DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE
Taxol
A2780/DDP-S A2780/DDP-R
Taxotere
Control 2.5 5.0 10 20 40 80 160 320nM 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 DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE
Taxol
A2780/DDP-S A2780/DDP-R
Taxotere Proliferation What?
A cycling phenomena regulated by check point controls
Control 2.5 5.0 10 20 40 80 160 320nM Control 2.5 DNA5.0 10 20 40 duplication 80 160 320nM followed by segregation into two new entities 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?
A2780WT A2780DDP
0 Confluent 0 Confluent 200 200 400 400 Logaritmic Logaritmic 600 600 800 FL2-A: PI Sparse FL2-A: PI 800 Sparse 1000 1000 FlowJo Software Cell Cycle Times and Numbers
• DNA content and population distributions 24h
Number of cells =100 G1 S G2/M 50% 30% 20%
24h Cell cycle 12hrs 7.2hrs 4.8hrs
24h Number of cells =125 G1 S G2/M new cycle 40% 38% 22%
18h Cell cycle 7.2 hrs 6.8 hrs 3.9hrs 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
FlowJo The Question of Doublet Discrimination Software
Alternatively Area vs. Height is also a way to discriminate doublets and higher clumps Cell Cycle Profile of Single Cells
FlowJo Gated vs. non gated populations Previous plots Software
single cells Single cells 400 1500 single cells 600 %G1 28.9 %G1 75.5 %G1 21.8 %S 22.6 %S 14.9 %S 48.9 300 %G2 38.2 %G2 3.84 1000 %G2 25.4 400 %
500 200 100
0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A: DNA-PI FL2-A: FL2-Area FL3-A: DNA-7AAD
Ungated Ungated 400 Ungated %G1 26.3 1500 %G1 64.5 600 %G1 12.4 %S 17.3 %S 15.1 300 %S 26.5 %G2 30.1 %G2 7.35 1000 %G2 23.8 % # % 0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A: FL2-Area 0 200 400 600 800 1000 FL2-A: DNA-PI FL3-A: DNA-7AAD 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 1000 single 400 single single %G1 47.7 %G1 18.4 %G1 22.8 600 800 %S 35.1 %S 31.5 %S 32.2 300 %G2 14.5 %G2 36.8 %G2 35.7 600 400 Cells Cells Cells 200 # # # 400 100 200 200 0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A: DNA-PI FL2-A: DNA-PI FL2-A: DNA-PI 1000 single 400 single single %G1 48.8 %G1 22.3 %G1 25.1 800 600 %S 26.2 %S 28.3 %S 29.8 300 %G2 20.6 %G2 40.5 %G2 37.5 600 400 Cells Cells Cells # # 200 # 400 200 100 200 0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A: DNA-PI FL2-A: DNA-PI FL2-A: DNA-PI 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 8hrs release 10000 10000 10000 10000 1000 1000 1000 1000 100 100 100 100 FL1-H FL1-H FL1-H FL1-H 10 10 10 10 1 1 1 1 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A FL2-A FL2-A FL2-A FlowJo A2780S software 10000 10000 10000 10000 1000 1000 1000 1000 100 100 100 100 FL1-H FL1-H FL1-H FL1-H 10 10 10 10 1 1 1 1 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A FL2-A FL2-A FL2-A 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 DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE Taxol A2780/DDP-S A2780/DDP-R Taxotere Proliferation Disturbances Arrest Control 2.5 5.0 10 20 40 80 160 320nM 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-S A2780/DDP-R Taxotere Titrations Control 2.5 5.0 10 20 40 80 160 320nM Control 2.5 5.0 10 20 40 80 160 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? LnCaP A2780 Camptothecin Camptothecin 100nM 100nM 30nM 30nM 10nM 10nM 0 3nM 0 3nM 200 200 400 1nM 400 1nM 600 600 800 Control none 800 Control none FL2-A: DNA-PI 1000 More titrations FL2-A: DNA-PI 1000 FlowJo Software 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 Camptothecin IC50 = 10 nM • Effects of BMS-250749 and CellQuest camptothecin Control 0.1x 1x 5x 10x 20x IC50 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 HCT116(VM46) HCT116 HCT116In (VM46) Arrest Profiles DNA Cell Cycle Profiles A second parameter reinforced your observations Cyclin B1 Profiles 488nm laser 633nM laser 488nm laser DNA 633nM laser Cyclin B1 Cyclin E Cyclin A Control cells 1000 1000 800 800 600 600 FL1-Height FL2-Height 400 400 FL1-H: FL2-H: 200 200 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 FL4-A: FL4 A FL4-A: FL4 A Camptothecin treated 1000 1000 800 800 600 600 FL1-Height FL2-Height 400 400 FL1-H: FL2-H: 200 200 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 FL4-A: FL4 A FL4-A: FL4 A Comparison between PI and ToPro3 Blue= 488nm Red=633nm 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 DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE Taxol A2780/DDP-S A2780/DDP-R Taxotere Arrest Leads into Apoptosis Control 2.5 5.0 10 20 40 80 160 320nM 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 104 104 104 104 apoptosis apoptosis apoptosis apoptosis single cells single cells single cells single cells 103 103 103 103 102 102 102 102 FL1-H: dUTP-FITC FL1-H: dUTP-FITC FL1-H: dUTP-FITC FL1-H: dUTP-FITC 101 101 101 101 100 100 100 100 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FL2-A: DNA-PI FL2-A: DNA-PI FL2-A: DNA-PI FL2-A: DNA-PI Dot Plot Overlays Flow Jo software p85PARP on Drug Effects Localization of populations engaged in apoptosis Vinblastin 5nM Colchicine 10nM 10000 10000 1000 1000 100 100 FL1-H: P85-FITC FL1-H: P85-FITC 10 10 1 1 0 200 400 600 800 1000 FL2-A: PI-DNA 0 200 400 600 800 1000 FL2-A: PI-DNA Flow Jo software The Sub-G1 Fraction * * * 0 High dose 200 400 Low dose 600 800 FL2-A: DNA-PI Control 1000 * Apoptosis by Sub-G1, an increasingly growing population DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE Taxol Taxol A2780/DDP-S A2780/DDP-R A2780/DDP-S A2780/DDP-R Taxotere 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 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 non- proliferating 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 Mechanism of Action of BMS-214662 A P+A A P+A A P+A P P P Control Cisplatin 500nM BMS-214662 9uM Red = non proliferating Blue = proliferating Green = apoptotic non proliferating P+A = proliferating cells engaged in apoptosis 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 Software Panel of cell lines growing at logarithmic growth rates HCT116 HCT116(VP35) HCT116(VM46) 0 A2780/DDP-R 200 400 A2780/Tax 600 800 A2780/DDP-S FL2-A: PI 1000 Why are times at logarithmic rate of growth taken as optimal? Results on BMS-250749 Six cell lines two time points plus two control drugs HCT116 (24h) HCT116 (48h) HCT116/TopoII (24h) HCT116/TopoII (48h) P388 (24h) P388 (48h) P388/Topo I (24h) P388/Topo I (48h) A2780mutp53 (24h) A2780mutp53 (48h) A2780mutp53 (24h) A2780mutp53 (48h) Camptothecin Camptothecin Camptothecin Camptothecin Camptothecin Camptothecin IC50= 10nM IC50= 10nM IC50= 10nM IC50= 10nM IC50= 10nM IC50= 10nM Etoposide Etoposide Etoposide Etoposide IC50= 1.2uM Etoposide Etoposide IC50= 1.2uM IC50= 1.2uM IC50= 1.2uM IC50= 1.2uM IC50= 1.2uM BMS-250749 BMS-250749 BMS-250749 BMS-250749 BMS-250749 IC50=3nM BMS-250749 IC50=3nM IC50=3nM IC50=3nM IC50=3nM IC50=3nM Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50 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 A Camptothecin Sensitive Cell line Control Camptothecin Etoposide TAS-103 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