Cell Proliferation and Death - MRC Centre for Regenerative ... · PDF fileCell Proliferation and Death Derek Davies, ... Apoptosis. Why is apoptosis important? Evading ...
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Cell Proliferation and Death
Derek Davies, Cancer Research UK
http://www.london-research-institute.org.uk/technologies/120
Proliferation signals
Proliferation
Senescence
Apoptosis
Cell death
A cell
DNA analysis
• Propidium Iodide
• Ethidium Bromide
• Hoechst dyes
• Cyanine dyes eg TO-PRO-3, SYTO/SYTOX dyes
• Acridine Orange
• Pyronin Y
• Styryl Dyes eg LDS-751
• Mithramycin, Chromomycin
• 7 Aminoactinomycin D (7AAD)
• Diamino-2-phenylindole (DAPI)
• DRAQ5, DRAQ7
We can use DNA dyes in two ways:
• As viability dyes
• To measure DNA content and monitor cell cycle and its regulation
Exclusion of dead cells during analysis and sorting
Identification and quantification of apoptotic cells
DNA analysis
Dead cell discrimination
Dead Dead
Note log scale! 0 200 400 600 800 1000
Forward Scatter
1
10
100
1000
10000
Pro
pid
ium
Io
did
e
We can use DNA dyes in two ways:
• As viability dyes
• To measure DNA content and monitor cell cycle and its regulation
Exclusion of dead cells during analysis and sorting
Identification and quantification of apoptotic cells
DNA analysis
G1: Gap 1 S: Synthetic G2: Gap 2 M: Mitosis G0: cells that cease division
G1
M G
2
S G0
The mammalian cell cycle
G1
M G
2
S G0
Cdk2/cyclin E
Cdk2/Cyclin A
P21, p27, p57
Cdk2/Cyclin A
Cdc2/Cyclin A
Cdc2/Cyclin B
RB Dephosphorylation
RB Phosphorylation
Cdk4/Cyclin D
Cdk6/Cyclin D
INK4, p21, p27, p57
The mammalian cell cycle
Cell cycle analysis by flow cytometry
Cells must be permeable - can use detergent or fixation (ethanol is best)
Basic protocol - fix, wash twice, remove RNA and stain with DNA-binding dye
DNA in cells can be stained with a fluorescent dye DNA probes like PI are stochiometric and increase fluorescence on binding Dyes either intercalate or bind specific base pairs So we can measure how much DNA is in a cell
Increase in Fluorescence Intensity
# o
f E
vent
s
In an ideal world…….
Increase in Fluorescence Intensity
# o
f E
vent
s
CV: SD/mean x 100.
In the real world…….
DNA stained with propidium iodide
DNA Content
0 200 400 600 800 1000
Propidium Iodide
0
200
400
600
800
1000
Ce
ll co
un
t
G1
S Phase
G2/M
Note linear scale!
DNA stained with propidium iodide
DNA Content
0 200 400 600 800 1000
Propidium Iodide
0
200
400
600
800
1000
Ce
ll co
un
t
We can quantitate the percentage of cells in each phase of the cell cycle and monitor the effect of treatments
G1 S G2
Example 1: Compare cycles
Example 2: S phase block
G1 S G2
Example 3: M block
G1 S G2
DNA analysis in a clinical situation
Many tumours show altered DNA content
Diploid index may have prognostic significance
Many tumours show increased proliferation
S phase fraction may have prognostic significance
DNA Index = 1.32
Aneuploid G1
0 200 400 600 800 1000
Propidium Iodide - Area
0
200
400
600
Ce
ll co
un
t
0 200 400 600 800 1000
Propidium Iodide - Area
0
300
600
900
1200
Ce
ll co
un
t
0 200 400 600 800 1000
Propidium Iodide - Area
0
20
40
60
80
100
Ce
ll co
un
t
DI=1.36
Diploid G1
Aneuploid G2
Analysis of DNA histograms - pitfalls and a better approach…
0 200 400 600 800 1000
Propidium Iodide
0
200
400
600
800
1000
# C
ells
0 200 400 600 800 1000
Propidium Iodide
0
300
600
900
1200
# C
ells
Mathematical modeling is a better approach but still not ideal!
G1 43% S 45% G2 10%
G1 54% S 27% G2 18%
The use of markers gives a good indication but is only an estimate!
G0-G
1
S
G2-M
Fluorescence Intensity
Cell N
umber
DNA analysis by a single fluorochrome can only take us so far!
• Thymidine analog
• Taken up by cycling cells
• Use for comparative growth rates, length of cell cycle, pulse labelling
• Staining procedure involves unwinding DNA
• Combine with Propidium iodide
Cell cycle analysis - Bromodeoxyuridine (BrdU) method
Typical dual parameter plot
Propidium Iodide
Ant
i-B
rdU
FIT
C
0 200 400 600 800 1000
Propidium Iodide
1
10
100
1000
10000
Brd
U-F
ITC
G1 G2/M
S Phase
Compare comparative growth rates
Control Drug-treated
MCF10A Breast cancer cell line
0 200 400 600 800 1000
Propidium iodide
100
101
102
103
104
BrdU
FIT
C
0 200 400 600 800 1000
Propidium iodide
100
101
102
103
104
BrdU
FIT
C
50% 33%
Measuring Proliferation by dye dilution
Dye must be taken up by live cells
Dye must have low toxicity
Dye must be compatible with flow cytometric set-up
Dye must be equally apportioned between daughter cells
Lipophilic dyes that label cell membrane
Succinimidyl dyes that label intracellular proteins
Measuring Proliferation by dye dilution
Divisions:
3 2 1 0
0 102
103
104
105
440/40 Violet-A
0
300
600
900
1200
# C
ells
0 102
103
104
105
440/40 Violet-A
0
100
200
300
# C
ells
Serum free
0 102
103
104
105
440/40 Violet-A
0
300
600
900
1200
# C
ells
+ Serum
0 102
103
104
105
440/40 Violet-A
0
20
40
60
80
100
% o
f M
ax
Overlay
Measuring Proliferation by dye dilution
CFSE
B-C
ell M
arke
r
Measuring Proliferation by dye dilution
The other side of the coin…
• “Falling off”
• Distinct from ‘necrosis’ and ‘oncosis’
• “Programmed cell death”
• Kerr, Wyllie and Currie BJC (1972), 26:239
apoptosis
Normal development Normal tissue turnover Negative selection in immune system T cell killing Exposure to certain conditions
Where is apoptosis seen?
Normal development Normal tissue turnover Negative selection in immune system T cell killing Exposure to certain conditions
Alzheimer’s Disease Parkinson’s Disease Autoimmune disorders Neurodegenerative disease Cancer
Where is apoptosis seen?
Necrosis Apoptosis Affects groups of cells Affects individual cells Non-physiological induction Physiological induction (viral, poison, ischemia) (lack of signals, changes) Phagocytosis by macrophages Phagocytosis by macrophages
or other cells Inflammatory response No inflammatory response
• Cell Shrinkage
• Cell shape change
• Condensation of cytoplasm
• Nuclear envelope changes
• Nuclear fragmentation
• Loss of cell surface structures
• Apoptotic bodies
• Cell detachment
• Phagocytosis of remains
• Free Ca2+ rise
• bcl2/bax interaction
• Cell dehydration
• Loss of mitochondrial membrane potential
• Enzyme activation (caspases)
• Phosphatidylserine externalisation
• Lamin B proteolysis
• DNA denaturation
• 50-300kb cleavage
• Intra-nucleosomal cleavage
• Protein cross-linking
Morphological Functional
Apoptosis
Why is apoptosis important?
Evading apoptosis
Sustained angiogenesis
Cancer
Self-sufficiency in growth signals
Tissue invasion and metastasis
Limitless replicative potential
Insensitivity to anti-growth signals
Hanahan, D. and Weinberg, R.A. 2000. Cell. 100:57.
Put simply….
http://www.nature.com/reviews/poster/apoptosis
Major Apoptotic Pathways in Mammalian Cells
Mitochondrial Pathway Death Receptor Pathway
D D D D
Fas/Apo1 /CD95
FADD
Procaspase 8
DISC
Caspase 8
Bcl-2 D
Procaspase 9
Apaf-1
dATP
Apaf -1 Caspase 9
apoptosome
dATP
Cytochrome c
oxidants ceramide others
DNA damage
FasL
Caspase 3
BID
Procaspase 3
Cellular targets
The road to commitment
How can apoptosis be detected?
DNA Laddering Comet assay
Electron microscopy Flow cytometry
Light scattering/cell permeability (PI, DAPI, To-Pro-3)
Apoptosis detection by Flow Cytometry
0 200 400 600 800 1000
Forward Scatter
100
101
102
103
104
Pro
pid
ium
Io
did
e
0 200 400 600 800 1000
Forward Scatter
100
101
102
103
104
Pro
pid
ium
Io
did
e
Untreated Treated
Dead Dead
Live Live
Changes to the mitochondria (TMRE, CMX dyes, JC-1)
Apoptosis detection by Flow Cytometry
100
101
102
103
104
CMXRos
100
101
102
103
104
TO
-PR
O-3
100
101
102
103
104
CMXRos
100
101
102
103
104
TO
-PR
O-3
Untreated Treated
Live Live
Dead Dead
Apoptotic Apoptotic
Changes to the cell membrane (Annexin binding)
Apoptosis detection by Flow Cytometry
100
101
102
103
104
Annexin V-FITC
100
101
102
103
104
Pro
pid
ium
Iodid
e
100
101
102
103
104
Annexin V-FITC
100
101
102
103
104
Pro
pid
ium
Iodid
e
Untreated Treated
Dead Dead
Live Live
Apop Apop
Changes in enzyme expression (Caspases 3, 8 and 9)
Apoptosis detection by Flow Cytometry
100 101 102 103 104
Cleaved Caspase-3 FITC
0
200
400
600
800
Cell
count
100 101 102 103 104
Cleaved Caspase-3 FITC
0
200
400
600
800
Cell
count
100 101 102 103 104
Cleaved Caspase-3 FITC
0
20
40
60
80
100
Cell
count
Untreated Treated
Changes in cellular DNA (Fragmentation and strand breaks)
Apoptosis detection by Flow Cytometry
0 200 400 600 800 1000
Propidium Iodide
0
100
200
300
Counts
0 200 400 600 800 1000
Propidium Iodide
0
200
400
600
Counts
Treated Untreated
Integration of apoptosis methods
TMRE, Annexin, 7-AAD, Hoechst33342
100
101
102
103
104
Annexin V-FITC
100
101
102
103
104
7-A
AD
0 200 400 600 800 1000
Hoechst 33342
100
101
102
103
104
TM
RE
0 200 400 600 800 1000
Hoechst 33342
100
101
102
103
104
TM
RE
0 200 400 600 800 1000
Hoechst 33342
100
101
102
103
104
TM
RE
What is the question?
Cell type? Cultured cells? Suspension or adherent? Primary cells?.
What has happened to the cells? Treatment? Time course?
What other information is being sought e.g. concurrent phenotyping.
Are there any technical restrictions e.g. lasers.
Cost, simplicity and number of samples.
Expertise available
Which method should I use to assess proliferation and death?
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