pro-inflammatory response in pure apoptosis. Apoptosis can in fact be anti-inflammatory through its recruitment of macrophages via “find-and-eat-me” signals; macrophages in turn produce anti-inflammatory TGF-β. Pro-inflammatory forms of cell death include necrosis, necroptosis, and pyroptosis.

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13
Q

Which of the statements is TRUE regarding the activation of one type of apoptotic pathway?

A. Apoptosis is initiated by PARP
B. Fas ligand binding its receptor initiates apoptosis
C. Caspases involved in the execution of apoptosis are also involved in the execution of necrosis
D. Bcl2 is a pro-apoptotic protein.
E. Anti-apoptotic Bax dimerizes and translocates to the mitochondria.

A

B

Fas ligand binds its receptor and triggers the external death receptor pathway.

Cleavage of PARP-1 by caspases is considered to be a biochemical hallmark of apoptosis (Answer Choice A).

Different caspases play different roles in the initiation and execution of apoptosis and are not involved in necrosis. Necrosis is the unregulated digestion of cellular components as a result of external factors (Answer Choice C).

Bcl-2 inhibits apoptosis while Bax stimulates apoptosis (Answer Choices D and E).

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