WIXLIB

A2780/DDP-RControl 2.5 5.0 10 20 40 80 160 320nMCarmen Raventos-Suarez istol-Myers Squibb

When We Study CellsAt the microscope we can visualize many structures inside the cellsFACS Answers presented on this workshop Proliferation: What When and How Arrest: Why “When” is a key feature Apoptosis: Dealing with the kinetics of adisappearing populationPhotographs from Molecular Probes web site

SA2780/DDP-RProliferationWhat?A cycling phenomena regulated by check point controlsDNA duplication followed by segregation into two new entitiesControl 2.5 5.0 10 20 40 80 160 320nM

ProliferationWhen 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 ComponentsA picture of DNA SynthesisCell QuestG0/G1softwareSG2/MSub-G1TetraploidDNA (area)

Cell Cycle Profiles:A Personal Signature of CellsDNA histograms gives a first hint about cells proliferation potentialSlow growthFast growthDNA-PI

Proliferation FeaturesWhat does “Cell Cycle Profiles” really L2-A: PI400Logaritmic600Logaritmic600800FL2-A: PISparse1000800Sparse1000FlowJoSoftware

Cell Cycle Times and Numbers DNA content and population distributions24hNumber of cells 10024h Cell cycleG150%S30%12hrs7.2hrs24hNumber of cells 12518h Cell cycleG140%7.2 hrsS38%6.8 hrsG2/M22%3.9hrsG2/M20%4.8hrsnew cycle6h additional hours of growth

When A Cell DividesAddressing cells one at a timeSG1G2/MModified from Cell Signaling Web site

Getting The Components RightThe Question of Doublet DiscriminationAlternatively Area vs. Height is also a way to discriminate doublets and higher clumpsFlowJoSoftware

Cell Cycle Profile of Single CellsFlowJoGated vs. non gated populations Previous plots40028.9%G175.5%S22.6%S14.9%G238.2%G23.84% G110.6% G14.05% G2-0.47% G20.821000single cells300200%G121.8%S48.9%G225.4% G10.8% G23.4950020010000200400600FL2-A: DNA-PI800100000200400600FL2-A: % G118% G29.241000200400600FL3-A: DNA-7AAD400%G164.5%S15.1%G230.1# Cells6008007.35% G17.14% G24.588001000Ungated300# Cells0# Cells400%G1# Cells# Cells600Single cells1500# Cellssingle cellsSoftware200%G112.4%S26.5%G223.8% G12.05% G235.9500200100000200400600FL2-A: DNA-PI80010000200400600FL2-A: FL2-Area800100000200400600FL3-A: DNA-7AAD8001000

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

Cell Cycle Software PackagesGetting Numbers to Fit Your Data Multicycle Modfit FlowJoAll are packages that contain algorithms withdifferent restrictions bound to their calculations.Unfortunately, no algorithms are consistently reliable in fitting all thedistributions you may encounter. You may have to experiment with differentoptions and constraints in finding your best results. (copied FlowJo quote)

Getting To See The NumbersWatson Pragmatic vs. Dean Jett Fox in FlowJo 60018.4600# Cells300# Cells# 40040020010020000200400600FL2-A: DNA-PI1000200400600FL2-A: DNA-PI80010000200400600FL2-A: .53008001000single%G1# Cells600001000single800# Cells800600# 0400600FL2-A: DNA-PI80010000200400600FL2-A: DNA-PI80010000200400600FL2-A: DNA-PI8001000Alice 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 AnalysisHow 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 instrumentSearch for probes that allow to you the best combinations on added color– When you are able to set up your own configurationFirst pick up different lasers then change your dichroics and band filters

Proliferation and BrdUS phase and Doubling TimesK56230min pulse5hrs L2-A80010008hrs -A8001000

Calculating the NumbersGetting the mathematics to work for youFrom: 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 inCell biology 33: 1-12Vindelov.L.L.,Cristensen.IJ. An integrated set of methods for routine flow cytometry DNAAnalysis. 1990. Methods in Cell biology 33: 127-137 Cell Lines (whole cells)––Pozarowwski.P., Darzynkiewiwicz.Z. Analysis of Cell Cycle by Flow Cytometry. 2004. MethodsMol Biol 281:301-311.Traganos.F.,Juan.G.,Darzynkiewiwicz.Z. Cell Cycle Analysis of Drug treated Cells 2001. MethodsMol Biol 95:229-240. Overall–––Rabinovitch.P.S. Practical considerations for DNA Content and Cell Cycle Analysis in ClinicalFlow Cytometry. Principles and applications. Williams and Wilkins-1993Pham.NA., dley.D.W. 2004. The dietaryisothiocyanate sulforaphane targets pathways of apoptosis,cell cycle arrest, and oxidative stress inhuman pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficientmice. Mol.Cancer.Ther.3:1239-1248Bagwell.B. et al. 2004. Optimizing Flow Cytometric DNA ploidy and S phase Fraction asnNegative prosnostic Markers for Node-Negative Breast Cancer Specimens Cytometry 46:121-135

SA2780/DDP-RProliferation DisturbancesArrestControl 2.5 5.0 10 20 40 80 160 320nMA Way to Cope with Stress

ARRESTWhat is it? A slowdown in cell cycle progression? A reversible or irreversible block in one phase ofthe 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 ArrestHow Do You Identify Specific Growth Arrest?ControlCampthothecinCisplatinPaclitaxelCell growth arrest induced by stress or chemical compounds areefficiently identified when cells are growing at their optimallogarithmic rates and inducers are at an equilibrium concentrationwhere cells are halted to activate repair mechanisms or apoptosisCell Questsoftware

Identification of ArrestDRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSUREPaclitaxel a model drug for G2/M eControl 2.55.010204080160320nMControl 2.55.010204080160320nMLaidlaw, 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 arrestand induction of apoptosis.91st Annual meeting of the AACR. San Francisco, April 1-5. 2000.CellQuestsoftware

Identification of ArrestCamptothecin a Model drug for S M30nM30nM10nM010nM3nM20001nM400600800FL2-A: DNA-PI4001nM600Control none10003nM200800More titrationsFlowJoSoftwareFL2-A: DNA-PIControl none1000

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 ofgrowth at a time– Preliminary titration curves done at a 24h time pointprovide good hints of activity

Arrest As An AssayMechanism of Action The Story of BMS-250497– BMS-250497 is the first non camptothecin compounddescribed to be specific for Topoisomerase I inhibition.– Because of the side effects of camptothecin anotherefficient compound with this kind of activity have beenactively pursued by the pharmaceutical industry– Biochemical assays required confirmation at the cellularlevel to reinforce the value of this compound as a candidatefor the clinic

Results on BMS-250749BMS-250749IC50 3 nM Flow cytometryanalysis of A2780cells with wt p53Effects ofBMS-250749 andcamptothecinCamptothecinIC50 10 nMControl0.1x1x5x10x20x IC50CellQuestsoftware-Raventos-Suarez,C., Class. K., Buczek,J,L.,Balasubramanian,N., Long, B., and Menendez, A,T. 2001 Topoisomerase I is theTarget 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 TopoisomeraseActivities Using FACS Analyses.2ISAC XXII International Congress on Analytical Cytology. May22-27 2004 Montpellier,France. Cytometry 58A: p115 Abst# 96207

HCT116(VM46)In Arrest ProfilesHCT116 (VM46)DNA Cell Cycle ProfilesA second parameter reinforced your observationsDNA633nM laserCyclin B1 ProfilesCyclin B1Cyclin EFL2-H: FL2-HeightControl cellsCyclin A10001000800800FL1-H: FL1-Height488nm laser600400600400200200000200400600FL4-A: FL4 A80001000200400600FL4-A: FL4 A8001000Camptothecin treated10001000800800FL1-H: FL1-HeightFL2-H: FL2-HeightrHCT116600400000Blue 488nmRed 633nm400200200Comparison between PI and ToPro3600200400600FL4-A: FL4 A80001000200400600FL4-A: FL4 A8001000

FlowJo Allows Line Graphs Over DNA ProfilesA second parameter always reinforced your observationsCyclin ECyclin 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 efficientlyconfirm at the cellular level effects ofknown activities from a biochemical assay

A2780/DDP-RArrest Leads into ApoptosisControl 2.5 5.0 10 20 40 80 160 320nM

ApoptosisAn active metabolic processdevised to dismiss stressed cellsunable to cope with the insultAn active act of disappearance

Morphology of ApoptosisControlApoptotic

Apoptosis PicturesApoptosis a disappearing population

Evaluation MethodsBest When Linked to DNA profiles Annexin VTdT Tunelp85PARPCaspase 3Many other caspasesOther approaches- The Sub G1 fraction

Tunel AssayProvides Specificity of Phase104104104apoptosissingle cells10101020200400 600FL2-A: DNA-PI8001000102101101010100Dot Plot Overlays200400600FL2-A: DNA-PI8001000103FL1-H: dUTP-FITC102apoptosissingle cells103FL1-H: dUTP-FITC103FL1-H: dUTP-FITCFL1-H: dUTP-FITCapoptosissingle cellsapoptosissingle cells103101041021010100200400 600FL2-A: DNA-PI800100000200400 600FL2-A: DNA-PI8001000Flow Josoftware

p85PARP on Drug EffectsLocalization of populations engaged in apoptosisVinblastin 5nMColchicine 10nM1000010001000FL1-H: P85-FITCFL1-H: P85-FITC10000100100101010200400600FL2-A: PI-DNA800100010200400600FL2-A: PI-DNA8001000Flow Josoftware

The Sub-G1 Fraction***0High dose200400Low dose600FL2-A: DNA-PI800Control1000* Apoptosis by Sub-G1, an increasingly growing population

STaxolA2780/DDP-RA2780/DDP-STaxotereWhen Proliferation, Arrestand Apoptosis MeetControl 2.5 5.0 10 20 40 80 160 320nMControl 2.5 5.0 10 20 40 80 160 320nM

The Story of BMS-214662– BMS-214662 is a farnesyl transferase inhibitor withhigh apoptosis induction capabilities.– Targets on the farnesyl transferase cascade are notexpected to produce any apoptosis in short periods oftime but this compound did it– An assay needed to be devised to stain forproliferation and apoptosis simultaneously to directlyanswer 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 AnalyticalCytology .May 2002 San Diego, California. Cytometry supp11: p72