Cell Growth and Diff

Question 1

Hyperplasia

Answer 1

increase in cell numbers

Question 2

Hypertrophy

Answer 2

Increase in cell size

Question 3

What is a good example of hypertrophy in the human body?

Answer 3

postnatally the heart has grown by hypertrophy alone

• heart contains lots of actin and contractile filaments
• exercise increases demand –> larger heart allows increased pumping of blood

Question 4

How is it determined what type of growth a . cell undergoes?

Answer 4

dependant on 4 main factors

1. integration of intra and extracellular signals
2. checks on cellular physiology
3. growth and inhibitory factors
4. cell adhesion; to nearby cells or basal membrane

Question 5

what is the most common type of growth?

Answer 5

hyperplasia in tissue & organs

Question 6

the term ‘ cell growth’ refers to…

Answer 6

growth in terms of size but also cell division

Question 7

name all the phases of the cell cycle, and the no. of checkpoints (also what is the purpose of the checkpoints)

Answer 7

G1, S, G2 and M phase
3 checkpoints
- progression of cell growth control also known as restriction points.

Question 8

How is loss of cells coordinated and why, give examples of real life scenarios?

Answer 8

programmed cell death
(apoptosis)
- requires the expenditure of ATP
- as the cell is aware is becoming deleterious for the whole organism.
- separation of digits, involution, immune and nervous sys. development

Question 9

How is Apoptosis distinct from necrosis?

Answer 9

phagocytosis occurs in apoptosis to end cell dismantling.

Question 10

What triggers apoptosis?

Answer 10

DNA damage and viral infection

Question 11

What are mitogens?
what are the different types of mitogens?
A common misconception about them?
Give an example of a proliferation inhibiting mitogen?

Answer 11

proteins that stimulate proliferation and maintain survival are known as mitogens.

usually named after originally identified target e.g. EGF, FGF, Interleukins (IL2 & IL4), NGF
but see also PDGF (platelet-derived GF) and IGF1 (Insulin-like GF – the main effector of pituitary growth hormone)
- stimulate differentiation and inhibit proliferation e.g. TGFbeta
- induce apoptosis e.g. TNFα and other members of the TNF family (tumor necrosis factor)

Question 12

Where do these mitogens come from?

Answer 12

Three broad classes:

Paracrine: produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor

Autocrine: produced by a cell that also expresses the appropriate cell surface receptor

Endocrine: like conventional hormones, released systemically (into BV –> acts on distant target organ) for distant effects

Question 13

Explain the cell population growth graph?

- give an example of an application in a real life scenario

Answer 13

Log2 cell numbers
Plot number of days observing cell population.
pDGF -> increases cell division cell numbers

Stop receiving pDGF -> cells wont proliferate -> same cell number over time -> cell not growing

Again PDGF -> increase in cell numbers
TGFB -> growth inhibitor -> population starts growing
Same cell number over days-> no increase in cell division

TNFa -> decrease in number of cells after exposure to this death signal

Lab; working with numerous cells -> numerous signals -> sometimes a way to follow the actual the number of cells over time when they are exposed to diff proteins or diff. number of proteins.

Question 14

what factor does the number of chromsomes in the cell cycle change by and when?

Answer 14

G1 – 2N
2 x number of chromosmes
After synthesis duplication –> 4n - twice as much DNA content.

Question 15

At what point in the cell cycle does the fate of a cell change and if it does then what happens?
give a relevant example

Answer 15

After mitosis -> production of 2 daughter cells
Fate; one re-enters cycle and one withdraws and becomes arrested -> called quiescent cell -> G0 phase of cell cycle.
- Tgf beta -> promotes terminal differentiation of cells

Gut cells > epithelial cells in gut shed > undergo cell death > cells will be replenished by having quiescent cells that will be differentiating
So after generating gut cells > some will start cycling to replenish these kind of gut cells. > therefore produce more gut cells to produce more tissue. In response to shredding and apoptosis.

Question 16

Name each phase of cell cycle and what happens in each one?

What is interphase?

Answer 16

Interphase; G1, S and G2, Cells grow in size
- Synthesis and accumulation of macromolecules proteins enzymes lipids that are needed for having the right cell size and for actually going through the cell cycle

Synthesis phase > DNA replicated into 2 copies and incorporation of thymidine
G1 > cell after division will start growing in cell size
G2 > in between S & M phase
After mitosis > production of 2 daughter cells

Question 17

How does the fluorescence activated cell sorter work? what does it tell us?
Any applications?

Answer 17

Use a DNA stain that is actually fluorescent
in machine we have a tube; cell that’s coming through the tube at a time
The laser will measure the fluorescence of that tube.

S peak can be used to analyse tells you about the number of cells in the S phase

two different profiles provided;
cells dividing rapidly + cells dividing slowly

Useful tool to analyse cell population
In lab > use cells to do first profile without GF and second with GF
Tell GF is increasing or decreasing cell growth of that population

Question 18

Summarise DNA replication, what is required, what is fidelity and why is DNA synthesised from 5’ to 3’ ?

Answer 18

DNA is replicated semiconservatively (daughter cells inherit one parental and one new strand)

dsDNA x1 -> ssDNA x2

New DNA is synthesized in the 5’ to 3’ direction from deoxynucleotide triphosphate precursors at a replication fork by a multienzyme complex (a replication machine)

Fidelity is determined by base pairing (A=T, G≡C) and presence of a proof reading enzyme in DNA polymerase

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)

Fidelity of a DNA polymerase refers to its ability to accurately replicate a template.

Question 19

What are the main stages of mitosis? ( 4 main stages)

what colour stains are used mainly to show these stages?

What is the difference in visibility in comparison to interphase?

what is the significance of γ-tubulin in mitosis?

Answer 19

Blue - DNA
DNA blue, γ-tubulin red, CHEK2 green - used to contrast

Interphase all blue throughout nucleus as DNA not in chromosomes

Gamma tubulin – protein that forms part of the centrosome

• Centrosomes being the structure which will start accumulating microtubules
• Microtubules will separate the chromosomes into opposite poles of the cell during cell division

Prophase (1)
Nucleus becomes less definite
Microtubular spindle apparatus assembles
Centrioles (yellow) migrate to poles

Prometaphase
Nuclear membrane breaks down
Kinetochores attach to spindle in nuclear region

Metaphase (2)
Chromosomes (blue) align in equatorial plane

Anaphase (3)
Chromatids separate and migrate to opposite poles

Telophase (4)
Daughter nuclei form

Cytokinesis
Division of cytoplasm
Chromosomes decondense

Question 20

Action of 5-Fluorouracil, Bromodeoxyuridine, Colchicine, Vinca alkaloids, Paclitaxel?
what are they all used for?

Answer 20

5FU - analogue of thymidine blocks thymidylate synthesis
inhibits the production of thymidine by cells
If cells don’t produce thymidine then can’t replicate DNA into 2 copies
Cell is arrested in the S phase of the cell cycle.
Essentially preventing DNA duplication

BDU - analogue of nucleotides
Incorporated into DNA by DNA polymerase I
Antibodies can acc recognise this molecule
Can do staining and identify what cells passing through the S phase

Colchicine (stabilizes free tubulin, preventing microtubule polymerization and arresting cells in mitosis – used in karyotype analysis)

Vinca alkaloids (similar action to colchicine)
Both drugs prevent tubulin form forming polymers  cannot form microtubules  required to separate the chromosomes also required for mitosis
Cells which can’t form microtubules arrest in the M phase as they cannot divide

Paclitaxel (Taxol, stabilizes microtubules, preventing de-polymerization)  into single tubulin proteins
Not destabilize  cells can’t physically separate after the M phase

Use the staining to test drugs and cells
Tamoxifen – antagonist of oestrogen
Oestrogen required for cells to grow
Treat the cells with tamoxifen then do BrdU staining
After treating the breast cancer cells with tamoxifen – the number of cells that are stained and are acc dividing are quite small.

This method of staining shows that whether a compound is promoting cell division or whether a compound halts cells proliferation

5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen are used in treatment of cancer
Essentially used to prevent cell division and therefore the growth of tumors.

Question 21

what are the different checkpoints used in the cell cycle?

what is significant about G1?

Answer 21

Before M - DNA completely replicated,
DNA not damaged

in M - chromosomes aligned on spindle + two copies on chromosomes on each side of spindle

Before S - Restriction point:
DNA not damaged, Cell size,
Enough metabolite/nutrient stores, macromolecules to allow efficient cell cycle

G1 phase  under control of growth factors of extracellular signals is the g1 phase
Phase where cell is responsive to GF mentioned before  therefore main site of control for cell growth

Sg2 and M phase are refractory to control from Gf or cell signals

Question 22

Role of CDK and action?

Answer 22

• controls cell cycle progression
• CDK to be active need to complex with the cyclin
  Then you have functional CDK which can then go on and phosphorylate substrates
  Binds to recruiter substrate protein and then the protein will be phosphorylated

Question 23

How is CDK activity regulated?

Answer 23

• mainly at the level of gene expression

Cyclical synthesis (gene expression) and destruction (by proteasome).

Post translational modification by phosphorylation – depending on modification site may result in activation, inhibition or destruction

Dephosphorylation – opposite molecular events in contrast to phosphorylation

Binding of cyclin-dependent kinase inhibitors – can also regulate the activity of these complexes by the expression of cyclin dependant kinase inhibitors

Mainly we can regulate the expression of CDKs and cyclins at the level of gene expression
Therefore we can make more RNA and protein or we can actually inhibit the expression of these genes at the level of transcription
Regulate the protein by targeting for destruction with the amount of proteasome
Increase the synthesis via translation
We can regulate how much protein we have around by promoting the degradation of this CDK
Play with the phosphorylation and dephosphorylation of proteins with kinases and phosphatases and then that will actually target these complexes either for activation or for inhibition

Question 24

CDK v/s Cyclin gene types?

Answer 24

CDK
Catalytic subunit
10 genes - diff types

Cyclin
Regulatory subunit
> 20 genes/ diff types

Question 25

Role of Retinoblastoma, what it is?, which parts of the cycle it acts on and what it does?

Answer 25

RB – retinoblastoma – tumor suppressor

The retinoblastoma protein is a key substrate ofentry into G1 and G1/S cyclin-dependent kinases

The retinoblastoma protein is a key substrate ofentry into G1 and G1/S cyclin-dependent kinases

Cells that are in G0, G1 is binding the TF called E2F
E2F in this scenario is inhibited

These cells in G0 receive a signal
Signal activates the expression for cyclin D or Cyclin E  one of the targets of the CDK is the RB protein
Kinases will phosphorylate substrate
In this scenario we have a phosphorylated RB protein
When RB is phosphorylated it can no longer bind to E2F

Now you have dissociation of this inactive complex
Phosphorykated RB and E2F dissociation
E2F can now bind the promoter of doff genes and induce the expression of target genes.

Question 26

What does E2F stimulate the expression of?

Answer 26

Released E2F stimulates increased expression of more Cyclin E and
S-phase proteins e.g. DNA polymerase (need to copy the DNA), thymidine kinase (required for expression of thymidine), PCNA (protein involved in DNA rep.) etc.
DNA replication starts.

Question 27

What is a Cyclin-dependent kinase inhibitors?
what are the 2 families?
What is the difference in expression stimulation?

Answer 27

Inhibit cell cycle progression

1 - CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP) family (now called CDKN1)

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

2 - Inhibitor of Kinase 4 family (INK4) (now called CDKN2)

Expression stimulated by TGF

Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors)

Question 28

what is CDKN1’s role and TP53?

Answer 28

CDKN1
Inhibit cell cycle progression
Some induced at the level of transcription by TGF beta – inhibits cell growth by inhibiting expression of CDKN1

Another strong regulator of CDKN1 – TP53 – tumor suppressor protein involved in DNA damage
Guardian of the genome
It activates the expression of these inhibitors of kinases

Question 29

How do Growth factors induce cyclin expression?

Answer 29

Growth factor binding to the growth factor receptor
Start activating a series of intracellular signal transducers
As a result of activating these signalling pathways
Effect in nucleus
In the nucleus end up activating either expression of p21 or expression of cyclin/cdk4
Induce response in nucleus in response to growth factors

Question 30

What is the cascade of events that occur due to the binding of growth factors?

Answer 30

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)

E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)

G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F

E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)

S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.

Question 31

how is DNA damage detected in the cell cycle?

name the enzymes involved?

Answer 31

DNA damage can acc be detected at diff. checkpoints
Cell first arrests in response to dna damage
Stop cell cycle -Stop the cycle
(cyclin dependent kinase inhibitors, CHEK2 etc.)

Attempt to repair DNA - Attempt DNA repair
(nucleotide or base excision enzymes, mismatch repair etc.)

2 outcomes
Fully repaired – cell re enters the cell cycle and starts dividing
If DNA repair not acahieved the DNA obvs contains mutations  cell doesn’t want to transmit thei mutation into daughter cell
Can produce cancer
If DNA not properly reapired - undergoes programmed cell death or apoptosis
- Programmed Cell Death if repair impossible
(BCL2 family, caspases)

Question 32

What is the role of TP53?
Is it expressed in all cell types?
In a lab env. in the absence of DNA damage will we find it’s proteins?
what kinases are involved in it’s job?

Does TP53 play a part in DNA repair?

Answer 32

Tumor suppressor P53
All cell types express it and make protein
However in the absence of damage the p53 is destroyed by proteasome

Look at cell pop.  detect RNA for p53  it is expressed  however if you want to detect protein it is unlikelya s it is constantly being degraded
So cells with p53 but its not active

In response to DNA damage
Let’s say you have a mutagen
Incorporate a mutation in that part of the DNA
This DNA damage can be read and detected by kinases
Mainly ATM ATR kinases
Kinases become active and one of the substrates for the kinase is p53
Kinase adds phosphate or more than 1 phosphate group to p53 so as a result of this the p53 can no longer be degraded so therefore as a result of DNA damage you start accumulating P53 in cells

P53 is a TF
It will activate the expression of different genes
Cdkn1 inhibitor of CDK and as a result of that p53 stops cells from dividing by expressing the CDKN1

P53 will aslso try to repair the DNA by activating the expression of quite a few different genes that come from proteins that are involved in DNA reapir for instance quite a few proteins involved in acquisition of that protein there are actually regulated by p53 protein

If DNA reapir outcome is good  repair DNA and cell cycling again
If not possible then P53 will induce expression of genes required to promote apoptosis

Question 33

Summary of cell growth lec

Answer 33

Growth factors binding to receptors induce gene expression

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)

E2F released, stimulating expression of genes required for S-phase

Cell replicates DNA (expression of S-phase Cyclin-CDK complexes)

If all DNA replicated, G2/M Cyclin-CDK complexes cause cell to enter mitosis. If chromosomes aligned on spindle, exit from mitosis is triggered

If process fails, TP53 initiates apoptosis