L7- Cellular senescence, apoptosis & maintaining genome stability II

what is apoptosis

Allows cell to die in restrictive manner without affecting environment  

• Contrast to necrosis- accidental cell death 

when is apoptosis important

Important in embryogenesis and normal tissue homeostasis

• Provides a self-destruct mechanism for damaged cells and is a crucial anti-cancer mechanism

how does apoptosis occur

• A series of biochemical events that lead to characteristic cell changes and cell death / “recycling”. These changes include blebbing,

• cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation.

• Form apoptotic bodies that are then taken up 

why does Unrepaired or excessive DNA damage leads to apoptosis

This prevents damaged DNA converting to mutations in progeny cells

• i.e. prevents the propagation of deleterious mutations that might otherwise activate oncogenes or inactivate tumour suppressor genes.

why do Inappropriate growth signals lead to appoptosis

Inappropriate growth signals (such as those resulting from oncogene activation) can lead to apoptosis.

• So activation of a growth-promoting oncogene can be pro-apoptotic (archetype c-MYC).

what is the Bcl-2 family

A family of proteins that have BH homology domain that allows them to interact with each other  

• Cell fate determined by the balance of these family members 

• Many cancers upregulate antiapoptotic bcl2 proteins  

what s the function of BAX (a Bcl-2 protein)

BAX promotes apoptosis by forming oligomers in the outer mitochondrial membrane, creating pores that release cytochrome c and activate caspases.

what are the members of the Bcl-2 family

how do Bcl-2 proteins relate to apoptosis

The balance between pro- and anti-apoptotic Bcl-2 family proteins determines whether apoptosis occurs.

• Under normal conditions anti-apoptotic proteins inhibit BAX/Bak,

• but after DNA damage BH3-only proteins block the anti-apoptotic proteins, allowing BAX/Bak to trigger apoptosis.

what is Venetoclax

a BH3 mimetic 

what are caspases

A family of cysteine proteases

• Effector caspases require cleavage by initial caspases to become active  

1. initiator caspases- 8, 10, 9, 2

2. Effector caspases – “executors of apoptosis” 3, 7, 6

what are the 2 key apoptosis pathways

2 key pathways  

1. Intrinsic- important in cancer, intertwined with pathways that sense DNA damage  

2. Extrinsic- linked to extracellular signalling e.g. death signalling. Ultimately lead to activation of intracellular pathway 

• Cross talk via activation of BID 

what is the role of SMAC in the apoptosis pathway

inhibits inhibitor of apoptosis proteins- ensures activation of caspase (initiation caspase) 

what is the role of the disc complex in the apoptosis pathway

Disc complex- leads to activation of caspase 8 (initiator) that leads to effectors 

what is the cell intrinsic apoptosis pathway

1. DNA damage causes activation of p53

2. p53 drives expression of BH3 only members (eg puma, noxa

3. bind and inhibit action of anti apoptotic Bcl-2 proteins leaving BAX and BAX free to form pores/oligomers in outer mitochondrial membrane

4. cytochrome c released from mitochondria and binds to APAF1

• SMAC/diablo also releases, inhibits inhibitor of apoptosis proteins to ensure activation of activator caspase 9

5. APAf1 binds caspase 9(initiator

6. activation of initiator caspase to activate effector caspase (3,7)

7. cleave cell contents, cell shrinks, cell death

what is the cell extrinsic apoptosis pathway

1. receptor, ligand and death receptor fuse

2. forms DISC complex with recruits pro caspases to activate caspase 8

3. leads to signalling down to effector caspases

cross talk and ability for 2 pathways to join via activation of BID, causing BAX and BAK activation, forming oligomers in outer mitochondrial membrane

what are the most notable triggers of apoptosis

1. DDR- Cause phosphorylation of p53 

2. hyperactive oncogenic signalling- Due to oxidative stress or ROS 

• Evading apoptosis is a key step during carcinogenesis and therapy resistance

• Numerous mechanisms to resist cell death

what is one main issue of cancer therapies

Fundamental rationale of cancer therapies - DNA damaging

• Not selective to only cancer cells 

• Harms cells that proliferate fast

what are some mechanisms to resist cell death

1. impaired receptor signalling pathway

• reduced expression of death receptor and death signal

2. defects/mutations in p53

3. disrupted balance of Bcl-2 family

• underexpression of proapoptotic proteins

• overexpression of antiapoptotic proteins

4. overexpression of IAP5

5. reduced caspase activity

give an overview of the p53 pathway

• P53 induces expression of negative regulator mdm2 which drives the degradation of p53- very controlled negative feedback loop 

• High p14 levels regulates mdm2 levels  

  Wip1- direct target of p53- phosphatase and removes any phosphates on p53 

what is the role of ATM and ATR

• ATM and ATR signalling to respective proteins CHK1/2 and activate by phosphorylation 

• Then phosphorylate p53 to prevent it form binding mdm2- activating it  

P53 normally has short half life when not activated 

describe the findings from p53 KO mice

p53 KO mice develop tumours ~4.5 months

• mdm2 KO mice die early in development

• Mice deficient for both mdm2 and p53 develop normally and are viable

what are the 4 mechanisms of p53 signalling inhibition

1. TP53 mutations (~50%)

2. MDM2/MDMX amplification or overexpression

3. p14ARF deletion or promoter hypermethylation

4. WIP1 overexpression

describe the relevance of p53 mutations in cancer

• P53 inactivated by mutation in 50% of cancers 

• These pathways don’t have to happen simultaneously 

• mutations don’t always inactivate it, may be gain of function that drive cancer development 

where is the most common area for p53 mutations

• Hotspots normally in DNA binding domain of p53- between exons 5-8 

• P53 acts as tetramer- has a dominant negative effect 

Accumulation of p53 indicates a mutation that increases its half life as mdm2 doesn’t negatively regulate it  

what is the pathway of oncogene induced apoptosis

• In response to oncogenic hyperactive signalling- can lead to apoptosis 

1. sent by p14- ARF (aRF upregulation)

2. upreg of p53

3. drive apoptosis

what is genomic instability, what are the 2 forms

Characteristic of most if not all cancers

• Accumulation of instability that deviate from the norm 

1. Chromosomal level (CIN) structural and numerical- Gaining a portion or an entire set  

2. Non-CIN (DNA level)

describe non CIN (DNA level) genomic instability

• Microsatellite instability (MIN) composed of short nucleotide repeat sequences resulting from impaired DNA mismatch repair

• Increased frequency of single/few bp substitutions, insertions, deletions

what are the 2 types of chromosomal instability (CIN)

1. numerical changes (aneuploidy)

2. structural changes

vast majority of human tumours exhibit CIN (~60-80%)

what is chromothripsis

Shattering of one or more chromosomes 

• All pieces randomly put together 

• Catastrophic and rare 

what are some sources of DNA instability

Sources:

• telomere dysfunction

• various exogenous and endogenous sources of DNA damage

• replication or segregation errors

• telomere dysfunction

• loss of DDR

what are the fundamental mechanisms to maintain genomic stability

• Activation of DDR

• Repair of any DNA damage

• Induction of senescence or apoptosis to prevent propagation of cells with damaged DNA

give an overview of DNA repair mechanisms

DSB- most cytotoxic DNA damage- can initiate with translocation and change DNA 

what is mismatch repair (MMR)

Detects any errors in replication 

post replicative repair pathway

• Repairs for example base mismatches that have arisen during replication in S phase

• Increased fidelity of replication by 100-fold

• Strand specific

• Aberrations can lead to MIN

• Lynch syndrome (AKA hereditary nonpolyposis colorectal cancer HNPCC) with increased susceptibility to colon cancer

what is the process of MMR

1. MutSⱭ detects change and recruits MutLⱭ

2. recruits additional proteins to cleave mismatch

3. elongation with DNA polymerase

4. then ligated

Must be able to recognise newly synthesised (inaccurate) strand  

why are MutSⱭ and MutLⱭ given these names

called mut due to homology with E.coli proteins where originally studied

what are MutSⱭ and MutLⱭ made up of

MutSⱭ- heterodimers of Msh2,3 and 6 proteins

MutLⱭ- heterodimers of mlh1 and pms 2

What is base excision repair (BER)

BER remove inaccuracies to the bases themselves- has glycosylases specific to the bases  

• Generates a ssDNA break- so the single strand base repair mechanism works in tandem  

What is nucleotide excision repair (NER)

Much more broad spectrum of damage  

• repair bulky lesions which distort the DNA- defective in Xeroderma

  Pigmentosum

• Pyrimidine dimer that causes distortion 

• Recognised and cleaved 

• DNA polymerase recruited to synthesis new section, then ligated 

• On new synthesised strand 

what are the 2 types of DSB repair

NHEJ and HR

what are some features of DBS repair

• DSBs most cytotoxic lesions

• Caused by cancer therapies and stalled and collapsed replication forks

• 2 main pathways: Homologous recombination (HR) and Non-homologous end-joining (NHEJ).

• Vary in fidelity of repair and phase of cell cycle

• BRCA1/2 and HR: hereditary risk of breast and ovarian cancer

what does DBS occur in response to

Can occur in response to ionisation and stalled replication forks  

• Fork can be fragile, if held for a long time can break 

• A ss break can cause a ds break, which can then cause a translocation 

describe NHEJ DSB repaur

NHEJ- large proteins detect DSB, processing to remove any single strands 

• 2 ends are simply ligated together 

• Can happen anywhere in cell cycle 

• Not as high fidelity- joined whether they should or not 

• That’s why end of telomeres are protected  

describe HR DSB repair

HR in S or G2 phase- use info from sister chromatid 

• Recruitment of BRCA and Rad51- causes invasion of sister chromatid so an be used as a template 

• Higher fidelity 

what are some examples of hereditary cancers

Mutations in caretaker genes leads to increased cancer risk due to greater risk of DNA mutations

how is oncogene driven replication stress a key driver of DNA instability

Key initiator of genomic instability in sporadic cancer is oncogenic driven replication stress 

• Stalling replication forks 

• Replication forks crashing with transcription mechanisms