NES
NLS
Cyclin E
CDK2
S967-P
S
NES
NLS
UbC
EGFP
PSLD
Sensor Characterisation
Time lapse imaging of a U-2 OS cell line exhibiting stable
expression of the G1S CCPM demonstrated that the sub-cellular
distribution of the sensor and other cellular characteristics can be
used to define up to 4 separate phases of the cell cycle (Figs 2 and
3). After mitosis and cytokinesis lasting 1h, there is a phase of 2 to
5hrs when the sensor exhibits predominantly nuclear distribution.
Rapid changes in sub-cellular distribution associated with this
phase are indicative of increasing Cdk2/cyclin E activity (Fig 3).
G1
G1/S
4h
S
7h30m
G2
21h
M
24h
1.6
1.4
siRNA cyclin E
siRNA PLK
siRNA MCM4
7h30m
1.2
1.0
21h
0.8
2h30min
0.6
0.4
nocodazole
0.2
olomoucine
colcemid
taxol
0.0
0
5
10
15
20
25
Time (hours)
Figure 3. Analysis of time lapse images of G1S CCPM stable cell line. Graph of subcellular distribution (nuclear:cytoplasmic ratio in green channel) of G1S CCPM sensor for a
single cell passing through one complete cell cy cle (images taken at 30 min intervals on IN
Cell Analyzer 3000). The sub- cellular distribution of the sensor exhibits 4 distinct phases
that correlate with reported lengths for M, G1 , S and G2 -phases in U-2 OS cells.
The correlation between G1S CCPM phenotype and DNA complement
provides further evidence that the cytoplasmic relocation of the sensor
is cell cycle related (Fig 4).
2n
4n
1.40 G 1
M
1.35
1.30
1.25
1.20
1.15
1.10
1.05
1.00
S
0.95
4.7
G2
4.8
4.9
5.0
5.1
5.2
5.3
Log (Integrated Nuclear Hoechst Intensity)
Figure 4. Correlation between sub-celllular
distribution of the G1S CCPM (nuclear:
cytoplasmic ratio) and DNA content per
nucleus (indicated by Integrated blue
fluorescence due to Hoechst staining) for
individual cells. As expected, cells with a low
integrated Hoechst intensity (indicative of 2n)
have a high N:C ratio and are in G0/G1 . Cells
exhibiting DNA replication from 2n to 4n have
a low G1S CCPM N:C ratio. G 2 cells have the
lowest G1S CCPM N:C ratio and are 4n.
Mitotic cells are 4n and have a high G1S
CCPM N:C ratio due to cell rounding. Fixed
cell images and analysis were carried out after
48hr growth on IN Cell Analyzer 1000 with IN
Cell Analyzer 1000 Morphology Analysis
Module (GE Healthcare).
In addition, cells that demonstrate BrdU incorporation indicative of
active DNA replication consistently exhibit even or predominantly
cytoplasmic distribution of the G1S CCPM sensor (Fig 5), confirming
that the sensor is nuclear in G 1-phase and is exported from the nucleus
prior to DNA replication and entry into S -phase.
450
S
400
350
Figure 6. Effect of phasespecific chemical and
siRNA-induced cell cycle
arrest on the G1S CCPM
stable cell line. Cells were
untreated, or transfected
with siRNA pools or drugs
for 24 hrs. Cells in top
figures also indicate BrdU
incorporation (red). Fixed
cell images from IN Cell
Analyzer 1000 (GE
Healthcare). Where
appropriate blocks were
confirmed using propidium
iodide staining and flow
cytometry (data not shown).
Cell Cycle Assignment
Cell cycle status can be determined by measurement of G1S CCPM
fluorescence intensity, sub-cellular distribution and other cellular
characteristics (see Fig 2 schematic) using high-throughput imaging
and analysis with the G1S Cell Cycle Trafficking Analysis Module (GE
Healthcare; Fig 7). Image analysis outputs (Table 1) for individual
cells and the total population include: cell number, classification, cell
rounding, nuclear area, nuclear, cytoplasmic and cellular intensity
values for the blue, green and red channel, and relative nuclear to
cytoplasmic distribution.
a
GFP BrdU
G1
100
0.8
1
1.2
1.4
500
1.6
N:C ratio Green
Figure 5. Multiplex assay with G1S CCPM sensor and Bromodeoxyuridine (BrdU)
incorporation. A U-2 OS cell line exhibiting stable expression of the G1S CCPM sensor was
incubated with BrdU for 1h and fixed in 4% formaldehyde (right top panel). Nuclear BrdU
incorporation was detected with mouse anti-BrdU/DNAase and Cy™5 anti-mouse
antibodies (right bottom panel; Cell P roliferation Fluorescence Assay, GE Healthcare). The
graph (left panel) depicts analysis of the image (bottom right) and demonstrates that
individual cells with a predominantly nuclear distribution of the G1S CCPM sensor (high
nuclear:cytoplasmic ratio of green signal) do not exhibit BrdU incorporation (nuclear
intensity red signal) indicative of DNA replication and cells in S-phase (few M-phase cells
visible using current fixation protocol). Images from IN Cell Analyzer 1000 were analysed
with the Morphology analysis module (GE Healthcare).
80
Cell count
g1
S
G2
60
50
300
40
Log BrdU:Cy5 Fluorescence
Log BrdU:Cy5 Fluorescence
30
20
20
100
10
10
0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
60
40
200
30
100
70
50
70
Cell count
g1
S
G2
200
400
0
3.5
0
-4.0
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
0
1.0
log [Nocodazole] µM
log [Roscovitin]µ M
Figure 8. Dose response curves demonstrating effects of roscovitine and nocodazole on
cell proliferation and cell cycle distribution determined using the G1S CCPM stable cell line
and IN Cell Analyzer 3000 G1S Cell Cycle Trafficking Analysis Module. Cells were treated for
24 hrs prior to fixation and imaging (n=4).
Treatment of the G1S CCPM cell line with nocodazole, an inhibitor of
microtubule assembly, resulted in a significant increase in the percentage
of cells with a G2 -phenotype (EC50 298 nM; few M-phase cells were evident
with fixed assay protocol) and a reciprocal effect on cells with a G1phenotype (Fig 6 and 8 right panel).
500
400
50
40
200
30
Inhibitor (120 nM)
20
100
10
0
-3.0
0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
log [Inhibitor A] µM
Figure 7. Partial screenshot of IN Cell Analyzer 3000 G1S Cell Cycle Trafficking
Analysis Module. The module can be used to classify cells into different phases of the
cell cycle based upon a number of user defined cellular characteristics from the blue,
green and red channel (right panel). The module provides interactive update of the
bitmap overlay and projects cell classification directly onto the image (panels a and b).
Chara cteristic G1 arrest resulting from treatment with a relatively high concentration
of roscovitine (333 µ M; panel a) is clearly demonstrated when compared to a control
population (panel b). In the current figure the G1S CCPM has been multiplexed with
BrdU incorporation.
Roscovitin (µM)
Cell count
M%
G1 %
S%
G2 %
333
162.3 (20.7)
5.0 (2.8)
84.3 (4.1)
2.6 (1.5)
8.1 (2.9)
Control
228.3 (38.2)
0.6 (0.5)
38.9 (6.1)
46.1 (6.0)
14.5 (1.9)
Table 1. Output from IN Cell Analyzer 3000 G1S Cell Cycle trafficking Analysis
Module. The table shows the effect of roscovitin at 333 µ M (results are from images
including panels a and b in Fig 8; mean of 4 wells, SD in brackets).
Figure 9. Dose response curve and images demonstrating effect of cmpd A on cell
proliferation (cell count) and cell cycle distribution (%) determined using the G1S CCPM
stable cell line and IN Cell Analyzer 3000 G1S Cell Cycle Trafficking Analysis Module. Cells
were treated for 48 hrs with a range of concentrations prior to fixation and imaging (n=2).
However, an approximate doubling in the nuclear size of cells treated with
cmpd A (Fig 9 and 10) would seem to be consistent with the phenotype of
G2, and not G1, cells (see Fig 2) suggesting that this cmpd does not induce
conventional G1 arrest. In addition, cmpd A produced a decrease in the
symmetry of nuclei and an increase in extranuclear inclusions that stain
with Hoechst (see images in Fig 9 and 12, and data in Fig 10) indicating
problems with chromosome alignment/ segregation during mitosis or
DNA fragmentation resulting in irregular nuclear morphology.
Nuc Area
Nuc asymmetry
Extranuclear inclusion area
Effect of Cell Cycle Inhibitors
Treatment of the G1S CCPM stable cell line with a relatively high
concentration of roscovitine, a purine derivatised dual Cdk 1/2
inhibitor, resulted in a population with predominantly nuclear
distribution of the G1S CCPM sensor, indicative of Cdk2 inhibition
and arrest in G1 (Fig 7 and Table 1). This was confirmed by an
absence of BrdU incorporation (Fig 7). A dose response curve for
roscovitine treatment (Fig 8) highlighted the bi-modal effect of this
compound.
500
1.6
1.2
400
0.8
300
0.4
200
0.0
-4
-3
-2
-1
log [cmpd A] µ M
0
1
4n
4.85
5.1
8n
2n
3.8
5.35
5.6
4.6
Replicating cells
4n
4.85
5.1
8n
5.35
5.6
Log DNA:Hoechst Fluorescence
When the G1S CCPM sensor was multiplexed with BrdU
incorporation and analysed multiparametrically for DNA
complement, DNA replication activity and cell cycle phase it was
evident that exposure to cmpd A caused endoreduplication
resulting in polyploidy and arrest in G1-phase (Fig 11) with 4n
and 8n DNA complement.
60
Cell count
% G1
% G2
4.4
Figure 11. Multiplex assay showing that cmpd A causes endoreduplication
after 24 hours and arrest in G1-phase at 4n and 8n after 48 hours. The G1S
CCPM stable cell line was grown for 24 or 48 hrs in the pre sence (red) or absence of
cmpd A (blue), pulse incubated with BrdU for 1h, fixed and processed accordingly.
Graph shows an object plot of individual cells imaged on the IN Cell Analyzer 1000
and analysed twice with the Morphology Analysis Module (GE Healthcare). The
DNA replication activity (BrdU incorporation) per nucleus is indicated on the y-axis
and is a measure of ht e log of the integrated red fluorescence per nucleus;
replicating cells are shown above the dotted line. The DNA content per nucleus is
indicated on the x-axis and is a measure of the integrated blue fluorescence (due
to Hoechst 33342) per nucleus; nuclear DNA complement has been indicated at
2n, 4n and 8n. The cell cycle phase of each cell is indicated by the size of each
point and is a measure of the nuclear:cytoplasmic ratio of G1S CCPM sensor.
Larger dots are G1 -phase cells, smaller dots are S-phase and G 2 -phase cells (few
M-phase cells are evident using current fixation protocol). Cells exhibiting DNA
replication from 2n to 4n are clearly visible in cont rol populations at 24 and 48 hrs
and cell cycle distribution is normal. Treated cells exhibit reduplication from 4n to
8n at 24 hrs and many cells seem to have arrested in G1 -phase at 4n or 8n after 48
hrs.
Control
70
4.6
4
2n
Log DNA:Hoechst Fluorescence
Treatment of the G1S CCPM cell line with a novel compound (A) for 48 hrs
produced a cellular population exhibiting predominantly nuclear
distribution of the G1S CCPM sensor, indicative of arrest in G 1 (Fig 9; EC5 0 7
nM for G1%).
300
200
G2
100
300
b
300
250
400
90
Control
Count
Nuc:Cyt ratio (green)
M
24h
Count
G2
1.8
Nuclear asymmetry
and inclusion area
Figure 2. Schematic and live time lapse images of U-2 OS cell line exhibiting
stable expression of the G1S CCPM. Relative brightness and size of cells and subcellular compartments are shown for each phase of the cell cycle. Images acquired
on IN Cell Analyzer 3000 (GE Healthcare).
S
4h
150
2h
Cdk 2/ cyclin A?
G1
At concentrations between 5 and 15 µ M, roscovitine produce d a
significant increase in the percentage of cells with a G2-phenotype (and a
reciprocal effect on G1%), whilst at higher concentrations a G1-phenotype
predominated (EC50 60 µM, S:N 6.2). This result is consistent with the
reported EC50 values for roscovitine against purified Cdk1/cyclin B (0.45
µM) and Cdk2/cyclin E (0.7 µM) 2, respectively and confirms that the low
cellular permeability of roscovitine impedes efficacy.
5
4.8
0h
2h
5h
0h
4.5h
5h
25h
26.5h
28h
26.5h
27h
28h
10h
23.5h
Figure 8. Dose response curves
demonstrating
effects
of
olomoucine,
roscovitin
and
10h on cell proliferation
25h
nocodazole
and cell cycle distribution
determined using the G1/S
CCPM stable cell line. Cells were
treated for 24 hours prior to fixation
and imaging (n=4).
32h
38h
32h
38h
%
For DNA replication studies, cells were incubated in BrdU for 1h,
fixed in 4% formaldehyde. BrdU incorporation was detected using
the Cell Proliferation Fluorescence Assay (GE Healthcare). To
analyse the effects of known chemical cell cycle inhibitors, cells in
96-well plates were exposed for up to 72hrs. For gene knockdown
studies, siRNA pools from the Dharmacon siARRAY ™ (25nM) were
transfected into cells in 96-well plates for 4hrs using
Lipofectamine™ 2000 (Invitrogen), followed by media change and
further incubation for 20hrs. DNA was stained with Hoechst™ (2
µM) and plates were imaged on the I N Cell Analyzer 1000 or IN Cell
Analyzer 3000 (GE Healthcare).
Cdk 2/ cyclin E
M
2.0
The phase specific sub-cellular localisation of the G1S CCPM sensor
(Figs 2, 3, 4 and 5) was characterised further using chemical and
siRNA-based arrest (Fig 6). Agents known to effect phase-specific
arrest in G1 resulted in a cellular population with predominantly
nuclear distribution of the sensor (Fig 6), whilst cells that had been
arrested in G2-phase (few M-phase cells are visible using current
fixation protocol) exhibited predominantly cytoplasmic distribution
(Fig 6).
5.2
4.2
4
Control
S967
G1
4.4
Compound A 48h
5.4
Treated
PSLD
Figure 1. Schematic of human helicase B and
development of the G1S CCPM sensor
containing the phosphorylation dependent
subcellular localisation control domain
(PSLD). In late G1 -phase S967 of human
helicase B is phosphorylated by the Cdk2/ Cyclin
E complex (putative CDK phosphorylation sites
are shown in yellow). This event unmasks a revtype nuclear export sequence (NES) resulting in
translocation of the endogenous protein from the
nucleus to the cytoplasm around the G 1/S
boundary. The G1S CCPM sensor is a fusion of
the helicase B PSLD region to the C-terminus of
EGFP (via a short flexible amino acid linker
region). The sensor is expressed from the human
ubiquitin C promoter (UbC).
4.6
Control
1087
4.8
Control 48h
5.6
Treated
S967
Walker sites
5
4.6
The sensor demonstrates a slower progressive export from the nucleus
over the following 10 to 14hrs, and is exclusively cytoplasmic for a
period of 2 to 5hrs. The length of each of these phases correlates with
the reported lengths of G1, S, G2 and M-phase for rapidly dividing
U-2 OS cells (Fig 3).
5.8
4.2
Count
HDHB
5.2
3.8
Mean nuclear Area
1
Healthcare, The Maynard Centre, Whitchurch, Cardiff,UK, CF14 7YT.
2Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232 USA
%
Human helicase B has been shown to contain a phosphorylation
dependent subcellular localisation domain (PSLD; Fig 1) that directs
translocation of the endogenous protein from the nucleus to the
cytoplasm at the G1/S boundary1. The PSLD was fused to EGFP ,
and U-2 OS cell lines exhibiting stable expression of these
constructs were generated and characterised in further studies
(Fig 1).
1GE
%
Experimental Approach
Control 24h
Compound A 24h
5.6
Simon Stubbs 1*, Suzanne Hancock1, Hayley Tinkler1, Nick Thomas 1, Paul Michael1, Stephen Capper1, Michael
Kenrick1, Adrian Cushing1, Iain Bye1, Dwayne Dexter1, Jinming Gu 2 and Ellen Fanning 2
G1S CCPM N:C ratio
(Green)
GE Healthcare has previously demonstrated the use of functional
elements from the Cyclin B1 promoter and gene in the
development of a live cell, non-perturbing sensor of G2 and M
phases termed, the G2M cell cycle phase marker (G2M CCPM). The
current paper describes the use of functional elements from the
human helicase B gene to develop a similar sensor of G1/S
transition (G1S CCPM). We have used high-throughput sub-cellular
imaging in conjunction with known cell cycle inhibitors and siRNAs
to validate cell lines exhibiting stable expression of the sensor.
5.8
5.4
Inuc Red
Eukaryotic cell division proceeds through a regulated cell cycle
comprising the consecutive phases: gap1 (G1), synthesis (S), gap 2
(G2) and mitosis (M). Dividing cells are subject to a number of
control mechanisms that are carried out at specific checkpoints.
These mechanisms maintain genomic integrity by arresting
progress through the cell cycle or inducing destruction of aberrant
cells. Consequently, accurate determination of the position of a
cell within the cell cycle can be used to: screen for antiproliferative agents, establish whether a compound has adverse
effects upon the cell cycle, or determine the effect of cell cycle
position on a separate process.
Replicating cells
G1/S Checkpoint Reporting
Introduction
Figure 10. Dose response
curve showing effect of 48h
treatment with cmpd A on
nuclear asymmetry, nuclear
area and extranuclear DNA
inclusion area of G1S CCPM
stable cell line. EC 50 20nM for
nuclear area, 15nM for nuclear
asymmetry and 60nM for
inclusion area. Fixed cells were
imaged on IN Cell Analyzer 1000
and analysed using the
morphology or dual area object
analysis module (GE Healthcare).
Figure 12. Live time lapse images of G1S CCPM stable cell line treated with
cmpd A (120 nM). The arrow s highlight representative G 1 cells for treated and
control populations monitored over 38hrs. Cells demonstrate a temporally normal
cell cycle and enter mitosis around 25hrs but the treated population fails to
demonstrate cytokinesis. The resulting polyploid cell exhibit intense nuclear
distribution of the G1S CCPM sensor indicative o f G1 -phase arrest . Images
acquired on IN Cell Analyzer 3000 (GE Healthcare).
The current data and time lapse images of the G1S CCPM cell line
treated with cmpd A (Fig 12) indicate the occurrence of a
temporally unimpeded but aberrant mitosis with failed
cytokinesis, resulting in polyploidy, possible endoreduplication to
8n and/ or ‘pseudo G 1’ arrest.
Conclusions
• The G1/S CCPM sensor provides a sensitive and dynamic
phenotypic cellular assay and is a powerful tool for
functional analysis of the cell cycle.
• High-throughput sub-cellular imaging and automated
multi-parameter image analysis enables the effects of a
broad range of agents upon the cell cycle to be monitored.
References
Gu J, Xia X, Yan P, Liu H, Podust VN, Reynolds AB, Fanning E. Mol. Biol. Cell.
2004 (7) 3320-32.
2 De Azevedo WF, Leclerc S, Meijer L, Havlicek L, Strnad M, Kim SH. Eur. J.
Biochem. 1997 (243) 518-26.
1
© General Electric Company 2005 - All rights reserved. GE and the GE monogram are trademarks of General Electric Company . Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA U.K. Amersham Biosciences AB SE-751 84 Uppsala Sweden. Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA. Amersham Biosciences Europe GmbH Munzinger Strasse 9 D-79111 Freiburg. Amersham Biosciences KK Sanken Buiding 3 -25-1 Hyakunicho Shinjuku Ku Tokyo 169-0073 Japan. The above companies are all Ge neral Electric companies going to market as GE Healthcare.
The G1S Cell Cycle Phase Marker assay is the subject of international patent application numbers PCT/GB2005/002876, PCT/GB2005/002884 and PCT/GB2005/002890 in the name of Amersham Biosciences and Vanderbilt University. The G2M Cell Cycle Phase Marker Assay is the subject of patent applications AU 2002326036, CA 2461133, EP 02760417.2, IL 160908, JP 2003-534582 and US 10/491762 in the name of Amersham Biosciences and Cancer Research Technology. GFP: This product is the subject of patent applications US
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and Japanese patent JP 3283523 and other pending and foreign patent applications. Columbia University under US patent numbers 5 491 084 and 6 146 826. University of Florida under US patents 5 968 750, 5 874 304, 5 795 737, 6 020 192 and other pending and foreign patent applications. The IN Cell 1000 is the subject of US patents 6,563,653 & 6345115 and US patent application number 10/514925, together with other granted and pending family me mbers, in the name of Amersham Biosciences Niagara, Inc. The IN Cell
Analyzer 3000 is the subject of US patents 6,400,487 and 6,388,788 and US patent application number 10/227552, together with other granted and pending family members, in the name of Amersham Biosciences Corporation. The IN Cell Analyzer 1000 and 3000 and associated analysis modules are sold under license from Cellomics Inc. under US patent Nos 6573039, 5989835, 6671624, 6416959, 6727071, 6716588, 6620591 6759206; Canadian patent No 2328194, 2362117, 2,282,658; Australian patent No 730100; European patent
No 1155304 and other pending and foreign patent applications. *To whom all correspondence should be addressed. This poster was presented at the 11 th Annual Conference of the Society for Biomolecular Screening, Geneva, Switzerland (11 - 15 September 2005).