A2780/DDP-R
Control 2.5 5.0 10 20 40 80 160 320nM
Carmen Raventos-Suarez Ph.D.
[email protected] (609-252-6492)
Bristol-Myers Squibb
When We Study Cells At the microscope we can visualize many structures inside the cells
FACS Answers presented on this workshop Proliferation: What When and How Arrest: Why “When” is a key feature Apoptosis: Dealing with the kinetics of a disappearing population Photographs from Molecular Probes web site
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Proliferation What? A cycling phenomena regulated by check point controls DNA duplication followed by segregation into two new entities
Control 2.5 5.0 10 20 40 80 160 320nM
Proliferation When do you measure it?
• Basic science research – signal transduction evaluation
• Pharmaceutical industry – biochemical to cellular assays
• Clinical follow up – cancer therapeutic efficiencies – inflammatory cascades identification – immune activation responses
Cell Cycle Profile Components A picture of DNA Synthesis Cell Quest
G0/G1
software
S
G2/M
Sub-G1
Tetraploid
DNA (area)
Cell Cycle Profiles: A Personal Signature of Cells DNA histograms gives a first hint about cells proliferation potential
Slow growth
Fast growth
DNA-PI
Proliferation Features What does “Cell Cycle Profiles” really mean? A2780DDP
A2780WT
0
Confluent
0
Confluent 200
200 400 FL2-A: PI
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Logaritmic
600
Logaritmic
600 800
FL2-A: PI
Sparse 1000
800
Sparse 1000
FlowJo Software
Cell Cycle Times and Numbers • DNA content and population distributions 24h Number of cells =100
24h Cell cycle
G1 50%
S 30%
12hrs
7.2hrs
24h Number of cells =125
18h Cell cycle
G1 40% 7.2 hrs
S 38% 6.8 hrs
G2/M 22% 3.9hrs
G2/M 20% 4.8hrs
new cycle 6h additional hours of growth
When A Cell Divides Addressing cells one at a time
S
G1
G2/M Modified from Cell Signaling Web site
Getting The Components Right The Question of Doublet Discrimination
Alternatively Area vs. Height is also a way to discriminate doublets and higher clumps
FlowJo Software
Cell Cycle Profile of Single Cells FlowJo
Gated vs. non gated populations Previous plots
400
28.9
%G1
75.5
%S
22.6
%S
14.9
%G2
38.2
%G2
3.84
%G2
0.82
1000
single cells
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200
%G1
21.8
%S
48.9
%G2
25.4
%G2
3.49
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400 600 FL2-A: DNA-PI
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Ungated 26.3
%S
17.3
%G2 400
1000
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Ungated
%G1
1500
%G2
9.24
1000
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400 600 FL3-A: DNA-7AAD
400
%G1
64.5
%S
15.1
%G2
30.1 # Cells
600
800
7.35
%G2
4.58
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1000
Ungated
300 # Cells
0
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# Cells
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Single cells
1500
# Cells
single cells
Software
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%G1
12.4
%S
26.5
%G2
23.8
%G2
35.9
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Nuclei vs. Whole Cells What is better?
Nuclei
Whole cells
•Ploidy paraffin blocks
•Ploidy fresh samples
•Apoptosis problems
•Apoptosis efficiency
•Nuclear localization
•Cytoplasmic markers.
Cell Cycle Software Packages Getting Numbers to Fit Your Data
• Multicycle • Modfit • FlowJo All are packages that contain algorithms with different restrictions bound to their calculations. Unfortunately, no algorithms are consistently reliable in fitting all the distributions you may encounter. You may have to experiment with different options and constraints in finding your best results. (copied FlowJo quote)
Getting To See The Numbers Watson Pragmatic vs. Dean Jett Fox in FlowJo software single
%G1
47.7
%G1
%S
32.2
%S
35.1
%G2
14.5
%G2
36.8
600
18.4 600
# Cells
300
# Cells
# Cells
800
single
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single
1000
200
%G1
22.8
%S
31.5
%G2
35.7
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400 200
100 200 0
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400 600 FL2-A: DNA-PI
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400 600 FL2-A: DNA-PI
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1000
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400 600 FL2-A: DNA-PI
single
400
48.8
%G1
22.3
%S
26.2
%S
28.3
%G2
20.6
%G2
40.5
300
800
1000
single
%G1
# Cells
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0 0
1000
single
800
# Cells
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600
# Cells
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%G1
25.1
%S
29.8
%G2
37.5
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400 200
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Alice Givan previously showed the Mod Fit variations on their selective options
Precautions-Tissue Culture Monolayers
• Learn the metabonomics of your cells – Doubling times, density optimization
• Cell Culture Stocks Maintenance – Rigid protocol for split times/avoid overgrowth
• Protocol for Experimental Tests – Standardized number of cells & scheduling – Efficiently trypsinize to a unicellular suspension
Beyond DNA Quantitative Analysis How to add more significance to your data?
• Adding a second marker to your cells – Cell identification markers (CD’s or signal transduction probes) – DNA doubling features, BrdU incorporation
• Adding a third or more markers to your cells – When preset configuration is fixed in your instrument Search for probes that allow to you the best combinations on added color
– When you are able to set up your own configuration First pick up different lasers then change your dichroics and band filters
Proliferation and BrdU S phase and Doubling Times K562
30min pulse
5hrs release 10000
10000
1000
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FL1-H
FL1-H
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8hrs release
FL1-H
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FlowJo software
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FL1-H
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Calculating the Numbers Getting the mathematics to work for you
From: Cell Cycle Kinetics Estimated by Analysis of Bromodeoxyuridine Incorporation. Terry N.H.A., and White A. Methods in cell Biology 63:355-374 (1994)
Overall messages • When to do DNA? – Maybe every time you address cells
• Watch out! Single cells exclusively – Cell preparation. Avoid clumping
• How to address specific questions? – Cell culture conditions. Use tight protocols – Most appropriate software. Up to you
References • Solid Tumor dissociation and storage (nuclei) – –
Cerra.R., Zarbo R.J., and Crissman. JD.,1990 Dissociation of cells from Solid Tumors. Methods in Cell biology 33: 1-12 Vindelov.L.L.,Cristensen.IJ. An integrated set of methods for routine flow cytometry DNA Analysis. 1990. Methods in Cell biology 33: 127-137
• Cell Lines (whole cells) – –
Pozarowwski.P., Darzynkiewiwicz.Z. Analysis of Cell Cycle by Flow Cytometry. 2004. Methods Mol Biol 281:301-311. Traganos.F.,Juan.G.,Darzynkiewiwicz.Z. Cell Cycle Analysis of Drug treated Cells 2001. Methods Mol Biol 95:229-240.
• Overall – –
–
Rabinovitch.P.S. Practical considerations for DNA Content and Cell Cycle Analysis in Clinical Flow Cytometry. Principles and applications. Williams and Wilkins-1993 Pham.NA., Jaccobberger.JW.,Schimmer.A.D.,Cao.P.,Gronda.M.,Hedley.D.W. 2004. The dietary isothiocyanate sulforaphane targets pathways of apoptosis,cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice. Mol.Cancer.Ther.3:1239-1248 Bagwell.B. et al. 2004. Optimizing Flow Cytometric DNA ploidy and S phase Fraction as nNegative prosnostic Markers for Node-Negative Breast Cancer Specimens Cytometry 46:121-135
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Proliferation Disturbances
Arrest Control 2.5 5.0 10 20 40 80 160 320nM
A Way to Cope with Stress
ARREST What is it?
• A slowdown in cell cycle progression? • A reversible or irreversible block in one phase of the cycle? • An induced effect of stress or toxic insult? • A response of the cell genetic makeup? • All of the above?
The Meaning of Cell Cycle Arrest How Do You Identify Specific Growth Arrest? Control
Campthothecin
Cisplatin
Paclitaxel
Cell growth arrest induced by stress or chemical compounds are efficiently identified when cells are growing at their optimal logarithmic rates and inducers are at an equilibrium concentration where cells are halted to activate repair mechanisms or apoptosis Cell Quest software
Identification of Arrest DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE
Paclitaxel a model drug for G2/M arrest
Taxol
A2780/DDP-R
A2780/DDP-S
Titrations
Taxotere
Control 2.5
5.0
10
20
40
80
160
320nM
Control 2.5
5.0
10
20
40
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160
320nM
Laidlaw, J., Raventos-Suarez,C., Fairchild, C.R., Peterson, R.W., and Menendez, A.T. Taxol and taxotere have similar potency in cytotoxicity assays, cell selectivity, bcl-2 phosphorylation, G2/M arrest and induction of apoptosis.91st Annual meeting of the AACR. San Francisco, April 1-5. 2000.
CellQuest software
Identification of Arrest Camptothecin a Model drug for S arrest? A2780 Camptothecin
LnCaP Camptothecin
100nM
100nM
30nM
30nM
10nM 0
10nM
3nM
200
0
1nM
400 600 800 FL2-A: DNA-PI
400
1nM
600
Control none 1000
3nM
200 800
More titrations FlowJo Software
FL2-A: DNA-PI
Control none 1000
How to Evaluate a Compound • Preliminary Requirements – Cytotoxic tests provide the best tool to identify dosage
• Cell Cycle Titration Curves – Cell cycle profiles need to be address one cycle of growth at a time – Preliminary titration curves done at a 24h time point provide good hints of activity
Arrest As An Assay Mechanism of Action
• The Story of BMS-250497 – BMS-250497 is the first non camptothecin compound described to be specific for Topoisomerase I inhibition. – Because of the side effects of camptothecin another efficient compound with this kind of activity have been actively pursued by the pharmaceutical industry – Biochemical assays required confirmation at the cellular level to reinforce the value of this compound as a candidate for the clinic
Results on BMS-250749 BMS-250749 IC50 = 3 nM
•
•
Flow cytometry analysis of A2780 cells with wt p53
Effects of BMS-250749 and camptothecin
Camptothecin IC50 = 10 nM
Control
0.1x
1x
5x
10x
20x IC50
CellQuest software
-Raventos-Suarez,C., Class. K., Buczek,J,L.,Balasubramanian,N., Long, B., and Menendez, A,T. 2001 Topoisomerase I is the Target Responsible for Cytotoxicity of BMS-250749: Confirmation by Flow Cytometry 92th Annual Meeting, New Orleans March 24-28; Proc. AACR 2001 42:103 Abs 562 -Raventos-Suarez,C., Class, K., Wild, R., Menendez, A., Long, B. 004 IN VITRO Specificity Profiling of Cellular Topoisomerase Activities Using FACS Analyses.2ISAC XXII International Congress on Analytical Cytology. May22-27 2004 Montpellier, France. Cytometry 58A: p115 Abst# 96207
HCT116(VM46)
In Arrest Profiles
HCT116 (VM46) DNA Cell Cycle Profiles
A second parameter reinforced your observations
DNA
633nM laser
Cyclin B1 Profiles
Cyclin B1
Cyclin E
FL2-H: FL2-Height
Control cells
Cyclin A
1000
1000
800
800
FL1-H: FL1-Height
488nm laser
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Camptothecin treated 1000
1000
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800
FL1-H: FL1-Height
FL2-H: FL2-Height
r
HCT116
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0 0
Blue= 488nm
Red=633nm
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Comparison between PI and ToPro3
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FlowJo Allows Line Graphs Over DNA Profiles A second parameter always reinforced your observations
Cyclin E
Cyclin A
Results on BMS-250749 • We used a panel of 3 paired cell lines: sensitive and resistant to identify activity • It takes several comparisons to efficiently confirm at the cellular level effects of known activities from a biochemical assay
A2780/DDP-R
Arrest Leads into Apoptosis
Control 2.5 5.0 10 20 40 80 160 320nM
Apoptosis An active metabolic process devised to dismiss stressed cells unable to cope with the insult An active act of disappearance
Morphology of Apoptosis Control
Apoptotic
Apoptosis Pictures
Apoptosis a disappearing population
Evaluation Methods Best When Linked to DNA profiles
• • • • • •
Annexin V TdT Tunel p85PARP Caspase 3 Many other caspases Other approaches- The Sub G1 fraction
Tunel Assay Provides Specificity of Phase 104
104
104
apoptosis single cells
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1000
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101
101
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Dot Plot Overlays
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400 600 FL2-A: DNA-PI
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1000
103 FL1-H: dUTP-FITC
102
apoptosis single cells
103 FL1-H: dUTP-FITC
103 FL1-H: dUTP-FITC
FL1-H: dUTP-FITC
apoptosis single cells
apoptosis single cells
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Flow Jo software
p85PARP on Drug Effects Localization of populations engaged in apoptosis Vinblastin 5nM
Colchicine 10nM 10000
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FL1-H: P85-FITC
FL1-H: P85-FITC
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Flow Jo software
The Sub-G1 Fraction * * * 0
High dose
200 400
Low dose
600 FL2-A: DNA-PI
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Control
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* Apoptosis by Sub-G1, an increasingly growing population
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Taxol A2780/DDP-R
A2780/DDP-S
Taxotere
When Proliferation, Arrest and Apoptosis Meet Control 2.5 5.0 10 20 40 80 160 320nM Control 2.5 5.0 10 20 40 80 160 320nM
The Story of BMS-214662 – BMS-214662 is a farnesyl transferase inhibitor with high apoptosis induction capabilities. – Targets on the farnesyl transferase cascade are not expected to produce any apoptosis in short periods of time but this compound did it – An assay needed to be devised to stain for proliferation and apoptosis simultaneously to directly answer this question -Raventos-Suarez,C., Class. K. and Lee F. 2002 The pro-apoptotic FT-inhibitor BMS-214662 selectively targets nonproliferating tumor cells. Demonstration by a new flow cytometric method. ISAC XXI International Congress on Analytical Cytology .May 2002 San Diego, California. Cytometry supp11: p72
Building An Assay • Proliferation by BrdU Uptake and evaluation by Dnase treatment: modify the BrdU Flow Kit from BD cat#552598” • Cell cycle profiles by DNA 7AAD stain • Apoptosis by p85 PARP “use the Anti-PARP p85 fragment Ab from Promega” cat#G734A”
Proliferation and Apoptosis A
Mechanism of Action of BMS-214662 P+A A P+A A
P
P
Control
Cisplatin 500nM
Red = non proliferating Blue = proliferating Green = apoptotic non proliferating P+A = proliferating cells engaged in apoptosis
P+A
P
BMS-214662 9uM
Take Home Messages • Cell Cycle Profiles are the best handle in addressing cell behavior, capacity to respond to an stimulus and possible engagement in apoptosis • Careful preparation of cells is essential • Multiparameter analysis linked to cell cycle profiles provide a more complete picture of cellular activities
Back up data
Cell Cycle Profile of Cells Getting to see the numbers When algorithms don’t seem to fit you can force constrains
FlowJo Software
Cell Cycle Profile of Cells A cell specific signature FlowJo
Panel of cell lines growing at logarithmic growth rates
HCT116 HCT116(VP35) HCT116(VM46) 0
A2780/DDP-R
200 400
A2780/Tax
600 FL2-A: PI
800
A2780/DDP-S 1000
Why are times at logarithmic rate of growth taken as optimal?
Software
Results on BMS-250749 Six cell lines two time points plus two control drugs
HCT116 (24h)
HCT116 (48h)
HCT116/TopoII (24h)
Camptothecin IC50= 10nM
Control 0.1x 1x 5x 10x 20xIC50
HCT116/TopoII (48h)
P388 (24h)
Etoposide IC50= 1.2uM
BMS-250749 IC50=3nM
BMS-250749 IC50=3nM
Control 0.1x 1x 5x 10x 20xIC50
Control 0.1x 1x
5x 10x 20xIC50
Control 0.1x 1x 5x 10x 20xIC50
P388/Topo I (24h)
Etoposide IC50= 1.2uM
Control 0.1x 1x 5x 10x 20xIC50
BMS-250749 IC50=3nM
Control 0.1x 1x 5x 10x 20xIC50
Control 0.1x 1x 5x 10x 20xIC50
A2780mutp53 (24h)
A2780mutp53 (48h) Camptothecin IC50= 10nM
Etoposide IC50= 1.2uM
BMS-250749 IC50=3nM
Control 0.1x 1x 5x 10x 20xIC50
A2780mutp53 (24h)
P388/Topo I (48h) Camptothecin IC50= 10nM
Camptothecin IC50= 10nM
Camptothecin IC50= 10nM
Etoposide IC50= 1.2uM
P388 (48h)
Control 0.1x 1x 5x 10x 20xIC50
A2780mutp53 (48h) Camptothecin IC50= 10nM
Etoposide IC50= 1.2uM
Etoposide IC50= 1.2uM
BMS-250749 IC50=3nM
BMS-250749 IC50=3nM
Control 0.1x 1x 5x 10x 20xIC50
Control 0.1x 1x 5x 10x 20xIC50
Control 0.1x 1x 5x 10x 20xIC50
It takes a panel of comparisons to address population distribution effects Shown are Cells in 3 pairs:sensitive/resistant (TopoII, TopoI and p53)
Proliferation What and When Beyond DNA quantitative analysis
• Proliferation activities require more than DNA measurements to account for disturbances – BrdU assay • will identify the S phase and determine doubling times.
– Mitotic markers: • will allow discrimination of G2 and S phases
– Cyclins: • will determine how far into a cell cycle phase cells have been able to go before stop growing
– Specific altered functions by induced by resistance or mutation mechanisms
More on Mechanism of Action Specificity on Topo II A Camptothecin Resistant Cell line
Control
Camptothecin
Etoposide
TAS-103
Control
Camptothecin
Etoposide
TAS-103
A Camptothecin Sensitive Cell line
Long, B.H., Fairchild, C.R., Raventos-Suarez,C., Cornell, L., and Menendez, A.T. Cytotoxic mechanism of TAS-103 is related to topoisomerase II mediated DNA cleavage. 91st Annual meeting of the AACR. San Francisco, April 1-5, 2000.
Cell Quest software