lec6: Cell Death and Signalling

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Cell death is a natural process T or f

True

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Cell death is necessary for

Homeostasis, development and immune function

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Most cell death occur through a process of ……………

programmed cell death

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Programmed cell death is responsible for

balancing cell proliferation and maintaining constant cell numbers in tissues undergoing cell turnover.

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The defense mechanism by cell death helpsin

preventing the accumlation of dead or unnecessary cells that could lead to disease

  1. virus infected cells to limit the spread of viruses

  2. DNA damage to eliminate cells with potentially harmful mutations that might lead to cancer

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Programmed cell death can lead to diseases and tissue damage t or f

false, excessive or unregulated cell death

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Examples of unregulated cell death

  1. cancer: mutations in genes that regulate cell death

  2. neurodegenrative: excessive death of neurons

  3. ischemic heart disease: blood supply is blocked to the heart, causing necrotic cell death (infarction)

  4. Autoimmune disease: ex:rheumatoid arthiritis, immune system attacks health cells, leading to excessive cell death

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Major types of cell death

  1. Apoptosis

  2. Autophagy

  3. Necrosis

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Apoptosis

programmed cell death

chromosomal DNA fragmentation, chromatin condensation, break up of nucleaus to small pieces, cell shriking to small membrane fragments called apoptotic bodies.

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cell itself shrinks and breaks up into membraneenclosed fragments called ……….

apoptotic bodies

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excutioners of apoptosis

caspases (proteases)

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How does caspases activation happen in apoptosis

when apoptosis is triggered, initiator caspases are activated (8,9), thus activating effector caspases (3,6,7), carry the responsibility of cell destruvtion

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effector caspases are activated via

proteolytic cleavage

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caspases

a family of proteases that cleave specific proteins in the cell

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caspases activation is …………

irreversible switch for cell death

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types of apoptosis

  1. intrinsic apoptosis signaling

  2. extrinsic apoptosis signaling

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Intrensic apoptosis

signals are initiated from inside the cell

triggered by DNA damage, cellular stress or oncogene activation

BCL-2 family pathway (act at mitochondria)

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mitochondria dependant

Mitochondria induce apoptosis when pro-apoptotic factors of the bcl2 factor outnumber anti--apoptotic factors (mitochondria storage site for apoptosis-regulating molecules) apoptosis-regulating

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steps of intrinsic apoptosis

Step 1: Increase in the balance of proapoptotic (Bax/Bak of Bcl2 family) to antiapoptotic factors

Step 2: Release of cytochrome C, formation of apoptosome

Step 3: Activation of caspase 9 → activation of effector caspase 3 → apoptosis

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……………. is a particularly dangerous form of cell stress

DNA damage

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cells with damaged genomes may have suffered …………… that can lead to the development of ………….

mutations, cancer

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DNA damage activates ………………, which ………….

ATM and Chk2 protein Kinases, phosphorylate p53

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intrinsic apoptosis, DNA damage

➢ DNA damage activates the ATM and Chk2 protein kinases, which phosphorylate p53

➢ Accumulation of activated p53 leads to transcriptional activation of genes encoding the proapoptotic regulatory proteins

➢ This leads to activation of PUMA, release of cytochrome c from mitochondria, and activation of caspase-9 → activation of caspase 3→ apoptosis

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Mutations in the …….. and ………genes are common in cancer which contribute to the ………..

P53, BCL2, Cell death resistance

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PI3K/AKT

major regulator of cell survival and cell death in response to growth factor signaling by regulating both pro and antiapoptotic Bcl-2 family members

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PI3K/AKT steps

➢ PI 3-kinase phosphorylates the membrane phospholipid PIP2 to PIP3

➢ PIP3 activates the serine/threonine kinase Akt

➢ One key substrate for Akt is the proapoptotic regulatory protein called Bad.

➢ Akt inhibits Bad→ inhibits apoptosis and promotes cell survival

➢ Bad is similarly a substrate for the Ras/Raf/MEK/ERK pathway

➢ Growth factor deprivation → activation of Bad → activates the intrinsic pathway of apoptosis.

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Extrinsic apoptosis signaling

Initiated from outside the cell

from other cells from the extracellular matrix

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Most well-known extrinsic pathways

Fas/fasl

tnf/tnfr

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extrinsic pathway steps

➢ Step 1: Ligand (e.g. TNF) binds to cell death receptor (e.g. TNF receptor)

➢ Step 2: Activation of Caspase 8

➢ Step 3: caspase-8 can directly cleave and activate effector caspases (e.g. caspase 3)

➢ Step 4 (in some cases): caspase-8 cleaves the proapoptotic proteins of BCL-2, which activates the mitochondrial pathway of apoptosis, leading to caspase-9 activation → activate effector caspases (e.g. caspase 3)

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Apoptosis induced by activation of Fas is responsible for

killing target cells of the immune system, such as cancer cells or virus infected cells.

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Response to caspases

Caspases bring about the events of apoptosis by cleaving more than 100 different target proteins, resulting in DNA fragmentation and cell condensation.

  1. inhibitor of DNases: DNA fragmentation

  2. Nuclear lamins: nucleus frag

  3. Cytoskeletal: disruption, cell frag, membrane blebbing

  4. golgi matrix: golgi frag

  5. scramblase: translocation of phosphatidylserine to the cell surface

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Apoptotic cells and cell fragments are efficiently recognized and ………….by both …………..and …………..

Phagocytosed, macrophages, neighboring cells

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The removal of apoptotic cells is mediated by the expression of socalled ……. on cell surface

eat me signals

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……………… is usually in the inner leaflet of plasma membrane, but translocates outside during apoptosis and recodnized by ………..

phosphatidylserine, phagocytic cells

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T or F cells that die by apoptosis are rapidly removed from the tissue

True

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How can apoptosis be detected?

Annexin V: Fluorescently labeled Annexin V protein can be used to detect PS that is exposed on the outside of apoptotic cells (by fluorescent microscope, plate reader, flow cytometry…etc)

caspases expression whether gene or protein

live cell imaging of caspase 3 with fluorscent dye

DNA fragmentation (tunnel assay)

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Medication target for apoptosis

  1. chemotherapy

  2. radiptherapy

  3. Bcl-2 inhibitors

  4. immunosuppressives

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chemotherapy for apoptosis

ex: doxorubicin

severe DNA double stranded breaks

ATM activation and the phosphorylation of p53 resulting in capsapse 9 activation and apoptosis in cancer cells

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Radiotherapy

cause DNA damage

→ ATM activation → p53 phosphorylation → Caspase 9 activation → apoptosis

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Bcl2 inhibitors

ventoclax, apoptosis in leukemia cells

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immunosuppressive meds

for autoimmune disease ex: arthritits, rheumatoid, lupus

ex: anti-TNF drugs as infliximab

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Autophagy

Cell eats its own content

remove unnecessary or dysfunctional components in a lysosome dependant regulated mechanism

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Importance of autophagy

  1. survival during nutrient deprivation: autophagy increased degradation of cellular proteins and other macromolecules, allowing their components to be degraded as a source of energy or reutilized for essential functions

  2. quality control: remove defective organelles or proteins

  3. pathogen removal/ defense: during infection by Toll-like receptors that recognize pathogenassociated molecular patterns (PAMP), allow autophagy to kill pathogens to the innate and acquired immunity

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nutrient rich conditions

mTOR active = no autophagy

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nutrient deprivation

mTOR inhibited= autophagy

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t or f mTOR is a positive regulator of autophagy

false

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…………….. is a common antitumor medication that inhibits mTOR

Rapamycin

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mTOR full name

mamilian target of rapamycin

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function of rapamycin

inhibitor of the protein kinase mTOR → promoting cell death via autophagy in cancer cells

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Mechanism of autophagy

  1. initiation: nutrient deprivation, stress, infection

  2. autophagosome formation: nucleauation and elongation of a phagophore

  3. fusion with lysosome: form autolysosome

  4. degradation of cellular components: by enzymes in lysosome

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autophagosome is formed via

nucleation and elongation of a phagophore (double membraned vesicle)

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function of autophagosome

engulf cellular components, such as organelles, macromolecules, and bacteria.

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What happens if autophagy is impaired

  1. Neurodegenerative disease: es: parkinson’s. accumlation of damaged organelles or macromolecules in neurons

  2. Cardiovascular disease: ex: atheroscelerosis, accumlation of damaged cells in the heart or blood vessels

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Necrosis

passive due to injury or stress, infection, toxins

unregulated breakdown of cells and tissues

accompanied by inflammation

ex: ischemia/ lack of oxygen

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necrosis happen in ……. conditions while apoptosis in ……….. or ……….

pathological, physiological, pathological

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…….. is the body’s response to injury or infection

inflammation

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main feautures of necrosis

  • Cell swelling

  • cell membrane disruption

  • cellular leakage

  • inflammatory responses

  • DAMPS activation

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DAMPS

Damage-Associated Molecular Patterns are molecules released from dying cells as a result of necrosis.

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summary

knowt flashcard image
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Necrosis vs apoptosis

knowt flashcard image
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Experimental work to answer the research question

•Detection of activated caspases, such as caspase-3 and caspase-7: qPCR, western blot

•Analysis of DNA fragmentation using techniques like the TUNEL assay : fluorscent microscope

•Measurement of phosphatidylserine externalization using Annexin V staining