Proliferation, Arrest and Apoptosis

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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A2780/DDP-R

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

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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

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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

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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

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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

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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

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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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A2780/DDP-R

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

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Control 2.5

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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

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More titrations FlowJo Software

FL2-A: DNA-PI

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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

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Flow cytometry analysis of A2780 cells with wt p53

Effects of BMS-250749 and camptothecin

Camptothecin IC50 = 10 nM

Control

0.1x

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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

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488nm laser

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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

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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

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apoptosis single cells

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p85PARP on Drug Effects Localization of populations engaged in apoptosis Vinblastin 5nM

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The Sub-G1 Fraction * * * 0

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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

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Control

Cisplatin 500nM

Red = non proliferating Blue = proliferating Green = apoptotic non proliferating P+A = proliferating cells engaged in apoptosis

P+A

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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

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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


P388/Topo I (24h)



BMS-250749 IC50=3nM



A2780mutp53 (24h)

A2780mutp53 (48h) Camptothecin IC50= 10nM


BMS-250749 IC50=3nM


A2780mutp53 (24h)

P388/Topo I (48h) Camptothecin IC50= 10nM




P388 (48h)


A2780mutp53 (48h) Camptothecin IC50= 10nM



BMS-250749 IC50=3nM

BMS-250749 IC50=3nM




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