Whole summary oncology
Lecture 1 Apoptosis
Definition of apoptosis
- Apoptosis: regulated and orderly destruction of a cell through a genetically encoded process
also known as programmed cell death (PCD)
- Apoptosis is a type of “cell suicide” that is intrinsic to cell due to an intrinsic or extrinsic signal
- Process is genetically encoded by genes
Apoptotic process
- Apoptosis is structurally organized, neat, and tidy, leaving behind little evidence of the pre-
existing cell: no induction of inflammation
- The cell undergoing apoptosis is swept clean during phagocytosis by macrophages,
neighbouring cells that recognize molecular flags (phosphatidyl serine)
o Phosphatidyl serine: first on the inside of the cell when apoptotic flips to the outside
and can be recognized and phagocytized
Function of apoptosis
- Programmed cell death: recognized during embryogenesis,
- syndactyly: dysregulation apoptosis: fingers will not be properly divided
- Active ATP dependent process
- Physiological in many organs
o developmental morphogenesis
o controls cell numbers during homeostasis
o removal of damaged cells removed by apoptosis by p53: example sunburn
o negative and positive selection of lymphocytes: e.g. lymphocytes recognizes self
antigens
o cytotoxic effect of radio- and chemotherapy
Apoptosis in humans
- Estimation of apoptosis in a human: of about 70 kg
o 25 x 106 mitoses per second
o 25 x 106 apoptosis per second
o 2,2 kg cells per day
Apoptosis versus Necrosis
Apoptosis: controlled cell death with specific Necrosis: cell death involved in heart attacks
morphology
Cell shrinkage Cell swells
Membrane blebbing Leaky membranes
Organelles intact Organelles damaged
Apoptotic bodies Cell lyses
Chromatin condensation and fragmentation Chromatin damaged
No inflammation Inflammation
,Apoptosis: electron microscopy
- Dark points: apoptotic bodies with condensation of chromatin
Apoptosis signalling
- Induction of apoptosis:
o Programmed: embryology
o Loss of growth factors, of adhesion, contact needed with cells for survival
o Death receptors of the TNFR family
o T- and B cell antigen receptors
o CTLs
o DNA damage (irradiation, chemotherapy)
o Stress conditions
- Mitochondrial changes
- Activation of the caspase family
- Proteolytic cleavage of structural and functional proteins actin microfilaments e.g.
- Induction of apoptosis morphology
Caspases
- Machinery apoptosis
- Caspases are cysteine-proteases synthesized as zymogens
- Requirement for an aspartatic acid at the P1 position
- 14 family members cloned
- Caspases 2, 3, 6, 7, 8, 9 and 10 are involved in apoptosis
- Divided into:
o initiators 2, 8, 9 and 10: activate effectors
o effectors 3, 6 and 7
o inflammatory 1,4 and 5: neurodegenerative diseases
Caspase activation
- Build of domains:
o Prodomain: big part
o Large subunit connected to small by spacer
o Specific sequence: in large subunit: different
in each caspase: important because these are used fr activation
- Step 1: small is transferred
- Step 2 prodomain removed
- Activation of caspases is done mostly by other caspases using ASP and cleavage
four Apoptosis pathways
1. Majore Intrinsic/stress-induced or mitochondrial apoptosis pathway internal signals
a. Most researched
2. Majore Extrinsic/Death receptor mediated apoptosis pathway
3. Granzyme B mediated apoptosis pathway
, 4. ER mediated apoptosis pathway: neurodegernative: inflammatory caspases
Intrinsic and Extrinsic apoptosis pathway
Extrinsic:
- induced by cytotoxic t lymphocytes
- ligands bind to receptor
o Fas ligand binds to the one receptor
Trimerizes, has death domain, bound
o Tnf: ligand, will bind tradd proteins
o Trill ligand more receptors, R1 and r2 induce
caspase 8
- caspase 8 is recruited: death induceing signalling process
- activated: activate other caspase 3/6/7
- apoptosis
intrinsic
- induced due to internal signals
- p53 is stabilized
- leads to activation of bcl 2
- from cytoplasm to mitochondria
- form homodimers or heterodimers
- result in formation of channels of mitochondria
- leads to release of cytochrome c
- presence of atp leads to complex formation of
apoptisome same ad death complex
- activation of caspase 9 and cascade
Breakdown of the cell
- induction of caspase 3 and 7: macrophages will remove the cell
- active 3 6 7 caspase will cleave protein
- bind specific proteins results is cleavage of important proteins leads to finishing of apoptosis
- Results from the proteolysis of target proteins:
o nuclear lamins
o Cytoskeletal proteins (actin)
o Intermediate filaments (cell structure)
o Specific kinases for cell signaling
o Other enzymes (caspase-activated DNAse)
Regulators of the apoptosis pathways
- Bcl-2 family: upstream intrinsic mitochondria
o Inhibitors: Bcl-2, Bcl-XL
o Activators: Bax, Bid
- C-Flip: extrinsic caspase 8
o c-Flip short: inhibitor of activation of caspase 8
o c-Flip long: controversial, inhibitor/inducer of apoptosis
, - IAPs (inhibitors of apoptosis proteins): inhibiting downstream both pathways
o cIAP1, cIAP2, XIAP
c-FLIP inhibition
- c flip: same protein as caspase 8, misses domain to activate caspase 8
- caspase 8 forms homodimer to activate but c flip can compete with the caspase
o will result in no activation
- c flip long: debated inhibitor or limiter
o little activation will occur leads to 2 proteins one will inhibit the other will activate
- c flip short
o inhibits caspase 8 and apoptosis
- p43flip
o NF-kbeta activation
Intrinsic apoptosis pathway
- Induced by DNA damage or other factors,
- Activated bax and bid
- Bax will lead to translation to the outside of the mitochondria results in presence procaspase
- cytochrome released forms heptameric complex, procaspase 9 activated when apoptosome
is formed, activates 3 6 and 7
Bcl-2 family
- A large family of proteins with sequence homology BH domains ‘
- Form 3 groups
- Group 1: bl-2 Anti-apoptotic
- Group 2: bax Pro-apoptotic
- Group 3: bid and bik (BH3 only)
- Form hetero – or homodimers through shared domain (Bcl-2 homology domein ((BH)3)
- Regulate mitochondrial permeabilization
Balance Bcl-2 family
- BH3-sensitizers: binding and inhibiting the anti-BCL-2 family members downstream BAD,nox
o Important for immune therapy
o Downstream: e.g.g BAX NOX
o When this happens the family members can bind bax or bac: apoptosis will occur
- BH3-activators: inducing activity of pro-apoptotic molecules,
o bim bid puma, can activate bax and bac and will help with translocation towards the
mitochondria
- Pro-apoptotic BCL-2 family members (BAX/BAK): forming pores in the outer mitochondrial
membrane are dimers
o Bax: present cytoplasm, cells get triggered, bh3 activation, induces bax/bac formation
in mitochondria outer membrane: degradation and lysation
o : inhibited forms heterodimer
- Anti-BCL-2 family members: Bind and sequester pro-apoptotic members to inhibit apoptosis
Lecture 1 Apoptosis
Definition of apoptosis
- Apoptosis: regulated and orderly destruction of a cell through a genetically encoded process
also known as programmed cell death (PCD)
- Apoptosis is a type of “cell suicide” that is intrinsic to cell due to an intrinsic or extrinsic signal
- Process is genetically encoded by genes
Apoptotic process
- Apoptosis is structurally organized, neat, and tidy, leaving behind little evidence of the pre-
existing cell: no induction of inflammation
- The cell undergoing apoptosis is swept clean during phagocytosis by macrophages,
neighbouring cells that recognize molecular flags (phosphatidyl serine)
o Phosphatidyl serine: first on the inside of the cell when apoptotic flips to the outside
and can be recognized and phagocytized
Function of apoptosis
- Programmed cell death: recognized during embryogenesis,
- syndactyly: dysregulation apoptosis: fingers will not be properly divided
- Active ATP dependent process
- Physiological in many organs
o developmental morphogenesis
o controls cell numbers during homeostasis
o removal of damaged cells removed by apoptosis by p53: example sunburn
o negative and positive selection of lymphocytes: e.g. lymphocytes recognizes self
antigens
o cytotoxic effect of radio- and chemotherapy
Apoptosis in humans
- Estimation of apoptosis in a human: of about 70 kg
o 25 x 106 mitoses per second
o 25 x 106 apoptosis per second
o 2,2 kg cells per day
Apoptosis versus Necrosis
Apoptosis: controlled cell death with specific Necrosis: cell death involved in heart attacks
morphology
Cell shrinkage Cell swells
Membrane blebbing Leaky membranes
Organelles intact Organelles damaged
Apoptotic bodies Cell lyses
Chromatin condensation and fragmentation Chromatin damaged
No inflammation Inflammation
,Apoptosis: electron microscopy
- Dark points: apoptotic bodies with condensation of chromatin
Apoptosis signalling
- Induction of apoptosis:
o Programmed: embryology
o Loss of growth factors, of adhesion, contact needed with cells for survival
o Death receptors of the TNFR family
o T- and B cell antigen receptors
o CTLs
o DNA damage (irradiation, chemotherapy)
o Stress conditions
- Mitochondrial changes
- Activation of the caspase family
- Proteolytic cleavage of structural and functional proteins actin microfilaments e.g.
- Induction of apoptosis morphology
Caspases
- Machinery apoptosis
- Caspases are cysteine-proteases synthesized as zymogens
- Requirement for an aspartatic acid at the P1 position
- 14 family members cloned
- Caspases 2, 3, 6, 7, 8, 9 and 10 are involved in apoptosis
- Divided into:
o initiators 2, 8, 9 and 10: activate effectors
o effectors 3, 6 and 7
o inflammatory 1,4 and 5: neurodegenerative diseases
Caspase activation
- Build of domains:
o Prodomain: big part
o Large subunit connected to small by spacer
o Specific sequence: in large subunit: different
in each caspase: important because these are used fr activation
- Step 1: small is transferred
- Step 2 prodomain removed
- Activation of caspases is done mostly by other caspases using ASP and cleavage
four Apoptosis pathways
1. Majore Intrinsic/stress-induced or mitochondrial apoptosis pathway internal signals
a. Most researched
2. Majore Extrinsic/Death receptor mediated apoptosis pathway
3. Granzyme B mediated apoptosis pathway
, 4. ER mediated apoptosis pathway: neurodegernative: inflammatory caspases
Intrinsic and Extrinsic apoptosis pathway
Extrinsic:
- induced by cytotoxic t lymphocytes
- ligands bind to receptor
o Fas ligand binds to the one receptor
Trimerizes, has death domain, bound
o Tnf: ligand, will bind tradd proteins
o Trill ligand more receptors, R1 and r2 induce
caspase 8
- caspase 8 is recruited: death induceing signalling process
- activated: activate other caspase 3/6/7
- apoptosis
intrinsic
- induced due to internal signals
- p53 is stabilized
- leads to activation of bcl 2
- from cytoplasm to mitochondria
- form homodimers or heterodimers
- result in formation of channels of mitochondria
- leads to release of cytochrome c
- presence of atp leads to complex formation of
apoptisome same ad death complex
- activation of caspase 9 and cascade
Breakdown of the cell
- induction of caspase 3 and 7: macrophages will remove the cell
- active 3 6 7 caspase will cleave protein
- bind specific proteins results is cleavage of important proteins leads to finishing of apoptosis
- Results from the proteolysis of target proteins:
o nuclear lamins
o Cytoskeletal proteins (actin)
o Intermediate filaments (cell structure)
o Specific kinases for cell signaling
o Other enzymes (caspase-activated DNAse)
Regulators of the apoptosis pathways
- Bcl-2 family: upstream intrinsic mitochondria
o Inhibitors: Bcl-2, Bcl-XL
o Activators: Bax, Bid
- C-Flip: extrinsic caspase 8
o c-Flip short: inhibitor of activation of caspase 8
o c-Flip long: controversial, inhibitor/inducer of apoptosis
, - IAPs (inhibitors of apoptosis proteins): inhibiting downstream both pathways
o cIAP1, cIAP2, XIAP
c-FLIP inhibition
- c flip: same protein as caspase 8, misses domain to activate caspase 8
- caspase 8 forms homodimer to activate but c flip can compete with the caspase
o will result in no activation
- c flip long: debated inhibitor or limiter
o little activation will occur leads to 2 proteins one will inhibit the other will activate
- c flip short
o inhibits caspase 8 and apoptosis
- p43flip
o NF-kbeta activation
Intrinsic apoptosis pathway
- Induced by DNA damage or other factors,
- Activated bax and bid
- Bax will lead to translation to the outside of the mitochondria results in presence procaspase
- cytochrome released forms heptameric complex, procaspase 9 activated when apoptosome
is formed, activates 3 6 and 7
Bcl-2 family
- A large family of proteins with sequence homology BH domains ‘
- Form 3 groups
- Group 1: bl-2 Anti-apoptotic
- Group 2: bax Pro-apoptotic
- Group 3: bid and bik (BH3 only)
- Form hetero – or homodimers through shared domain (Bcl-2 homology domein ((BH)3)
- Regulate mitochondrial permeabilization
Balance Bcl-2 family
- BH3-sensitizers: binding and inhibiting the anti-BCL-2 family members downstream BAD,nox
o Important for immune therapy
o Downstream: e.g.g BAX NOX
o When this happens the family members can bind bax or bac: apoptosis will occur
- BH3-activators: inducing activity of pro-apoptotic molecules,
o bim bid puma, can activate bax and bac and will help with translocation towards the
mitochondria
- Pro-apoptotic BCL-2 family members (BAX/BAK): forming pores in the outer mitochondrial
membrane are dimers
o Bax: present cytoplasm, cells get triggered, bh3 activation, induces bax/bac formation
in mitochondria outer membrane: degradation and lysation
o : inhibited forms heterodimer
- Anti-BCL-2 family members: Bind and sequester pro-apoptotic members to inhibit apoptosis
