A2780/DDP-R. Carmen Raventos-Suarez Ph.D. ( ) Bristol-Myers Squibb

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1 A2780/DDP-R Control nM Carmen Raventos-Suarez Ph.D. ( ) Bristol-Myers Squibb

2 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

3 A2780/DDP-R Proliferation What? A cycling phenomena regulated by check point controls DNA duplication followed by segregation into two new entities Control nM

4 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

5 Cell Cycle Profile Components A picture of DNA Synthesis G0/G1 Cell Quest software S G2/M Sub-G1 Tetraploid DNA (area)

6 Cell Cycle Profiles: A Personal Signature of Cells DNA histograms gives a first hint about cells proliferation potential Slow growth Fast growth DNA-PI

7 Proliferation Features What does Cell Cycle Profiles really mean? A2780WT A2780DDP Confluent Confluent FL2-A: PI Sparse Logaritmic FL2-A: PI Sparse Logaritmic FlowJo Software

8 Cell Cycle Times and Numbers DNA content and population distributions 24h Number of cells =100 G1 50% S 30% G2/M 20% 24h Cell cycle 12hrs 7.2hrs 4.8hrs 24h Number of cells =125 G1 40% S 38% G2/M 22% new cycle 18h Cell cycle 7.2 hrs 6.8 hrs 3.9hrs 6h additional hours of growth

9 When A Cell Divides Addressing cells one at a time S G1 G2/M Modified from Cell Signaling Web site

10 Getting The Components Right The Question of Doublet Discrimination FlowJo Software Alternatively Area vs. Height is also a way to discriminate doublets and higher clumps

11 Cell Cycle Profile of Single Cells Gated vs. non gated populations Previous plots FlowJo Software # Cells single cells %G1 %S %G2 %<G1 %>G # Cells Single cells %G1 %S %G2 %<G1 %>G # Cells single cells %G1 %S %G2 %<G1 %>G FL2-A: DNA-PI 0 FL2-A: FL2-Area 0 FL3-A: DNA-7AAD # Cells Ungated %G1 %S %G2 %<G1 %>G # Cells Ungated %G1 %S %G2 %<G1 %>G # Cells Ungated %G1 %S %G2 %<G1 %>G FL2-A: DNA-PI 0 FL2-A: FL2-Area 0 FL3-A: DNA-7AAD

12 Nuclei vs. Whole Cells What is better? Nuclei Ploidy paraffin blocks Apoptosis problems Nuclear localization Whole cells Ploidy fresh samples Apoptosis efficiency Cytoplasmic markers.

13 Cell Cycle Software Packages Multicycle Modfit FlowJo Getting Numbers to Fit Your Data 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)

14 Getting To See The Numbers Watson Pragmatic vs. Dean Jett Fox in FlowJo software # Cells single %G1 %S %G # Cells single %G1 %S %G # Cells single %G1 %S %G FL2-A: DNA-PI 0 FL2-A: DNA-PI 0 FL2-A: DNA-PI 1000 single 400 single single # Cells %G1 %S %G # Cells %G1 %S %G # Cells %G1 %S %G FL2-A: DNA-PI 0 FL2-A: DNA-PI 0 FL2-A: DNA-PI Alice Givan previously showed the Mod Fit variations on their selective options

15 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

16 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

17 Proliferation and BrdU S phase and Doubling Times K min pulse 5hrs release 8hrs release FL1-H 100 FL1-H 100 FL1-H 100 FL1-H FL2-A A2780S FL2-A FL2-A FL2-A FlowJo software FL1-H 100 FL1-H 100 FL1-H 100 FL1-H FL2-A 1 FL2-A 1 FL2-A 1 FL2-A

18 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: (1994)

19 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

20 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 Methods in Cell biology 33: Cell Lines (whole cells) Pozarowwski.P., Darzynkiewiwicz.Z. Analysis of Cell Cycle by Flow Cytometry Methods Mol Biol 281: Traganos.F.,Juan.G.,Darzynkiewiwicz.Z. Cell Cycle Analysis of Drug treated Cells Methods Mol Biol 95: 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 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: Bagwell.B. et al Optimizing Flow Cytometric DNA ploidy and S phase Fraction as nnegative prosnostic Markers for Node-Negative Breast Cancer Specimens Cytometry 46:

21 A2780/DDP-R Proliferation Disturbances Arrest Control nM A Way to Cope with Stress

22 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?

23 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

24 Identification of Arrest Taxol Paclitaxel a model drug for G2/M arrest DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE A2780/DDP-S A2780/DDP-R Taxotere Titrations Control nM Control nM 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.91 st Annual meeting of the AACR. San Francisco, April CellQuest software

25 Identification of Arrest Camptothecin a Model drug for S arrest? LnCaP Camptothecin A2780 Camptothecin 100nM 100nM 30nM 30nM FL2-A: DNA-PI 1nM Control none nM 10nM More titrations FL2-A: DNA-PI 1nM Control none nM 10nM FlowJo Software

26 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

27 Arrest As An Assay The Story of BMS Mechanism of Action BMS 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

28 Results on BMS BMS IC 50 = 3 nm Flow cytometry analysis of A2780 cells with wt p53 Camptothecin IC 50 = 10 nm Effects of BMS and camptothecin 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 Topoisomerase I is the Target Responsible for Cytotoxicity of BMS : Confirmation by Flow Cytometry 92th Annual Meeting, New Orleans March 24-28; Proc. AACR :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. May Montpellier, France. Cytometry 58A: p115 Abst# 96207

29 HCT116 HCT116(VM46) In Arrest Profiles HCT116 (VM46) DNA Cell Cycle Profiles 488nm laser Control cells A second parameter reinforced your observations 633nM laser Cyclin B1 Profiles DNA Cyclin B1 Cyclin E Cyclin A FL2-H: FL2-Height FL1-H: FL1-Height Camptothecin treated 0 0 FL4-A: FL4 A FL4-A: FL4 A FL2-H: FL2-Height FL1-H: FL1-Height FL4-A: FL4 A FL4-A: FL4 A Comparison between PI and ToPro3 Blue= 488nm Red=633nm

30 FlowJo Allows Line Graphs Over DNA Profiles A second parameter always reinforced your observations Cyclin E Cyclin A

31 Results on BMS 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

32 A2780/DDP-R Arrest Leads into Apoptosis Control nM

33 Apoptosis An active metabolic process devised to dismiss stressed cells unable to cope with the insult An active act of disappearance

34 Morphology of Apoptosis Control Apoptotic

35 Apoptosis Pictures Apoptosis a disappearing population

36 Evaluation Methods Best When Linked to DNA profiles Annexin V TdT Tunel p85parp Caspase 3 Many other caspases Other approaches- The Sub G1 fraction

37 Tunel Assay Provides Specificity of Phase apoptosis single cells apoptosis single cells apoptosis single cells apoptosis single cells FL1-H: dutp-fitc 10 2 FL1-H: dutp-fitc 10 2 FL1-H: dutp-fitc 10 2 FL1-H: dutp-fitc FL2-A: DNA-PI FL2-A: DNA-PI FL2-A: DNA-PI FL2-A: DNA-PI Dot Plot Overlays Flow Jo software

38 p85parp on Drug Effects Localization of populations engaged in apoptosis Vinblastin 5nM Colchicine 10nM FL1-H: P85-FITC 100 FL1-H: P85-FITC FL2-A: PI-DNA 1 FL2-A: PI-DNA Flow Jo software

39 The Sub-G1 Fraction * * * 0 * Low dose 600 FL2-A: DNA-PI 800 Control 1000 Apoptosis by Sub-G1, an increasingly growing population High dose

40 Taxol A2780/DDP-R A2780/DDP-S Taxotere When Proliferation, Arrest and Apoptosis Meet Control nM Control nM

41 The Story of BMS BMS 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 The pro-apoptotic FT-inhibitor BMS 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

42 Building An Assay Proliferation by BrdU Uptake and evaluation by Dnase treatment: modify the BrdU Flow Kit from BD cat# Cell cycle profiles by DNA 7AAD stain Apoptosis by p85 PARP use the Anti-PARP p85 fragment Ab from Promega cat#g734a

43 Proliferation and Apoptosis A Mechanism of Action of BMS P+A A P+A A P+A P P P Control Cisplatin 500nM BMS uM Red = non proliferating Blue = proliferating Green = apoptotic non proliferating P+A = proliferating cells engaged in apoptosis

44 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

45 Back up data

46 Cell Cycle Profile of Cells Getting to see the numbers When algorithms don t seem to fit you can force constrains FlowJo Software

47 Cell Cycle Profile of Cells A cell specific signature Panel of cell lines growing at logarithmic growth rates FlowJo Software HCT116 HCT116(VP35) HCT116(VM46) FL2-A: PI 800 A2780/DDP-R A2780/Tax A2780/DDP-S 1000 Why are times at logarithmic rate of growth taken as optimal?

48 Results on BMS 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 IC50= 10nM Camptothecin IC50= 10nM Camptothecin IC50= 10nM Camptothecin IC50= 10nM Camptothecin IC50= 10nM Camptothecin IC50= 10nM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM Etoposide IC50= 1.2uM BMS IC50=3nM BMS IC50=3nM BMS IC50=3nM BMS IC50=3nM BMS IC50=3nM BMS 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)

49 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

50 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. 91 st Annual meeting of the AACR. San Francisco, April 1-5, Cell Quest software