Báo cáo khoa học: The in vitro effects of CRE-decoy oligonucleotides in combination with conventional chemotherapy in colorectal cancer cell lines potx

CDOhad a potent anti-proliferative effect in colorectal cell lines, yet, a similar enhancement of cell death was not observed.. The aims of the present study were threefold: first, to expl

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 CDOalone and in combination with a range of

common chemotherapeutic agents in colorectal cancer cell

lines CDOhad 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 CDOwith 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 p21waf1in 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 Clinically, the cytostatic ability of CDOcould improve and enhance the antiproliferative effects of conventional cyto-toxic agents

Keywords: cAMP response element; colorectal cancer; oligo-nucleotide decoy factors; synergy

The regulation of transcription by using short sequence

oligonucleotides has been a focus for developing new drug

therapies[1–5].Thisisbasedupontheprinciplethatrepression

of key genes associated with malignancy might provide novel

therapeutic targets Also, dysregulation of response elements

within promoter regions of genes has been implicated in

neoplastic transformation, thus restoring correct and

appro-priate function may reverse the aberrant phenotype [1]

There are two approaches using oligonucleotides to

achieve this First, the development of dominant mutants

with dysfunctional activation domains, which compete with

wild-type counterparts in binding to target genes [2] This

antisense oligonucleotide approach results in agents that,

through their ability to bind specific RNA and DNA

sequences are highly selective However, this genomic

approach has only met with a limited degree of success, as there have been conflicting reports to suggest that the efficacy of these antisense oligonucleotides may not exclu-sively be a result of sequence-binding, but to some other yet unknown mechanism predominant in cells that sensitizes them to cell killing [6,7] In addition, it is also possible that the reagents used to maximize delivery of these oligonucle-otides to the target cell may actually directly interfere with cellular processes, resulting in nonspecific effects [8,9] Another consideration for the use of generic antisense 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: wai.liu@icr.ac.uk

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

oligonucleotides; BrdU, 5-bromo-2¢-deoxyuridine; SA-b-gal,

senescence-associated b-galactosidase.

*Present address: Section of Medicine, Institute of Cancer Research,

Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey SM2 5NG,

UK.

(Received 18 February 2004, revised 22 April 2004,

accepted 7 May 2004)

genes [13] The promoter region of several of these genes has

been studied, and a common CRE sequence has been noted

upstream of the transcriptional start site [14] All of the

cAMP responsive gene promoter regions have the same

eight-base enhancer sequence, the CRE, which is the

palindromic sequence 5¢-TGACGTCA-3¢ [13] Proteins that

bind to these CREs have been identified that are 43 kDa in

size, and contain a basic leucine zipper DNA-binding motif

[15] Functional studies have shown that this transcription

factor, termed the CRE-binding protein (CREB), couples

gene activation to a wide variety of cellular signals [14], and

thus coordinates a multitude of genes that regulate

numer-ous cellular processes, including cell growth and

differenti-ation [16]

The ubiquitous nature of the CRE consensus site makes it

a good target for chemotherapy Indeed, it has been shown

that a palindromic trioctamer of this sequence can interfere

with CREB binding, and specifically inhibit PKA subunit

expression, interfering with the CRE-PKA pathway [17]

This causes specific inhibition of growth in cancer cells, and

although the CRE-regulated genes are common in all cell

types, surprisingly, CRE-decoy oligonucleotides (CDOs)

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 CDOalone in a panel of three

colorectal cancer cell lines; second, to investigate the effects

of combining CDOwith 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 GEOcolorectal

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 GEOcell lines were both wild-type p53, and SW620

lines were mutant p53 No antibiotics were used in our

experiments, and all cell lines were incubated in a humidified

atmosphere with 5% (v/v) CO2in air at 37C

Transfection with CDO

CDOand scrambled mismatch oligonucleotides (SO) were

gifts from Y S Cho-Chung (National Cancer Institute,

Bethesda, MD, USA), and were phosphothiorated for

stability [21,22] They were trioctamers of the CRE consen-sus site, and their complete sequences were: CDO, 5¢-TGACGTCATGACGTCATGACGTCA-3¢; SO , 5¢-TGT GGTCATGTGGTCATGTGGTCA-3¢

Cells (1· 105mL)1) were plated into 6-well plates, and allowed to adhere for 24 h Cells were rinsed in Hank’s buffered salt solution (Sigma) and refreshed with serum-free medium before the addition of CDOwith Oligofectamine reagent according to the manufacturer’s protocol (Invitro-gen Ltd) CDOwas added at a 50–200 nMfinal concentra-tion After 4 h of incubation, culture medium supplemented with 20% (v/v) fetal bovine serum was added to make the volume up to 5 mL At this stage, SN38, 5-FU or VP16 (all from Sigma) could be added Aliquots were removed daily for assessment of cell number and viability by staining with 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-G1(apoptotic fraction, cells with a reduced propidium iodide stain but similar morphology), G1, S and G2/M phases were determined using the cell cycle analysis program

WINMDIv2.4

Flow cytometric analysis of BrdU incorporation The degree of incorporation of the thymidine analogue 5-bromo-2¢-deoxyuridine (BrdU; Sigma) in HCT116 and GEOcells was measured by flow cytometry Following culture in CDOand drugs, cells (5· 105mL)1) were transferred into fresh culture medium containing 10 lM

BrdU for 30 mins before fixing with ice-cold 70% (v/v) ethanol and permeabilization in 2M HCl with 0.5% (v/v) Triton X-100 Samples were washed and incubated with fluorescein isothiocyanate-conjugated mouse anti-BrdU according to the manufacturer’s instructions (PharMingen) The cell cycle distribution was resolved by staining with propidium iodide, and BrdU fluorescence specifically within the S-phase was measured by using the FACSCalibur Ten-day clonogenic assays

Cells were harvested from initial cell cultures and resuspended in DMEM Cells (1· 105mL)1) were plated

in semisolid cultures containing 0.9% (w/v) methylcellulose and 30% (v/v) fetal bovine serum (Stem Cell Tech) Culture dishes were incubated at 37C in a humidified atmosphere with 5% (v/v) CO2 The number of colonies containing more than 50 cells was assessed on day 10

Immunoblotting and immunoprecipitation analysis For immunoblot analysis, total cellular protein was solubi-lized and resolved by SDS/PAGE using 15% acrylamide with a 5% stacking gel as described previously [23] Pri-mary antibody probing was performed with anti-p21waf1 (0.2 lgÆmL)1), anticyclin D (2 lgÆmL)1), anti-CDK 4

(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 mMTris pH 7.6,

150 mM NaCl), horseradish peroxidase-conjugated

anti-species IgG1 was used as the secondary antibody (1 : 1000;

DAKOLtd) 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 mMTris, 250 mMNaCl,

5 mM EDTA, 50 mM NaF, 10 lgÆmL)1

phenylmethane-sulfonyl fluoride, 0.5 lgÆmL)1leupeptin, 2 lgÆmL)1soybean

trypsin inhibitor, 0.5 lgÆmL)1aprotinin, 2 lgÆmL)1

N-tosyl-L-phenylalanine chloromethyl ketone, 0.1% (v/v) Triton

X-100; all Sigma), and clarified by centrifugation Protein was

used for immunoprecipitation with either anticyclin D or

cyclin B and protein A-sepharose (Amersham) Resultant

immune complexes were washed twice with RIPA buffer,

denatured in Laemmli buffer, and resolved by SDS/PAGE

(15% acrylamide)

Analysis of SA-b-gal activity

Cellular senescence-associated b-galactosidase (SA-b-gal)

activity was assessed as previously described by this group

[24] Briefly, cells were washed twice in ice-cold NaCl/Pi,

before fixing in 2% (v/v) formaldehyde and 0.2% (v/v)

glutaraldehyde Cells were then washed twice in ice-cold

NaCl/Pi, before overnight incubation at 37C in X-Gal

staining solution (1 mgÆmL)1 5-bromo-4-chloro-3-indolyl

b-D-galactoside in 40 mM citric acid/sodium phosphate

pH 6, 5 mM potassium ferricyanide, 5 mM potassium

ferrocyanide, 150 mMsodium chloride, 2 mMmagnesium

chloride) Samples were then washed twice in ice-cold NaCl/

Piprior to assessing the percentage of cells staining positive

for SA-b-gal activity by light microscopy

Statistical analysis

All statistical analyses were performed using MINITAB

version 10 (State College, PA, USA) Data was normally

distributed as established by Shapiro–Wilk testing, and parametric analyses were used throughout Differences between variables and control cultures, as determined by analysis of variance, were further characterized by paired Student’s t-tests

Results

Exposure to single-agent CDO

A concentration-dependent reduction in cell number and cell viability was observed in HCT116 and GEOcell lines cultured with CDO However, no changes to cell number or viability was observed in the cells treated with the SO control (Fig 1) Conversely, in SW620 cells that are intrinsically more resistant to cytotoxic agents in general, CDOhad no effect on cell proliferation at equi-molar concentrations (cells per mL and percentage viability with 1.6 lMCDO: 1.1· 106and 89.7% vs 1.7· 106and 91.3%

in SO-cultured control cells) Flow cytometric analysis revealed concomitant increases in the sub-G1population of cells, indicative of apoptosis (Fig 1)

Combination with other chemotherapeutic agents Preliminary experiments indicated that SW620 cells were resistant to both CDOand chemotherapy at the concen-trations studied, and so were excluded from the combina-tion studies These simple combinacombina-tion studies involved culturing cells simultaneously with each agent at the concentration that reduced cell numbers by 25% (IC25) Culturing HCT116 and GEOcells with these equi-toxic drug concentrations resulted in different responses

in these cell lines Specifically, combining CDOwith 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 GEOcells cultured with CDO and 5-FU; by simply comparing the total reduction in cell number in cultures treated with CDOand 5-FU together (relative to the SOcontrol) with the expected reduction in

Fig 1 The effect of CDO in HCT116 and GEO cell lines on day 3 The activity of CDOin the sensitive cell lines was fitted a standard E max model Representative DNA histograms following culture with CDOin 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.

cell number (calculated as the numerical sum of the

reductions in cell number seen in the cultures with the two

agents separately [25] (· 105 cellsÆmL)1: )22.3 ± 1.8 vs

)13.8 ± 2.6; P < 0.001) These results were confirmed by the flow cytometric data, which showed no enhancement of the apoptotic fraction (sub-G population) of HCT116 cells

Fig 2 The effect of combining CDO with cytotoxic agents in HCT116 and GEO cells Cells were cultured with CDO(C) alone or in combination with 5-FU, SN38 or VP16 There were significant reductions in cell number in GEOcells that was not seen in HCT116 cells (A) Representative DNA histograms of GEO cells cultured with VP16, 5-FU and SN38 in the presence or absence of CDO (B) Individ-ual fractions of events within the sub-G 1

population (apoptosis) Each data point represents the mean and SDs of six separate experiments; P-values were calculated from paired Student’s t-tests.

cocultured with CDOand any cytotoxic agent (Fig 2B).

This was similarly established by comparing the size of the

sub-G1 population in the combination sample with the

numerical sum of the separate sub-G1populations seen in

cells treated with the individual agents Additionally, flow

cytometry revealed no apparent blockades in the G1, S or

G2phases 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

GEOcells cocultured with CDOand cytotoxic drugs

compared to those treated with drugs separately (Fig 3)

This was most apparent for BrdU incorporation in cells cocultured with CDOand 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 GEOcells only, as inhibition of cell proliferation was not seen in the HCT116 cells At the end of the treatment schedules, the surviving fractions of GEOcells were plated in short-term semisolid cultures in the absence

of drug, and colony formation was assessed on day 10 The total number of colonies seen in control plates containing untreated cells was 254.2 ± 18.9, which was not signifi-cantly different from the number seen in plates pretreated with SOalone (251.9 ± 32.1; P¼ 0.809) However, the number of colonies in the CDO-treated samples was significantly less than that observed in the control plates (180.3 ± 33.1; P < 0.001; a reduction of around 71 colonies) Colony numbers were also reduced in the plates

Fig 3 Effect of combining CDO with

cyto-toxic agents in HCT116 and GEO cells The

numerical sum of BrdU incorporation into

cultures containing CDOor any of the

cyto-toxic agents alone (expected) was compared to

the observed level of incorporation in cultures

with the two agents used simultaneously

(observed) For example, the extent of BrdU

incorporation into cells treated with CDO

alone and into cells treated with VP16 alone

was summed, and compared to the extent of

BrdU incorporation into cells treated with

both CDOand VP16 together Each column

represents the mean and SDs of at least three

separate experiments; P-values were

calcula-ted from paired Student’s t-tests.

pretreated with cytotoxic drugs (e.g 163.7 ± 23.4 in

SN38-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 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 CDOalone (22.5 ± 5.2%; P¼ 0.027 vs SO-treated cells) Similarly, coculturing cells with cytotoxic drugs and SOalone also increased SA-b-gal staining slightly compared to the SO-control (Fig 5) Concurrent culture of CDOwith 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 CDOand cytotoxic drug in inducing senescence (Fig 5) Cyclin-associated CDK protein levels are reduced Whole cell lysates from GEOand HCT116 cells treated with drugs were separated by electrophoresis and immuno-probed for p21waf1, cyclin B, cyclin D, CDK 1 and CDK 4 Combining CDOwith any chemotherapeutic drug resulted

in changes in protein levels that appeared to be similar, irrespective of the chemotherapy used Consequently, the effects of 5-FU on the cell lines are presented, which were most representative of the effects seen with any of the three drugs studied

p21waf1 levels did not change after culturing with any combination of drug in HCT116 cells, but were significantly increased in GEOcells treated with the combination of CDOand 5-FU (Fig 6A) In both HCT116 and GEOcells, there were no significant changes in the levels of CDK 4, cyclin D or CDK 1 in response to drug combinations Only cyclin B appeared to be affected, its level being reduced in combination cultures (Fig 6B) This observation was con-fusing, did not correlate with the flow cytometry data (no G2-block was observed; Fig 2B), and was not associated with a respective change in its partner CDK 1 As the association of CDKs with their partner cyclins is crucial to function, we sought to resolve this by measuring the levels of CDK 1 and CDK 4 coimmunoprecipitating with the respective anticyclin antibody (Fig 6C) GEOcells (blots: i–ii) and HCT116 cells (blots: iii–iv) were cultured with 5-FU in the presence or absence of CDO, and results showed that the amount of each CDK precipitating with their respective cyclin was significantly reduced only in the GEOcell line

Discussion

This study was undertaken to determine whether combining CDOwith conventional chemotherapeutic drugs might have synergistic anticancer effects in colorectal cancer cell lines We confirmed that CDOwas cytotoxic in two of the cell lines studied at nanomolar concentrations Additionally,

we showed that combining CDOwith chemotherapeutic drugs resulted in enhanced inhibition of cell proliferation, which was associated with an increase in p21waf1expression, 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 CDOon cell viability and growth IC50 values showed that CDOwas an effective cytotoxic drug in HCT116 and GEOcells (300 n and

Fig 4 Effect on colony formation of combining CDO with cytotoxic

agents GEOcells were cultured for 4 days with CDOin the presence

or absence of SN38 (S), 5-FU or etoposide Equal number of cells were

removed from each of these cultures and plated onto methylcellulose

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

and observed.

360 nM, respectively), but ineffective in the more resistant

SW620 cells (>1600 nM) Results showed dose-dependent

decreases in cell number and concomitant decreases in cell

viability in the sensitive cell lines Flow cytometric analysis

showed that this cell death was not specific to any particular

phase of the cell cycle, and was associated with an increase

in the sub-G1(apoptotic) portion of the cell cycle

We next investigated the effect of combining CDOwith

the chemotherapeutic drugs 5-FU, SN38 and etoposide in

the two CDO-sensitive cell lines There was neither

enhancement of cell death nor a greater reduction in the

number of HCT116 cells when CDOwas combined with

any chemotherapeutic drug However, CDO/chemotherapy

combinations in GEOcells resulted in significant reductions

in cell number This was further investigated by assessing

BrdU incorporation Results confirmed the synergistic

reduction in cell number, and were in agreement with the

observation that CDOhas widespread effects on genes

controlling cell proliferation [26]

We then investigated the extent of the cell growth

inhibition in longer-term clonogenic assays As the

syner-gistic arrest in cell growth was observed only in GEOcells,

these studies were performed in this cell line only Results

confirmed significantly enhanced decreases in the number of

colonies cultured with both CDOand cytotoxic drugs,

compared to the reductions observed in cells cultured with

the drugs individually This suggested a protracted effect of

CDOin combination with chemotherapy, so we stained

cells for SA-b-gal activity, and showed that CDOalone did

not increase the extent of staining However, in cells that

had been cocultured with CDOand a cytotoxic drug,

staining was significantly increased, indicating the presence

of senescence Our data are consistent with a model in which

CDOinduces a sustained arrest (senescence) Others have

shown that senescence is mediated in part by p21waf1

activation [24,27] Therefore, we assessed p21waf1levels in

GEOand HCT116 cell lines, and showed that combination

therapy induced p21waf1in the GEOcell line only (the cell

line in which synergistic effects were observed) A possible

explanation for this difference could be the higher basal

p21waf1levels in the HCT116 cell line compared to the GEO

cell line, suggesting a possible fault in their pathway Hence treatment with CDOwould make HCT116 cells both less likely to respond with an increase in p21waf1, or to mount a functional p21waf1response This requires further investigation

The correct binding of cyclins to CDKs is required for successful cell cycle progression [28,29]; for example, the correct formation of cyclin D/CDK 4 complex is required for pRb hyper-phosphorylation, in order for it to release transcription factors necessary for G1/S transition Cells arrest if this complex is not formed It has been suggested that CDOreduces pRb hyper-phosphorylation through p53 stabilization [18] This would cause an increase in p21waf1 and induce cell cycle arrest through its CDK-inhibitory function [30] Our results showed that absolute CDK 1 and CDK 4 levels in HCT116 and GEOcells were unchanged after treatment with CDOand cytotoxic drug Small changes were seen in cyclin B levels in some

of the treatments; the significance of which was unclear However, as it is generally accepted that the heterodime-rization of catalytic CDKs with the cyclin subunits is the major determinant of cell cycle fate, rather than their absolute numbers in isolation [31,32], we next performed immunoprecipitation assays in an attempt to clarify the relationship between CDKs, cyclins and cell cycling follo-wing combination treatments Results showed that levels

of CDKs associated with their respective cyclins were significantly reduced, but only in the GEOcell lines where p21waf1 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 CDOon 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 CDOon p21waf1 were not investigated, making direct comparison with our data more difficult

Fig 5 Effect of CDO and cytotoxic agents on

SA-b-gal staining GEOcells were cultured

with VP16, 5-FU or SN38 and CDOor 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.

In summary, these data provide a possible model of drug

interaction in GEOcells, which involves the complex

interaction of proteins involved in cell cycle regulation To

recapitulate, combining CDOwith cytotoxic chemotherapy

reduced CDK activity This ultimately resulted in reduced

cell cycling, which was manifest as a general reduction in cell

proliferation and the appearance of senescence

characteris-tics The activation of p21waf1 appeared to play an

important role in mediating this effect, as we also showed

that an inability to upregulate this protein, as seen in

HCT116 cells, resulted in the absence of enhanced cell growth inhibition The central role of p21waf1in mediating senescence is currently being investigated in isogenic cell lines by gene expression profile methodologies, and will form the basis of a future publication Nevertheless, it appears that clinically, the cytostatic ability of CDOcould improve and enhance the conventional cytotoxic effect of other chemotherapies in some cancers Any synergistic effect however, may be independent of pathways controlling p21waf1expression

Fig 6 The effect of CDO and 5-FU on cell cycle regulating proteins The cytotoxic agents appeared to have similar effects on the proteins studied; therefore, immunoblots from only the CDOand 5-FU combination are presented The patterns of protein expression following culture with CDO and 5-FU together were similar in GEOand HCT116 cells (A) The only protein that was expressed differently in the cell lines was p21 waf1 , which was increased in GEObut unchanged in HCT116 cells (B) Standard immunoblots for cyclin and CDK levels in GEOcells (C) Immuno-precipitation experiments highlighting cyclin/CDK interactions in the GEOcell 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.

We thank Prof Yoon Cho-Chung for supplies of the CRE decoy

oligonucleotides and Dr Gianpaolo Tortora for the provision of the

GEOcell line We also thank Dr Simon Joel for helpful discussions.

This work was supported by the New Drug Study Group discretionary

fund.

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