2.8 - Cell Replication

Question 1

The cell cycle

Answer 1

• orderly sequence of events in which a cell duplicates its contents and divides in two
• duplication, division, coordination
• G1 + S + G2 = interphase
• M = mitosis
• cell cycle is incredibly busy as cell must replicate 3 billion bp DNA, double in size, tear itself apart in a controlled fashion

Question 2

Cell division in specific cells

Answer 2

Different cells divide at different rates:

• embryonic vs adult cells
• complexity of system
• necessity for renewal
• state of differentiation (some cells never divide e.g. neurons and cardiac myocytes)
• tumour cells?

Question 3

G0

Answer 3

• G0 = quiescent phase
• in the absence of stimulus, cells go into G0
• most cells in the body which are differentiated to perform specific functions
• if a cell is damaged
• if cells are not dormant, but non-dividing e.g. neurons, skeletal muscle, hepatocytes

Question 4

Cell cycle checkpoints

Answer 4

• control at multiple checkpoints guards against disastrous progression through the cycle
• monitoring of external environment - is it favourable?
• in terms of nutrients, growth factors
• DNA can undergo repair, or the cell can undergo apoptosis
• G1 checkpoint - before entry into S phase - is environment favourable? is there damaged DNA?
• G2 checkpoint - before entry into mitotic phase - is all DNA replicated? is all DNA damage repaired?
• mitosis checkpoint - before anaphase - are all chromosomes properly attached to the mitotic spindle?
• a variety of checkpoints can arrest the cell cycle - if checkpoint not met

Question 5

How / why do cells leave G0?

Answer 5

Signalling cascades:

• response to extracellular factors - growth factors stimulate entry from G0 into G1
• signal amplification
• signal integration / modulation by other pathways
• Ras / Raf / MEK / ERK
• growth factor –> growth factor receptor –> intracellular signalling pathway –> protein synthesis increased + protein degradation decreased –> cell growth

Question 6

Cell cycle entry - role of c-Myc

Answer 6

• growth factor stimulation of signalling pathways promotes G0 to G1 transition
• growth factor signalling pathways induces the expression of c-Myc
• c-Myc is a transcription factor that promotes G0 to G1 transition
• stimulates the expression of cell cycle genes
• oncogene that is overexpressed in many tumours

Question 7

Cyclin dependent kinases (Cdks)

Answer 7

• cell cycle control based on cyclically activated and expressed proteins
• require cyclin to function
• levels of Cdks are fairly stable - it is the levels of cyclin that drive the active process
• present in proliferating cells throughout the cell cycle, BUT only active when a cyclin is bound
• activity is regulated by: interaction with cyclins, phosphorylation
• serine / threonine / tyrosine residues phosphorylated/dephosphorylated by Cdks
• become sequentially active and stimulate synthesis of genes required for next phase
• growth factor –> C-Myc –> cyclin D –> cyclin D:Cdk4/6 complex (cyclin bound to Cdk) –> cyclin E = gives direction and timing to the cycle
• e.g: Cdk1, Cdk2, Cdk4, Cdk6
• cell cycle entry requires Cdk 4/6:cyclin D complex

Question 8

Cyclins

Answer 8

• named because their concentrations within the cell fluctuate/cycle
• transiently expressed at specific points in the cell cycle
• regulated at level of expression
• produced/synthesised, then degraded (cyclic nature)
• only the cyclin:Cdk complex is active during mitosis
• cyclins susceptible to degradation, hence cyclical activation
• e.g: CyclinA, CyclinB, CyclinD, CyclinE

Question 9

Protein kinase cascades

Answer 9

• frequently, the protein regulated by a kinase is another kinase
• leads to signal amplification, diversification and opportunity for regulation
• regulation = dephosphorylation
• phosphorylation by kinases (activation)
• phosphorylation reversed by phosphatases (deactivation)

Question 10

Sequential phosphorylation and dephosphorylation activates Cdks

Answer 10

1. cyclin binds to Cdk, forming an inactive cyclin-Cdk complex (needs phosphorylation) - has an inhibitory phosphate and an activating phosphate
2. protein kinase activates activating phosphate
3. activating protein phosphatase dephosphorylates inhibitory phosphate
4. active cyclin-Cdk complex

Question 11

Active Cdks

Answer 11

• S-Cdk = controls S phase (when active S-Cdk is present, S phase is triggered, S cyclin falls as M phase begins)
• M-Cdk = controls M phase (when active M-Cdk is present, mitosis is triggered, M cyclin falls before G1 begins)
• positive feedback drives the cell cycle forwards - more active M-Cdk = more phosphatase (Cdc25) produced, reinforcing production of active M-Cdk
• cyclins are turned off by ubiquitination - method of degrading proteins
• active cyclin-Cdk complex –> ubiquitination of cyclin (ubiquitin attaches) –> destruction of cyclin –> inactive Cdk

Question 12

Retinoblastoma

Answer 12

• a molecular ‘brake’
• first identified through studies of a childhood eye tumour
• retinoblastoma protein (Rb) - essential for sequential progression - either missing or inactive
• tumour suppressor - inhibits cell proliferation and tumour development + often lost/inactivated in tumours, removing negative regulation of proliferation –> tumour
• abundant in all nucleated cells
• resting cell - active Rb is tightly bound/sequesters a transition factor in an inactive form (e.g. E2F family of TFs) –> TFs cannot turn on genes needed for cell cycle progression e.g. DNA polymerase, thymidine kinase
• in a proliferating cell, activation of intracellular signalling leads to production of G1-Cdk and G1/S-Cdk complexes, which can phosphorylate Rb inducing the inactivation of Rb and release of TF = target genes now activated
• E2F family members regulate the expression of several genes needed for cell cycle progression

Question 13

Growth factor (mitogen) signalling

Answer 13

• for the cell cycle to progress, the cell needs to double in size
• intracellular signalling pathways drive protein synthesis
• also protein degradation is inhibited
• net increase in protein synthesis

Question 14

P53

Answer 14

• p53 (transcription factor + tumour suppressor) arrests cells with damaged DNA in G1
• active p53 induces the p21 gene: stable active p53 binds to regulatory region of p21 gene –> p21 mRNA by transcription
• p21 family members are inhibitors of cyclin:Cdk complexes - p21 codes for inhibitors which bind to Cdks and inactivate them
• in absence of DNA damage, p53 is degraded in proteasomes

Question 15

Cell cycle regulatory proteins and cancer - oncogenes

Answer 15

• oncogenes are genes with the potential to cause cancer
• overexpression leads to cancer
• EGFR/HER2 - mutationally activated / overexpressed in breast cancers - Herceptin antibody for the treatment of HER2+ metastatic breast cancer
• Ras - proto-oncogene mutationally activated in many cancers
• Cyclin D1 - overexpressed in 50% of breast cancers
• C-Myc - overexpressed in many tumours

Question 16

Cell cycle regulatory proteins and cancer - tumour suppressors

Answer 16

• tumour suppressors inhibit protein synthesis - under expression leads to cancer
• Rb - loss of function mutations in 80% of small cell lung cancers
• p53 - loss of function mutations in over 50% of all human cancers