Cell Determination and Senescence

Question 1

What are the two general mechanisms for memory in cell determination?

Answer 1

• Chromatin Remodelling: states of DNA methylation and histone modification can be copied to daughter cells
• Positive Feedback

Question 2

Describe positive feedback.

Answer 2

A signal is initiated that causes A to be made (or activated).
A then causes B to be made (or activated), and B in turn causes A to be made (or activated).
This means that, even if the signal is removed later on, the process will still continue.

Question 3

What are two examples of positive feedback in master gene regulators in differentiation?

Answer 3

• Melanocytes, MITF (MIcrophthalmia-associated Transcription Factor)
• Skeletal Muscle, MYOD1 family

Question 4

RECAP: What is a master gene regulator?

Answer 4

It is a transcription factor that coordinately regulates many/all of the specialised genes expressed by a particular cell type.

Question 5

Describe the positive feedback of melanocytes.

Answer 5

MSH-MC1R signalling causes an increase of cAMP.
This, via a CREB activation, causes MITF to be made.
The MITF made then, via a MC1R transcription, causes more cAMP to be made.

MC1R also has some basal activity (without ligand MSH), so once MC1R is present, some cAMP and MITF continue to be made even if MSH not present.

Question 6

Describe the process of the MSH-MC1R signalling that causes MITF to be made.

Answer 6

The Melanocyte-Stimulating Hormone (MSH/ aka Melanocortin 1)) binds to the Melanocortin 1 receptor (MC1R).
The MC1R is a G-protein coupled receptor, coupled to a αGs subunit. This subunit activates Adenylate Cyclase, which increases the conversion of ATP to cAMP.
The cAMP activates Protein Kinase A (PKA). The PKA then phosphorylates the CRE-Binding protein (CREB).
CREB is a transcription factor, and it then travels into the nucleus and binds to the cAMP- Responsive Element (CRE) on the DNA. This then induces the transcription of several genes, including MITF.

Question 7

What do mutations in the MITF gene cause?

Answer 7

When homozygous, they cause a loss of all melanocytes in the body (skin, hair, eyes). The eyes become small, and there’s a loss of pigmented retinas.

The Waardenburg Syndrome 2 is a mutation of one copy of the MITF genes in humans. It causes deafness and congenital patchy loss of pigment (in the skin, can include irises)

Question 8

Describe the other positive feedback in melanocyte differentiation.

Answer 8

A Stem Cell Factor (SCF) is the ligand of a receptor tyrosine kinase, KIT.
The KIT then acts as the signal, causing MAPK to be made. Then, via CREB activation, MITF is caused to be made.
However, at this point, instead of the MITF causing more MAPK to be made, it instead causes more KIT to be made, via KIT transcription.

This positive feedback will work only so long as SCF is around (KIT has no basal activity).

Question 9

What are Myogenic Factors?

Answer 9

They are master gene regulators in skeletal differentiation.

Question 10

What are the four known myogenic factors?

Answer 10

• MYOD
• MYOG
• MRF4
• MYF5

Question 11

What are E-Proteins?

Answer 11

They are widely expressed transcription factors (myogenic factors normally work in dimers with E-proteins).

Question 12

What is ID1 (inhibitor of differentiation 1)?

Answer 12

It’s a protein in myoblasts (muscle precursor cells), which can strongly bind E-proteins, but not DNA.

Question 13

Describe how the situation in normal muscles is, then how it changes with ID1.

Answer 13

MYOD1, MYF5, etc. bind and activate muscle gene promoters, working as dimers with E-proteins.
However, ID1 bind strongly to E-proteins, and prevents activation. ID1 has no DNA-binding domain.

So ID1 inhibits differentiation.

Question 14

What effect does the MYOD family have on skeletal muscle differentiation in the embryo?

Answer 14

While they are migrating myoblasts, they have MYOD1 or MYF5 myogenic factors, but they also have ID1, which ‘steal’ away the E-proteins from the factors, inhibiting differentiation.
As they reach their destination, there is a different environment with low levels of Fibroblast Growth Factor (FGF) and Insulin-like Growth Factor (IGF). The ID1 is destabilised, meaning the myogenic factors are now free to bind and make dimers (active complexes) with the E-proteins. These go on to activate different muscle genes, such as myosin, actin, desmin, muscle kreatine kinase, etc.

Question 15

What is cell senescence?

Answer 15

It is permanent cell growth arrest, following extended cell proliferation.

Question 16

What is the Hayflick Limit?

Answer 16

It is the finite amount that a cell can proliferate before arresting.

Question 17

What is the cell lifespan?

Answer 17

It is the total number of doublings that a cell population goes through before senescence. It is normally measured from the time of explantation into cell culture.
We can’t track the cell proliferation that occurs before explantation, so the culture lifespan is only comparative. We can, however, distinguish immortal cells from those that senesce like this.

Question 18

List some morphological features of a senescent cell.

Answer 18

• they are large, flat cells
• they have prominent nucleoli
• the edge of the nucleus is hard to see (due to lamin loss)

Question 19

What are the two best known molecular markers of senescent cells?

Answer 19

• Lysosomal β-galactosidase
  (all cells have some lysosomes, but senescent cells have very many)
• Protein P16, a cell cycle inhibitor

Question 20

What are telomeres?

Answer 20

They are thousands of repeats of a hexamer sequence (TTAGGG) found at chromosome ends.
3’end of linear DNA can’t be replicated normally, because and RNA primer has to bind beyond the part to be replicated. So, the enzyme telomerase is needed to maintain telomere length.

Question 21

Describe telomerase.

Answer 21

The enzyme telomerase, a protein-RNA complex, can replicate telomeric DNA by reverse-transcribing DNA hexamers (TTAGGG) from its own RNA sequence, and joining them to the chromosome end.
TERT: telomerase reverse transcriptase (the protein part, = catalytic subunit).
TERC (or TR): telomerase RNA component

Question 22

How do telomeres come into cell senescence?

Answer 22

In humans, most somatic cells express no TERT, so no telomerase activity, so telomeres shorten as cells divide.
Replicative senescence is triggered in normal cells when telomere(s) get quite short. About 1-5 short telomeres is sufficient.

Question 23

How is a cell considered ‘immortal’?

Answer 23

Normal germline cells (oocytes, sperm, and their diploid progenitors) do express TERT, so they maintain full-length telomeres. Hence the germline is immortal - cells can divide forever.
Cancer cell lines in culture nearly all (~90%) express TERT, so they are immortal.

Question 24

Describe the established effector pathways (2) of cell senescence.

Answer 24

1) The telomeres shorten
2) The DNA damage signal phosphorylates p53 (a tumour suppressor), activating it
3) The p53 stimulates the expression of p21 (a growth inhibitor)
4) P21 inhibits CDK 1/2/4/6
5) That means that there is no phosphorylation of pRB, meaning that it remains bound to E2F, blocking transcription, thus effectively arresting cell division.

1) Raditaion, oxidative stress or DNA damage occurs.
2) This activates p53 (and that continues down the same path as before) and p16
3) P16inhibits CDK 4/6
4) That also continues down the same path as before.

Question 25

What are some of the commonest abnormalities found in cancer cells that lead to defective senescence, and thus immortality?

Answer 25

• expression of TERT
• p53 defects
• p16 defects

Question 26

What are the three types of stem cells?

Answer 26

• UNIPOTENT – can form only one functional cell type
• PLURIPOTENT – can form several functional cell types
• TOTIPOTENT – can form ALL functional cell types including placenta

Question 27

What cells are totipotent?

Answer 27

The zygote is totipotent but is not normally considered to be a stem cell (it doesn’t divide to make more zygotes).
But cells of the inner cell mass of the early mammalian embryo can act as totipotent stem cells – called embryonic stem cells.

Question 28

The zygote is totipotent but is not normally considered to be a stem cell (it doesn’t divide to make more zygotes).
But cells of the inner cell mass of the early mammalian embryo can act as totipotent stem cells – called embryonic stem cells.

Answer 28

Some of them have some telomerase activity, but in general too little to make the cells immortal.
In other words, telomeres shorten less per division in somatic stem cells than in other somatic cells, but they do shorten. So, somatic stem cells do senesce gradually.

Question 29

What are some examples of connections between cell senescence and ageing symptoms?

Answer 29

• Older people show decreased immunity, increased bone marrow failure and decreased success rate as bone marrow donors. There is a reduced proliferative ability of their marrow stem cells.
• Hair greying is linked to decreased melanocyte stem cell maintenance in hair follicles (data from mice).