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

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. Clinically, the cytostatic ability of CDO could 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) Eur. J. Biochem. 271, 2773–2781 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04208.x 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 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 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) CO 2 in air at 37 °C. Transfection with CDO CDO and 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 · 10 5 mL )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 CDO with Oligofectamine reagent according to the manufacturer’s protocol (Invitro- gen Ltd). CDO was added at a 50–200 n M final 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-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) ethanol and permeabilization in 2 M 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 · 10 5 mL )1 )wereplated in semisolid cultures containing 0.9% (w/v) methylcellulose and 30% (v/v) fetal bovine serum (Stem Cell Tech). Culture dishes were incubated at 37 °C in a humidified atmosphere with 5% (v/v) CO 2 . 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-p21 waf1 (0.2 lgÆmL )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, 5m M EDTA, 50 m M NaF, 10 lgÆmL )1 phenylmethane- sulfonyl fluoride, 0.5 lgÆmL )1 leupeptin, 2 lgÆmL )1 soybean trypsin inhibitor, 0.5 lgÆmL )1 aprotinin, 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/P i , 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 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 GEO cell 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, CDO had no effect on cell proliferation at equi-molar concentrations (cells per mL and percentage viability with 1.6 l M CDO: 1.1 · 10 6 and 89.7% vs. 1.7 · 10 6 and 91.3% in SO-cultured control cells). Flow cytometric analysis revealed concomitant increases in the sub-G 1 population of cells, indicative of apoptosis (Fig. 1). 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 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 1 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 GEO cells 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. 2776 W. M. Liu et al. (Eur. J. Biochem. 271) Ó FEBS 2004 cocultured with CDO and any cytotoxic agent (Fig. 2B). This was similarly established by comparing the size of the sub-G 1 population in the combination sample with the numerical sum of the separate sub-G 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 was not seen in the HCT116 cells. At the end of the treatment schedules, the surviving fractions of GEO cells 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 SO alone (251.9 ± 32.1; P ¼ 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 CDO or 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 CDO and VP16 together. Each column represents the mean and SDs of at least three separate experiments; P-values were calcula- tedfrompairedStudent’st-tests. Ó FEBS 2004 CRE-decoys in colorectal cancer cells (Eur. J. Biochem. 271) 2777 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 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 , cyclin B, cyclin D, CDK 1 and CDK 4. Combining CDO with 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. p21 waf1 levels did not change after culturing with any combination of drug in HCT116 cells, but were significantly increased in GEO cells treated with the combination of CDO and 5-FU (Fig. 6A). In both HCT116 and GEO cells, 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). GEO cells (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 GEO cell line. Discussion 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 and observed. 2778 W. M. Liu et al. (Eur. J. Biochem. 271) Ó FEBS 2004 360 n M , respectively), but ineffective in the more resistant SW620 cells (>1600 n M ). 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-G 1 (apoptotic) portion of the cell cycle. We next investigated the effect of combining CDO with 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 CDO was combined with any chemotherapeutic drug. However, CDO/chemotherapy combinations in GEO cells 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 CDO has 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 GEO cells, 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 ,orto mount a functional p21 waf1 response. 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 G 1 /S transition. Cells arrest if this complex is not formed. It has been suggested that CDO reduces pRb hyper-phosphorylation through p53 stabilization [18]. This would cause an increase in p21 waf1 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 GEO cells were unchanged after treatment with CDO and 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 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 interaction of proteins involved in cell cycle regulation. To recapitulate, combining CDO with 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 p21 waf1 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 p21 waf1 in 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 CDO could improve and enhance the conventional cytotoxic effect of other chemotherapies in some cancers. Any synergistic effect however, may be independent of pathways controlling p21 waf1 expression. 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 CDO and 5-FU combination are presented. The patterns of protein expression following culture with CDO and 5-FU together were similar in GEO and HCT116 cells. (A) The only protein that was expressed differently in the cell lines was p21 waf1 ,which was increased in GEO but unchanged in HCT116 cells. (B) Standard immunoblots for cyclin and CDK levels in GEO cells. (C) Immuno- 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 chronic myeloid leukaemia patient cells by a non-antisense mechanism. Br. J. Haematol. 96, 377–381. 8.Juliano,R.L.,Alahari,S.,Yoo,H.,Kole,R.&Cho,M. (1999) Antisense pharmacodynamics: critical issues in the trans- port and delivery of antisense oligonucleotides. Pharm. Res. 16, 494–502. 9. Juliano, R.L. & Yoo, H. (2000) Aspects of the transport and delivery of antisense oligonucleotides. Curr. Opin. Mol. Ther. 2, 297–303. 10. Gewirtz, A.M. (1999) Oligonucleotide therapeutics: clothing the emperor. Curr. Opin. Mol. Ther. 1, 297–306. 11. Jahagirdar, B.N., Miller, J.S., Shet, A. & Verfaillie, C.M. (2001) Novel therapies for chronic myelogenous leukemia. Exp. Hematol. 29, 543–556. 12. Tomita, N., Morishita, R., Tomita, T. & Ogihara, T. (2002) Potential therapeutic applications of decoy oligonucleotides. Curr. Opin. Mol. Ther. 4, 166–170. 13. Roesler, W.J., Graham, J.G., Kolen, R., Klemm, D.J. & Mcfie, P.J. (1995) The cAMP response element binding protein synergizes with other transcription factors to mediate cAMP responsiveness. J. Biol. Chem. 270, 8225–8232. 14. Shaywitz, A.J. & Greenberg, M.E. (1999) CREB: a stimulus- induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68, 821–861. 15. Montminy, M.R. & Bilezikjian, L.M. (1987) Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 328, 175–178. 16. De Cesare, D., Fimia, G.M. & Sassone-Corsi, P. (1999) Signaling routes to CREM and CREB: plasticity in transcriptional activa- tion. Trends Biochem. Sci. 24, 281–285. 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, S.P. (2002) DNA damage is able to induce senescence in tumor cellsinvitroandinvivo.Cancer Res. 62, 1876–1883. 25. Liu, W.M., Stimson, L.A. & Joel, S.P. (2002) The in vitro activity of the tyrosine kinase inhibitor STI571 in BCR-ABL positive CML cells: Synergistic interactions with anti-leukaemic agents. Br.J.Cancer86, 1472–1478. 26. Cho, Y.S., Kim, M.K., Cheadle, C., Neary, C., Park, Y.G., Becker, K.G. & Cho-Chung, Y.S. (2002) A genomic-scale view of the cAMP response element-enhancer decoy: a tumor target-based genetic tool. Proc. Natl Acad. Sci. USA 99, 15626–15631. 27. Han, Z., Wei, W., Dunaway, S., Darnowski, J.W., Calabresi, P., Sedivy,J.,Hendrickson,E.A.,Balan,K.V.,Pantazis,P.&Wyche, J.H. (2002) Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin. J. Biol. Chem. 277, 17154–17160. 28. Sherr, C.J. (1993) Mammalian G1 cyclins. Cell 73, 1059–1065. 29. Nurse, P. (1994) Ordering S phase and M phase in the cell cycle. Cell 79, 583–589. 30. El-Deiry (1998) p21/p53, cellular growth control and genomic integrity. Curr. Top. Microbiol. Immunol. 227, 121–237. 31. Mcdonald, E.R. & El-Deiry, W.S. (2000) Cell cycle control as a basis for cancer drug development. Int. J. Oncol. 16, 871–886. 32. Murray, A.W. (2004) Recycling the cell cycle: cyclins revisited. Cell 116, 221–234. Ó FEBS 2004 CRE-decoys in colorectal cancer cells (Eur. J. Biochem. 271) 2781 . The in vitro effects of CRE-decoy oligonucleotides in combination with conventional chemotherapy in colorectal cancer cell lines Wai M. Liu*, Katherine. 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