Application Note

HORIZON DISCOVERY

IntroductionApoptosis or programmed cell death is a highly regulated process,essential for the development and homeostasis of all multicellularorganisms. It is also an important mechanism of tumour suppression:the failure of cells to undergo apoptosis is a key element oftumorigenesis. Conversely, an excess level of apoptosis is a feature ofpathologies such as neurodegenerative disease and autoimmunity.

Two main pathways are known to trigger apoptosis: an intrinsic and anextrinsic pathway. The key event downstream of both pathways is theactivation of the proteolytic caspases cascade, which causes specificmolecular changes together with morphological changes, such as cellshrinkage, nuclear condensation and fragmentation, membraneblebbing and the production of apoptotic bodies. In healthy tissueswhere levels of apoptosis are low, dying cells are efficientlyphagocytosed. In diseases where levels of apoptosis are high, not allapoptotic cells are cleared by phagocytosis and these cells undergosecondary necrosis.

In this study, we analysed the ability of two drugs, staurosporine andetoposide, to induce apoptosis in different cancer cell lines.

Silvia Vivarelli and Annette Little

Detecting the onset and kinetics of apoptosis

Case Study: Detecting Apoptosis in vitroIn this study we used three different readouts to assess the kinetics ofapoptosis in response to staurosporine and etoposide:• IncuCyte live cell imaging to track the appearance of a fluorescent

caspase-3/7 dye• Multiplexing a fluorescent cell viability dye with an apoptosis-

caspase-3/7 luminescent dye in a plate reader-based assay• Analysing early and late apoptotic cell markers using western blotting

and flow cytometry

Results and DiscussionMonitoring apoptosis using live cell imaging

The Cell Player Kinetic Caspase-3/7 reagent (Essen Bioscience) couplesthe activated caspase-3 and caspase-7 amino acid recognition motif(DEVD) to a green fluorescent DNA intercalating dye. This substrate isnon-fluorescent unless the DEVD sequence is cleaved by activecaspase-3 and/or caspase-7 , so it can be added directly to the cellswithout perturbing their growth or morphology. Once in the medium,the substrate crosses the cell membrane where it is cleaved byactivated caspase-3 and/or caspase-7 resulting in the release of the dyeand green fluorescent staining of nuclear DNA. Therefore, the kineticactivation of caspase-3 and/or caspase-7 can be monitoredmorphologically following the variation of the green florescence withlive cell imaging using an IncuCyte microscope.

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Caspase 3/7 (Cell Player) Phase objects count

Figure 1. MDA-MB-231 cells were treated with staurosporine (A,C)

or etoposide (B,D) in the presence of CellPlayer Caspase-3/7. The

plate was imaged every 4 h using the IncuCyte Zoom timelapse

microscope.

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Cell Line Genotype Cat. No.

MDA-MB-231 Parental HD PAR-402

HCT116 Parental HD PAR-007

HCT116 CHEK2-/- HD R02-017

HCT116 CHEK2-/-; TP53-/- HD R02-018

Cell Lines Used

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0 hours 24 hours 48 hours 72 hours

Figure 2. MDA-MB-231 cells were treated with staurosporine 1 mM

(A) or etoposide 10 mM (B) in the presence of CellPlayer Caspase-

3/7. Representative images taken at 0, 24, 48 and 72 h after

treatment (IncuCyte Zoom microscope images of cells in individual

wells).

A

B

The fluorescent cell count increases with increasing drug doses (A, B),compared with the confluence of the cells detected using phasecontrast (C, D).

Both staurosporine (A) and etoposide (B) induce apoptosis, but withdiffering kinetics. Staurosporine induces detectable caspase-3/7activity after 24 hours (A), whereas fluorescent cells are not visibleuntil 48 hours post treatment with etoposide (B).

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Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

+ 44 (0)1223 655580

+ 44 (0)1223 655581

info@horizondiscovery.com

www.horizondiscovery.com

ConclusionHorizon Discovery offers several assays and techniques that can assessthe induction of apoptosis. We offer live cell monitoring to assess thekinetics of apoptosis induced by a compound of interest. Moreover,Horizon can multiplex assays, such as cell viability, cell proliferation andapoptosis, to attain more data from an individual experiment. Finally,Horizon can include a number of additional assays to detect early andlate apoptotic markers (flow cytometry, western blotting and qPCR, forexample).

Other Assays of Interest• 2D and 3D cell proliferation assays

• Confocal imaging service

• HT drug combination screening service

• Immuno-Oncology Assays

© 2016 v-01

Analysing apoptotic markers using flow cytometry and western blot

The AnnexinV–Alexa Fluor 488 kit detects the external exposure ofphosphatidylserine on the cell membrane, an early apoptotic changethat occurs before the integrity of the cell membrane is compromised.Propidium iodide is an intercalating DNA dye that can only cross the cellmembrane once its integrity has been lost, thus the level of signal isproportional to the degree of cell membrane integrity. Using bothmarkers allows early and late markers apoptotic cell membraneintegrity to be measured.

Cleaved poly ADP-ribose polymerase (PARP) and caspase 3, detectedusing western blot, are middle-to-late markers of apoptosis that aregenerated as a consequence of activation of the caspase cascade.

Multiplexing apoptosis with viability detection

Multiplexing cell viability and apoptosis endpoints requires twosequential steps. First, the CellTiter-Blue Viability Assay (Promega) isused to determine the number of viable cells. When added to cells inculture, the resazurin substrate is reduced to resorufin, which is thefluorescent end substrate of the assay. The number of metabolicallyactive cells is determined by the level of resazurin reduction. Second,the Caspase 3/7-Glo Assay (Promega) is used to measure caspase 3/7activity using a DEVD tagged luciferine substrate. Adding the reagent tothe wells results in cell lysis, followed by caspase cleavage of thesubstrate. This cleavage liberates free aminoluciferin, a substrate forluciferase, resulting in the generation of a luminescent signal that isproportional to the caspase 3/7 activity present.

Figure 3. MDA-MB-231 cells were treated with etoposide (0, 1, 3 and

10 mM). After 72 hours, cells were incubated with CellTiter-Blue to

detect viability and with Caspase-3/7 Glo to detect apoptosis

(caspase-3/7 positive cells) in two sequential steps.

Viability

Apoptosis

Figure 4A. Flow cytometry - MDA-MB-231 cells were treated with

staurosporine (0, 1 mM). After 24 h cells were harvested and stained

using the Annexin V - Alexa Fluor 488 conjugate (apoptosis) and

Propidium iodide (Pi) Staining Solution (cell death). Data were

collected using a FACS-verse instrument (BD Biosciences).

Figure 4B. Western blot - MDA-MB-231 cells were treated with

staurosporine (0, 0.1, 1 mM). After 24 h cells were harvested and

total protein extracts probed for both PARP (full length versus

cleaved) and activated caspase 3.

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DMSO STAUROSPORINE 1mM

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β-actin

Full Length PARP

Cleaved PARP

Cleaved Caspase 3

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β-actin

Full Length PARP

Cleaved PARP

Cleaved Caspase 3

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HCT116 PAR

HCT116 CHEK2-/-

HCT116 CHEK2-/-TP53-/-

STAUROSPORINE

Figure 5. HCT116 parental and isogenic cell lines were treated with

staurosporine (0, 0.1, 1 mM). After 24 h cells were harvested and

total protein extracts probed for both PARP (full length versus

cleaved) and activated caspase 3.

The addition of increasing concentrations of etoposide decreases cellviability and increases apoptosis.

Flow cytometry: After 24 hours of drug exposure, levels of both annexinV and propidium iodide increase, indicating the presence of an early andlate apoptotic cell population.

Western blot: After 24 hours of drug exposure, both cleaved PARP andcleaved caspase 3 increase in a dose dependent manner.

After 24 hours of treatment with staurosporine, both cleaved PARP andcleaved caspase 3 levels increase in a dose dependent manner. Theabundance of the cleaved proteins varies in the isogenic cell linescompared with the parental (PAR) cells (stronger in CHEK2-/- cells andweaker in CHEK2-/- TP53-/- cells).