carmen raventos-suarez ph.d. bristol-myers squibb...carmen raventos-suarez ph.d....

Carmen Raventos-Suarez [email protected]

(609-252-6492)

Bristol-Myers Squibb

A2780/DDP-R

DRUG-CELL CYCLE PROFILES AFTER 24HRS EXPOSURE

A2780/DDP-S

Taxol

Taxotere

Control 2.5 5.0 10 20 40 80 160 320nM Control 2.5 5.0 10 20 40 80 160 320nM

When We Study Cells

FACS Answers presented on this workshopProliferation: What When and HowArrest: Why “When” is a key featureApoptosis: Dealing with the kinetics of a

disappearing population

At the microscope we can visualize many structures inside the cells

Photographs from Molecular Probes web site

ProliferationWhat?

A cycling phenomena regulated by check point controlsDNA duplication followed by segregation into two new entities

A2780/DDP-R


A2780/DDP-S

Taxol

Taxotere


Proliferation

• Basic science research– signal transduction evaluation

• Pharmaceutical industry– biochemical to cellular assays

• Clinical follow up– cancer therapeutic efficiencies– inflammatory cascades identification– immune activation responses

When do you measure it?

Cell Cycle Profile Components

G0/G1

S G2/M

DNA (area)

Cell Questsoftware

Sub-G1 Tetraploid

A picture of DNA Synthesis

Cell Cycle Profiles:A Personal Signature of Cells

DNA histograms gives a first hint about cells proliferation potential

DNA-PI

Slow growth Fast growth

Proliferation Features

What does “Cell Cycle Profiles” really mean?

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A2780WT

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FlowJo Software

• DNA content and population distributions

G150%

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24h Cell cycle 12hrs 7.2hrs 4.8hrs

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18h Cell cycle 7.2 hrs 6.8 hrs 3.9hrs 6h additional hours of growth

Number of cells =100

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Cell Cycle Times and Numbers

When A Cell Divides

Modified from Cell Signaling Web site

G1

S

G2/M

Addressing cells one at a time

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

Gated vs. non gated populations Previous plots

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FlowJo Software

Nuclei vs. Whole Cells

Nuclei

•Ploidy paraffin blocks

•Apoptosis problems

•Nuclear localization

Whole cells

•Ploidy fresh samples

•Apoptosis efficiency

•Cytoplasmic markers.

What is better?

Cell Cycle Software Packages

• 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 thedistributions you may encounter. You may have to experiment with differentoptions and constraints in finding your best results. (copied FlowJo quote)

Getting Numbers to Fit Your Data

Getting To See The NumbersWatson Pragmatic vs. Dean Jett Fox in FlowJo software

Alice Givan previously showed the Mod Fit variations on their selective options

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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

• 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

Beyond DNA Quantitative Analysis

How to add more significance to your data?

Proliferation and BrdU

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A2780S

30min pulse 5hrs release 8hrs release

FlowJosoftware

S phase and Doubling Times

Calculating the Numbers

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)

Getting the mathematics to work for you

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: 1-12

– Vindelov.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. 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 inhuman pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficientmice. Mol.Cancer.Ther.3:1239-1248

– Bagwell.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

Proliferation Disturbances

Arrest

A Way to Cope with Stress

A2780/DDP-R


A2780/DDP-S

Taxol

Taxotere


• 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?

ARRESTWhat is it?

The Meaning of Cell Cycle 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 Questsoftware

How Do You Identify Specific Growth Arrest?

A2780/DDP-R


A2780/DDP-S

Taxol

Taxotere


CellQuest software

Identification of Arrest

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.

Titrations

Paclitaxel a model drug for G2/M arrest

FlowJo Software

LnCaPCamptothecin

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A2780Camptothecin

More titrations

Camptothecin a Model drug for S arrest?

Identification of Arrest

• 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

How to Evaluate a Compound

Arrest As An Assay

• 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 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

Mechanism of Action

Results on BMS-250749

Control 0.1x 1x 5x 10x 20x IC50

• Flow cytometryanalysis of A2780cells with wt p53

• Effects ofBMS-250749 andcamptothecin

CamptothecinIC50 = 10 nM

BMS-250749IC50 = 3 nM

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

In Arrest Profiles

488nm laser 633nM laser 488nm laser 633nM laser

HCT116 HCT116 (VM46)

DNA Cell Cycle Profiles

HCT116 HCT116(VM46)

Cyclin B1 Profiles

Comparison between PI and ToPro3 Blue= 488nm Red=633nm

DNA Cyclin B1 Cyclin E Cyclin A

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A second parameter reinforced your observations

FlowJo Allows Line Graphs Over DNA Profiles

Cyclin E

Cyclin A

A second parameter always reinforced your observations

• 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

Results on BMS-250749

Arrest Leads into Apoptosis

A2780/DDP-R


A2780/DDP-S

Taxol

Taxotere


Apoptosis

An active metabolic processdevised 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

• Annexin V• TdT Tunel• p85PARP• Caspase 3• Many other caspases• Other approaches- The Sub G1 fraction

Best When Linked to DNA profiles

Tunel Assay

Flow Josoftware

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Dot Plot Overlays

Provides Specificity of Phase

p85PARP on Drug Effects

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Vinblastin 5nM Colchicine 10nM

Flow Josoftware

Localization of populations engaged in apoptosis

Apoptosis by Sub-G1, an increasingly growing population

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The Sub-G1 Fraction

When Proliferation, Arrestand Apoptosis Meet

A2780/DDP-R


A2780/DDP-S

Taxol

Taxotere


A2780/DDP-R


A2780/DDP-S

Taxol

Taxotere


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 non-proliferating tumor cells. Demonstration by a new flow cytometric method. ISAC XXI International Congress on AnalyticalCytology .May 2002 San Diego, California. Cytometry supp11: p72

Building An Assay

• Proliferation by BrdU Uptake andevaluation by Dnase treatment: modify theBrdU 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”

Cisplatin 500nM BMS-214662 9uM

Proliferation and Apoptosis

P

A P+A A P+A

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Red = non proliferatingBlue = proliferatingGreen = apoptotic non proliferatingP+A = proliferating cells engaged in apoptosis

Control

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Mechanism of Action of BMS-214662

Take Home Messages

• Cell Cycle Profiles are the best handle inaddressing cell behavior, capacity to respond toan stimulus and possible engagement inapoptosis

• Careful preparation of cells is essential

• Multiparameter analysis linked to cell cycleprofiles provide a more complete picture ofcellular activities

Back up data

Cell Cycle Profile of CellsGetting to see the numbers

When algorithms don’t seem to fit you can force constrains

FlowJo Software

Cell Cycle Profile of CellsA cell specific signature

Why are times at logarithmic rate of growth taken as optimal?

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HCT116

HCT116(VP35)

HCT116(VM46)

A2780/DDP-R

A2780/Tax

A2780/DDP-S

Panel of cell lines growing at logarithmic growth ratesFlowJo Software

Control 0.1x 1x 5x 10x 20xIC50 Control 0.1x 1x 5x 10x 20xIC50

P388 (24h) P388 (48h)

Camptothecin IC50= 10nM

BMS-250749 IC50=3nM

Etoposide IC50= 1.2uM


P388/Topo I (48h)P388/Topo I (24h)


BMS-250749 IC50=3nM



HCT116/TopoII (24h) HCT116/TopoII (48h)


BMS-250749 IC50=3nM



HCT116 (48h)


BMS-250749 IC50=3nM


HCT116 (24h)


A2780mutp53 (24h) A2780mutp53 (48h)


BMS-250749 IC50=3nM



A2780mutp53 (24h) A2780mutp53 (48h)


BMS-250749 IC50=3nM


Results on BMS-250749 Six cell lines two time points plus two control drugs

It takes a panel of comparisons to address population distribution effectsShown are Cells in 3 pairs:sensitive/resistant (TopoII, TopoI and p53)

• Proliferation activities require more than DNAmeasurements 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 beenable to go before stop growing

– Specific altered functions by induced by resistance ormutation mechanisms

Proliferation What and WhenBeyond DNA quantitative analysis

Control Camptothecin Etoposide TAS-103

Control Camptothecin Etoposide TAS-103

More on Mechanism of Action Specificity on Topo II

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.

A Camptothecin ResistantCell line

A Camptothecin SensitiveCell line

Cell Questsoftware

carmen raventos-suarez ph.d. bristol-myers squibb...carmen raventos-suarez ph.d....

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