The
in vitro
effects of CRE-decoy oligonucleotides in combination
with conventional chemotherapy in colorectal cancer cell lines
Wai M. Liu*, Katherine A. Scott*, Sipra Shahin and David J. Propper
New Drug Study Group, Barry Reed Oncology Laboratory, St. Bartholomew’s Hospital, London, UK
The cAMP response element consensus sequence directs the
transcription of a wide range of genes. A 24-mer single-
stranded cAMP response element decoy oligonucleotide
(CDO) has been shown to compete with these sequences for
binding transcription factors and therefore interferes with
cAMP-induced gene transcription. We have examined the
effect of this CDO alone and in combination with a range of
common chemotherapeutic agents in colorectal cancer cell
lines. CDO had a potent anti-proliferative effect in colorectal
cell lines, yet, a similar enhancement of cell death was
not observed. Simple drug–drug interaction studies showed
that combining CDO with chemotherapy resulted in an
enhancement of the antiproliferative effects. Furthermore,
this cytostatic effect was protracted and associated with an
increase in senescence-associated b-galactosidase activity at
pH 6. There is a possible role for p21
waf1
in mediating this
effect, as the enhancement of cell growth inhibition was not
observed in cells lacking the ability to correctly upregulate
this protein. Additionally, significant decreases in cyclin-
dependent kinase (CDK) 1 and CDK 4 function were seen
in the responsive cells. These data provide a possible model
of drug interaction in colorectal cell lines, which involves the
complex interplay of the molecules regulating the cell cycle.

oligonucleotides is the diversity and number of possible
fusion sequences in cancer, which can actually prevent a
particular disease from being treated successfully with just
a single agent. For example, the bcr-abl translocation in
chronic myeloid leukaemia can have as many as seven
distinct junctional sequences that would require their own
antisense oligonucleotide [10,11]. Consequently, treatment
would have to be adapted for each individual patient,
making the concept of using oligonucleotides less attractive.
The second approach involves the use of short strands of a
nucleotide sequence as a decoy factor, which competes with
the response elements within the promoter regions of genes
that bind transcription factors [1,12]. In a similar manner to
the first approach, specificity is achieved through sequence
binding. However, this is enhanced further, as relevant
transcription factors are specifically sequestered by the decoy
oligonucleotides, resulting in an effect that is both sustain-
able and nongenomic in nature. Additionally, as protein–
protein interactions would be distal from native enhancer
sites, nonspecific interference of these sites would be reduced.
The cAMP response element (CRE) consensus sequence
is intimately involved in the transcription of a wide range of
Correspondence to W. M. Liu, Drug Resistance Team, Section of
Medicine, Institute of Cancer Research, Haddow Laboratories,
15 Cotswold Road, Sutton, Surrey SM2 5NG, UK.
Fax: + 44 208 661 3541, Tel.: + 44 208 722 4429,
E-mail:
Abbreviations: CRE, cAMP response element; CREB, CRE-binding
protein; CDK, cyclin-dependent kinase; CDO, CRE-decoy
oligo-nucleotide; 5-FU, 5-fluorouracil; SO, scrambled mismatch

has no significant effect in normal cells. Furthermore, in
animal studies, CDOs induced tumour shrinkage without
obvious toxicity [17]. The mechanism by which CDOs
inhibit cell growth has not been elucidated, although it has
been shown that CRE-decoy treatment reduces cyclin D1
and cyclin-dependent kinase (CDK) 4 levels and retino-
blastoma protein (Rb) phosphorylation. CDO-induced
growth inhibition was independent of p53 status [18,19],
and accompanied by the hallmarks of apoptosis [20], which
together suggests a more profound interaction.
The aims of the present study were threefold: first, to
explore the in vitro effects of CDO alone in a panel of three
colorectal cancer cell lines; second, to investigate the effects
of combining CDO with etoposide (VP16), 5-fluorouracil
(5-FU) or SN38 on cell growth and viability; and third, to
elucidate the cellular mechanisms underlying any synergistic
effects seen in the drug combinations.
Materials and methods
Cell culture
HCT116 and SW620 colorectal cell lines were obtained
from the Cancer Research UK laboratories, and were
maintained in Dulbecco’s modified Eagle’s medium supple-
mented with 10% (v/v) fetal bovine serum. GEO colorectal
cancer cell lines were a gift from G. Tortora (Dipartimento
di Endocrinologia e Oncologia Molecolare e Clinica,
Universita
`
di Napoli, Italy), and were maintained in
McCoy’s 5A with 10% (v/v) fetal bovine serum. HCT116
and GEO cell lines were both wild-type p53, and SW620

Trypan blue, and cell cycle distribution by flow cytometry.
DNA analysis
The distinct phases of the cell cycle were distinguished by
flow cytometry, according to methods described previously
[23]. The acquisition of data was performed within 1 h using
a FACSCalibur (BD Biosciences). Five thousand cells were
analysed for each data point, and the percentages of cells in
sub-G
1
(apoptotic fraction, cells with a reduced propidium
iodide stain but similar morphology), G
1
,SandG
2
/M
phases were determined using the cell cycle analysis program
WINMDI
v2.4.
Flow cytometric analysis of BrdU incorporation
The degree of incorporation of the thymidine analogue
5-bromo-2¢-deoxyuridine (BrdU; Sigma) in HCT116 and
GEO cells was measured by flow cytometry. Following
culture in CDO and drugs, cells (5 · 10
5
mL
)1
)were
transferred into fresh culture medium containing 10 l
M
BrdU for 30 mins before fixing with ice-cold 70% (v/v)

)1
), anticyclin D (2 lgÆmL
)1
), anti-CDK 4
2774 W. M. Liu et al. (Eur. J. Biochem. 271) Ó FEBS 2004
(2 lgÆmL
)1
), anticyclin B (2 lgÆmL
)1
), or anti-CDK 1
(2 lgÆmL
)1
) (all from PharMingen). Anti-(b-actin) Ig was
used to confirm equal sample loading (1 : 2000; Oncogene
Research Products). Following a washing step in 0.1% (v/v)
Tween in Tris-buffered saline (Sigma; 100 m
M
Tris pH 7.6,
150 m
M
NaCl), horseradish peroxidase-conjugated anti-
species IgG1 was used as the secondary antibody (1 : 1000;
DAKO Ltd). Bands were visualized by the ECL plus
detection system (Amersham Biosciences Ltd).
For the analysis of cyclin–CDK interaction, cells were
lysed in a modified RIPA buffer (50 m
M
Tris, 250 m
M
NaCl,

,
before fixing in 2% (v/v) formaldehyde and 0.2% (v/v)
glutaraldehyde. Cells were then washed twice in ice-cold
NaCl/P
i
, before overnight incubation at 37 °CinX-Gal
staining solution (1 mgÆmL
)1
5-bromo-4-chloro-3-indolyl
b-
D
-galactoside in 40 m
M
citric acid/sodium phosphate
pH 6, 5 m
M
potassium ferricyanide, 5 m
M
potassium
ferrocyanide, 150 m
M
sodium chloride, 2 m
M
magnesium
chloride). Samples were then washed twice in ice-cold NaCl/
P
i
prior to assessing the percentage of cells staining positive
for SA-b-gal activity by light microscopy.
Statistical analysis

Combination with other chemotherapeutic agents
Preliminary experiments indicated that SW620 cells were
resistant to both CDO and chemotherapy at the concen-
trations studied, and so were excluded from the combina-
tion studies. These simple combination studies involved
culturing cells simultaneously with each agent at the
concentration that reduced cell numbers by 25% (IC
25
).
Culturing HCT116 and GEO cells with these equi-toxic
drug concentrations resulted in different responses
in these cell lines. Specifically, combining CDO with a
chemotherapeutic drug had no significant effect in HCT116
cells, but significantly reduced cell numbers in GEO
cultures. Also, this effect was greater than expected
(hyper-additive) (Fig. 2A). This can be illustrated most
clearly with the results for GEO cells cultured with CDO
and 5-FU; by simply comparing the total reduction in cell
number in cultures treated with CDO and 5-FU together
(relative to the SO control) with the expected reduction in
Fig. 1. The effect of CDO in HCT116 and GEO cell lines on day 3. The activity of CDO in the sensitive cell lines was fitted a standard E
max
model.
Representative DNA histograms following culture with CDO in HCT116 cells are also shown. Each point represents the means and SD of at least
three separate experiments. A, Apoptosis; SO, scrambled mismatch oligonucleotide control.
Ó FEBS 2004 CRE-decoys in colorectal cancer cells (Eur. J. Biochem. 271) 2775
cell number (calculated as the numerical sum of the
reductions in cell number seen in the cultures with the two
agents separately [25] (· 10
5

1
populations seen in
cells treated with the individual agents. Additionally, flow
cytometry revealed no apparent blockades in the G
1
,Sor
G
2
phases of the cell cycle, suggesting that the reduction in
cell number may have been the result of a general and
simultaneous blockade of all three phases of the cell cycle.
Cell proliferation is reduced
The reduction in cell number may have been a result of an
inhibition of cellular proliferation. Therefore, at the end of
each of the culture schedules, cells were pulsed with BrdU
for 30 mins. The extent of BrdU incorporation was then
measured by flow cytometry. In HCT116 cells, there were
no significant differences in the measured level of BrdU
incorporation and the expected level (Fig. 3). In contrast,
there was a significant reduction in BrdU fluorescence in
GEO cells cocultured with CDO and cytotoxic drugs
compared to those treated with drugs separately (Fig. 3).
This was most apparent for BrdU incorporation in cells
cocultured with CDO and 5-FU (% BrdU incorporation
normalized to control cells with SO: 72.6 ± 4.2% in cells
treated with both drugs vs. 90.2 ± 4.6% and 98.9 ± 0.8%
in cells treated with the two individually).
Cell growth arrest is protracted
The extent of treatment-induced growth-arrest was investi-
gated in GEO cells only, as inhibition of cell proliferation

treated cells; P < 0.001; a reduction of around 88 colonies)
(Fig. 4A). Therefore the expected reduction in colony
number caused by coculturing with the two agents was
159. However, the observed number of colonies was actually
49 ± 13.4—a reduction of around 202 that was signifi-
cantly greater than the calculated expected reduction,
consistent with an enhanced suppression in growth
(Fig. 4B).
Combining CDO with cytotoxic drugs increases
SA-b-gal activity
As combination treatment induced a protracted reduction
in cell number, and did not induce significantly more
apoptosis, we sought evidence for senescence by assessing
SA-b-gal activity. In control and SO-treated cells, % SA-
b-gal positive cells after a 3-day culture were 13.3 ± 5.2%
and 11.7 ± 4.1%, respectively, and increased slightly
following culture with CDO alone (22.5 ± 5.2%; P ¼
0.027 vs. SO-treated cells). Similarly, coculturing cells with
cytotoxic drugs and SO alone also increased SA-b-gal
staining slightly compared to the SO-control (Fig. 5).
Concurrent culture of CDO with any cytotoxic drug
resulted in further increases in SA-b-gal staining that were
significantly greater than those seen in cells cultured with SO
and drug (all P < 0.001), indicating a synergistic effect of
CDO and cytotoxic drug in inducing senescence (Fig. 5).
Cyclin-associated CDK protein levels are reduced
Whole cell lysates from GEO and HCT116 cells treated with
drugs were separated by electrophoresis and immuno-
probed for p21
waf1

This study was undertaken to determine whether combining
CDO with conventional chemotherapeutic drugs might
have synergistic anticancer effects in colorectal cancer cell
lines. We confirmed that CDO was cytotoxic in two of the
cell lines studied at nanomolar concentrations. Additionally,
we showed that combining CDO with chemotherapeutic
drugs resulted in enhanced inhibition of cell proliferation,
which was associated with an increase in p21
waf1
expression,
loss of CDK function, and the generation of cells with
senescence characteristics.
In the first part of the investigation, we determined the
effect of continuous exposure to CDO on cell viability and
growth. IC
50
values showed that CDO was an effective
cytotoxic drug in HCT116 and GEO cells (300 n
M
and
Fig. 4. Effect on colony formation of combining CDO with cytotoxic
agents. GEO cells were cultured for 4 days with CDO in the presence
or absence of SN38 (S), 5-FU or etoposide. Equal number of cells were
removedfromeachoftheseculturesandplatedontomethylcellulose
for assessment of colony numbers on day 10. (A) Typical magnitude of
colony numbers seen in plates, using SN38 as an example. (B) The
differences in colony number (respective to controls) following treat-
ment were calculated (expected), and compared with the actual
(observed) numbers. Each column represents the mean and SDs of
at least four separate experiments. *P<0.05, between the expected

these studies were performed in this cell line only. Results
confirmed significantly enhanced decreases in the number of
colonies cultured with both CDO and cytotoxic drugs,
compared to the reductions observed in cells cultured with
the drugs individually. This suggested a protracted effect of
CDO in combination with chemotherapy, so we stained
cells for SA-b-gal activity, and showed that CDO alone did
not increase the extent of staining. However, in cells that
had been cocultured with CDO and a cytotoxic drug,
staining was significantly increased, indicating the presence
of senescence. Our data are consistent with a model in which
CDO induces a sustained arrest (senescence). Others have
shown that senescence is mediated in part by p21
waf1
activation [24,27]. Therefore, we assessed p21
waf1
levels in
GEO and HCT116 cell lines, and showed that combination
therapy induced p21
waf1
in the GEO cell line only (the cell
line in which synergistic effects were observed). A possible
explanation for this difference could be the higher basal
p21
waf1
levels in the HCT116 cell line compared to the GEO
cell line, suggesting a possible fault in their pathway. Hence
treatment with CDO would make HCT116 cells both
less likely to respond with an increase in p21
waf1

of CDKs associated with their respective cyclins were
significantly reduced, but only in the GEO cell lines where
p21
waf1
expression was increased by treatment. This
suggests that protracted inhibition of cell growth was
mediated through reduced cyclin/CDK function. There
have only been two studies reporting CDO-induced
inhibition of cyclin/CDK operation and cell proliferation
[18,19], which were in concordance with our results, and
highlighted a modulatory effect of CDO on cell cycle
progression. However, in contrast with our results, these
studies showed a reduction in cyclin D and E expression.
Disappointingly, the specific interactions between CDKs
and cyclins were not assessed, and the effects of CDO on
p21
waf1
were not investigated, making direct comparison
with our data more difficult.
Fig. 5. Effect of CDO and cytotoxic agents on
SA-b-gal staining. GEO cells were cultured
with VP16, 5-FU or SN38 and CDO or the
scrambled mismatch oligonucleotide (SO)
control, before staining for SA-b-gal activity.
Each column represents the mean and SDs of
six separate; P-values were calculated from
paired Student’s t-tests.
Ó FEBS 2004 CRE-decoys in colorectal cancer cells (Eur. J. Biochem. 271) 2779
In summary, these data provide a possible model of drug
interaction in GEO cells, which involves the complex

precipitation experiments highlighting cyclin/CDK interactions in the GEO cell line (i–ii) and the HCT116 cell line (iii–iv). The results of
densitometry analyses are given in (A) and (B), and are expressed as a percentage of each individual control.
2780 W. M. Liu et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Acknowledgements
We thank Prof. Yoon Cho-Chung for supplies of the CRE decoy
oligonucleotides and Dr Gianpaolo Tortora for the provision of the
GEO cell line. We also thank Dr Simon Joel for helpful discussions.
This work was supported by the New Drug Study Group discretionary
fund.
References
1. Cho-Chung, Y.S., Park, Y.G. & Lee, Y.N. (1999) Oligonucleo-
tides as transcription factor decoys. Curr. Opin. Mol. Ther. 1,386–
392.
2. Wang, H., Prasad, G., Buolamwini, J.K. & Zhang, R. (2001)
Antisense anticancer oligonucleotide therapeutics. Curr. Cancer
Drug Targets 1, 177–196.
3. Cho-Chung, Y.S. (2002) Antisense DNAs as targeted therapeutics
for cancer: no longer a dream. Curr. Opin. Invest. Drugs 3,934–
939.
4. Cho-Chung, Y.S. (2003) Antisense DNAs as targeted genetic
medicine to treat cancer. Arch. Pharm. Res. 26, 183–191.
5. Stephens, A.C. & Rivers, R.P. (2003) Antisense oligonucleotide
therapy in cancer. Curr. Opin. Mol. Ther. 5, 118–122.
6. Bergan, R., Connell, Y., Fahmy, B., Kyle, E. & Neckers, L. (1994)
Aptameric inhibition of p210bcr-abl tyrosine kinase autopho-
sphorylation by oligodeoxynucleotides of defined sequence and
backbone structure. Nucleic Acids Res. 22, 2150–2154.
7. Smetsers, T.F., Linders, E.H., van de Locht, L.T., de Witte, T.M.
& Mensink, E.J. (1997) An antisense Bcr-Abl phosphodiester-
tailed methylphosphonate oligonucleotide reduces the growth of

17.Park,Y.G.,Nesterova,M.,Agrawal,S.&Cho-Chung,Y.S.
(1999) Dual blockade of cyclic AMP response element-(CRE) and
AP-1-directed transcription by CRE-transcription factor decoy
oligonucleotide. gene-specific inhibition of tumor growth. J. Biol.
Chem. 274, 1573–1580.
18. Lee, Y.N., Park, Y.G., Choi, Y.H., Cho, Y.S. & Cho-Chung, Y.S.
(2000) CRE-transcription factor decoy oligonucleotide inhibition
of MCF-7 breast cancer cells: cross-talk with p53 signaling path-
way. Biochemistry 39, 4863–4868.
19. Park, Y.G., Park, S., Lim, S.O., Lee, M.S., Ryu, C.K., Kim, I. &
Cho-Chung, Y.S. (2001) Reduction in Cyclin D1/CDK4/Retino-
blastoma Protein Signalling by CRE-Decoy Oligonucleotide.
Biochem. Biophys. Res. Commun 281, 1213–1219.
20. Alper, O., Bergmann-Leitner, E.S., Abrams, S. & Cho-Chung,
Y.S. (2001) Apoptosis, growth arrest and suppression of inva-
siveness by CRE-decoy oligonucleotide in ovarian cancer cells:
protein kinase A downregulation and cytoplasmic export of CRE-
binding proteins. Mol. Cell. Biochem. 218, 55–63.
21. Henry, S.P., Geary, R.S., Yu, R. & Levin, A.A. (2001) Drug
properties of second-generation antisense oligonucleotides: how
do they measure up to their predecessors? Curr. Opin. Invest.
Drugs 2, 1444–1449.
22. Fisher, A.A., Ye, D., Sergueev, D.S., Fisher, M.H., Shaw, B.R. &
Juliano, R.L. (2002) Evaluating the specificity of antisense oligo-
nucleotide conjugates: a DNA array analysis. J. Biol. Chem. 277,
22980–22984.
23. Liu, W.M., Oakley, P.R. & Joel, S.P. (2002) Exposure to low
concentration of etoposide reduces the apoptotic capability of
leukaemic cell lines. Leukemia 16, 1705–1712.
24. te Poele, R.H., Okorokov, A.L., Jardine, L., Cummings, J. & Joel,