Quantitative Analysis of Drug Effects on Cytoskeletal Structure, Cell Morphology and the Cell Cycle by High-Content Assays
Suk J. Hong and Richik N. Ghosh
We have developed a portfolio of Cellomics HCS kits to identify individual
cells’ cell cycle phase and proliferation state, using a fixed end-point HCS
assay based on immunofluorescence detection in cells grown on standard
high-density microplates. These kits all contain the DNA binding dye
DAPI which is used to determine the cell cycle phase in individual cells
by DNA content, and this is verified by correlating the expression level
or phosphorylation state of other cell cycle associated indicators. Cells
which have incorporated BrdU into DNA can be quickly detected by using
monoclonal antibody directed against BrdU and fluorophore-conjugated
secondary antibody. BrdU staining facilitates the identification of cells that
have progressed through the S-phase of the cell cycle during the BrdUlabeling period. The Thermo Scientific Cellomics BrdU Cell Proliferation
HCS kits have been specifically designed to enable simple BrdU detection
in nuclear DNA without extreme treatments to the cell. This enables
multiplexed detection of BrdU with other targets in the same cells. We also
developed several other cell cycle assay targets, which include Cyclin
B1, phosphorylated polo-like kinase 1 (phospho-PLK1), phosphorylated
retinoblastoma protein (phospho-Rb) and phosphorylated Histone H3.
These kits enable different options to directly determine individual cells’ cell
cycle phase, such as the G1, S phase (DNA content & BrdU) and G2/M
phase (Cyclin B1, Phospho-PLK1, Phospho-Rb and mitosis specific Histone
H3 phosphorylation).
The intracellular cytoskeletal meshwork is responsible for maintaining cell
shape, cell movement, cytokinesis, and the organization of organelles within
the cell. The dynamic network of the cytoskeleton also facilitates proper
function of other cellular proteins through direct binding, transportation,
repositioning and sequestration of these proteins. The structure of the
cytoskeleton is controlled by bundling, aligning and repositioning of the
filaments through cytoskeleton-associated proteins in response to the
external signaling. Alterations of cytoskeletal structure are often associated
with pathologies and cell death. Signaling defects in conjunction with
cytoskeletal rearrangement can also contribute to increased cell proliferation
rate and tumor cell motility which result in metastasis. Thus, the key players
controlling cytoskeletal rearrangement in the cell are potential therapeutic
targets.
Thermo Scientific, Inc. provides an integrated set of products that work
together to deliver a “total solution” platform for HCS. Integral components
of this set are the Thermo Scientific Cellomics HCS Reagent Kits. These
kits provide easy-to-use methods and reagents for preparing highquality samples for automated cell-based imaging assays. Using Thermo
Scientific Cellomics Cytoskeleton Rearrangement kit and other Cell
Cycle kits, we have developed quantitative HCS assays for assessing
the cytoskeletal rearrangement and the cell cycle phases in cells treated
with different compounds. The Cellomics HCS kits makes these assays
easy to implement, and provides a powerful set of multiplexed parameters
for monitoring the response of the cell due to drug treatment in these
key functional areas. All images and data shown were acquired and
quantitatively analyzed using Thermo Scientific’s Cellomics ArrayScan®
HCS Reader with associated Cellomics BioApplication image processing
software modules.
p-PLK1 Activation Assay
Cyclin B1 Activation Assay
Nucleus
Cyclin B1
M
G1 Phase
M Phase
M Phase
Control
Nuclei
p-Plk1
Cytochalasin D
G1
G2 Phase
G2
G2
S
G2 Phase
M
G2 Phase
M
B.
B.
Cytoskeleton Rerrangement by Cytochalasin D
Control
Microtubules Fiber
20
C.
Dose Response of Cyclin B1
Cytochalasin D
10
Figure 5: Aphidicolin and Cycloheximide treatment inhibit both BrdU and phospho-Histone
H3. Nocodazole increases phospho-Histone H3 positive cells, which indicates the cells are
in G2/M phase.
0
Dose Response of Phospho-Plk1
0
10
75
50
25
0
75
-5
-4
-3
-2
-1
0
1
2
50
3
Dose Response Curve
-4
-3
-2
-1
0
1
Log [M], Compounds
Vinblastine, EC50 = 9.35 nM (A549 cell)
Vinblastine, EC50 = 11.0 nM (HeLa cell)
Nocodazole, EC50 = 54.9 nM (A549 cell)
Nocodazole, EC50 = 43.6 nM (A549 cell)
ICRF-193, EC50 = 10.3 M (IMR-90 cell)
Multiparameter Quantification of Cell Structures
and Cell Cycle Markers
A.
75
50
Cytoskeleton Rearrangement Detection
Perimeter
F-actin
Microtubule
-3.5
-2.5
-1.5
-0.5
0.5
1.5
2.5
Z' factors
0.12
80
0.18
60
0
0.46
0.44
0.41
0
1
0.51
0.49
0.48
0.44
0.53
2
0.52
0.54
3
0.53
4
Time (h)
Log [  M], Cytochalasin D
IC50
0.37
0.42
40
20
0
-4.5
Figure 1: (A) A549 cells stained with the new Thermo Scientific Cellomics HCS kits for
Cyclin B1 and phospho-PLK1. The Cyclin B1 and phospho-PLK1 are shown in green, and
the DAPI staining is in blue. The individual cells’ cell cycle phases are determined by the
expression level (i.e. fluorescence intensity) and the localization of the cell cycle target
(Cyclin B1 or phospho-PLK1) correlated with the DNA content. (B) Population analysis of
Cyclin B1 and phospho-PLK1 fluorescence intensity vs DNA content in individual cell levels
after Nocodazole treatment. Nocodazole treatment causes cells to be blocked at G2/M, and
have double the DNA content and high levels of Cyclin B1 and phospho-PLK1. (C) Dose
response analyses of Cyclin B1 and phospho-PLK1 for various drugs that cause inhibiton at
G2/M phase.
Cell Cycle Kit I – BrdU
100
100
25
SK&F96365, EC50 = 2.74 M (A549 cell)
Population Analysis of the Changes in Cell Cycle
Phase After Cell Cycle Inhibitory Drug Treatments
Time Course Response, 10 μM Cytochalasin D
120
-5
Log [M], Compounds
30
C.
25
-6
20
F-actin Fiber
100
100
% Control
Cell stress, cell proliferation and the characterization of agents that either
promote or inhibit cell proliferation are particularly of interest in cell biology
and drug-discovery research. High content analysis (HCA) involves a
fluorescence cell-based assay where the cells are automatically imaged and
analyzed using quantitative fluorescence microscopy. HCA can be used to
quantify the cell proliferation activity by measuring their DNA content, the
state of cell cycle-associated proteins, and morphological and structural
changes in individual cells and in cell populations.
Cell Cycle Markers, BrdU and Phospho-Histone
H3 are Affected by Cell Cycle Inhibitory Drugs
A.
% Control
Introduction:
Analysis of Cellular Structures Followed by
Cytochalasin D (F-actin Drug) Treatment
A.
% Control
Being fundamental and ubiquitous features of proper cellular function,
cytoskeletal rearrangement and cell cycle progression are sensitive
indicators of cell growth and cell stress. Previously, we developed a
number of multiplexed, quantitative, cell-based, high-content screening
(HCS) assays to monitor these key cellular functions using a Thermo
Scientific Cellomics ArrayScan® HCS Reader. For example, the Thermo
Scientific Cellomics Cytoskeletal Rearrangement HCS Kit simultaneously
detects changes in cytoskeletal structures (F-actin and microtubule fibers),
nuclear DNA content, and nuclear and cellular morphologies using bright
fluorescent probes. The Thermo Scientific Cellomics Cell Cycle I HCS Kit
characterizes cell cycle progression by simultaneously quantifying DNA
content, BrdU incorporation and Histone H3 phosphorylation. Here we
demonstrate the value of using these two multiparameter kits together. By
assessing the cell cycle using BrdU and phospho-Histone H3 antibodies
while simultaneously monitoring cytoskeletal rearrangements and cell
morphological changes, we established a multiparameter profile of different
drugs affecting cell cycle, cell growth, cell differentiation and cell death. A
variety of cells were treated with different actin or tubulin affecting-drugs
or cell cycle inhibitors, and then monitored the drug effect on cellular and
nuclear shape, cytoskeletal structure, and cell cycle phases. Evaluation
of drugs using these multiplexed cytoskeletal and cell cycle assays is a
simple and easy-to-implement method for compound toxicity profiling in high
content screening.
New HCS Assays for Cyclin B1 Activation and
Phospho-PLK1 Activation
% Control
Abstract:
Thermo Fisher Scientific • 3747 N. Meridian Rd., PO Box 117, Rockford, Illinois U.S.A. 61105
Perimeter = 0.751 ± 0.024 μM
F-actin = 0.104 ± 0.010 μM
Tubulin = 0.510 ± 0.066 μM
Cell Cycle Kit I – Phospho-Histone H3
Figure 3: (A) Cell images of F-actin (green), microtubules (red), and nuclei (blue) in NIH 3T3
cells with or without Cytochalasin D treatment (20 μM, 3 hrs). (B) The numbers of F-actin
fibers and microtubule fibers are decreased by Cytochalasin D treatment (20 μM, 3 hrs) –
individual cell are plotted. (C) Dose response and time course response of Cytochalasin D
on the cell morphology, and the number of F-actin and microtubule fibers.
Cytoskeletal Structures are Affected by Cell
Cycle Inhibitory Drugs
The Effects of Cell Cycle Inhibitors on Cytoskeleton Structure
Control
Nocodazole
Vinblastine
Figure 6: Inhibition of BrdU incorporation in cells by the G1/S blocker, Aphidicolin (strong
inhibition) and the G2/M blocker, nocodazole (weak inhibition) after 2 hours and 16 hrs
treatment, respectively. Nocodazole causes a strong increase in cells with phospho-Histone H3.
Combined Analysis of Cytoskeleton
Rearrangement and Cell Cycle Markers
Whole Cell Stain
B.
Actin Fibers
Microtubules
Cell Cycle Marker Detection
Hydroxyurea
Cycloheximide
Mimosine
Figure 7: Hierarchical clustering of the quantitative results from the previous figures shows
that the changes in cell structures and cell cycle markers are closely related to each other
when treated with cell cycle inhibitory drugs. The combined parameters of these two assays
are good indicators of the cell’s response to a drug treatment.
Summary:
DAPI
BrdU
• N
ew Cell Cycle Marker analysis provide comprehensive
information on cell cycle phases (Kits: Cyclin B1:#8404401&2,
p-PLK1:#8404801&2, p-Rb:#8404501&2 and BrdU/p-Histone
H3:#8404601&2).
Phospho-Histone
Figure 2: (A) Cell morphology, F-actin fibers and microtubule fibers in NIH 3T3 cells
are visualized with the Cytoskeleton Rearrangement kit, imaged with the ArrayScan V TI
HCS Reader using a 20X objective lens, and analyzed using the Morphology Explorer
BioApplication. (B) DNA content, BrdU and phospho-Histone H3 in A549 cells are stained
with the Cell Cycle Kit I, imaged with the ArrayScan V TI HCS Reader using a 10X objective
lens, and analyzed by the Target Activation BioApplication. For both panels, the top row
shows the acquired fluorescence image, and the colored traces in the bottom row shows
the features identified and analyzed by the respective BioApplication.
Figure 4: NIH 3T3 cells were labeled with the Cytoskeleton Rearrangement kit (F-actin
= green, microtubules = red, and nuclei = blue). Hierarchical clustering analysis was
performed with the data of drug effect on microtubules, F-actin and cell morphology, which
indicates that Nocodazole and Vinbalstine disrupt microtubule structure but Hydroxyurea,
Cycloheximide and Mimosine increase F-actin fibers (stress fibers).
• C
ytoskeletal rearrangement (#8402401&2) and cell cycle marker
detection allow to evaluate and profile the multiparameter
cellular effect of cell cycle inhibitory drugs.
• T hermo Scientific Cellomics HCS Reagent Kits and ArrayScan
HCS Reader offer the powerful tools for High Content Cell-Based
Screening and Analysis