Cell Determination and Cell Senescence

Question 1

What does it mean by a cell having ‘memory’?

Answer 1

Once a cell differentiates, it remembers this state without any external inducing signal.

Question 2

What are the mechanisms for memory in cell determination?

Answer 2

Chromatin remodelling

Positive feedback

Question 3

What is chromatin remodelling?

Answer 3

It is when states of DNA methylation and histone modification can be copied to daughter cells.

Question 4

What is positive feedback?

Answer 4

It is when a signal causes a cell to make a product and that product causes another product to be made.
For example a signal causes A to be activated and made and A then can cause B to be activated and made. However B can also cause A to be activated and made.

Question 5

What is a master gene regulator?

Answer 5

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

Question 6

Provide an example for positive feedback for memory in cell differentiation.

Answer 6

Melanocytes signal from outside the cell promotes cAMP to activate MITF ( a master transcription factor). This MITF can up-regulate the receptor (MC1R) which amplifies the cAMP signal.

Question 7

What is MITF?

Answer 7

Microphthalmia-associated transcription factor. MITF is needed for all normal melanocytes development.

Question 8

If a mutation occurs in the MITF, what can happen?

Answer 8

Sever mutations in the MITF gene when homozygous, causes the loss of all melanocytes in the body and eyes becomes small as there is a loss of pigmented retina (microphthalmia).
Not only can this occur in mice, but in humans too where people are heterozygous. Mutation of a 1 copy of the MITF gene in humans produces the Waardenberg syndrome 2. These people are deaf as the pigmented cells in the ear that are needed for hearing are reduced in number. They also may have contigential patchy loss of pigment in skin and can include irises- the eye colour being blue as they have no melanin in the iris.

Question 9

How is MITF upregulated in melanocytes by MC1R-cAMP?

Answer 9

The MSH binds to MC1R which switches Ad cyclase to make cAMP which goes through this pathway to switch off MITF.
The MITF gene is switched on making proteins and various pigment cells including the receptor called MC1R.
Positive feedback works as more MC1R is made the more cAMP and MITF and MITF forms more MC1R.
*This also work when the ligand MSH is not present as once MC1R is present, it has some basal activity of its own and so some cAMP and MITF continue to be made.
*Another example of melanocyte differentiation:
A receptor calle KIT has a ligand called SCFF found in skin. This switches another pathway. This feedback can only work as long as SCF is around as KIT has no basal activity.

Question 10

What are the key transcriptional regulators needed for muscle differentiation?

Answer 10

-Myogenic factors
-Master gene regulators in skeletal muscle
differentiation
-MYOD1 (MYOD), MYF5, MYOG and MRF4
-E proteins
-Widely expressed transcription factors
-Myogenic factors normally work as dimers with E
proteins
-ID1 family
-Inhibitor of differentiation 1
-A protein in myoblasts (muscle precursor cells) which can strongly bind to E proteins but not DNA.
*In normal skeletal muscle: A MYOD1 molecule and E-
protein bind onto promoter region on DNA which
helps activate transcription.
*In myoblasts expressing ID1: the ID1 binds strongly to
E-proteins and so it cannot bind to DN and so
transcription is not activated. Therefore ID1 inhibits
differentiation.

Question 11

LOOK AT NOTES TO SEE HOW THE MYOD FAMILY IN SKELETAL MUSCLE DIFFERENTIATION WORKS.

Answer 11

LOOK AT NOTES TO SEE HOW THE MYOD FAMILY IN SKELETAL MUSCLE DIFFERENTIATION WORKS.

Question 12

What is cell senescence?

Answer 12

Permanent cell growth arrest followed by extended cell proliferation.

Question 13

Why is cell senescence important?

Answer 13

• Major defence against cancer
• Strongly implicates in symptoms of ageing
• Understanding is still recent so there is still things we do know.

Question 14

Who discovered cell senescence?

Answer 14

Leonard Hayflick who found that cells have divided for a very long time and cells do not divide anymore
Cells have a finite lifespan and this is measured in population doublings. This is called the ‘Hayflick Limit’
Some cells are immortal and have a finite lifespan.

Question 15

What is a cell lifespan?

Answer 15

It is the total number of doublings that a cell goes through before senescence. This is measured from the time of explanation into cell culture.

Question 16

Why is cell culture lifespan only comparative?

Answer 16

As we cannot track the cell proliferation that occurs before explantation, however we can distinguish immortal cells from those that senesce like this.

Question 17

What are the molecule markers for senescent cells?

Answer 17

There are many but not all cells have them. The two best markers are:

• Lysosomal b-galactosidase: (All cells have some lysosomes, but senescent cells have very many)
• Protein p-16: a cell cycle inhibitor.

Question 18

What are the basic pathways of cell senescence?

Answer 18

Telomeres, p53, p16 and cancer.

Question 19

How are telomeres associated with cell senescence?

Answer 19

• Telomeres: 1000’s of repeats of a hexamer sequence (TTAGGG) at chromosome ends.
• 3’ ends of linear DNA can’t be replicated normally, because an RNA primer has to bind beyond the part to be replicated.
• So the enzyme telomerase is needed to maintain telomere length.

Question 20

How does telomerase work?

Answer 20

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. I.e. make DNA from RNA.
Telomeres has a molecule called TERC: telomerase RNA component.
The protein section is the catalytic subunit (yellow) aka TERT: telomerase reverse transcriptase.
This makes DNA at the 3’ end of DNA by copying from sequence on the RNA. It transcribes the hexamers (TTAGGG) from its own RNA sequence.
Telomerase activity is highest in germ cells (among normal cells), which thus have the longest telomeres.
NOTE:
In humans, most somatic cells express no TERT, so no telomerase activity, so telomeres shorten as cells divide.

Question 21

How is replicated senescence triggered?

Answer 21

It gets triggered in normal cells when telomeres get quite short.

Question 22

How are germline cells able to be immortal?

Answer 22

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

Question 23

How are cancer cells immortal?

Answer 23

Cancer cell lines in culture nearly all (~90%) express TERT, so they are immortal.
Cell senescence now known to form an important barrier to cancer.

Question 24

What are the established effector pathways of cell senescence (simplified): p53 and p16?

Answer 24

The telomere shortens and causes DNA damage signalling which switches on P53 (tumour suppressor protein) and growth inhibitor called p21. It is an inhibitor of CDKase – CDK1/2
Other factors trigger cell senescence e.g Radiation, oxidative stress, DNA damage which causes DNA damage signalling and it can take that route. Or it can switch on another tumour suppressor protein called p16. It is also a growth inhibitor of CDKase.

Question 25

What are the most common abnormalities found in cancer cells?

Answer 25

Advanced cancer cells have usually bypassed cell senescence. Some of the commonest abnormalities found in cancer cells are those leading to defective senescence & immortality:

• Expression of TERT
• p53 defects*
• p16 defects
• p53 has other anti-cancer functions too, not covered here.

Question 26

What pieces of evidence is accumulating that cell senescence is also behind many of the symptoms of normal ageing?

Answer 26

• Telomere length (measured in blood cells), variable, but on average falls with age. Typically very short in people aged >100.
• p16 and other senescence-associated proteins are expressed increasingly in ageing tissues.
• Telomere length at birth varies between people: genetically linked to age at death.
• Defective genes for telomerase subunits give syndromes with premature ageing and early death.
• p16 (CDKN2A) locus also genetically associated with human senile defects – cardiovascular disease, frailty, type II diabetes, neurodegeneration, cancer.

Question 27

What are stem cells?

Answer 27

A cell that is capable of both self-replication (division into more cells like itself), and differentiation into one or more kinds of specialized, functional cells.

Question 28

What are the different types of stem cells?

Answer 28

• Unipotent – can form only one functional cell type.
  (Latin, uni- = one)
• Pluripotent – can form several functional cell types. (Latin, pluri- = several.)
• Totipotent – can form ALL functional cell types including placenta. (Latin, toti- = all.)
  o The zygote is totipotent but is not normally
  considered to be a stem cell (it doesn’t divide to
  make more zygotes).
  o But cells of the inner cell mass of the early
  mammalian embryo can act as totipotent stem cells
  – called embryonic stem cells.

Question 29

What are embryonic stem cells?

Answer 29

• ES cells also express TERT/telomerase – they are naturally immortal too.
• They can be considered germ-line cells, since they can form all cell types including gametes.
• Totipotent, though sometimes also called pluripotent.

Question 30

What are somatic stem cells?

Answer 30

Somatic stem cells - Or postnatal stem cells – those that remain as a proliferative reservoir after birth, e.g. in gut, skin, bone marrow, prostate. (Also called adult stem cells.)

Question 31

Do human somatic stem cells have telomerase?

Answer 31

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 32

What are the examples of connections between cell senescence (including stem cell senescence) and ageing symptoms?

Answer 32

• Bone marrow: Older people show decreased
  immunity, increased bone marrow failure, decreased
  success rate as bone marrow donors. Reduced
  proliferative ability of marrow stem cells.
• Hair greying linked to decreased melanocyte stem
  cell maintenance in hair follicles (data from mice).
• Reduced healing ability of skin with age, increased
  risk of skin ulcers. Senescence in dermal fibroblasts.
• However epidermal stem cells have very little
  telomere shortening & remain able to divide
  throughout life.