L6- Cellular senescence, apoptosis & maintaining genome stability I

what are the barriers replicative immortality

1. senescence- non proliferative, viable state

2. crisis- cell death

what is the Hayflick limit

Leonard Hayflick - normal human cells in culture have limited capacity to divide, after which they become senescent

• Phenomenon now known as the ‘Hayflick limit’

• State of permanent cell cycle exit, not proliferating but viable

• Associated with morphological, metabolic and biochemical changes (pathway changed)

• Potent anti-cancer mechanism (together with apoptosis)

• Replicative senescence is linked to telomere erosion

what is a passage in cell culture

Passage- when you split the cells 

Stop growing at phase II 

• Discovered process of senescence 

• Limit where cell stops proliferating- Hayflick's limit  

• Irreversible no matter how many nutrients given  

Cells seen as very large and very flat 

• Various conditions that stress a cell and cause the cell to enter 

• Eg oncogene, chemo 

what are telomeres

Telomere (Greek word telos “an end”)

• Essential for maintaining genetic integrity

• Specialised DNA-protein structures (“protective caps”) that protect the linear ends of chromosomes from being recognised as damaged DNA

• Comprised of thousands of copies of tandem repetitive hexanucleotide sequence 5’ –TTAGGG – 3’

• Protein complex - Shelterin

what is the function of telomeres

To protect DNA- maintain genomic integrity and stability 

Hexanucleotide repeats 

• With each cycle of replication- get shorter 

• Eventually get so short to trigger replicative senescence 

what happens to telomeres when a component of the shelterin complex is knocked out

Knockout of key component of shelterin protein complex 

• Dysfunction where chromosomes no longer stable, start to fuse together  

what are the different features of telomeric DNA

1. non telomeric DNA of chromosome (many megabase pairs long)

2. subtelomeric DNA- telomeric like sequences (2-3 kbp long)

3. double-stranded region of telomeric DNA (5-10 kbp long)

4. single stranded 3’ overhang of G rich strand of telomeric DNA (several hundred bases long)

what is the function of the T loop

• To protect free DNA- g rich strand loops around and tucks its end back in to form complementary pairing with c strand 

In doing so displaces a portion of G strand DNA to make a D loop 

• Forms loop that hides free ends of DNA from damaging agents  

what proteins make up telomeres

Telomeres also made of protein

• shelterin- 6 member protein complex that binds to telomeric repeats and help to stabilise T loop, so ends stay protected  

• Loops needed for shelterin to bind and protect structure 

what proteins make up shelterin

6 member complex

1. RAP1

2. telomeric binding factor 1 (TRF1)

3. TRF2

4. TERF1- interacting nuclear factor 2 (TIN2)

5. TPP1

6. protection of telomeres protein 1 (POT1)

what does Shelterin do

• Engages both double-stranded DNA (dsDNA) and single- stranded DNA (ssDNA) regions of a telomere.

• POT1 binds telomeric ssDNA.

• RAP1 binds at junction between ss and ds DNA

• TRF1 and TRF2 are protein homodimers that bind telomeric dsDNA

• Prevents telomeres being recognised as damaged DNA- may cause DNA to bind together to be repaired 

what is the end replication problem

the shortening of telomeres 

what happens due the end replication problem

Due to end replication problem 

• in replication- lagging strand has rna primers that ultimately need to be removed 

• Always have an area where the primer has been bound and hasn’t been replicated- 

• If primer bound right at end of telomeric sequence- sequence is very similar, if primer didn’t prime right at end- the region it hasn’t primed will be lost  

• To maintain overhand- exonucleases cleave that so loop can be made- also shortens  

what happens to telomeres with age

Increasing age is a risk factor to developing cancer- telomere shortening may be a factor 

why do cells enter senescence

At some point in cell some chromosomes become so short that they enter senescence 

• Not all chromosomes shorten at the same time 

• Takes 1 or 2 chromosomes to start activating senescence 

what do cells do if they avoid senescence

If avoid senescence, they get critically short so no longer protect chromosome- through repair mechanisms, get joining of chromosomes (or cell death) 

• This is the 2nd barrier in most cases 

• This is recognised and in most cases causes cell death to prevent carcinogenesis 

 

However if cell is able to activate telomerase it can re stabilise the cell 

what is the senescence activation pathway (when telomeres are too short)

• When telomeres become critically short, Shelterin can no longer protect them effectively.

• Short telomeres activate DNA damage response pathways.

• p53 is activated, which activates p21 and causes cell-cycle arrest.

• p16 is also activated.

• p16 inhibits cyclin D/E-CDK activity, keeping Retinoblastoma protein active.

• Active Rb binds E2F and prevents progression through the cell cycle.

• Result: permanent cell-cycle arrest (senescence).

what happens f the senescence activation pathway is bypassed

If bypassed- through loss of p53 or Rb, common in cancer, escape senescence- telomeric DNA critically short, enter crisis to avoid instability 

• Need to stabilise- they ay develop this mechanism before catastrophic 

what is the breakage-fusion-bridge (BFB) cycle

During crisis- repeated cycles of BFB event 

• all chromosomes have different telomere length 

• Sister chromatids usually the same length 

• Free ends of DNA join together making a fused chromosome 

• When pulled apart in mitosis- leads to chromosome snapping at a weak point 

• One slightly smaller and one larger chromosome 

Doesn’t always happen in sister chromatids can be in any chromosome 

• Cycle repeats itself 

 

what does telomere maintenance mechanisms allow for

Allow telomeres to be extended- doesn't occur in normal cells, upregulated in lots of cancers 

• TMM must be developed by cancer cells if they want to be replicative immortal 

what are the 2 telomere maintenance mechanisms (TMM)

1. increased telomerase expression; ~85-90% of cancers

2. Alternative Lengthening of Telomeres (ALT) ~10-15%

describe increased telomerase expression (a TMM)

• increased telomerase expression; ~85-90% of cancers

Ribonucleoprotein complex

• Catalytic subunit hTERT - enzyme telomerase reverse transcriptase

• RNA subunit – provides template hexameric repeats- Non-coding human telomerase RNA (hTR)

Expression highest in in stem cells, progenitor cells, germ cells and very low or absent in normal somatic cells

describe Alternative Lengthening of Telomeres (ALT) (a TMM)

• Alternative Lengthening of Telomeres (ALT) ~10-15%

• Telomerase-independent telomere maintenance mechanism

• Recombination mediated synthesis

• Phenotypic characteristics: C-circles and ALT-associated promyelocytic leukaemia nuclear bodies

• Associated with mutations of ATRX and DAXX (death domain-associated protein) – complex partner of ATRX (chromatin remodelling members)

which TMM occurs in cells that dont express telomerase

ALT

in small amount of cancer cells - most common in mesenchymal origin cancer cells 

• Similar to recombination mediated synthesis 

• Telomere template from a different cell used for elongation 

• Diff to telomerase as these templates are thousand of base pairs linger 

what are HeLa cells

Generated from Henrietta lacks 

• Fist immortal cell lines in culture 

Can get hTERT-immortalised cells for research to avoid Hayflick limit issue 

what are the 5 inducers of cellular senescence

1. oxidative stress

2. telomere dysfunction

3. oncogene activation

4. DNA damage

5. inflammation

describe oncogene induced senescence

Key concept  

When cells overexpress oncogenes- can lead to sate of oncogene induced senescence 

Senescence is a barrier to  

• Via p16 and p14- lead to oncogene induced senescence as a barrier of carcinogenesis 

what are the markers of cellular senescence

• Flat, spread out and large 

• Can use beta galactosidase- stained blue as a marker 

• Look for upregulation of biochemical markers like p16 

• Look for downregulation of apoptotic markers