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BCH 4125Regulation of cell proliferation in

mammalians

Prof: Alexandre Blais, Ph.D. – BMI Department, Faculty of Medicine (RGN Hall)

1. G1/S transition: the restriction point, cyclins and CDKs

2. G1/S transition: growth factors and cell signaling,

3. G1/S transition: convergence on E2F and Rb

4. Cell cycle arrest

5. Cell cycle and cancer

6. Cell proliferation and development

7. Programmed cell death

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Availability

• By appointment:– alexandre.blais@uottawa.ca– 613-562-5800 x8463

Course format

• Basic concepts are introduced• Presentation of the research articles that led to key

discoveries• Explanation of laboratory experiments performed• Explanation of old or new laboratory techniques that

enabled these discoveries

• Suggestions for further reading (e.g. original research articles, review articles, book chapters, websites)

Examination

• Final exam: designed to take 1h30 to complete, but you will have a full 3 hours to finish it.

• Questions may be on all aspects of the material covered in class

• Questions will not test your memorization skills, but remembering facts is essential

• Questions will verify your understanding of the key concepts seen in class, your ability to propose experiments to test a hypothesis, and your ability to interpret experimental data.

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Today: G1/S transition the restriction point, cyclins and CDKs

1. What drives a cell to proliferate or cease to proliferate?

2. The Restriction point

3. Cyclins and CDKs

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The eukaryotic cell cycleThe eukaryotic cell cycle

SSynthesisynthesis

G2G2G1G1

MMitosisitosis

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The eukaryotic cell cycleThe eukaryotic cell cycle

SSG2G2

G1G1MM

G0G0GrowthGrowthfactorsfactors

++++

++++

RR (To divide or(To divide ornot to divide ?)not to divide ?)

In higher eukaryotes, the Restriction point is the equivalent of START, in yeast

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G0 phase?

• The G0 phase corresponds to a quiescent state, when proliferation is impossible or is not desired– also referred to as a post-mitotic state, since it may

follow the M phase– differentiated cells (e.g. neurons, muscle) do not

proliferate after differentiation, and remain in G0 for ever

– other cell types constantly proliferate (e.g. intestinal epithelium cells, certain blood cells)

SSG2G2

G1G1MM

G0G0

RR

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What drives a cell to proliferate or cease to proliferate?

• Regulatory mechanism to shift cells between proliferative and quiescent states– suboptimal nutritional conditions

• Regardless of the cause of the block to proliferation, – cells enter the same state of quiescence– cells re-enter the cycle at a precise point (R) when nutrition is restored

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What drives a cell to proliferate or cease to proliferate?

• Conditions that lead to quiescence– nutrients deprivation– growth factor deprivation– high density: contact inhibition

• Are these conditions all different or similar in some way?• How can we position cells, in between M and the next S

phase?– there are no landmark events ! (well, in 1974 they were

unknown…)

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Thymidine incorporation assay

Grow cells in rich medium

(all nutrients, plus plenty of fetal calf serum, for growth factors)

Switch to nutrient-poor medium

(remove serum, or certain essential amino acids)

Return cells to rich medium, plus radioactive thymidine

(will be incorporated in DNA of dividing cells)

Day 0 Day 1 Day 4

Harvest at different time points, extract DNA, and count radioactivity

CPM: counts per minute, a measure of radioactive decay of the 3H isotope

Time (h) in rich medium

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Findings:

1. All “poor” conditions prevent cell cycle progression

2. Cells already blocked by one “poor” condition will not be “released” when switched to a different “poor” condition.

Findings: This is also true with contact inhibition:

Confluent cells stop proliferating

When re-seeded at lower densities in “poor” medium, cells do NOT resume growth

No matter what the block is, cells end up in the same state

Block

Releas

e

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Findings:

Applied first or second, these poor conditions do not allow proliferation

Therefore they must arrest cells all at the same state, at the Restriction point

Possibility #2 is the correct one

Second condition

G1 SB1 B2Possibility #1: Poor conditions arrest cells at different points of the cycle.

If we apply the Blocking condition B2 first and then switch to condition B1, cells will go through S phase (they are released)

Possibility #2: Poor conditions arrest cells at the same point of the cycle.

No matter which poor condition is used first, cells will NOT enter the cycle

G1 SB1B2

% thymidine incorporation

R !

G2M

G2M

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Responsiveness to growth factors

• Only in G0/G1 is a cell responsive to mitogens– Once DNA synthesis is

initiated, and at any time until the end of mitosis, the cycle must proceed!

SSG2G2

G1G1MM

G0G0

RR

MitogensMitogens

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Is there an “R-protein” ?

1. Controlling the passage through R

2. Synthesized in response to mitogenic stimuli

3. Short-lived in normal cells

4. Stabilized in transformed, cancerous cells

Reminder: Transformed =~ cancerous

Normal cells that have integrated DNA from certain tumor viruses become transformed, they acquire the cancerous phenotype.

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What is the R-protein ?Some short-lived protein must control the passage

through the restriction point

1. Serum starve cells

2. Return to rich medium, with various concentrations of CHX, in the presence of 3H-thymidine

3. Fix the cells at various time points and autorad. to count number of labeled cells

Reminder: CHX is cycloheximide

Inhibits translational elongation

blocks or slows down protein synthesis

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1. Serum starve cells

2. Return to rich medium, with various concentrations of CHX, in the presence of 3H-thymidine

3. Fix the cells at various time points and autorad. to count number of labeled cells

Finding: blocking protein synthesis prevents passage through R

The R protein is labile

No CHX

Highest [CHX]

Time after addition of serum to starved cells

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Normal vs Cancer cells

Normal fibroblasts

Transformed fibroblasts

Finding: Transformed “cancer” cells are NOT sensitive to CHX and therefore the R protein is not labile in these cells, but stable instead

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Reminder: cyclins and CDKs• Cyclins are proteins with cyclical

expression profiles during the cell cycle !

• Function as the regulatory subunit for protein kinases, the cyclin-dependent kinases (CDKs)

• Specific cyclins associate with specific CDKs

• The cyclin subunit dictates substrate specificity of the CDK

http://www.celldiv.com/content/1/1/6/figure/F3?highres=y

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Cyclins A or B as the R-proteins•Associate with CDK1 (Cyclin B) or both Cdk1 and Cdk2 (cyclin A)

•Cyclin B expressed from mid-S into mitosis

•Cyclin A expressed from the early S phase into mitosis

Does that match the definition of the R-protein ?

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Cyclins E as the R-proteins•There are two cyclins E: E1 and E2

•Associate with CDK2

•Expressed from mid-G1 to mid-S

Does that match the definition of the R-protein ?

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E-type Cyclins as the R-proteins1. Knock-out BOTH cyclin E genes in the

mouse (E1 and E2)

2. Allow double-KO embryos to develop, and harvest embryonic fibroblasts (MEFs)

3. Grow MEFs in vitro in rich medium and perform growth curve analysis and PI vs BrdU FACS

Finding: The E-type cyclins are not required for constant cell cycle progression (PI vs BrdU plots are similar)

Maybe the other cyclins can fill in…

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MEFs: Mouse embryonic fibroblasts

1. Sacrifice pregnant female mouse, usually around day 13, but can be done earlier

2. dissect out the uterus

3. separate the embryos from placenta and membranes

4. mince (razor blade)

5. dissociate cells with trypsin (a protease that digests connective tissue)

6. centrifuge the cells and remove debris

7. seed the cells on a tissue culture plate, in rich medium

8. do your experiments !

© Michael F. McElwaine

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PI: Propidium iodide

DNA intercalating dye, fluoresces at ~570 nm

BrdU: 5-bromo-2-deoxyuridine

Synthetic analogue of thymidine

Incorporated into DNA in S phase

Can be detected with anti-BrdU antibody coupled to FITC, which fluoresces at ~510 nm

BrdU and PI analysis1. Grow MEFs

2. Pulse with BrdU for 1 hour

3. Fix cells in formaldehyde

4. Incubate with anti-BrdU-FITC (fluorescent)

5. Incubate with PI

6. FACS analysis and plot FITC vs PI

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BrdU and PI analysis

PI staining only

http://science.cancerresearchuk.org/sci/facs/facs_major_apps/cell_cycle_analysis/?version=1

Just entered in G1, or in G1 for a long time?

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BrdU and PI analysis

PI staining onlyPI plus BrdU pulse

http://science.cancerresearchuk.org/sci/facs/facs_major_apps/cell_cycle_analysis/?version=1

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E-type Cyclins as the R-proteins1. Knock-out BOTH cyclin E genes in the

mouse (E1 and E2)

2. Allow double-KO embryos to develop, and harvest embryonic fibroblasts (MEFs)

3. Grow MEFs in vitro in rich medium and perform growth curve analysis and PI vs BrdU FACS

Finding: The E-type cyclins are not required for constant cell cycle progression (PI vs BrdU plots are similar)

Maybe the other cyclins can fill in…

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Cyclins E as the R-proteins1. Knock-out BOTH cyclin E genes in the

mouse (E1 and E2)

2. Allow double-KO embryos to develop, and harvest embryonic fibroblasts (MEFs)

3. Serum-starve the MEFs in vitro and perform 3H-thymidine incorporation assay

Finding: The E-type cyclins are essential for cell cycle re-entry and passage through the restriction point.

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Cyclins D as the R-proteins•There are three cyclins D: D1, D2 and D3

•They are considered the G1 cyclins

•Associate with CDK4 and CDK6

•Expressed in response to growth factor stimulation, peak somewhat at the G1/S transition

Does that match the definition of the R-protein ?

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D-type Cyclins as the R-proteins

1. Knock-out all three cyclin D genes (D1, D2, D3) in the mouse

2. Allow triple-KO embryos to develop, and harvest protein lysates (they die at E16.5)

3. Analyze cyclin protein expression levels by western blot

Finding: When the D-type cyclins are wiped out, the expression of Cyclins E and A are normal, and cyclin E- and cyclin-A associated CDK activity are normal

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Measuring CDK activity

1. Lyse cells, remove debris

2. Immunoprecipitate the CDK of your choice OR the cyclin of your choice

3. Incubate with [32P]-ATP and the protein substrate (e.g. purified Histone H1)

4. Run on SDS-PAGE to separate by size

5. Autoradiograph to determine to what extent the substrate was phosphorylated.

E Cdk2bead

Histone H1[32P]-ATP

Histone H1P

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D-type Cyclins as the R-proteins1. Knock-out all three cyclin D genes (D1,

D2, D3) in the mouse

2. Allow triple-KO embryos to develop, and harvest MEFs

3. Serum-starve the MEFs in vitro and perform 3H-thymidine incorporation assay

Findings: Cell cycle profile of triple-KO MEFs is not grossly altered

Triple-KO MEFs require higher serum amounts to pass the restriction point

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How can Cyclin D triple KOMEFs go through R?

• Compensation by Cyclin E+Cdk2 ?– over-expression of p16, a CDK4 and

CDK6 inhibitor, does NOT arrest triple KO MEFs

– over-expression of p27, an inhibitor of CDK2 and CDK4/6, arrests the cells

– knock-down of CDK2 by RNA interference arrests the triple KO MEFs

• FINDINGS: Cdk2 and cyclin E and/or Cyclin A compensate for the loss of D-type cyclins

• How could you improve on these experiments? Which of Cyclin E or Cyclin A is most important for the rescue?

CDK2-A and CDK2-C: two siRNA molecules that target the same CDK2 mRNA

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Conclusion: is there only 1 R-protein?

• In constantly cycling cells, Cyclins D or E are dispensable

• In quiescent cells, Cyclin D expression comes BEFORE cyclin E

• Quiescent cells absolutely need Cyclins E to go through R

• Quiescent cells manage to go through R without cyclins D

E-type cyclins compensate for loss of D-type cyclins

D-type cyclins do NOT compensate for loss of E-type Cyclins

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…but let’s keep going with more KO!

Reminder:

CDK4 and CDK6 are the two CDKs that function with D-type cyclins

They are the “G1 CDKs”

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G1 CDKs are not essential to go through R !

1. Knock-out both G1 CDKs (CDK4 and CDK6) in the mouse

2. Allow double-KO embryos to develop, and harvest MEFs (they die at E18.5)

3. Serum-starve the MEFs in vitro and look at S-phase re-entry, or look at continuous growth

Findings: Double-KO MEFs cycle more slowly than WT

Double-KO MEFs manage to go through R (just like the triple D-type cyclin KO MEFs) WT

CDK4 KOCDK6 KODouble KO

WT

CDK4 KOCDK6 KODouble KO

Growth in rich medium

Return from quiescence

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…the ultimate KO!

Reminder:

CDK1 is the mitotic CDK, it is active at the G2/M passage and in mitosis

All other CDKs are called interphase CDKs

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One CDK is all you need !

1. Knock-out all interphase CDKs (CDK2, CDK4 and CDK6) in the mouse

2. Allow triple-KO embryos to develop, and harvest MEFs (they die at E12.5)

3. Serum-starve the MEFs in vitro and look at S-phase re-entry

Findings: Triple-KO MEFs cycle more slowly than WT…BUT…

Triple-KO MEFs manage to go through R !

Return from quiescence

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Weird stuff happens !

1. Extract proteins from embryos (WT or KO)

2. Perform Immuno-precipitation with anti-cyclin antibodies

3. Separate by SDS-PAGE

4. Detect CDK1 by western blot

Findings: D-type and E-type cyclins associate with CDK1 in triple-KO cells much more than in WT cells

Kinase assay shows that even without CDK4/CDK6/CDK2, cyclins D and E are associated with a kinase activity

That could explain how these cells manage to proliferate

CO-IP on embryo extracts

Kinase assays

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You do need AT LEAST one CDK!

1. Grow TKO MEFs or controls

2. Use RNAi to knock-down CDK1 (the last CDK remaining in the TKO cells!!!)

3. Serum starve the cells

4. Assay for S-phase entry after serum stimulation

Findings: You need at least one CDK to go into S phase!

Removing only CDK1 from MEFs doesn’t prevent passing the R point.

Also, knocking out ONLY CDK1 prevents development past the 2-to-4 cell stage, right after fertilization of the egg.

Control MEFs TKO MEFs

Cdc2a: the name of the gene that encodes the CDK1 protein

Source: Henry Gray's Anatomy of the Human Body

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What do you think of this statement?

• Observation:Cells with only CDK1 are able to pass the

restriction point • Therefore:CDK1 is normally involved in making cells pass the

restriction point, when returning from quiescence

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Knock-outs vs real-life4 roommates cooking mashed potatoes

Peel Cook Mash Wash the dishes…

PeelCookMashWash the dishes…

X X Xhttp://images2.fanpop.com/images/photos/3200000/Twilight-Cartoon-Version-of-Characters-twilight-movie-3204468-451-660.jpg

@%#&!

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What do you think of this statement?

• Observation:

Deleting all three D-type cyclins does not prevent passing the restriction point

• Therefore:

D-type cyclins serve absolutely no purpose

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To summarize• The restriction point is a time of the cell cycle:

– before which cells arrest if conditions are not favorable for cell division– before which cells are responsive to stimulation by growth factors (after

that, no growth factors are required)– that is deregulated/abrogated in cancer cells– where several cyclins and CDKs exert their effects to enable exit from

quiescence

• D-type Cyclins are the first ones to be expressed upon serum stimulation– ...but the other cyclins can compensate for their loss

• E-type cyclins are induced later, and function at the G1/S boundary– their loss prevents exit from quiescence (no compensation)

• Of all CDKs, only CDK1 is absolutely essential– embryonic development reaches at least E12.5 when any other CDK is

knocked-out.

Exam(ple) question

• Draw a BrdU-PI plot that you would get from analyzing the following cell population. Explain in 2-3 lines your rationale.– MEFs are grown in high mitogen concentration medium, then

mitogens are removed for 2 days (48 hours). Two hours prior to harvest, BrdU is added to the cells (2h long pulse, from 46 to 48h).

• Answer: the cells are all quiescent, so they mostly have a 2n DNA content (all in G0/G1) and are all negative for BrdU, since none of them were in S phase during the pulse.

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Next week:Mitogens and cell signaling

http://www.toxikologie.uni-mainz.de/ak-dietrich/research.jsp