Mechanisms of Disease I: Cell growth and differentiation

Question 1

Name the 3 groups of diseases related to cell growth and differentiation

Answer 1

Developmental conditions
Can be related to cell growth or differentiation (or both)
E.g. Neural tube defects like spina bifida
Neoplasia (and metaplasia)
E.g. cancer, tumours
Others, e.g. Cardiac hypertrophy

Question 2

Name the 2 forms of cell growth

Answer 2

hyperplasia (increase in cell number)
hypertrophy (increase in cell size). hyperplasia is the most common!

cell death balances this

Question 3

How does hypertrophy work and is this more or less rare?

Answer 3

• hypertrophy is caused by an increase in the synthesis of macromolecules (proteins, membrane etc.)
• driven by increased protein synthesis
• can be pathological, like in the heart

Question 4

Explain what is meant by co-incidence detectors

Answer 4

Intracellular and extracellular signals both converge on promoters to either affect the cell cycle in cell growth or to affect the differentiation of the cell - these promoters are known as co-incidence detectors. When the right signals bind, the promoter has a binary decision to make or not make the transcript as well as the amount to produce

Question 5

Name the 3 broad classes of extracellular signalling

Answer 5

Paracrine
Endocrine
Autocrine

Question 6

Explain how extracellular signals may influence cell growth and differentiation

Answer 6

may be proteins that stimulate proliferation and promote survival - so are mitogens (growth factors and interleukins)
induce differentiation and inhibit proliferation (like TGFbeta) some ligands (like WnT ligands can do either)
induce apoptosis (like TNFalpha and other TNFs)

Question 7

Explain what interphase is and what happens to the cell in this phase

Answer 7

This it all of the phases that are not M (mitosis) - so is G1, S and G2

the cells grow in size as macromolecules are synthesised continuously

Question 8

At what stage does DNA replication occur?

Answer 8

S (between the 2 gap stages of G1 and G2)

so G1 has 2N
and G2 has 4N

Question 8

What are quiescent cells?

Answer 8

When a cell leaves the cell cycle to be in G0 - these cells can be here indefinitely but can re-join in G1 or will begin differentiation

Question 9

Describe the expected trace of the FACS in a more proliferative cell type - so essentially, how does the % of cells in each phase of the cell cycle change?

Answer 9

there is more S phase and less in G1! G2 is basically the same

Question 10

What property allows us to measure how many cells we have in the cell cycle? Name the process we use to do this

Answer 10

The fact that G1 is 2N, S is between 2 and 4N and G2 is 4N

so we can use flow cytometry (FACS) to measure the DNA content with a DNA stain and its strength to measure the amount of DNA

Question 11

Describe how we can use fluorescence microscopy in mitosis

Answer 11

Yellow is when there is red (gamma-tubulin) and green (CHEK2) in the same place

these are the centrioles

Question 12

Describe fully the processes in mitosis

Answer 12

• Prophase
• Prometaphase
• Metaphase
• Anaphase
• Telophase
• Cytokinesis

Question 13

Name all of the cell cycle checkpoints (3)

Answer 13

Restriction point (end of G1)
G2 M-phase checkpoint (end of G2)
In the middle of mitosis

Question 14

Which is the most important checkpoint, what does it check?

Answer 14

The restriction point - this is at the end of G1, and it regulates the entry into the cell cycle before duplication of DNA, including any G0 cells returning into the cell cycle

checks for no DNA damage but also other stuff such as size and energy stores

Question 15

Describe the second checkpoint

Answer 15

G2 M-phase checkpoint (end of G2) this checks that the DNA is completely replicated and that the DNA is not damaged. This is just before the entry into mitosis.

Question 16

Describe the third checkpoint

Answer 16

This is in the middle of mitosis and checks that the chromosomes are aligned on the mitotic spindle

Question 17

What key enzyme types allow for cell cycle checkpoints?

Answer 17

Phosphatases and kinases

Question 18

Which cell cycle phase do the growth factors act on?

Answer 18

G1 is when the cell is responsive to growth factors so before the restriction point

Question 19

Describe what CDK is

Answer 19

Cyclin-dependant kinases

humans have 10 genes encoding them
cyclin proteins (over 20 genes) are regulatory elements

Question 20

So explain how growth factors affect the cell cycle (mechanism)

Answer 20

Growth factors regulate the expression of cyclin (over 20 genes for it) which regulates (is a regulatory subunit) to CDK

regulates expression by synthesis and degradation by proteasome and also its post translational modification (phosphorylation of cyclin can activate it)

Question 21

What is the function of CDKs

Answer 21

phosphorylate specific substrate proteins to cause progression through the cell cycle

Question 22

What are CDKIs?

Answer 22

Cyclin dependant kinase inhibitors - are another way to regulate (inhibit) cyclin-CDK expression

Question 23

Describe what retinoblastoma protein is (RB)

Answer 23

RB is a key substrate of CDKs that operate in G1 and G1/S

Question 24

What is RB normally bound to and what does this cause?

Answer 24

RB is bound to E2F (a transcription factor) this prevents E2F from promoting transcription of genes that are needed in S phase when bound to E2F, RB is unphosphorylated

Question 25

So explain what CDKs do to RB bound to this substrate and the significance of this

Answer 25

The CDKs will phosphorylate RB meaning that it dissociates from E2F so that E2F can stimulate the transcription of key genes in S phase E2F also stimulates transcription of more cyclin E and cyclin D

Question 26

Name 2 CDKs in this process (that phosphorylate Rb)

Answer 26

Cyclin D-CDK4 Cyclin E-CDK2

Question 27

Explain how this (CDKs and Rb and E2F) is a positive feedback loop

Answer 27

As when RB dissociates, E2F increases the transcription of cyclin E (as well as the proteins needed for S phase) cyclin E can allow CDK2 to be active and cause dissociation of RB from E2F to allow synthesis of S phase proteins as well as more cyclin E and so on ...

Question 28

Which phosphatase dephosphorylates RB at the end of the cycle (mitosis)?

Answer 28

PP1

Question 29

Fully explain the use of cyclin D

Answer 29

cyclin D has its transcription increased by TFs that are increased in transcription themselves by mitogens. So increased cyclin D means more Cyclin D-CDK4/6 complexes which phosphorylate RB a bit - enough to have some activity of E2F which will then increase cyclin E expression as well as expression of E2F responsive genes needed in S phase!!

Question 30

What would happen if there is DNA damage detected at the first or second checkpoint (3)?

Answer 30

stop of cell cycle (driven by expression of CDKI (cyclin dependant kinase inhibitors)) attempt DNA repair with enzymes or mismatch repair programmed cell death

Question 31

Describe what TP53 is

Answer 31

Tumour protein 53 (AKA P53) is the ‘guardian of the genome’ as it is an important tumour suppressor gene

Question 32

How does TP53 function in a non-damaged vs damaged DNA cell?

Answer 32

If the cell is NOT damaged DNA, then TP53 will be constantly being degraded by proteasome so that the levels are very very low. If there is DNA damage, some protein kinases will be activated that phosphorylate Tp53 so that it can no longer be destroyed by a proteasome so that it accumulates

Question 33

Name the effects of TP53

Answer 33

increase expression of CDKIs (for cell cycle arrest) activate DNA repair activate apoptosis if damage is too much

Question 34

Describe the role of TP53 in cancer

Answer 34

Loss of function mutations are some of the most frequently observed mutations in cancer so there is a loss of cell cycle arrest, loss of apoptosis and loss of DNA repair

Question 35

With the loss of TP53 function there is also a loss of DNA repair in the tumour, why is this actually beneficial to the growth of the tumour?

Answer 35

the loss of DNA repair function causes a more heterogeneous tumour due to more mutations in different cells which increases its chances of surviving by adapting

Question 36

How do traditional (S-phase) chemotherapeutic drugs work such as cisplatin?

Answer 36

act on the cell cycle to stop proliferation and induce apoptosis S-phase drugs cause DNA damage (so 5-fluorouracil prevents synthesis of thymidine) and cisplatin binds to DNA causing damage and blocks repair

Question 37

How do M-phase chemotherapeutic drugs work such as Vinca alkaloids?

Answer 37

Targets the mitotic spindle - stabilise free tubulin so that microtubules to do polymerise and form so the cells will be stuck in mitosis Taxol drugs are another class of M-phase chemotherapeutic drugs, they stabilise microtubules which prevents de-polymerisation which prevents mitosis and so they arrest in M-phase