L10 - The Cell Cycle

Question 1

What are the 5 different causes of cancer?

Answer 1

Environmental causes and carcinogens
Viral infection –> Rous Sarcoma Virus
Inherited factors –> Retinoblastoma
Genetic instability –> oncogenes and tumour suppressor genes
Many cancers arise from defects in the machinery that regulates cell growth or cell death

Question 2

What causes most normal cells to proliferate?

Answer 2

Most normal cells do not proliferate unless stimulated by extrinsic factors
Other extracellular signalling proteins can overrule these stimulatory factors and halt proliferation
- Induce a post-mitotic, differentiated state

Question 3

What is the master governed to integrate all the signals a cell receive?

Answer 3

Cell cycle clock

Signals have to be integrated to make the decision to proliferate, be quiescent or differentiate

Question 4

What is the cell cycle clock?

Answer 4

Network of interacting proteins that receives signals from outside and inside the cell, integrates them and decides the cell’s fate
- Proliferation –> cell cycle of growth and division
- Quiescence –> non-proliferative state
Operates in the nucleus

Question 5

What must the decision of growth versus quiescence must take into consideration?

Answer 5

Neighbouring cells

Question 6

How are messages about proliferation communicated between neighbouring cells/

Answer 6

Mediated by growth factors
- Small proteins released by some cells
- Travel through intercellular space and convey messages to other cells
Also called mitogens - induce cells to proliferate

Question 7

What cells are the exception to cells requiring signals to grow and divide?

Answer 7

Embryonic stem cells
In vitro mouse ES cells drive their own proliferation through internally generated signals
Only WT cells able to generate a benign tumour when injected in adult - tumorigenic

Question 8

What is the cell clock influenced by?

Answer 8

Cancer-associated proteins (oncogenes and tumour suppressors) which disrupt normal control mechanisms

Question 9

What are the alternative pathways that cancer cells use to cause proliferation?

Answer 9

Production of growth factors by themselves –> autocrine proliferative stimulation
Signals to stimulate surrounding normal cells to produce growth factors
Deregulation of growth factor receptor signalling –> elevated level of receptors or ligand-independent firing
Constitutive activation of signalling downstream of growth factor receptors
Disruption of negative feedback mechanisms

Question 10

What are the characteristics of the Go - growing phase of the cell cycle?

Answer 10

Withdrawal from cell cycle can be actively induced by growth-inhibiting factors - TGFbeta
Quiescent state can be
- Reversible - mitogens can stimulate cell proliferation again
- Irreversible - post-mitotic cells (neurons in the brain)

Question 11

What are the characteristics of the G1 - first gap phase of the cell cycle?

Answer 11

Proliferation versus quiescence decision
Decision to proliferate
- Doubling of cell’s macromolecular constituents
- Accumulation of cellular constituents - cell growth

Question 12

What are the characteristics of the S - synthesis phase of the cell cycle?

Answer 12

Length depends on how much DNA needs to be replicated

Question 13

What are the characteristics of the G2 - second gap phase of the cell cycle?

Answer 13

Cell growth continues
Cell prepares itself for mitosis
4 hours long

Question 14

What happens during prophase?

Answer 14

Chromosome condensation
Spindle fibres emerge from centrosomes
Nuclear envelop breaks down
Centrosomes move towards opposite poles

Question 15

What happens during metaphase?

Answer 15

Chromosome line up at metaphase plate

Each sister chromatid is attached to a spindle fibre originating from opposite poles

Question 16

What happens during anaphase?

Answer 16

Sister chromatids pulled towards opposite poles

Certain spindle fibres begin to elongate the cell

Question 17

What happens during telophase and cytokinesis?

Answer 17

Chromosomes arrive at opposite poles and begin to decondense
Nuclear envelop surrounds each set of chromosomes
The mitotic spindle breaks down
Spindle fibres push poles apart
Division of the cytoplasm of the mother cell –> 2 daughter cells

Question 18

What is the main method used to study the cell cycle?

Answer 18

Flow cytometry

Fast and quantitative

Question 19

How does flow cytometry work?

Answer 19

Analysed by measuring DNA content and imaging

• Cells treated with a fluorescent dye - labels DNA quantitatively
• As DNA content doubles during S phase - intensity of fluorescence increases in proportion
• • Cells in G0 and G1 phase have half the fluorescence as those in G2 or M phase
• In dividing cell the peak for G2 fluorescence on the graph is greater than in a quiescent cell

Question 20

What method is used to study mitosis?

Answer 20

Monitored using immunofluorescence and epifluorescence microscopy
Not fully quantitative

Question 21

When is the small window during G1 where they have to consult the extracellularly environment?

Answer 21

Onset of G1 to 1 hour before G1-S transition

Question 22

G1 decision making machinery shown in responses of cells to extracellular signals

Answer 22

Serum and growth factors removed before cells have completed 80-90% of G1
- Fail to proceed further and revert to G0 state
Serum and growth factors removed in final hour of G1
- Proceed to S, G2 and M phase

Question 23

Where is the restriction point?

Answer 23

Restriction point – at the end of G1
Where the decision is made to proliferate or enter quiescence
Deregulation of R-point decision-making machinery accompanies formation of cancer cells

Question 24

What were the 1970 nuclear fusion experiments?

Answer 24

Rao and Johnson

Mixed nuclei together in same cytoplasm to determine if they could influence one another

Question 25

What were the conclusions from mixing S nucleus and G1 nucleus?

Answer 25

S phase nucleus contains a diffusible factor that will induce replication

Question 26

What were the conclusions from mixing S nucleus and G2 nucleus?

Answer 26

G2 nucleus is resistant to S phase promoting factor

Question 27

What were the conclusions from mixing G1 nucleus and G2 nucleus?

Answer 27

G1 and G2 do not influence each other

Question 28

What were the conclusions from mixing interphase nucleus and M nucleus?

Answer 28

Mitotic nuclei release mitosis-promoting factor that affects all interphase nuclei

Question 29

How was the circuit controlling cell cycle progression studied?

Answer 29

3 principal model systems
Genetic manipulations in yeast – easy
Biochemical analysis of embryonic cell division in frog eggs - big dividing embryos
Sea urchin embryo – many embryos

Question 30

Who carried out genetic isolation of cell division cycle mutants in yeast?

Answer 30

1970s – Paul Nurse and Leland Hartwell

Question 31

Genetic isolation of cell division cycle mutants in yeast method?

Answer 31

Mutants affecting cell cycle arrested in different stages of the cell cycle

• Cell division cycle mutants
• Wee mutants - based on size at time of arrest
• Temperature-sensitive mutants
• • At permissive temperature (25˚C) - behave as WT
• • At restrictive temperature (37˚C) – inactive
• • Temperature sensitive cdc mutants can grow but not divide at restrictive temperature

Question 32

Who carried out studies on frog embryos?

Answer 32

1970s – Masui

Question 33

Who carried out studies on sea urchin embryos?

Answer 33

1980s – Tim Hunt

Question 34

Studies on frog embryos method?

Answer 34

Injection of substance from cytoplasm of mature egg into oocyte induced entry into M-phase
Maturation Promoting Factor - Mitosis Promoting factor
Later shown to induce mitosis on all eukaryotic cells
- Linked to protein kinase activity oscillating during cell cycle

Question 35

Studies on sea urchin embryos method?

Answer 35

Unfertilised sea urchin eggs spontaneously start dividing if dunked in soapy water
Eggs labelled with radioactive methionine  proteins run on a gel
Cyclin protein accumulated as the cells got ready to divide and disappeared as the cells split

Question 36

What were the conclusions from the experiments carried out on yeast, frogs and sea urchins?

Answer 36

Injection of cyclin sufficient to induce maturation of frog oocyte and activation of MPF
MPF activity
- Binding of cyclin to Cdc2 kinase
- Phosphorylation of Cdc2