2- MoD; Cell Growth and Differentiation

Question 1

define cell division

Answer 1

the process by which a parent cell divides into two or more daughter cells

often precedes differentiation though there is overlap

Question 2

describe the two main forms of cell growth

Answer 2

hypertrophy and hyperplasia

1. hypertrophy = increased cell SIZE through increased macromolecule synthesis (proteins and lipids)
2. hyperplasia = more cells
   - through cell division and proliferation

Question 3

define cell differentiation

Answer 3

the process where cells become specialized and acquire specific functions

Question 4

how does differentiation occur?

Answer 4

cell fully exists the cell cycle and commits to terminal differentiation

elicits a tissue-specific gene expression which changes its shape and function

becomes a specific cell type = called a ‘post-mitotic’ cell

Question 5

what is the similarity between cell division and differentiation?

Answer 5

they’re both governed by intracellular and extracellular signal which converge on gene promoters (called ‘co-incidence detectors’)

combinations of signals are integrated for regulating growth/ differentiation - promoter make a binary yes or no decision for the (level of) gene expression

Question 6

how do extracellular signals work? - type of signals, their effects on growth & differentiation?

Answer 6

there are three kinds - paracrine, autocrine and endocrine = ligand signal binds to receptor, stimulating an intrac. cascade

induce effects for cell growth and differentiation such as:
- stimulating proliferation and promoting survival = e.g. mitogens
- inducing differentiation and inhibiting proliferation = e.g. TGF-beta
- induce apoptosis = e.g. TNF-alpha

Question 7

how do extracellular signals induce gene expression? - mechanism?

Answer 7

growth factor/ protein binds to cell surface receptor = activates a signal transduction pathway via kinase cascade

signal transducers activate transcription factors in the nucleus - TFs drive exp. of downstream genes

mRNA produced and exported from nucleus to cytoplasm = translated into protein

protein can:
- remain in the cytoplasm and exerts its effects
- go to cell surface membrane and act as TF for other proteins
- affect other downstream genes

Question 8

define paracrine, autocrine and endocrine signals

Answer 8

paracrine = signal produced locally by neighbouring cell to stimulate a different cell type with the appropriate cell surface receptor

autocrine = produced by a cell that also expresses the appropriate cell surface receptor - signal acts on the cell that produced it

endocrine = conventional hormones, released systemically for distant effects

Question 9

describe the four stages of the cell cycle in terms of events and chromosome number

Answer 9

G1, S, G2 phases are interphase stages - cell grows larger, synthesises macromolecules, makes copies of its DNA

M phase - mitosis; cell divides into two haploid daughter cells (2N)

G1 phase - cell grows physically larger, copies organelles (between 2N-4N as it’s in the process of replicating its genome)

S phase - DNA synthesis, making copies of every chromosome and duplicating centrosomes (between 2N-4N)

G2 phase - cell grows more, makes proteins and organelles, reorganises contents for mitosis (4N)

Question 10

what are the three main checkpoints of the cell cycle?

Answer 10

G1 restriction point
G2 checkpoint
M phase spindle checkpoint

Question 11

describe the three checkpoints of the cell cycle - when they occur, what it checks for, what does that ensure?

Answer 11

three checkpoints - G1 restriction checkpoint, G2 checkpoint and M phase checkpoint

1. G1 restriction checkpoint = occurs at the end of G1
   - checks for DNA damage so errors don’t carry forward
   - cell size, nutrient availability and metabolite stores
   - ensures cell has sufficient resources and is in a suitable environment for DNA replication and cell division
2. G2 checkpoint = occurs at the end of G2
   - checks for DNA damage, success of DNA synthesis and replication before mitosis
   - ensures cell only progresses to mitosis if DNA is undamaged and correctly replicated
3. M phase spindle checkpoint = occurs during metaphase of mitosis
   - checks physical positioning of of chromosomes attached to mitotic spindle before separation
   - ensures proper chromosome segregation so each daughter cell gets the correct number of chromosomes

Question 12

what is FACs?

Answer 12

fluorescence activated cell signalling

fluorescence flow cytometry technique that analyses and measures DNA content within a cell population

Question 13

describe the difference in FAC flow cytometry traces between highly proliferative and less proliferative cell populations

Answer 13

highly proliferative - rate of cell division is high
- lower proportion of cells in G1 though it’s still the highest
- slightly more in S compared to a less proliferative popl
- G2/M relatively the same as its the time limited stage of cell division

less proliferative - rate of cell division is low
- G1 has the highest proportion of cells
- S phase has the least
- G2/M phase has the same as for a highly proliferative popl

Question 14

how many genes encode for CDK proteins?

Answer 14

10

Question 15

how many genes encode for cyclin proteins?

Answer 15

more than 20

Question 16

what are CDKs?

Answer 16

cyclin-dependent kinases, regulate the cell cycle and work with cyclin proteins (regulatory proteins)

Question 17

what is cyclin?

Answer 17

regulatory protein - forms a complex with CDK which activates a kinase cascade, phosphorylating specific substrates
cyclin expression is influenced by external signals - e.g. mitotic signals from growth factors

Question 18

how is cyclin-CDK pathway regulated?

Answer 18

cyclin proteins have a high rate of turnover- are constantly synthesised, expressed and destroyed

post-transcriptional modifications via phosphorylation and dephosphorylation affect kinase activity, and result in activation/ inhibition/ destruction

bindings of CDKIs/ cyclin-dependent kinase inhibitors will inhibit cyclin-CDK complex activity

Question 19

describe the RB protein, cyclin and CDK mechanism for cell cycle regulation

Answer 19

RB is a regulatory protein for G1 to S phase transition, and a key CDK substrate

unphosphorylated RB binds E2F TFs - prevents their transcriptional activity, suppresses expression of S phase proteins

specific CDK-cyclin complexes phosphorylate RB - cause a conformational change - dissociates from E2Fs

allows E2Fs to be transcriptionally active - drives expression of S phase proteins like DNA polymerase, and drives cyclin-E exp. forward (positive feedforward loop between E2Fs and cyclin-E)

Question 20

what are the specific CDK-cyclin complexes for RB phosphorylation?

Answer 20

cyclin-D and CDK-4
cyclin-E and CDK-2

Question 21

describe the sequential events of the cell cycle relating to gene expression and cyclin-CDK activity

Answer 21

mitogens in early G1 stimulate the expression of early genes, including TFs

TFs induce expression of delayed genes like cyclin-D

cyclin-D forms cyclin-CDK complexes with CDKs 4 & 6

cyclin D-CKD 4 hypo-phosphorylate RB protein = minimal level of phosphorylation = minimal repression of E2Fs, allows some activity = allows low level expression of cyclin-E

cyclin E - CDK 2 complexes activate - cause hyperphosphorylation of RB protein

RB can no longer repress E2Fs - E2F activity remains high throughout S phase, E2F responsive genes are activated

sequential activation of various CDK-cyclin complexes occurs:
- cyclin E -CDK 2 phosphorylates and activates cyclin-A and CDK-2 = activates cyclin A and CDK 1 = activated cyclin B and CDK-1
- S-G2/M phase cyclin-CDK complexes until cyclin B-CDK 1

at the end of M phase - RB dephosphorylates, can repress E2Fs again, mediated by PP1

Question 22

at what checkpoints is DNA damage checked at? what occurs if DNA damage is detected?

Answer 22

G1 to S restriction point
end of G2 checkpoint

detected - cell cycle stops, driven by CDKI/ cyclin-dependent kinase inhibitors and cell attempts to repair damage through repair mechanisms

if damage is too great - cell is reprogrammed for apoptosis through expression of apoptotic genes and enzyme activations

Question 23

what is TP53?

Answer 23

a tumour suppressor gene

continually destroyed by proteasomes and synthesised/ high turnover in a cell with viable/ normal DNA

Question 24

describe how TP53 is involved in DNA damage repair and apoptosis

Answer 24

DNA damage in a cell is picked up by complex processes - activates DNA-dependent kinases

TP53 is phosphorylated - prevents its destruction by proteasomes

TP53 accumulates in cell and exerts its biological effects:
- acts as a TF, drives CDKI expression = induces cell cycle arrest
- activates DNA repair mechanisms - if cell DNA is successfully repaired, it returns to the cell cycle
- if repair is unsuccessful = TP53 activates apoptotic mechanisms by driving apoptotic gene and enzyme expression

Question 25

what’s the significance of TP53 in cancer?

Answer 25

loss of function mutations in TP53 are common in cancer - frequently observed in cancer

effects:
- prevent apoptosis = no cell death
- prevent cell cycle arrest = faster proliferation, uncontrolled
- prevent DNA repair = DNA mutations accumulate from lack of DNA repair, contributed to cancer development and tumour heterogeneity which allows it to adapt to microenvironment and resist therapies - drives cancer progression

Question 26

how does 5-fluorouracil work as a chemotherapy drug?

Answer 26

S phase drug that targets DNA damage

prevents thymidine synthesis which is required to make more DNA

Question 27

how does cisplatin work as a chemotherapy drug?

Answer 27

S phase drug that targets DNA damage

binds to DNA causes damage and blocks repair mechanisms, induces cell death

Question 28

how do vinca alkaloids work as a chemotherapy drug?

Answer 28

M phase drug that targets mitotic spindle

binds to and stabilises free/monomeric tubulin

prevents microtubule polymerisation = can’t form the mitotic spindle

arrests cell in mitosis, apoptosis occurs

Question 29

how does Paciltaxel work as a chemotherapy drug?

Answer 29

M phase drug that targets mitotic spindle

binds to and stabilises polymerised/ multimeric microtubule complexes = prevents their depolymerisation

arrests cell in mitosis, causes a rest in M phase and induces apoptosis

Question 30

how does colchicine work as a chemotherapy drug?

Answer 30

a similar mode of action to vinca alkaloids - binds to and stabilises monomeric tubulin, prevents microtubule polymerisation, can’t form mitotic spindle

cell arrested in mitosis and apoptosis occurs

Question 31

what are the two types of chemotherapy drugs? what do they target?

Answer 31

S phase drugs - target DNA damage
- 5-fluorouracil
- cisplatin

M phase drugs - target the mitotic spindle
- vinca alkaloids
- Pacitaxel
- colchicine