Cellular Senescence and Immortalisation

Question 1

What is cellular senescence?

Answer 1

Stable growth arrest of cells - when cells enter a post-mitotic state and are unable to be induced to divide by any signal - a permanent quiescence. Their replicative potential is said to have been exhausted.

This is relatable to terminal differentiation into cells such as neurons and myofibres which are unable to divide.

Question 2

How is cellular senescence relevant to human ageing?

Answer 2

It has a role in the ageing of tissues by disrupting tissue renewal, repair and regeneration.

If you take cells from a younger person that they will take a number of divisions more than that of an older person: this can be seen in the healing capacity of children versus adults.

Question 3

Why is cell senescence not relevant in some model organisms?

Answer 3

Drosophila and C. elegans, bodies made up of only non-dividing cells – cell survival is more important for ageing . Every cell in C. elegans is post mitotic.

Question 4

What is the hayflick limit? How does it vary?

Answer 4

The number of divisions a cell population can divide before reaching senescence.

This is dependent on cell type and species of origin (human fibroblasts have a limit of 50, mouse ones 20-30) and on the age of the donor.

Question 5

In what manner do cells in a culture reach the hayflick limit?

Answer 5

They do so asynchronously, rather than simultaneously. Rather than reaching the limit at the same time, the ration of senescent to dividing cells will slowly increase.

Question 6

Why is senscence though to have evolved?

Answer 6

Due to Kirkwood’s theory of Antagonistic Pleiotropy - favouring of traits which offer benefit to an organism when of reproductive age despite their deleterious effects when older.

The replicative potential of cells is high enough for normal growth and development, but not so high that it leads to early-onset cancer. The exhausting of this potential in old age not only leads to slower healing but is also pro-cancerous due to the cell phenotypes induced.

Question 7

Why must the replicative potential of cells be carefully balanced?

Answer 7

If the limit is too low then growth and development would be prevented due to our cells becoming senscent too early.
If the limit is too high it increases the likelihood of tumour
development.

Question 8

How can senescence be used to regulate development?

Answer 8

(Munoz-Espin et al, 2013)
In the mouse inner ear, developmentally programmed senscence of certain cells triggers macrophage infiltration, cell clearance and subsequent tissue remodelling as part of the normal growth pathway.

(Storer et al, 2013)
Oncogene-induced senescence (OIS) is a normal developmental mechanism in mouse embryonic signalling centres (the apical ectodermal ridge and neural roof plate) through apoptosis and macrophage clearance of these cells.

Question 9

What is SAB?

Answer 9

Senescence-associate beta-galactosidase is a hypothetical hydrolase enxyme that catalyzes the hydrolysis of the beta galactosides into monosaccharides only in senescent cells.

Question 10

Why is SAB referred to as a hypothetical enzyme?

Answer 10

Its existence was proposed in 1995 by Dimri et al. following the observation that when beta galactosidase assays were carried out at pH 6.0 only cells in senescence state develop staining.

Question 11

What is the explanation for the evidence for SAB?

Answer 11

Endogenous lysosomal beta galactosidase is overexpressed and accumulates only in senescent cells.

Question 12

What is the use of SAB?

Answer 12

Its expression is not required for senescence, however it remains as the most widely used biomarker for senescent and aging cells, because it is easily detectable

Question 13

What biomarkers can be used to identify senescent cells?

Answer 13

SAB, heterochromatinised nuclei and ATM kinase activation level.

Question 14

What is Werner Syndrome?

Answer 14

Werner syndrome (WS) is a genetic premature aging disorder used as a model of normal human aging. WS individuals have several characteristics of normal aging, such as cataracts, hair graying, and skin aging, but manifest these at an early age. Additionally, WS individuals have high levels of inflammatory diseases, such as atherosclerosis and type 2 diabetes.

The in vivo aging in WS is associated with accelerated aging of fibroblasts in culture.

Question 15

What is the primary cause of WS?

Answer 15

WRN, which lies on chromosome 8 in humans, encodes the WRNp protein, a 1432 amino acid protein with a central domain resembling members of the RecQ helicases. Mutation of this is what causes the disease.

Question 16

What are RecQ helicases?

Answer 16

RecQ helicases are a special type of helicase that function at unique times during DNA repair of doubled stranded breaks, which are a form of DNA damage that results in a break of both strands of DNA.

Thus, RecQ helicases are important for maintaining DNA stability, and loss of function of these helicases has important implications in the development of Werner syndrome.

Question 17

What phenotype is shown in heterokaryons produced from fusion of senescent and non-senescent cells? What does this show?

Answer 17

The senescence is dominant. This demonstrates that senescence is regulated intrinsically/genetically, although extrinsic factors can also stimulate it.

Question 18

What are the two barriers to unlimited proliferation?

Answer 18

Senescence and crisis: when cells are propagated in culture, repeated cycles of cell division lead first to induction of senescence and then, for those cells that succeed in circumventing this barrier, to a crisis phase, in which the great majority of cells in the population die.

On rare occasion, cells emerge from a population in crisis and exhibit unlimited replicative potential. This transition has been termed immortalization

Question 19

What is the primary theory of how senescence and replicative potential is dictated?

Answer 19

The telomere theory. That consistent reduction in telomere length with each division eventually leads to cells with very short telomeres that are unable to protect the chromosomes and thus lead to senescence.

Question 20

What do telomeres protect chromosomes from?

Answer 20

end-to-end fusions; such fusions generate unstable dicentric chromosomes whose resolution results in a scrambling of karyotype that threatens cell
viability.

Question 21

By what mechanisms can senescence be induced?

Answer 21

In addition to telomere attrition, senescence can be activated by many types of stress, including aberrant activation of certain oncogenes, damage to chromatin structure, oxidative stress, DNA damage, and inadequate culture conditions. Collectively, they are referred to as stress-induced premature senescence.

Question 22

How does stress-induced premature senescence interact with telomere attrition?

Answer 22

Among the senescence-inducing stimuli, oxidative stress has been shown to accelerate telomere shortening, possibly by inducing telomeric single-strand breaks.

However, stress-induced premature senescence, unlike replicative senescence, is largely independent of the telomere length or the number of cell divisions.

Question 23

How much do telomeres shorten with each division?

Answer 23

Human somatic cells’ telomeres shorten about 50-100bp per doubling, due to the end replication problem.

Question 24

How are telomeres generated and maintained to prevent senescence?

Answer 24

By telomerase. This is expressed in germline cells to ‘reset the mitotic clock’.

Question 25

What are telomeres anyway?

Answer 25

Special DNA structures at ends of chromosomes with 1000s of tandem TTAGGG repeats, ending in a G-rich 3’ overhang of several hundred bases due to the end replication problem.

They protect the end of the chromosome from deterioration or fusion with other chromosomes

Question 26

Why do telomeres grow short?

Answer 26

During chromosome replication the DNAP cannot continue duplication all the way to the end of a chromosome because the synthesis of Okazaki fragments requires RNA primers attaching ahead of the lagging strand. This is called the end-replication problem.

Question 27

How are the ends of telomeres protected?

Answer 27

By the shelterin complex, which binds and arranges the DNA to prevent it from being recognised as a double strand break using the 3’ overhang.

Question 28

How does shelterin protect the telomere end?

Answer 28

It forms the DNA into a protective t-loop by curling the 3’ overhang around to anneal to an earlier section, displacing an earlier part of that strand into a bubble called the D-loop (displacement).

This hides the end of the 3’ overhang, so it is not recognised as a DSB.

Question 29

What are the components of the shelterin complex?

Answer 29

TRF1/2
POT1
TPP1
TIN2
Rap1

Question 30

What is the role of TRF1/2 and Rap1 in the shelterin complex?

Answer 30

These are homodimeric proteins that bind to double stranded TTAGGG repeats and anchor the shelterin complex.

Rap1 binds TRF2 and stabilises it.

Question 31

What is the role of POT1 in the shelterin complex, and how is it connected?

Answer 31

POT1 binds the single stranded 3’overhang, and is recruited by TPP1.

TIN2 binds to the TPP1/POT1 complex and also binds to the TRF1/2 complexes to link the two and facilitate t-loop formation.

Question 32

What DNA damage pathways are inhibited by the shelterin complex?

Answer 32

There are two main DNA damage signaling pathways that shelterin suppresses: the ATR pathways, blocked by POT1 and the ATM pathways blocked by TRF2.

Question 33

What happens when telomeres get too short?

Answer 33

When the telomeric length reaches a certain length, then it reaches a checkpoint signaling the hayflicks constant and stops dividing.

Question 34

How do short telomeres activate senescence?

Answer 34

Critically shortened telomeres lose the protection of telomere-binding proteins, leading to telomere “uncapping”.

Recent studies have revealed that DNA damage foci containing multiple DNA damage-response proteins, such as 53BP1, γH2AX, MDC1 and MRE11, are found at telomeres in senescent cells, suggesting that uncapped telomeres are recognized as DNA breaks and thus trigger a DNA damage response.

Question 35

What is telomerase?

Answer 35

This is an RNA-dependent DNA polymerase that synthesizes telomeric DNA sequences, overcoming the end replication problem to maintain/extend the telomere.

Question 36

Where is telomerase active?

Answer 36

Its activity is negligible in somatic cells, but expressed at functionally significant levels in the vast majority (90%) of spontaneously immortalized cells. It is often found in cancer cells, as well as in germline cells and some stem cells.

Recent research shows that hTERT is upregulatedat a low level upon entry into S phase which disappears as cells advance to G2. Since this is not sufficient for telomere maintenance it is assumed to be part of a different role.

Question 37

What are the components of active telomerase?

Answer 37

hTERT is the reverse transcriptase catalytic domain that polymerises the addition to the 3’ overhang.

hTR is Template RNA associated with the protein. This is complementary to the TTAGGG tandem repeat to allow for production of the telomere, but is 1.5x longer than one repeat to allow it to bind to the end of the overhang and extend beyond it for addition of a new repeat (catalysedby the hTERT component).

Question 38

How is telomerase repressed in somatic cells?

Answer 38

hTERT expression is repressed in soma, but hTR is expressed constitutively (hence activity is almost entirely dependent only on hTERT expression)

Question 39

What is seen when cell cultures are made to express hTERT?

Answer 39

Expression of the telomerase catalytic subunit (TERT) has been shown to prevent telomere shortening and extend the lifespan of human somatic cells up to five fold. Conversely, inhibition of telomerase in immortal cells has been found to limit their replicative lifespan.

Question 40

Why do cancer cells express telomerase?

Answer 40

Telomerase activity has been detected in approximately 90% of tumor samples. Expression of telomerase is sufficient for the escape of cells from the two barriers to proliferation (senescence and crisis) and for the immortalization of many cell types.

Question 41

How can telomeres be maintained other that with telomerase?

Answer 41

Using Alternative Lengthening of Telomeres (ALT), which is thought to occur using recombination with other chromosomes.

Question 42

What is notable about mouse telomeres/rase?

Answer 42

mTERT is expressed in some mouse soma. In inbred strains the mice have hugely long telomeres (as rat cells do), which allows telomerase-null mutants to be viable. However after around five generations of breeding the telomeres shorten sufficiently for premature ageing.

Outbred strains have much shorter telomeres.

Question 43

What effect does the telomeric limit upon replicative potential have on the potential of cells to produce tumours?

Answer 43

Due to the rapid proliferation of transformed cells, their telomeres shorten rapidly. They are thought to often be stopped at this stage due to telomere-induced crisis.

However, in cancer cells lacking in DNA damage repair systems due to p53 mutation, telomere erosion can actually encourage tumour progression due to the induction of breakage-fusion-bridge cycles until telomerase expressing cells are selected for. Transient telomere deficiency is thus thought to be important for malignant progression.

Question 44

What pathways are facilitate cell senescence?

Answer 44

Two major pathways that underlie senescence:

1. Activation of p53 pathway- induces p21CIP1 prevents cyclin/cdk complexes from phosphorylating the RB family of proteins, thereby activating RB tumour suppressor pathway – Activated by Oxidative Stress and and DNA damage
2. RB pathway can be activated independently of p53 upregulation of p16INK4A, inhibition of cyclinD/cdk4,6 kinases that also phosphorylate RB family of proteins

These are notable the same pathways that cause quiescence, differing only in their permanence.

Question 45

How do quiescent cells become senescent?

Answer 45

This is called geroconversion. It is generally driven by simultaneous cell cycle arrest due to p53/Rb and continued mitogenic growth signalling though mTOR.

Question 46

How can senescence be tumour promoting?

Answer 46

Due to the effect of mTOR, Geroconversion can lead to hyper-secretory, hypertrophic and pro-inflammatory cellular phenotypes, hyperfunctions and malfunctions.

Increased NF-kB signalling and secretion of SASP (Sen. Assoc. Secretory Phenotype) proteins such as VEGF. MMPs and ILs can promote tumours in nearby cells.

Question 47

What are immortalising genes?

Answer 47

These allow cells to overcome the limits of replicative potential, bypassing both barriers (sen. and crisis). Mutant p53 and Myc have immortalising potential, and reports vary on whether hTERT is (some success is seen when paired with the SV40 T antigen). Most however are viral.

Question 48

How do viral immortalising genes work?

Answer 48

Genes such as adenovirus E1A, HPV type 16/18 E7 and SV40 or polyoma virus large T antigen are capable of immortalising cells.

This generally works because they inhibit pRb family proteins via their CR2 domains through the viral genes’ conserved LXCXE binding pocket.

The SV40 T antigen and AD E1B 55K proteins stabilise p53, while the HPV E16/18 E7 gene targets p53 for ubiquitination and degradation.