Cellular Growth Regulation

Question 1

What are the two different types of cell growth?

Answer 1

“1. Hyperplasia:
- Increase in cell numbers.

2. Hypertrophy:
   - Increase in cell size.”

Question 2

How is cell growth regulated?

Answer 2

“This depends on on integration of intracellular and extracellular signals:

• Checks on Cellular Physiology: Growth and Inhibitory Factors.
• The level of Cell Adhesion to nearby cells or basal membrane.”

Question 3

List the main Cell Cycle Phases:

Answer 3

“G1: Growth Phase 1

S: Synthesis DNA Replication

G2: Growth Phase 2

M: Mitosis”

Question 4

How is the Cell Cycle regulated?

Answer 4

“There are Three Key Restriction Points:

• Involving specific protein kinases and phosphatases.
• Ensure the strict alternation of mitosis and DNA replication.”

Question 5

What is a Mitogen and what does it do?

Answer 5

“A substance that Initiates Mitosis:

• Stimulates proliferation.
• Maintains survival.”

Question 6

What factors control the Cell Cycle?

Answer 6

“1. Growth Factors

2. Interleukins
3. Cytokines”

Question 7

Where do these factors come from?

Answer 7

“There are Three Broad Classes:

1. Paracrine:
   Produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor.
2. Autocrine:
   Produced by a cell that also expresses the appropriate cell surface receptor.
3. Endocrine:
   Like conventional hormones, released systemically for distant effects.”

Question 8

Describe the progression of cell population growth over time:

Answer 8

“1. Initially there is growth in response to PDGF added.

2. Plateau phase as the amount of PDGF is removed.
3. PDGF is added again and the cell number increases exponentially.
4. This is until a Growth Inhibitor like TGF Beta is added and the cell number plateaus again.
5. Finally there is a death signal such as TNF Alpha and the cells undergo apoptosis so cell numbers fall again.”

Question 9

Describe the main phases of the cell cycle:

Answer 9

” 1. Interphase

• G1 Phase: Cell grows larger, copies organelles, makes molecular building blocks for later steps.
• S Phase: Cell synthesises a complete copy of DNA in its nucleus. Duplicates the centrosome.
• G2 Phase: Cell grows again making protein and organelles. Contents are reorganised.

2. The M phase/Mitosis:
   - Prophase
   - Prometaphase
   - Metaphase
   - Anaphase
   - Telophase
3. Cytokinesis:
   Cell split in two to form two new cells.”

Question 10

What cells are in the G0 Phase?

Answer 10

”- Cells may exit the G1 phase and enter a resting state called the G0 phase.

• These cells are not actively preparing to divide.
• They are just performing their functions.
• Quiescence is a permanent state for some cells.
• Some quiescent cells may re-start division if they get the right signals.”

Question 11

What is FACS?

Answer 11

“Fluorescence - Activated Cell Sorting (FACS):

• Specialized type of flow cytometry.
• Provides a method for sorting a heterogeneous mixture of biological cells.
• One cell at a time.
• Based upon the specific light scattering and fluorescent characteristics of each cell.”

Question 12

How can cellular DNA content be analysed using FACS?

Answer 12

“We can determine what fraction of the cells are dividing.
We can also determine the rate of cell division.

1. Rate of Cell Division is Low:
   - There will be a lot of cells in the G1 phase and not many in the S phase.
2. Rate of Cell Division is High:
   - There will be a lot more cells in the S phase. “

Question 13

Briefly describe DNA Replication:

Answer 13

“1. DNA is replicated semiconservatively.
Daughter cells inherit one parental and one new strand.

2. New DNA is synthesized in the 5’ to 3’ direction from deoxynucleotide triphosphate precursors.
   At a replication fork by a multienzyme complex.
3. Fidelity is determined by base pairing (A=T, G≡C).
   And presence of a proof reading enzyme in DNA polymerase.
4. Synthesis of the new DNA strand uses an RNA primer and occurs continuously on the leading strand.
   And discontinuously on the trailing strand.
   Giving rise to Okazaki Fragments, which are ligated together after removal of the RNA primer.”

Question 14

Describe the Phases of Mitosis:

Answer 14

“1. PROPHASE:

• Nucleus becomes less definite.
• Microtubular spindle apparatus assembles.
• Centrioles migrate to poles.

PROMETAPHASE:

• Nuclear membrane breaks down.
• Kinetochores attach to spindle in nuclear region.

2. METAPHASE:
   Chromosomes align in equatorial plane
3. ANAPHASE:
   Chromatids separate and migrate to opposite poles.
4. TELOPHASE
   - Daughter nuclei form.
5. CYTOKINESES:
   - Division of cytoplasm.
   - Chromosomes decondense.”

Question 15

What stages of the Cell Cycle do drugs act on?

Answer 15

“S PHASE
or
M PHASE”

Question 16

Which drugs are S - Phase Active?

Answer 16

“5-Fluorouracil:

• Analogue of Thymidine.
• Blocks Thymidylate Synthase enzyme which makes the nucleotide thymine

Bromodeoxyuridine (BRDU):

• Nucleotide analogue that may be incorporated into DNA.
• BRDU will be detected by antibodies that are added.
• This will help identify cells that have passed through the S - Phase.
• In this way you can stain for DNA that is being synthesised/replicated.
• Provides a way of visualizing cells that have been actively dividing DNA.
• Useful for monitoring cell growth.

Tamoxifen:

• Antagonist of the Oestrogen Receptor.
• Blocks entry into S Phase.
• Can be used for Cancer Treatment.”

Question 17

What do M - Phase drugs act on?

Answer 17

All M-Phase active drugs affect microtubules.

Question 18

Which drugs are M - Phase Active?

Answer 18

“Colchicine:

• Stabilizes free tubulin.
• Preventing microtubule polymerization.
• Used in karyotype analysis.

Vinca Alkaloid:
- Similar action to Colchicine.

Paclitaxel/Taxol:

• Stabilizes microtubules.
• Preventing microtubule de-polymerization.
• This inhibits the movement of chromosomes to separate poles.”

Question 19

What is the main use of M - Phase drugs?

Answer 19

“All of these drugs can be used in treatment of cancer.

By stopping the cancer cells from dividing.”

Question 20

Describe the 4 main Cell Cycle Restriction/Check Points?

Answer 20

“1. During the G1 Phase:

• Cells responsive to Growth Factors.
• Main site of control for Cell Growth.

2. Before the S Phase:
   - DNA not damaged.
   - Cell size is large enough.
   - If there is sufficient metabolite/nutrient stores.
3. Before the M Phase:
   - If the DNA is completely replicated,
   - To ensure that the DNA is not damaged.
4. During the M Phase:
   - Ensure that the chromosomes are aligned on the spindle.”

Question 21

What controls the progression of the Cell Cycle?

Answer 21

“Cyclin Dependent Kinases:

• The Kinases are useless unless Cyclin is bound.
• All Kinases phosphorylate proteins to trigger movement through the cell cycle.
• Cyclin presence varies depending on the stage of the cell cycle.”

Question 22

How is Cyclin - CDK activity regulated?

Answer 22

“1. Synthesis controlled through the level of gene expression.
Destruction by the Proteasome.

2. Post Translational modification by Phosphorylation.
   Depending on modification site may result in activation, inhibition or destruction.
3. Dephosphorylation by Phosphatases.
   Of the substrates that were initially Phosphorylated by the Active Cyclin-CDK complex
4. Negative regulation through the binding of Cyclin-Dependent Kinase Inhibitors”

Question 23

Describe the normal function of the the Retinoblastoma Protein (Rb)?

Answer 23

“Tumor Suppressor Protein:

• Key substrate of the G1 and G1/S Cyclin-Dependent Kinases.
• Unphosphorylated Rb binds E2F preventing its stimulation of S-phase protein expression
• To prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide.
• Recruiter of several chromatin remodeling enzymes such as methylases and acetylases.”

Question 24

How is the Retinoblastoma Protein (Rb) inactivated?

Answer 24

“1. Cyclin D binding to CDK4 and Cyclin E binding to CDK2.

2. Phosphorylate Rb so it is unable to bind to E2F.
3. This enables cell cycle progression.”

Question 25

What does free E2F do in the cell?

Answer 25

“4. The released E2F stimulates S-phase proteins e.g. DNA polymerase, thymidine kinase, PCNA etc.

5. As a result of this DNA replication starts.
6. It also stimulates the expression of further Cyclin E.”

Question 26

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

Answer 26

“1. CDK Inhibitory Protein/ Kinase Inhibitory Protein Family.
2. Inhibitor of Kinase 4 Family.”

Question 27

CDK Inhibitory Protein/ Kinase Inhibitory Protein Family:

Answer 27

”- Expression of members of this family stimulated weakly by TGF.

• And strongly by DNA damage (involving TP53).
• Inhibit all other CDK-cyclin complexes (late G1, G2 and M).
• Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs.”

Question 28

Inhibitor of Kinase 4 Family:

Answer 28

”- Expression stimulated by TGF.

- Specifically inhibit G1 CDKs.”

Question 29

How do growth factors induce cyclin expression?

Answer 29

“1. Growth Factor acts on Growth Factor Receptor on the cell.

2. Stimulates the release of signal transducers in the cytosol.
3. Activates a cascade of serine kinases within the cell.
4. Which lead to waves of transcription factor activation and specific protein synthesis occurs.”

Question 30

Describe how the level of protein Phosphorylation varies?

Answer 30

“1. G1 CDKs are activated in response to environmental signals.
G1 CDKs Hypophosphorylate.

2. Late CDKs by preceding kinase activities.
   Late CDKs Hyperphosphorylate.
3. Hyperphosphorylated Rb is dephosphorylated by Protein Phosphatase 1. “

Question 31

Outline the sequential activities in the Cell Cycle?

Answer 31

“1. Growth Factor Signalling activates early gene expression (transcription factors: FOS, JUN, MYC).
Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors).

2. E2F sequestered by binding to unphosphorylated retinoblastoma protein (Rb).
   G1 cyclin-CDK complexes hypophosphorylate Rb.
   Then G1/S cyclin-CDK complexes hyperphosphorylate Rb releasing E2F.
3. E2F stimulates expression of more Cyclin E and S-phase proteins.
   (e. g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)
4. CDKs can be synthesised but cannot act until post transcriptionally activated at various stages.
   So the S-phase cyclin-CDK and G2/M cyclin-CDK complexes will build up in inactive forms.
5. These switches are activated by post-translational modification or removal of inhibitors,
   Driving the cell through S-phase and mitosis.”

Question 32

What can DNA damage detected at checkpoints trigger?

Answer 32

“Cell Cycle Arrest:

• Stop the Cell Cycle.
• Cyclin Dependent Kinase Inhibitors, CHEK2.

DNA Repair:

• Attempt DNA Repair.
• Nucleotide or Base Excision Enzymes.
• Mismatch Repair.

Apoptosis:

• When DNA damage is so extensive repair won’t be possible and then apoptosis will need to occur.
• BCL2 family, Caspases.”

Question 33

How does TP53 respond to DNA damage?

Answer 33

“1. Tumour Protein 53 is rapidly degraded in most cells by proteasomes when not needed.

2. DNA damage occurs causing this TP53 to be stimulated.
   TP53 is phosphorylated by a kinase, this is kinase activation.
   The phosphorylation of TP53 stabilizes it.
3. TP53 arrests the cell cycle.
   And tries to repair DNA.
4. DNA repair is usually very efficient.
5. TP53 only triggers apoptosis when DNA damage is really extensive.
   And repair of the DNA is not possible.”

Question 34

Summary of key steps:

Answer 34

“1. Growth Factors binding to receptors induce gene expression.
2. G1 and G1/S Cyclin-CDK complexes phosphorylate Rb in the absence of inhibition by CKIs.
Expression of these is regulated by TP53 or TGF Beta.
3. E2F released, stimulating expression of genes required for S-phase.
4. Cell replicates DNA due to the expression of S-phase Cyclin-CDK complexes.
5. If all the DNA is replicated, G2/M Cyclin-CDK complexes cause the cell to enter mitosis.
6. If chromosomes aligned on spindle, exit from mitosis is triggered.
7. If process fails, TP53 initiates apoptosis.”