Cellular pathology: Cellular Growth Regulation

Question 1

What are the 2 main types of cell growth?

Answer 1

• Growth of a population of cells
• Growth at the cellular level (cell cycle)

Question 2

There are 2 ways in which cells within a population can grow, what are these 2 ways?

Answer 2

• Increase in the number of cells (Hyperplasia)
• Increase in cell size (Hypertrophy)

Question 3

For growth of cells at cellular level there are also 2 ways in which they can grow. What are they?

Answer 3

• Increase in size
• Increase in cell division

Question 4

What is apoptosis?

Answer 4

• A coordinated program of cell dismantling ending in phagocytosis
• Can be thought of as a “lack of cell growth”

Question 5

What proteins are able to affect cell growth?

Answer 5

• Growth factors
• Cytokines
• Interleukins

Question 6

What mechanisms do proteins such as cytokines use to affect cell growth? For each mechanism give an example of a protein that uses it to affect cell growth.

Answer 6

They are able to:

• Stimulate proliferation and maintain cell survival - e.g. EGF (Epidermal growth factor)
• Stimulate differentiation and inhibit proliferation - e.g.TGFβ (Transforming growth factor β)
• Induce apoptosis - e.g. TNFα (Tumour necrosis factor α)

Question 7

What is the general term used to describe proteins that are able to stimulate cell proliferation?

Answer 7

Mitogens

Question 8

What are the 3 broad classes of signalling that proteins such as cytokines and interleukins use to affect cell growth?

Answer 8

• Paracrine: Protein produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor
• Autocrine: Protein produced by a cell that also expresses the appropriate cell surface receptor
• Endocrine: Protein released systemically to produce effects on distant cells

Question 9

Describe what happens during each phase of the cell cycle

Answer 9

• M-phase (Mitosis) - Mitotic cell division occurs leading to production of two identical daughter cells from one parental cell
• G₁ (Gap phase 1) - Cell growth, mRNA and protein synthesis
• S-phase (Synthesis) - DNA replication occurs
• G₂ (Gap phase 2) - Cell growth, protein synthesis

Question 10

What is the G₀ phase of the cell cycle?

Answer 10

It occurs when a daughter cell produced during mitosis “leaves” the cell cycle and becomes arrested

Question 11

What are cells within the G₀ phase of the cell cycle known as?

Answer 11

Quiescent cells

Question 12

What can happen to quiescent cells once they leave the cell cycle?

Answer 12

• They can receive a mitogen which will cause them to re-enter the cell cycle
• They can also receive proteins that induce differentiation, e.g.TGFβ, which will result in these cells being in a state of terminal differentiation (cannot re-enter cell cycle)
• Terminally differentitated cells will eventually begin to shed

Question 13

State the number of sets of chromosomes (Ploidy) at each stage of the cell cycle

Answer 13

• G1 phase: 2N (2 sets of chromosomes)
• S phase: 2N (2 sets of chromosomes)
• G2 phase: 4N (4 sets of chromosomes)
• M phase: 4N (4 sets of chromosomes)

Question 14

Why do cells in G2 and M phase have 4 sets of chromosomes?

Answer 14

Because both occur after S phase and in S phase each one of the 2 sets of chromosomes are replicated resulting in the production of 4 set of chromosomes.

Question 15

Give a brief overview of the process of DNA replication

Answer 15

• DNA is replicated semiconservatively (daughter cells inherit one parental and one new strand)
• Parental DNA unwound to form replication fork
• New DNA strands synthesised in the 5’ to 3’ direction via the replication machinery
• Synthesis of leading strand is continuous, requires a single primer, as DNA polymerase working in 5’ to 3’ direction means it moves towards replication fork
• Synthesis of lagging strand discontinous, requires multiple primers, as DNA polymerase working in 5’ to 3’ direction means it moves away from replication fork
• Synthesis of lagging strand gives rise to Okazaki fragments which are ligated together after removal of the RNA primer

Question 16

Describe what happens during each of the stages of mitosis

Answer 16

• Prophase - Chromosomes condense, centrosomes move to opposite poles, mitotic spindle begins to form
• Prometaphase - Nuclear membrane breaks down, Kinetochores attach to mitotic spindle
• Metaphase - Centrosomes at opposite poles, chromosomes line up at the equator and attach to mitotic spindle
• Anaphase - Chromatids are seperated and migrate to opposite poles
• Telophase - Chromosomes decondense, nuclear envelope reforms thus froming the daughter nuclei

Question 17

What process occurs after the telophase of mitosis?

Answer 17

• Cytokinesis - Contractile ring constricts causing the cell to split into two daughter cells

Question 18

Give some examples of drugs that are active during the S-phase of the cell cycle. For each example explain the effect that they have on the S-phase

Answer 18

• 5-Fluorouracil - An analogue of thymidine that blocks the thymidylate synthesis. Without Thymidylate DNA unable to be replicated completely so cell will be stuck in S phase
• Bromodeoxyuridine (BrdU) - Another analogue that can be incorporated into DNA and be detected using fluorescence to identify which cells have gone through the S-phase of the cell cycle

Question 19

When you incorporate Tamoxifen into breast cancer cells that have been stained using Bromodeoxyuridine, the no. of breast cancer cells that go through the S-phase of the cell cycle decreases compared to when Tamoxifen hasn’t been incorporated. Why is this?

Answer 19

• Tamoxifen is an Oestrogen antagonist and competes with Oestrogen to bind to the Oestrogen receptor
• When tamoxifen binds to oestrogen receptor the tamoxifen-oestrogen receptor complex formed is unable to induce expression of genes that the breast cancer cells need to survive
• This means the no. of breast cancer cells will decrease which is why less breast cancer cells will have gone through the S-phase of the cell cycle

Question 20

Give some examples of drugs that are active during the M-phase of the cell cycle. For each example explain the effect that they have on the M-phase

Answer 20

• Colchicine - Stabalises free tubulin which prevents microtubule polymerisation. Without microtubules mitotic spindle is unable to form so chromosomes are unable to be seperated during mitosis
• Vinca alkaloids - Also stabalises free tubulin
• Paclitaxel - Stablises microtubules preventing de-polymerisation back into tubulin

Question 21

What are the different checkpoints throughout the cell cycle?

Answer 21

• G₁ checkpoint: Cell checks for DNA damage, if cell size is correct and whether it has sufficient metabolite nutrient stores to efficiently go through cell cycle
• G₂ checkpoint: Cell checks for DNA damage and if the DNA is completely replicated
• M-phase checkpoint: Cell checks that chromosomes are aligned properly on mitotic spindle

Question 22

What part of the cell cycle is the main site for cell growth? Why is this?

Answer 22

• G₁
• This is because it is the only part of the cell cycle in which cells can respond to growth factors

Question 23

What proteins are able to regulate the cell cycle?

Answer 23

• Cyclin dependent kinase (CDK) - Enzymes that phosphorylate the target proteins
• Cyclin - Regulators of CDK
• CDK needs to form a complex with cyclin in order to become active

Question 24

What are some of the ways in which CDK-cyclin activity can be regulated?

Answer 24

• Promote gene expression of Cyclins/CDKs or promote destruction via the proteasome
• Increase level of phosphorylation
• Increase level of de-phosphorylation
• Bind cyclin-dependent kinase inhibitors

Question 25

Explain how Cells are able to advance into the S-phase of the cell cycle once they reach the G₁ checkpoint

Answer 25

* Unphosphorylated retinoblastoma (RB) binds to E2F which prevents E2F from inducing expression of genes required for G1/S-phase progeression * Once cell realises it's adequate to progress into S-phase it will produce Cyclin-D * Cylin-D binds to CDK4 causing it to activate * Activated CDK4 phosphorylates retinoblastoma which causes it to dissociate from E2F * This allows E2F to induce expression of particular genes needed for the S-phase

Question 26

What are some of the S-Phase proteins that are expressed as a result of E2F?

Answer 26

* Cyclin E * DNA polymerase * Thymidine kinase * PCNA

Question 27

What are the 2 families of Cyclin-dependent kinase inhibitors?

Answer 27

* **CDK inhibitory protein/kinase inhibitory protein family (CIP/KIP) or CDKN1** - inhibit all other CDK complexes (late G₁, G₂ and M) * Gradually released by G₁ CDKs allowing for activation of later CDKs * **Inhibitor of kinase 4 family (INK4) or CDKN2** - specifically inhibit G₁ CDKs, e.g. CDK4

Question 28

What proteins are able to induce the expresion of each family of Cyclin-dependent kinase inhibitors?

Answer 28

* CIP/KIP or CDKN1 - Expression weakly stimulated by TGFβ and strongly stimulated by DNA damage (inloves TP53) * INK4 or CDKN2 - Expression stimulated by TGFβ

Question 29

Briefly explain how growth factors induce cyclin expression

Answer 29

* Growth factor will bind to growth factor receptors on cell surface * This causes the activation of a series of intracellular signal transducers * These will eventually lead to waves of transcription factor activation within the nucleus which results in the transcription of specific cyclin genes * mRNA produced from cyclin gene expression will be transcribed to produce the specific cyclin protein

Question 30

Describe the sequence of events within the cell cycle that are triggered by growth factors

Answer 30

* Growth factor signalling activates expression of early genes (FOS, JUN and MYC) * Early genes are transcription factors and induce transcription of delayed genes (Cyclin D, CDK4/6 and E2F transcription factors) * Retinoblastoma (RB) bind to E2F transcription factors * Cyclin D-CDK4 complexes form and as a result begin to hyperphosphorylate retinoblastoma and release E2F * Because E2F is released it induces the transcription of a third wave of genes, E2F responsive genes, e.g. Cyclin E and S-Phase proteins

Question 31

What sequence of events occur if DNA damage is detected at a specific checkpoint in the cell cycle?

Answer 31

* Cell cyle is stopped - done be cyclin -dpendent kinase inhibitors * An attempt is made to repair DNA damage - done be nucleotide or base excision enzymes * If the DNA damage can't be repaired programmed cell death occurs - done by bcl-2 family or caspases

Question 32

What gene is activated in response to DNA damage?

Answer 32

p53

Question 33

How is the p53 gene activated in response to DNA damage and what occurs as a result of its activation?

Answer 33

* Mutation causes DNA damage * DNA damage is detected by kinases which are activated in response and go on to phosphorylate, and therefore activate, p53 * Phosphorylated p53 induces the expression of CDKN1s which causes cell cycle arrest * Phosphorylated p53 also induces expression of DNA repair genes which will produce proteins that will try and repair DNA * If DNA repair not possible then p53 will induce expression of genes involved in apoptosis

Question 34

What happens to p53 if it isn't phosphorylated?

Answer 34

p53 is destroyed by the proteosome