Innovations Forum: Drug screening and cellular assays
siRNA screening of the cell cycle with
two dynamic GFP sensors
M. Kenrick, S. Hancock, S. Stubbs, and N. Thomas
GE Healthcare, The Maynard Centre, Cardiff, UK
Recent advances in siRNA methodologies and the
development of high-throughput image analysis platforms
such as the IN Cell Analyzer have revolutionized the
functional analysis of genes and proteins. Here, we describe
the application of two stable cell lines expressing green
fluorescent protein (GFP)✧ cell cycle sensors to screen a
library of siRNAs directed against key cell cycle control
genes. Imaging of GFP intensity and distribution within these
two cell lines allows cell cycle position to be assigned by
automated image analysis procedures and permits their
use in the screening of drugs that block the cell cycle.
Introduction
Recent developments in RNA interference (RNAi) and small
interfering RNA (siRNA) techniques for specifically modulating
gene expression in a diverse range of cells and organisms (1, 2,
3) have revolutionized the functional analysis of genes and
proteins. Advances in synthetic and virally encoded siRNA
methodologies (4, 5) have now reached a stage where large
scale RNAi screens can be applied to mammalian cells (6, 7, 8).
In addition to the provision of large numbers of validated
siRNAs, efficient mammalian siRNA functional screens will
require information-rich model systems that allow abstraction
of multi-parameter data at a level of throughput compatible
with large-scale projects. Fortunately, advances in the
capabilities of siRNA have been matched by the development of
sophisticated fluorescence imagers and software capable of
imaging and analyzing cellular events in live cells at highthroughput (9, 10). Such instrumentation enables study of
complex systems by combining data from fluorescent cellular
sensors with morphological parameters to provide a detailed
description of the phenotypic effects of siRNAs in cellular screens.
In this study we have used two stable cell lines expressing green
fluorescent protein cell cycle sensors (Fig 1) to screen a library
of siRNAs directed against key cell cycle control genes.
One cell line (11, 12) reports on G2 phase to M phase cell cycle
transition via dynamic expression and degradation of an EGFP
sensor that shadows endogenous cyclin B1 levels. The G2/M cell
cycle phase marker (CCPM) is switched on in late S phase,
switched off at the end of mitosis, and in the intervening period
translocates from the cytoplasm to the nucleus.
✧
See licensing information.
Fig 1. G2/M and G1/S cell cycle phase marker (CCPM) constructs and cell lines. The
expression, localization and degradation of GFP fusion proteins under the control of
well characterized cell cycle control and response elements permits determination of
cell cycle position in living cells. The G2/M CCPM is expressed under the control of the
cyclin B1 promoter initiating EGFP expression in late S phase. As cells progress through
G2, cytoplasmic EGFP intensity increases and at prophase the construct localizes to the
nucleus. GFP intensity reaches a maximum at mitosis, and is then rapidly degraded
under control of the cyclin B1 destruction box (D-box) such that the two resulting
daughter cells in G1 are nonfluorescent. The G1/S CCPM is expressed constitutively by
the ubiquitin C promoter giving low level fusion protein expression throughout the cell
cycle. In G1 cells the fusion protein is strongly localized to the nucleus and on transition
to S phase phosphorylation of the PSLD domain leads to export to the cytoplasm,
which continues through G2 phase completely reversing the nuclear/cytoplasmic
distribution of the fusion protein.
The second cell line reports on G1 phase to S phase transition
via translocation of an EGFP fusion protein incorporating the
phosphorylation-dependent sub-cellular location domain (PSLD)
of DNA helicase B (13). In this cell line a nuclear localization
sequence (NLS) within the PSLD retains the fusion protein within
the nuclei of G1 cells. Phosphorylation of serine967 within the
PSLD by CDK2/cyclin E during S phase unmasks a previously
inactive nuclear export sequence that predominates over the
NLS leading to export of the fusion protein into the cytoplasm
in S phase cells.
Imaging of GFP intensity and distribution within these two cell
lines allows cell cycle position to be assigned by automated
image analysis procedures, and permits their use in the
screening of drugs that block the cell cycle (Fig 2).
Discovery Matters Issue 1 2005 GE Healthcare 1
Innovations Forum: Drug screening and cellular assays
Fig 2. Effect of cell cycle inhibitors on the G1/S CCPM cell line. Treatment of cells with
the CDK2 inhibitors olomucine and roscovitine or application of a thymidine block
significantly increased the proportion of cells with nuclear GFP; indicative of G1 arrest.
Conversely, treatment of cells with the anti-microtubule agent nocodazole reduced the
number of cells showing nuclear GFP with the majority of cells showing cytoplasmic
GFP fluorescence, indicative of G2 arrest.
Fig 3. Measurement of siRNA transfection efficiency. U2OS cells were transfected with a
Cy5 labelled siRNA, imaged on IN Cell Analyzer 1000 and analyzed using object
intensity and granularity image analysis algorithms.
siRNA screening
Cell cycle gene knockdowns were carried out using a
Dharmacon siARRAY™ of 112 siRNA pools each comprising
four siRNAs directed against a single cell cycle related gene.
Additional scrambled sequence and Cy™5 labeled siRNAs were
used as controls and to determine transfection efficiency.
siRNAs were transfected into G2/M CCPM and G1/S CCPM
expressing U2OS cells in 96-well plates using Lipofectamine™
2000 (Invitrogen). Cells were transfected with siRNAs at 1, 5,
50, and 200 nM for 4 h followed by a media change and further
incubation for 24–48 h post transfection. Cells were fixed in
4% formalin and nuclei stained with DRAQ5™ (BioStaus).
Cellular imaging was performed on an IN Cell Analyzer 1000
using a 20× objective and 475BP20/535BP50 (GFP) and
620BP60/700BP75 (DRAQ5) excitation/emission filters. Image
stacks were converted to IN Cell Analyzer 3000 format and the
resulting images analyzed for cell number, cell cycle distribution,
and morphology.
Results
Cellular transfection efficiency was determined using a Cy5
labeled siRNA (Fig 3). Image analysis of Cy5 distribution and
intensity indicated 90% transfection efficiency. Transfection
efficiency was confirmed using an siRNA pool targeting cyclin
B1 sequences in the region of cyclin B1 used to construct the
G2/M CCPM fusion protein (Fig 4). Treatment of cells with this
siRNA pool ablated GFP fluorescence in over 90% of cells. Cells
transfected with siRNAs directed against sequences present
only in endogenous cyclin B1 retained EGFP expression and
their fluorescence increased indicating accumulation of cells in
2 Discovery Matters Issue 1 2005 GE Healthcare
Fig 4. Monitoring siRNA transfection efficiency and EGFP fusion protein knockdown.
G2/M CCPM expressing cells were transfected with two siRNA pools directed against
cyclin B1. One siRNA pool (V1) contained siRNA sequences directed against the
N-terminal region of cyclin B1, and were homologous to cyclin B1 sequences present
in the G2/M CCPM. The second siRNA pool (V3) contained siRNAs directed to cyclin B1
sequences downstream of the region incorporated into the G2/M CCPM.
G2 and M phase (see Fig 6, CCNB1). Measurement of cyclin B1
mRNA and EGFP fusion protein mRNA by RT-PCR and microarray analysis (data not shown) indicated a 70% reduction in
mRNA levels for both species, confirming the efficacy of the
siRNA transfection.
As would be expected following knockdown of a range of cell
cycle control genes, analysis of cell proliferation following siRNA
treatment revealed a number of wells in which cell numbers
were significantly larger or smaller than in control wells (Fig 5)
indicating arrest, slowing, or acceleration of the cell cycle. For
example, the effects of treating cells with siRNAs against the
DNA replication licensing factors MCM2-MCM7 (14) (Fig 5 row 7,
columns 1–6) resulted in cell cycle arrest very rapidly following
siRNA transfection.
Innovations Forum: Drug screening and cellular assays
%G1/S
Fig 5. siRNA effects on cell proliferation. G2/M CCPM cells were transfected with
siRNA pools directed against cell cycle associated genes and four pools of scrambled
non-specific siRNAs. Relative cell numbers for each well are indicated by the diameter
of spheres.
Similarly, analysis across all siRNA pools (Fig 6) showed a
diverse range of cell cycle distributions following gene
knockdown. For example, treatment of cells with cyclin A2 siRNA
(CCNA2) resulted in a significant accumulation of cells in
prophase and mitosis to a similar degree to that observed for
cyclin B1 (CCNB1), corresponding with the requirement for cyclin
A for G1/S and G2/M transitions. In confirmation of the
specificity of siRNA knockdown, cell cycle perturbation was not
observed for the germ line functional homologue cyclin A1
(CCNA1), which is not expressed in differentiated U2OS cells.
These changes correlated with additional changes in cellular
morphology described below.
Changes in cell cycle distribution around the G1-S boundary
following knockdown of cyclin E were not resolvable in G2/M
CCPM cells because the cyclin B1 fusion protein is not expressed
during this part of the cell cycle. Analysis of cyclin E siRNA
treated cells showed 76% of cells in G1 or S phases compared
with 74% in control siRNA treated cells. Analysis of G1/S CCPM
cells treated under the same conditions (Fig 7) allowed the
effects of cyclin E knockdown on G1 to S phase transition to be
quantitated. Control G1/S CCPM cells showed the same
proportion (76%) of cells in G1 or S phase as control G2/M CCPM
cells (74%), which was resolvable in G1/S CCPM cells to 9% G1
cells and 67% S phase cells. On knockdown of cyclin E the
proportion of cells in G1 or S phase remained constant at 76%,
as observed with G2/M CCPM cells. However the balance
%G2
%Prophase+Mitosis
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CONTROL
CYCPHL II
CYCPHL I
LAMIN III
LAMIN II
YWHAZ
TP73
TP63
TP53
TFDP2
TFDP1
STK12
SNK
SKP2
SKP1A
RPA3
RBP1
RBL2
RBL1
RBBP2
RB1
RAD9
RAD17
RAD1
PIN1L
PIN1
PLK
PCNA
ORC6L
ORC5L
ORC4L
ORC3L
ORC2L
ORC1L
MYC
MNAT1
MKI67
MDM2
MCM7
MCM6
MCM5
MCM4
MCM3
MCM2
MAD2L2
MAD2L1
LOC5153
JUNB
JUN
HUS1
HIPK2
FOS
E2F6
E2F5
E2F4
E2F3
E2F2
E2F1
CRI1
CNK
CHEK2
CHEK1
CENPH
CENPF
CEMPE
CENPC1
CENPB
CENPA
CDT1
CDKN2D
CDKN2C
CNDK2B
CDKN1C
CDK9
CDK8
CDK7
CDK6
CDK5
CDK4
CDK3
CDK2
CDK10
CDC7L1
CDC6
CDC45L
CDC37
CDC34
CDC27
CDC25C
CDC25B
CDC25A
CDC20
CDC2
CDC16
CCNT2B
CCNT2
CCNT1
CCNI
CCNH
CCNG2
CCNG1
CCNF
CCNE2
CCNE1
CCND3
CCND2
CCND1
CCNC
CCNB3
CCNB2
CCNB1
CCNA2
CCNA1
BTAK
ATR
ATM
APC2
0
25
50
75
100
Fig 6. siRNA effects on cell cycle distribution. G2/M CCPM cells transfected with siRNA
pools were analyzed for celI cycle distribution using automated image analysis. Results
are represented as % cells in G1/S, G2 and prophase + mitosis.
Discovery Matters Issue 1 2005 GE Healthcare 3
Innovations Forum: Drug screening and cellular assays
Fig 7. Cyclin E siRNA blocks G1 to S phase transition. G1/S CCPM cells were transfected
with an siRNA pool directed against cyclin E. Following culture for 24 h, cells were
pulsed with BrdU for 1 h, fixed and BrdU incorporation detected with the Cell
Proliferation Fluorescence Assay. G1 and S phase cells were quantitated using object
intensity image analysis to identify cells with green (G1) and red (S) nuclei.
between the two phases shifted significantly to 27% G1 cells
and 49% S phase cells, reflecting the critical role of cyclin E in
G1 to S transition.
Knockdown of Polo-like kinase (PLK) with siRNA has been
previously shown to inhibit cell proliferation, arrest cells in
mitosis, and induce apoptosis (15). Cell cycle analysis (Fig 6, PLK)
of G2/M CCPM cells treated with siRNA directed against PLK
showed a dramatic increase in mitotic cells 48 h after transfection
with 50 nM siRNA (Fig 8B). Extreme sensitivity to PLK knockdown
was confirmed by analysis of G1/S CCPM (Fig 8A) and G2/M
CCPM (data not shown) 24 h following transfection with 5 nM
siRNA, which showed an increase in G2 and M phase cells and
a corresponding decrease in G1 cells.
One key advantage of high content cellular analysis is the
ability to analyze high resolution images for multiple
parameters. In this case additional morphological analysis of
images derived from cells treated with Cyclin A2 siRNA revealed
a significant increase in nuclear area (395.3 ± 173.7 µm2)
compared with control cells (219.1 ± 95.7 µm2) and Cyclin A1
siRNA treated cells (229.8 ± 98.5 µm2).
Re-analysis of all image data from the siRNA library screen
using DNA granularity revealed that PLK siRNA gave the most
significant induction of apoptosis across the target genes in this
study (Fig 8C).
Conclusions
Perturbation of sensitive and dynamic phenotypic cellular
assays via siRNA provides a powerful tool for functional analysis
of the cell cycle. High-throughput sub-cellular imaging and
automated multi-parameter image analysis provides an
information rich environment to screen and study effects of
gene knockdown with siRNA.
4 Discovery Matters Issue 1 2005 GE Healthcare
Fig 8. Cell cycle arrest and induction of apoptosis by PLK siRNA. (A) G1/S CCPM cells
transfected with 5 nM PLK siRNA and pulsed with BrdU after 24h. (B) G2/M CCPM cells
transfected with 50 nm PLK siRNA and incubated for 48 h. (C) Measurement of DNA
granularity as a measure of apoptosis for all siRNAs used.
References
1. Hannon, G.J. RNA interference. Nature 418 (6894), 244–251 (2002).
2. Paddison, P.J. and Hannon G.J. siRNAs and shRNAs: skeleton keys to the human
genome. Curr. Opin. Mol. Ther. 5 (3), 217–224 (2003).
3. Medema, R.H. Optimizing RNA interference for application in mammalian cells.
Biochem. J. 380 (3), 593–603 (2004).
4. Kumar, R. Conklin DS, Mittal V. High-throughput selection of effective RNAi probes for
gene silencing. Genome Res. 13 (10), 2333–2340 (2003).
5. Luo, B. et al. Small interfering RNA production by enzymatic engineering of DNA
(SPEED). Proc. Natl. Acad. Sci. USA 101 (15), 5494–5499 (2004).
6. Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of
the p53 pathway. Nature 428 (6981), 431–437 (2004).
7. Paddison, P.J. et al. A resource for large-scale RNA-interference-based screens in
mammals. Nature 428 (6981), 427–431 (2004).
8. Mousses, S. et al. RNAi microarray analysis in cultured mammalian cells.
Genome Res. 13 (10), 2341–2347 (2003).
9. Ramm, P. and Thomas, N. Image-based screening of signal transduction assays.
Sci. STKE. 2003 (177), PE14.
10. Price, J.H. et al. Advances in molecular labeling, high-throughput imaging and
machine intelligence portend powerful functional cellular biochemistry tools.
J. Cell. Biochem. Suppl. 39, 194–210 (2002).
11. Thomas, N. Lighting the circle of life: fluorescent sensors for covert surveillance of
the cell cycle. Cell Cycle 2 (6), 545–549 (2003).
12. Thomas, N. et al. Characterization and gene expression profiling of a stable cell line
expressing a cell cycle GFP sensor. Cell Cycle 4 (1), (2005).
13. Gu, J. et al. Cell cycle-dependent regulation of a human DNA helicase that localizes
in DNA damage foci. Mol. Biol. Cell. 15 (7), 3320–3332 (2004).
14. Bailis, J.M. and Forsburg, S.L. MCM proteins: DNA damage, mutagenesis, and repair.
Curr. Opin. Genet. Dev. 14 (1), 17–21 (2004).
15. Liu, X. and Erikson, R.L. Polo-like kinase (Plk)1 depletion induces apoptosis in cancer
cells. Proc. Natl. Acad. Sci. USA 100 (10), 5789–5794 (2003).
Innovations Forum: Drug screening and cellular assays
Ordering Information
IN Cell Analyzer 1000
25-8010-26
IN Cell Analyzer 3000
25-8010-11
Cell Proliferation Fluorescence Assay
25-9001-89
(500 assays)
G2M Cell Cycle Phase Marker Assay,
25-8010-52
6 month assay evaluation (3 vials)
G2M Cell Cycle Phase Marker Assay,
25-9002-55
nonprofit research (3 vials)
To shop online, go to www.amershambiosciences.com
Discovery Matters Issue 1 2005 GE Healthcare 5