cell cycle

INTERPHASE (G2)

two centrosomes are visible comprising a pair of centrioles and associated microtubules (as centrioles are made up of microtubules)

the nucleus is intact and chromosomes are not visible by light microscopy - can be made visible by FISH (fluorescent in situ hybridisation)

how does FISH work in interphase

1. cells are grown on glass slides, fixed and permeabilised with detergent

2. and incubated with fluorescent oligonucleotide probes specific for individual chromosomes

3. the probes hybridise their targets

   • interphase chromosomes occupy distinct territories, their dispersed structure allows access of transcription factors to DNA, cells are actively making RNA and proteins

PROPHASE

early prophase:

• centrosomes move to opposite poles

• chromosomes condense and become visible

• nuclear membrane begins to disaggregate into small vesicles

• nucleus surrounded by interphase microtubules

late prophase:

• chromosome condensation = two sister chromatids held together at their centromeres

• spindle fibres attach to chromatids at kinetochores

• nuclear membrane is completely broken down

METAPHASE

• chromosomes align midway between the poles

• sister chromatids remain attached

ANAPHASE

• sister chromatids separate into separate independent chromosomes

• cell and spindle elongate

• cytokinesis begins: cleavage starts to form that separates the two daughter cells

TELOPHASE

• chromosomes uncoil and are less distinct

• nuclear structure is re-established: nucleoli and nuclear membranes re-form

• spindle fibres become less distinct and disappear

• cytokinesis = almost complete

INTERPHASE (G1)

• cytokenesis = complete

  • each (diploid) daughter cell enters a new interphase (G1)

summary of interphase

G1. S. G2.

G1 = daughter cells

S = DNA and centriole replication

G2 = prepare for mitosis

cyclin as a controller of mitosis

G1 = cyclin concentrations rise

G2 = high cyclin concentrations stimulate mitosis

late G2 = cyclin destruction: levels fall

what are cyclins

regulatory proteins that control progression through the cell cycle - the amount of cyclin determines whether the cell progresses into mitosis

cyclins act as switches by activating enzymes = cyclin-dependent kinases (CDKs)

action pathway of cyclin B

during G2 phase:

• cyclin B concentration gradually increases

• cyclin B binds CDK1 (cyclin dependent kinase) forms a cyclin B-CDK1 complex

when enough cyclin accumulates:

• CDK1 = fully activated

• cell enters mitosis

^^^ acts like a threshold switch

cyclin B-CDK1 complex phosphorylates target proteins that trigger mitotic events (eg chromosome condensation/ nuclear envelope breakdown/ spindle formation)

once Cyclin B-CDK1 has completed its action - complex is destroyed by proteolysis in proteasome

G1 cyclin-CDK complex

prepare the cell for S phase

S cyclin-CDK complex

controls chromosome replication

G2 cyclin-CDK complexes

phosphorylate their targets which activate spindle formation

exponential phase

retardation phase

stationary phase

exponential phase: the doubling time gives the mean length of the cell cycle

• the cell cycle runs at different rates under different conditions

• cell cycle runs at different rates by responding to changes in environment

retardation phase: growth rate slows, cell cycle runs slower than in exponential phase

stationary phase: cells stop dividing

• cells can leave the cell cycle and enter a quiescent phase (temp inactivity)

G₀ - quiescent phase

cells can withdraw from the cell cycle, enter into G₀ - so can differentiate into different tissues

cells can remain in G₀ indefinitely

G₀ - differentiation + proliferation

other differentiated cells can be stimulated to re-enter cell cycle + replicate

re-entry into the cell cycle requires mitogenic signals (external signals that stimulate a cell to begin dividing)

1. quiescent G₀ cells receive a mitogenic signals

2. cells pass through the RESTRICTION POINT

3. cells enter S phase

what is cancer due to in the cell cycle

uncontrolled proliferation

cancer cells can also leave the cell cycle and enter G₀ - these cells are resistant to radiotherapy and chemotherapy

EGF (epidermal growth factor) signalling pathway in relation to G0 and G1 in the cell cycle

1. growth signal starts: EGF binds to receptor EGRF (epidermal growth factor receptor)

2. activated EGRF bound by Grb2 and Sos which recurs and activate Ras

3. Ras recruits Raf to the membrane, signal is passed via intermediates to MAPK (MAP kinase)

   • signal transduction via an enzyme cascade - amplifies the signal

4. activated MAPK translocates into the nucleus and stimulates expression of early response genes (c-FOS + c-JUN)

5. c-FOS + c-JUN = transcription factors - induce expression of delayed response genes = G₁ cyclins and partner CDKs, triggering re-entry into G₁

growth signals - mutational activation of receptors

breat cancers = mutation in growth factor receptor

forms a dimer, then self activates auto-phosphorylating

• causing unregulated proliferation

growth signals - mutational activation of Ras

in cancer Ras is mutated = so Ras is always ACTIVATED

MAPK transduction cascade is stimulated

• causing unregulated proliferation

growth signals - mutational activation of Raf

in malignant melanomas a mutated Raf gene produces an always ACTIVATED Raf

• MAPK signal transduction cascade = stimulated

  • causing unregulated proliferation

growth signals - viral subversion

viral oncogenes make v-JUN and v-FOS MIMICKING the action of c-Jun and c-Fos = stimulating G₁, cyclin and CDK expression

• causing unregulated cell proliferation

restriction point

checkpoint in G₁ - gives time for DNA repair following mitosis

• key regulator: pRb (retinoblastoma protein)

childhood retinoblastoma

cancer affecting children

caused by retinoblastoma (RB) gene defects

hereditary retinoblastoma

sporadic blastoma

RB
pRb

RB = tumour suppressor gene

pRb = tumour-suppressor protein

• without pRb protein, retinal cells proliferate uncontrollably giving rise to retinoblastoma

• NORMALLY, pRb protein inhibits the formation of retinoblastoma - pRb encoded by RB

• pRb functions by regulating the restriction point in G1

how does pRb work

pRb controls the activity of E2F transcription factors (pRb acts like a brake on E2F)

E2F proteins control DNA replication and the synthesis of S phase proteins

G₁: pRb binds E2F and inactivates them, so the cell can’t make S phase proteins UNTIL G₁ cyclin/ CDK have accumulated enough to phosphorylate Rb and inactivate it, allowing S phase proteins to be made

what do S phase proteins do

activated during S phase of cell cycle when DNA replication occurs

main functions: replicate the genome, package newly synthesised DNA etc

TP53 > p53

loss of p53 creates genome instability = results in chromosome rearrangement

if TP53 genome damaged, tumour suppression is severely reduced

as p53 protein causes apoptosis, DNA repair, cell cycle restart

HPV - viral subversion

HPV - associated with cervical cancer

• HPV E6 protein inhibits p53 activity

• HPV E7 protein inhibits Rb

• inhibition of pRb and p53 drives cells from cell cycle to cell cycle

apoptosis vs. necrosis

apoptosis: dying cells shrink condense and fragment, releasing apoptotic bodies that are phagocytose by tither cells - inflammaition is not stimulated

necrosis: cells that die through tissue damage exhibit different morphological changes, dying cells swell and burst - inflammation stimulated and damage to neighbouring cells

triggers for apoptosis

internal: recognition of DNA damage by p53/ pRb

external: recognition of stress, heat, radiation, starvation, hypoxia

developmental: apoptotic programas remove the webbing between our fingers during foetal development and remove neurons