Cell Proliferation and Apoptosis: Two Sides of a Coin

Monisha Sundarrajan, PhDSenior Scientist

Research Applications Support

For Research Use Only. Not for use in diagnostic or therapeutic

procedures.

3

Cell Proliferation

Cell proliferation is defined as an increase in the number of cells as a result of cell growth and division.

Uncontrolled cell growth or proliferation is the hallmark of cancer cells.

4

The Cell Cycle and its Phases

MM

GG11

SS

GG00

cdk2

cyclin E

Cdk

4,6

cyclin D

cdc2

cyclin A,B

p27

p53

p21

GG22

5

Cell Proliferation Application Decision Tree

Cell proliferation

Bioimaging/IHC

BrdU incorporation

Ki-67, p-Histone H3,cyclins, etc

CBA Phosphorylations as indicators of cell proliferation/cytokine production

Live

BD Horizon™Violet Proliferation Dye 450

7-AAD and PI

Fixed

BrdU incorporation

Ki-67, p-Histone H3, PCNA, cyclins, RB, cytokines

Western blot/IP

Cell cycle markersp-Histone H3, PCNA

Fixed

BrdU incorporation, Ki-67, p-Histone, cyclins, etc

Intact cells Cell extracts Tissue sections

Treg suppression assay/functional assay suppression of effector cells assessed by CD25/CD69 expression

Phosphorylations as indicators of cell proliferation

Bioimaging/microscopy

Flow cytometry

6VPD 450

Day 1

Day 2

Day 3

Cell Proliferation Assessment Using Violet Proliferation Dye 450 (VPD 450)

Experimental design:

Enrich mouse spleen by positive selection via CD4+

enrichment.

Load isolated cells with VPD 450, 1 μM, for 10 minutes.

Harvest CD3/CD28 stimulated cells on the days indicated.

Analyze by flow cytometry.

7

Day 1

Day 2

Day 3

Condition: CD3/CD28

VPD 450

IL-2

Alexa

Fluor

®48

8

Experimental design:

Enrich Balb/c spleen by positive selection via CD4+

enrichment.

Load isolated cells with VPD 450, 1 μM for 10 minutes.

Stimulate cells with soluble antiwith soluble anti--

CD3/CD28 (1ug) in the presence of transport inhibitor.

Fix/perm stain cells.

Simultaneous Assessment of Cell Proliferation and IL-2 Secreted by Cells During T-cell Stimulation

8

Cell Cycle Analysis of Population Stained for Incorporated BrdU and Total DNA Levels (7-AAD)

Human PBMCs were stimulated with anti-CD3/CD28 for 48 hours and re-stimulated with PMA+Ionomycin for 4 hours, and BrdU was added for the final 1 hour. Cells were then harvested and stained using the BrdU staining protocol.

BrdU

-FIT

C

BrdU

-APC

Sub-G0/G1

S phaseS phase

G2+M phase

G0/G1

Sub-G0/G1

G2+M phase

G0/G1

9

Stimulated Splenocytes Assessed for Cell Proliferation Using VPD 450 and anti-BrdU Ab Simultaneously

Experimental design:

Mouse splenocytes were incubated with 1 μMVPD 450 for 10 minutes and stimulated with anti-CD3/CD28 for 48 hours. Cells were pulsedwith BrdU for 1 hour, prior to harvest.

Cells were harvested and stained using theBrdU

staining protocol and analyzed by flowcytometry.

VPD 450

VPD 450

BrdU

-APC

Condition: CD3/CD28 Stimulation for 48 hours

10

Ki-67: Another Marker for Cell Proliferation

Ki-67 is expressed in Go/G1 (P5 gated cells) and post mitotic G2/M phase (P4 gated) cells (data generated at BD Biosciences).

Ki-67 Alexa Fluor®

700

BrdU

Per

CP-C

y™5.

5

Ki-6

7 A

lexa

Flu

or®

700

DAPI-ADAPI-ASSC-A DAPI-A

BrdU

Per

CP-C

y5.5

FSC-

A

DA

PI-

W

G2/MG0/G1

S phase

11

Cell Cycle Analysis on HeLa Cells Treated with Aphidicolin (DNA Topisomerase α

Inhibitor) Monitored by BrdU

Staining

No treatment 1 μg Aphidicolin

BrdU

0

10

20

30

40

50

60

1.00E-09 1.00E-08 1.00E-07 1.00E-06 1.00E-05

Dose (on log scale)

% B

rdU

pos

itive EC50:

1.92E-07

The images were captured on a BD Pathway™

855 bioimaging system with a 20x objective and merged using BD Attovision™

software.

Hoechst

-

blueBrdU

-

redHistone

-

yellowTubulin

-

green

12

Cell Proliferation Assessed in Mouse Small Intestinal Sections by BrdU Staining

The images were captured on a BD Pathway™

435 bioimaging system with a 20x objective and merged using BD Attovision software.

Actin

-

greenβ-Tubulin-

redBrdU

–

yellowHoechst-

blue

13

•

Characteristics of Tregs:

proliferate very slowly

•

Hallmark assays to assess Treg functionsSuppress proliferation in effector cellsSuppress cytokine production by effector cells

•

Salient markers for Tregs–

Surface: CD4, CD25, CD127–

Intracellular: FoxP3

Regulatory T Cells (Tregs)

Regulatory T cells, also called “Tregs,”

play an important role in maintaining immunological unresponsiveness

to self antigens (self tolerance) and control of immune responses to foreign antigens.

14

Experimental design

Mouse splenocytes were stimulated with CD3/CD28

aand cultured

for 5 days. At the end of 5 days, cells were further cultured and then re-stimulated for 5 hours with PMA+Ionomycin. The cells were incubated with 100 μM BrdU for the final hour of culture and then harvested.

Following harvesting, the cells were stained with anti-CD4 PerCP-Cy5.5 and Foxp3 Alexa Fluor®

647 using the Foxp3 staining protocol.

Upon completion of FoxP3 staining, the cells were refixed and permeabilized

using BD Cytofix/Cytoperm™

buffer and stained with anti-BrdU Ab, allowing the detection of incorporated BrdU.

Staining of anti-BrdU with Foxp3 for Detection of Cell Proliferation in Mouse Tregs

BrdU FITC

Foxp

3–A

lexa

Flu

or®

647

2.3%

CD4 PerCP-Cy5.5

Foxp

3–A

lexa

Flu

or®

647

2.4% 0.4%

10.3%

15VPD

450

Foxp

3 Alexa

Fluor

®64

7

Day 1

Day 2

Day 3

Significant Insights into the Mechanism of Treg Proliferation as Assessed by Violet Proliferation

Dye 450 (VPD 450)

Experimental design

Enrich mouse splenocytes by positive selection via CD4+

enrichment.

Load isolated cells with VPD 450, 1 μM, for 10 minutes. CD3/CD28 stimulated cells were harvested on days as indicated

Fix and permeabilize cells using the Foxp3 staining protocol.

Condition: CD3/CD28

16

Expression of Ki-67 in Human Tregs

CD4+FoxP3+

2.9%

0.9%5.7% Ki-67+FoxP3+

Human PBMCs were stained for Ki-67 and FoxP3 using the FoxP3 staining protocol.

SSC

FoxP

3 PE

FSC

Ki-67 Alexa Fluor®

488

CD4 PerCP-Cy5.5

Ki-67-FoxP3+

CD4 PerCP-Cy5.5

FoxP

3 PE

SSC

17

Treg Suppression Assay Kit:Treg Suppression Assay Kit:

how the assay works

•

Tregs are sorted using CD4+, CD25+, CD127low/dim

and CD45RA+.

•

Cells are expanded in culture for 13 days.

•

Expanded Tregs are placed with effector cells (autologous PBMCs)

in the presence of T-cell specific stimulus (SEB, CD3/CD28, CD2/CD2R).

•

After 7 hours, the frequency of CD69-positive and/or CD154-positive effector T cells (response) is measured in the presence and absence of Tregs.

•

CD25 is used to identify and exclude Tregs during analysis.

•

The percent suppression of the response is calculated.

New Assay to Assess Treg Function: Suppression of Effector Cells

18

Treg-mediated Suppression of CD154 and CD69 in PBMCs Stimulated with CD3/CD28

CD4- CD4+

Tregs CD154

CD69

CD25

14.4%

34.1%MFI=6693

MFI=2597

CD154

CD69

22.6%

42.7%MFI=8943

MFI=3704

Cultured without Tregs Cultured with Tregs

19

Apoptosis

Definition:

The process leading to controlled self- destruction of a cell.

Cells undergo death neatly

without damaging their neighbors. Apoptosis is a “programmed event.”

20

Importance of Apoptosis•

Development–

Organs, appendages, patterning–

Thymic selection (lymphocyte development)•

Tissue homeostasis–

Tumor •

Cell termination–

Viral infection, cancer

21

Apoptotic Signaling Pathways

Fas

Procaspase 3

Caspase 8

Caspase 3

Bid

tBid

FADD

Procaspase 8

Bax/Bak

Procaspase 9Caspase 9

Procaspase 3

Caspase 3

Cytochrome c

Growth factor withdrawalIrradiationLoss of matrix contactGlucocorticoids

?

Bcl-2Bcl-X

DNA fragmentation

iCAD

CAD

Apaf-1↓Ψm

EXTRINSIC PATHWAY INTRINSIC PATHWAY

C-FLIPIAP’s

Smac

bdbiosciences.com/pathways

22

Hallmarks of Apoptosis• Plasma membrane alterations• Mitochondrial changes• Activation of caspases• DNA fragmentation

23

Apoptosis Application Decision TreeApoptotic cells

IHCActiveCaspase-3

Cleaved PARP

ELISA

ActiveCaspase-3

Cleaved PARP

Bioimaging/microscopyFlow cytometry

SpectrofluorometryCaspase activity

Live

Loss of membrane asymmetry-Annexin

V

Mitochondrial membrane potential-BD™

Mitoscreen

Fixed

Cleaved markers-Active Caspase3-Cleaved PARP

Caspase inhibitors-FAM-VAD-FMK

TUNEL/DNAfragmentation-APO-Direct-APO-BrdU

Western blot/IPApoptosis markers

Live

Annexin V

Fixed

Active Caspase-3Cleaved PARP

Caspase inhibitors-FAM-VAD-FMK

Intact cells Cell extracts Tissue sections

Caspase inhibitors-FAM-VAD-FMK

24

Apoptosis: Scatter Properties

Formation of apoptotic vesicles •Increases side scatter

Reduced refractive index of apoptotic cells •Decreases forward scatter

Cell shrinkage during apoptosis is associated with a decrease in

forward scatter. Analysis of light scatter is often combined with other assays.

Untreated Camptothecin treated

25

Annexin VAnnexin V is a surface marker and detects early membrane changes

associated with apoptosis.Pros:

Rapid confirmation of apoptosisUses live, unfixed cells

Applications•Flow cytometry (cells in suspension)•Fluorescence microscopy (adherent cells)

26

Detection of Membrane Changes by Annexin V Staining and Analysis by Flow Cytometry

V450-Annexin V

Rel

ativ

e C

ell N

umbe

r

Jurkat T cells were treated with 6 μM camptothecin for 4 hours. Cells were incubated with BD Horizon™

V450 Annexin V and analyzed by flow cytometry.

Untreated Camptothecin treated

V450-Annexin V

27

Detection of Changes in Mitochondrial Membrane Potential (JC-1)

JC-1: lipophilic cationic dye fluorescence is detected on a flow cytometer

J-aggregates (healthy cell)

monomers (apoptosis indicator)

J-aggregates (healthy cell)

monomers (apoptosis indicator)

28

Detection of Active Caspase 3: “Executioner”

of Apoptosis and its By-product: Cleaved PARP

TreatedTreatedUntreated Untreated

Jurkat T cells were treated with camptothecin, fixed and permeabilized with BD Cytofix/Cytoperm

buffer, and subsequently stained for active caspase 3 using anti-caspase Ab or cleaved PARP.

Untreated/control

Camptothecin treated (6 μM)

Caspase-3

Cleaved-PARP

29

Cleaved PARP Expression in Formalin-fixed, Paraffin-embedded Rat Lymph Node

Rat lymph nodes were stained with monoclonal cleaved PARP specific Ab F21-

852 using the biotin, streptavidin three-step detection method.

30

Detection of DNA Fragmentation During Apoptosis by “End Labeling”

or “TUNEL”

Using

the APO-DIRECT™

Kit

NonNon--

apoptotic apoptotic cellscells

NonNon--

apoptotic apoptotic cellscells

Positive apoptotic cells in SPositive apoptotic cells in S--

phasephase

31

Significant Tool in Drug Discovery Research: Assessing Cell Proliferation, DNA Damage, and

Apoptosis Using Flow Cytometry

`̀

3% 24%

14%59%

12% 31%

6%51%

Phos

H2A

X-A

PC

Untreated

15% 6%

19%60%

3% 1%

64%32%

Clea

ved

PARP

–APC

BrdU Alexa Fluor®

488

Camptothecin treatment, 5 μM

Experimental design

HeLa cells were untreated or treated with camptothecin 5 μM and BrdU for 4 hours.

Cells were then harvested and stained with anti-BrdU, H2AX, and cleaved PARP using the BrdU staining protocol.

32

Increase in Camptothecin Concentration and Incubation Time Leads to Increased H2AX and PARP Expression

and Loss of BrdU Incorporation

Phos

H2A

X A

PC

Clea

ved

PARP

APC

BrdU Alexa Fluor®

488

Camptothecin treatment 20 μM Experimental design

HeLa cells were untreated or treated with camptothecin,

20 μM, for 24 hours, further incubated for 48 hours post washing, and pulsed with BrdU for the final 1 hour.

Cells were then harvested and stained with anti-BrdU, H2AX, and cleaved PARP using the BrdU staining protocol.

32%0%

0%68%

49%

BrdU Alexa Fluor®

488

33

Acknowledgments

Jeanne Elia Jurg Rohrer Joyce Ruitenberg

Smita Ghanekar Christopher Boyce

Ravi Hingorani Cynthia Lane

Natalie Golts Martha Wilkinson

and others.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.Alexa Fluor® is a registered trademark of Molecular Probes, Inc.Cy™ is a trademark of Amersham Biosciences Corp.BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2010 BD23-12232-00