- Email: [email protected]
E2F decoy oligodeoxynucleotides effectively inhibit growth of human tumor cells
BBRC Biochemical and Biophysical Research Communications 310 (2003) 1048–1053 www.elsevier.com/locate/ybbrc
E2F decoy oligodeoxynucleotides effectively inhibit growth of human tumor cells Jong Deok Ahn,a,b Cheorl-Ho Kim,c Junji Magae,d Young Ho Kim,a Hye Jin Kim,a,b Kwan-Kyu Park,e SaHyun Hong,e Keun-Gyu Park,b In Kyu Lee,b,* and Young-Chae Change,* a Department of Microbiology, Kyungpook National University, Daegu 701-702, Republic of Korea Department of Internal Medicine, Keimyung University School of Medicine, Daegu 700-712, Republic of Korea National Research Laboratory for Glycobiology, Korean Ministry of Science and Technology, College of Oriental Medicine, Dongguk University, Kyungju City, Kyungbuk 780-714, Republic of Korea d Department of Biotechnology, Institute of Research and Innovation, 1201 Takada, Kashiwa 277-0861, Japan e Kidney Institute, Keimyung University School of Medicine, Daegu 700-712, South Korea b
c
Received 9 September 2003
Abstract Abnormal cell proliferation, largely dependent upon deregulation of cell-cycle regulatory proteins, is an important feature of several forms of human cancer. The transcription factor, E2F, plays a critical role in the trans-activation of several genes involved in cell-cycle regulation, thereby regulating cell growth. We have demonstrated that E2F decoy oligodeoxynucleotides (ODNs) with a circular dumbbell structure (CD-E2F decoy) corresponding to E2F binding sites effectively inhibit cell proliferation of primary cultured cells. Here we found that the E2F decoy ODNs inhibited serum-induced promoter activity of E2F-dependent genes in a sequence-specific manner in a RB-positive human osteosarcoma, U2OS, as well as in a RB-negative human cervical carcinoma, C33A. This E2F decoy ODN strongly inhibited gene expression of endogenous E2F1 and PCNA and proliferation of these cancer cells. Our results suggest that this decoy ODN strategy could represent a powerful investigative and potentially therapeutic strategy in the prevention and treatment of cancer. Ó 2003 Elsevier Inc. All rights reserved. Keywords: E2F; RB; Decoy; Dumbbell; Tumor growth inhibition; Osteosarcoma; Cervical carcinoma
It is now widely accepted that cancer is a disease characterized by defects in cell cycle regulation. Transcription factor E2F plays an important role in the regulation of cell cycle progression and entry into S phase [1–3]. E2F has been implicated in the periodic regulation of cellular genes required for transition through G1 and entry into the S phase including dihydrofolate reductase (DHFR), c-myc, DNA polymerase, cdc2, and proliferating cell nuclear antigen (PCNA) [4– 7]. E2F activity is regulated by interactions with RB family members. As cells progress toward the S phase, * Corresponding authors. Fax: +82-53-250-7892 (I.K. Lee), +82-53250-7095 (Y.-C. Chang). E-mail addresses: [email protected] (I.K. Lee), ycchang@ dsmc.or.kr (Y.-C. Chang).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.124
RB family proteins are phosphorylated by G1 cyclin– cdk complexes, resulting in the release of transcriptionally active E2F which leads to the activation of genes required for cell cycle progression [8–10]. Over-expression of various E2F proteins can induce cell cycle progression [11–14]. Several previous studies have shown that over-expression of various E2F proteins can induce cell cycle progression, and several intervention trials have been used to block the action of E2F proteins, including antisense methods, ectopic expression of dominant-negative mutants, and inhibition of upstream kinases [15,16]. An alternative approach to repress the action of transcription factors involves the use of double-stranded “decoy” oligonucleotides (ODNs) which inhibit the expression of genes by providing a competitor for the
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053
binding element of specific transcription factors such as E2F. Several studies have reported that synthetic double-stranded ODNs act as decoys against their target transcription factor when introduced into cells, and the decoys down-regulate the expression of several genes in a sequence-specific manner [17–20]. Therefore, decoy oligonucleotides have been proposed as a potential approach to block the action of transcription factors on gene expression. Moreover, we have demonstrated previously that treatment of E2F decoy ODN inhibits cell proliferation in vascular smooth muscle cells and mesangial cells [21,22]. Together with these findings led to us to investigate the inhibitory effect of E2F ODN decoys on cell cycle progression as a possible candidate for therapeutic intervention in proliferation of cancer cells. In this study, we developed and optimized E2F decoy ODN targeting activated E2F and found that circular dumbbell E2F decoy (CD-E2F decoy) ODN effectively inhibited transactivation of essential E2F-dependent cell-cycle regulatory genes (E2F1, PCNA) and proliferation of cancer cells. This study demonstrates the feasibility of E2F decoy ODN as a therapeutic strategy for human cancers. Materials and methods Cell culture. A human osteosarcoma, U2OS, and a human cervical carcinoma, C33A, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT) and 1% penicillin–streptomycin (Sigma, St. Louis, MO) at 37 °C in a 5% CO2 atmosphere. Construction of the circular dumbbell E2F decoy ODN. The sequences of circular dumbbell double-stranded ODN against the E2F binding site and mutated ODNs were as follows: CD-E2F decoy ODN (note: consensus sequences are underlined), 50 -ATGCGCGAA ACGCGTTTTCGCGTTTCGCGCATAGTTTTCT-30 ; mutated E2F decoy (M-E2F decoy) ODN, 50 -ATAATCTAAACGCGTTTTCGCG TTTAGATTATAGTTTTCT-30 . CD-E2F decoy ODN was predicted to form a stem-loop structure. Following the addition of T4 DNA ligase, the mixture was incubated for 24 h at 16 °C to generate a covalently ligated circular dumbbell decoy molecule. CD-E2F decoy ODN comprises two loops and one stem containing an E2F consensus sequence. In vitro gene transfer. Cells were fed with fresh culture medium the day before decoy was added and washed twice with Opti-MEM (Gibco-BRL), prior to the experiment. Cells were transfected with decoy ODN (100 nM) or plasmids combined with LipofectAMINE 2000 (molar ratio; DNA:lipid ¼ 1:3) (Gibco-BRL). The mixture was added dropwise to cells, according to manufacturer’s instructions. Transfection of plasmid expressing wild type RB protein (pCMV-RB) was carried out in a similar manner. Cells were incubated at 37 °C for 5 h. After the addition of fresh medium containing 10% FBS, cells were maintained in a CO2 incubator until use. The pSV-b-gal (Promega, Madison, WI, USA) was co-transfected to monitor the transfection efficiencies. Luciferase assay. [E2F]x4 luciferase reporter and a series of E2F1 reporter constructs were used in transient transfection assays as described previously [23]. Briefly, the [E2F]x4 luciferase reporter, which contains four E2F sites with the sequence TTTCGCGC and the TATA
1049
box from the adenovirus E1B gene, was generated by subcloning the promoter from [E2F]x4 chloramphenicol acetyltransferase into the reporter plasmid pGL3-Basic. For E2F1 promoter assays, E2F1-luc construct was supplied as a gift from N. Dyson of MGH Cancer Center [24]. The [E2F]x4-luc and E2F1 reporter gene plasmid were used to investigate the effects of E2F decoy ODN on promoter activity. To analyze luciferase expression, cells were washed twice with PBS and lysed with 200 ll of 1 Reporter lysis buffer (Promega, Madison, WI, USA). Each lysate (50 ll) was examined for luciferase activity and b-galactosidase activity as described previously [22]. Northern blot analysis. The radiolabeled probes used for Northern blot analysis were prepared by random primer labeling method with [a-32 P]dCTP using a kit (Amersham, Arlington Heights, IL, USA). After the labeling reaction, the radiolabeled probes were purified on a NAP-5 column (Pharmacia, Uppsala, Sweden). Total RNA for Northern blot analysis was extracted by RNeasy RNA extraction kit (Qiagen, Hilden, Germany). Ten micrograms of total RNA was applied to a 1% formaldehyde–agarose gel and transferred to a nylon membrane. The nylon membrane was hybridized in Express Hyb solution at 65 °C for 2 h with a radiolabeled E2F1 and PCNA cDNA probe and washed according to the manufacturer’s instruction. The membrane was exposed to X-ray film for 24–48 h and the mRNA expression was quantified with densitometric analysis. Loading differences were normalized using an 18s rRNA cDNA probe. Western blot analysis. Cells were washed twice with PBS and suspended in IPH lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 100 mM PMSF, leupeptin 1 mg/ml, aprotinin 1 mg/ml, and 1 M DTT]. Cells were extracted at 4 °C for 30 min. After centrifugation at 12,000 rpm for 20 min, the supernatant was obtained as cell lysate. Protein quantification was performed using a Bio-Rad protein assay system (Bio-Rad Laboratories). Cell lysates (30 lg/lane) were electrophoresed on 10% SDS–PAGE and transferred to Immobilon-P-membrane (Millipore, USA). The membrane was allowed to react with anti-E2F1 polyclonal antibody and anti-PCNA polyclonal antibody (Santa Cruz Biotechnology). Antibodies were detected by horseradish peroxidase-linked secondary antibody using the ECL Western blotting detection system, as specified by the manufacturer. Effect of decoy ODN on cell proliferation. Cells were seeded onto 96-well tissue culture plates. At 30% confluence, cells were rendered quiescent by incubation for 24 h in defined serum-free medium. LipofectAMINE 2000: decoy ODN (100 nM of decoy ODN) complex was added to the wells and cells were incubated at 37 °C for a further 5 h. After 48 h, an index of cell proliferation was determined with a WST cell counting kit (Wako, Osaka, Japan). Statistical analyses. Results are expressed as means SE. Analysis of variance was performed with Duncan’s test and used to determine significant differences in multiple comparisons. Values of p < 0:05 were taken as statistically significant. All experiments were performed at least three times.
Results Effect of E2F decoy ODNs on promoter activities E2F responsible promoter constructs were co-transfected with E2F decoy ODN alone or combined with pCMV-RB plasmids to investigate the effect on serumstimulated E2F responsible promoter activity. At first, we investigated the inhibitory effect of E2F decoy ODNs on the promoter activity of reporter gene plasmids [E2F]x4-luc, which contains four E2F binding sites in the promoter region (Fig. 1A), and E2F1 reporter gene plasmids (Fig. 1B) in U2OS and C33A cancer cell lines.
1050
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053
Effect of the E2F decoy ODNs on cellular gene expression Given the finding that E2F decoy ODN inhibits serum-induced promoter activity in a sequence-specific manner, we reasoned that the decoys would also reduce expression of endogenous cell cycle regulatory genes. As shown in Figs. 2 and 3, serum treatment stimulated the mRNA and protein expression levels of E2F1 and PCNA genes, representative downstream targets for E2F, in U2OS cells (p < 0:01). Transfection of the CDE2F decoy ODN, but not the M-E2F decoy ODN, resulted in the significant attenuation of serum-induced gene expression of E2F1 and PCNA (p < 0:01). In contrast, 18S rRNA expression was not affected by E2F decoy ODNs. In C33A cells, the basal E2F1 and PCNA expression levels were much higher than those of U2OS cells and the expression was slightly increased by treatment of serum (p < 0:001). Transfection of the CD-E2F decoy ODN or overexpression of RB protein significantly attenuated both E2F1 and PCNA gene expression (p < 0:01) and that gene expression was blocked much more efficiently by the combined treatment with CD-E2F decoy ODN (p < 0:001).
Fig. 1. Effect of E2F decoy ODN on serum-induced promoter activities in cancer cells. Cells (3 105 ) were cultured for 24 h in the presence of 10% serum, co-transfected with E2F decoy ODN (100 nM) and [E2F]x4-luc (A) and E2F1-luc (B) reporter plasmids, and further incubated in the presence of 0.1% or 10% serum. Expression of luciferase and b-galactosidase was determined 36 h after the transfection. Data are presented as means SE of five independent experiments after normalization of b-galactosidase activity. Statistical significance was determined as *p < 0:01 compared to U2OS control, # p < 0:001 compared to U2OS control, **p < 0:01 compared to U2OS control + 10% serum, ## p < 0:001 compared to C33A + 10% serum, and þ p < 0:01 compared to C33A + 10% serum. Abbreviations: Decoy, cells transfected with E2F decoy ODNs; CD, CD-E2F decoy ODN; and M, M-E2F decoy ODN.
Treatment with 10% serum resulted in a significant increase in the luciferase gene expression, compared to control (Fig. 1A, p < 0:01) in U2OS cells. As expected, transfection of CD-E2F decoy ODN significantly attenuated the promoter activity induced by serum (p < 0:01), whereas M-E2F decoy ODN did not affect the activity induced by serum. In C33A cells, the basal E2F activity was much higher than that of U2OS cell and the activity was not affected by treatment of serum. It might be due to the deficiency of RB function in C33A cells, because overexpression of RB protein significantly suppressed the promoter activity (p < 0:01). CD-E2F decoy significantly attenuated E2F1 promoter activity and the activity was blocked almost completely by the treatment of CD-E2F decoy ODN in conjunction with RB protein overexpression (p < 0:001).
Fig. 2. Effect of E2F decoy ODN on serum-induced mRNA expression in cancer cells. Cells (1 106 ) were cultured for 24 h in the presence of 10% serum and further incubated in the presence of 0.1% or 10% serum for 48 h. (A) Typical Northern blot results for E2F1 and PCNA. (B) Gene expression of E2F1 and PCNA in cancer cells was quantified by densitometric analysis. Values represent means SE of five independent experiments. Statistical significance was determined as *p < 0:01 compared to U2OS control, # p < 0:001 compared to U2OS control, **p < 0:01 compared to U2OS control + 10% serum, ## p < 0:001 compared to C33A + 10% serum, and þ p < 0:01 compared to C33A + 10% serum. Abbreviations: Decoy, cells transfected with E2F decoy ODNs; CD, CD-E2F decoy ODN; and M, M-E2F decoy ODN.
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053
1051
stimulated the growth of both U2OS (p < 0:01) and C33A (increase slightly but not significant) as compared to control cultures. Transfection of the CD-E2F decoy ODNs resulted in significant inhibition of cell growth stimulated by serum treatment (p < 0:01), while the ME2F decoy ODNs did not abolish this observed increase in cell proliferation. Combined treatment with CD-E2F decoy ODNs together with RB overexpression blocked the proliferation of C33A cells more efficiently (p < 0:001).
Discussion
Fig. 3. Effect of E2F decoy ODN on serum-induced protein expression in cancer cells. Cells (1 106 ) were cultured for 24 h in the presence of 10% serum and further incubated in the presence of 0.1% or 10% serum for 48 h. (A) Typical Western blot results for E2F1 and PCNA. (B) Protein expression of E2F1 and PCNA in cancer cell line U2OS and C33A was quantified by densitometric analysis. Values represent means SE of five independent experiments. Statistical significance was determined as *p < 0:01 compared to U2OS control, # p < 0:001 compared to U2OS control, **p < 0:01 compared to U2OS control + 10% serum, ## p < 0:001 compared to C33A + 10% serum, and þ p < 0:01 compared to C33A + 10% serum. Abbreviations: Decoy, cells transfected with E2F decoy ODNs; CD, CD-E2F decoy ODN; and M, M-E2F decoy ODN.
Effects of E2F decoy ODN on the inhibition of cell proliferation Since one of the major phenomena of cancer cell is abnormal growth and dividing in an unregulated fashion, E2F decoy ODNs were tested for their ability to inhibit cell proliferation (Fig. 4). Treatment of serum
Fig. 4. Effect of E2F decoy ODN on serum-induced cell proliferation in cancer cells. Cell proliferation was determined as described in Materials and methods. Values are presented as means SE of five independent experiments. Statistical significance was determined as *p < 0:01 compared to U2OS control, # p < 0:05 compared to C33A + 10% serum, ## p < 0:001 compared to C33A + 10% serum, and **p < 0:01 compared to 10% serum treated group in each cell line. Abbreviations: Decoy, cells transfected with E2F decoy ODNs; CD, CD-E2F decoy ODN; and M, M-E2F decoy ODN.
Recently, several new technologies have been introduced in the inhibition of target gene expression in a sequence-specific manner, such as antisense oligonucleotides, ribozymes, and RNA interference. A more successful oligonucleotide-based approach has been the use of synthetic double-stranded oligonucleotides containing a cis-transcription element (called as “decoy ODN”) that can penetrate cells, can bind sequence-specific DNA-binding proteins, and can interfere with eukaryotic transcription [17–22]. Decoy strategy targeting E2F was recently reported to be quite effective in inhibiting proliferation in gene therapy studies in vitro, in vivo or ex vivo [21,25,26]. To test the clinical potential of E2F decoy in cancer therapy, we introduced E2F decoy ODN to two human cancer cell lines, U2OS and C33A. Our E2F decoy effectively suppressed E2F-dependent promoter activity in reporter plasmids and gene expression of downstream targets for E2F, E2F1, and PCNA. Concomitant with the repression of E2F-dependent transcription, it suppressed the proliferative response of human cancer cell lines. We also examined the effect of inhibition on E2F decoy in the other cancer cell lines, HeLa and COS-1 cells. Transfection of both HeLa and COS-1 by the E2F decoy also suppressed the proliferation of these cells (data not shown). Our results demonstrated that E2F decoy strategy is clinically effective in cancer therapy. The therapeutic effectiveness of synthetic doublestranded ODNs in modulation of specific gene expression largely depends on several factors, including the stability, specificity, and the efficiency of ODN in tissue and cellular delivery. The main limitation of unmodified ODN is rapid degradation by nucleases prevalent in sera, cell, and tissue. To rectify this problem, ODNs have been chemically modified with sulfur ion, methyl group, or other foreign materials to enhance the stability against nuclease activity. Although the stability of ODNs is enhanced by these chemical modifications, other problems have been encountered that are attributed to foreign materials [27–30]. In this study, we employed CD decoy ODN instead of phosphorothioate modified decoy ODN to overcome the disadvantages of
1052
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053
the chemically modified form. We demonstrated previously that CD decoy ODN is structurally more stable and effective than chemically modified decoy ODN during in vitro as well as in vivo neointimal formation following vascular injury and mesangial cell proliferation [21,22]. The present study demonstrated that unmodified CD decoy ODN is also sufficiently stable and effective in cancer cells. In this study, we examined the effect of serum on trans-activity of E2F in cancer cells and its downstream genes. In U2OS cells, serum treatment stimulated promoter activities of the [E2F]x4 and E2F1 reporters as well as expression of endogenous downstream targets of E2F, E2F1, and PCNA. In contrast, basal E2F activity was significantly higher than that of U2OS cells and the stimulatory effect of serum was hardly observed in C33A cells. This difference could be due to the functional defect of a tumor suppressor, pRB, in C33A cells, because ectopic expression of pRB through transient transfection resulted in significant reduction of E2F activity and expression of E2F1 and PCNA as well as proliferation. CD-E2F decoy, but not M-E2F decoy, markedly attenuated proliferation and gene expression induced by the serum treatment in both cancer cell lines. These data suggest that CD-E2F decoy is effective even in the cancer cells deficient in pRB. Although E2F activates transcription of target genes, it also represses transcription of specific targets by recruiting pRB family proteins to their promoter [31]. When E2F serves as a transcription repressor, deprivation of E2F from the target gene should enhance the transcription. In our study, abrogation of E2F binding by E2F decoy suppressed the growth and expression of E2F1 and PCNA, and the co-transfection with pRB further enhanced the suppressive effect in C33A cells. These results suggest that E2F activates transcription of these target genes and pRB suppresses their transcription through the inactivation of E2F in the physiological condition. On the other hand, promoter analysis with reporter experiments suggests that E2F in E2F1 promoter serves as a transcription repressor [32–34]. Results of artificial reporter experiments are sometimes inconsistent with endogenous gene expression. Decoy strategy that removes the interaction of an endogenous transcription factor and an authentic promoter may provide important information about functions of the endogenous transcription factor in physiological relevance. In conclusion, the present study demonstrates that inhibition of trans-activity of E2F proteins using circular dumbbell E2F decoy ODN significantly decreased cell cycle regulatory gene expression and cell proliferation in cancer cell lines, U2OS and C33A. This new molecular strategy, using CD-E2F decoy ODN, could represent a powerful investigative and potentially therapeutic strategy in the prevention and treatment of cancer.
Acknowledgments We are grateful to H. Heintz (University of Vermont, USA), N. Dyson (MGH Cancer center, Boston, USA), and K. Helin (European Institute of Oncology) for plasmids and other reagents. This study was supported by a grant from Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (00-PJ3-PG6-GN07-001).
References [1] S.J. Weintraub, C.A. Prater, D.C. Dean, Retinoblastoma protein switches the E2F site from positive to negative element, Nature 358 (1992) 259–261. [2] R.A. Weinberg, The retinoblastoma protein and cell cycle control, Cell 81 (1995) 323–330. [3] J. DeGregori, T. Kowalik, J.R. Nevins, Cellular targets for activation by the E2F1 transcription factor includes DNA synthesis- and G1/S-regulatory genes, Mol. Cell. Biol. 15 (1995) 4215–4224. [4] S. Dalton, Cell cycle regulation of the human cdc2 gene, EMBO J. 11 (1992) 1797–1804. [5] K. Thalmeier, H. Synovzik, R. Mertz, E.L. Winnacker, M. Lipp, Nuclear factor E2F mediates basic transcription and transactivation by E1a of the human MYC promoter, Genes Dev. 3 (1989) 527–536. [6] S. Wagner, M.R. Green, A transcriptional tryst, Nature 352 (1991) 189–190. [7] R.J. Watson, P.J. Dyson, J. McMahon, Multiple c-myb transcript cap sites are variously utilized in cells of mouse haematopoietic origin, EMBO J. 6 (1987) 1643–1651. [8] A.B. Pardee, A restriction point for control of normal animal cell proliferation, Proc. Natl. Acad. Sci. USA 71 (1974) 1286–1290. [9] S.J. Weintraub, C.A. Prater, D.C. Dean, Retinoblastoma protein switches the E2F site from positive to negative element, Nature 358 (1992) 259–261. [10] M.G. Pagano, G. Draetta, J. Durr, Association of cdk2 kinase with the transcription factor E2F during S phase, Science 255 (1992) 1144–1147. [11] D.K. Strom, J.L. Cleveland, S. Chellappan, J. Nip, S.W. Hiebert, E2F-1 and E2F-3 are functionally distinct in their ability to promote myeloid cell cycle progression and block granulocyte differentiation, Cell Growth Differ. 9 (1998) 59–69. [12] R.J. Duronio, P.H. O’Farrell, J.E. Xie, A. Brook, N. Dyson, The transcription factor E2F is required for S phase during Drosophila embryogenesis, Genes Dev. 9 (1995) 1445–1455. [13] X.H. Yang, T.L. Sladek, Overexpression of the E2F-1 transcription factor gene mediates cell transformation, Gene Expr. 4 (1995) 195–204. [14] P. Singh, S.H. Wong, W. Hong, Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation, EMBO J. 13 (1994) 3329–3338. [15] R.J. Lee, C. Albanese, M. Fu, M. D’Amico, B. Lin, G. Watanabe, G.K. Haines 3rd, P.M. Siegel, M.C. Hung, Y. Yarden, J.M. Horowitz, W.J. Muller, R.G. Pestell, Cyclin D1 is required for transformation by activated Neu and is induced through an E2Fdependent signaling pathway, Mol. Cell. Biol. 20 (2000) 672–683. [16] A. Sala, N.C. Nicolaides, A. Engelhard, T. Bellon, D.C. Lawe, A. Arnold, X. Grana, A. Giordano, B. Calabretta, Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells, Cancer Res. 54 (1994) 1402–1406. [17] A. Bielinska, R.A. Shivdasani, L. Zhang, G.J. Nabel, Regulation of gene expression with double-stranded phosphorothioate oligonucleotides, Science 250 (1990) 997–1000.
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053 [18] R. Morishita, G.H. Gibbons, M. Horiuchi, Y. Kaneda, T. Ogihara, V.J. Dzau, Role of AP-1 complex in angiotensin IImediated transforming growth factor-b expression and growth of smooth muscle cells: Using decoy approach against AP-1 binding site, Biochem. Biophys. Res. Commun. 243 (1998) 361–367. [19] J.D. Ahn, R. Morishita, Y. Kaneda, K.U. Lee, J.Y. Park, Y.J. Jeon, H.S. Song, I.K. Lee, Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose- and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells, Diabetologia 44 (2001) 713–720. [20] Y. Sawa, R. Morishita, K. Suzuki, A novel strategy for myocardial protection using in vivo transfection of cis element ‘decoy’ against NFjB binding site, Circulation 96 (1997) II-280–II-285. [21] J.D. Ahn, R. Morishita, Y. Kaneda, H.S. Kim, Y.C. Chang, K.U. Lee, J.Y. Park, H.W. Lee, Y.H. Kim, I.K. Lee, Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia, Gene Ther. 9 (2002) 1682–1692. [22] K.K Park, J.D. Ahn, I.K. Lee, J.J. Magae, N.H. Heintz, J.Y. Kwak, Y.C. Lee, Y.S. Cho, H.C. Kim, Y.M. Chae, Y.H. Kim, C.H. Kim, Y.C. Chang, Inhibitory effects of novel E2F decoy oligodeoxynucleotides on mesangial cell proliferation by coexpression of E2F/DP, Biochem. Biophys. Res. Commun. 308 (2003) 689–697. [23] Y.C. Chang, S. Illenye, N.H. Heintz, Cooperation of E2F-p130 and Sp1-pRb complexes in repression of the Chinese hamster dhfr gene, Mol. Cell. Biol. 21 (2001) 1121–1131. [24] F.A. Dick, E. Sailhamer, N.J. Dyson, Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins, Mol. Cell. Biol. 20 (2000) 3715–3727. [25] R. Morishita, G.H. Gibbons, M. Horiuchi, K.E. Ellison, M. Nakama, L. Zhang, Y. Kaneda, T. Ogihara, V.J. Dzau, A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo, Proc. Natl. Acad. Sci. USA (1995) 5855–5859.
1053
[26] M.J. Mann, A.D. Whittemore, M.C. Donaldson, M. Belkin, M.S. Conte, J.F. Polak, E.J. Orav, A. Ehsan, G. Dell’Acqua, V.J. Dzau, Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomized, controlled trial, Lancet 354 (1999) 1493–1498. [27] T. Hosoya, H. Takeuchi, Y. Kanesaka, H. Yamakawa, N. Miyano-Kurosaki, K. Takai, N. Yamamoto, H. Takaku, Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides, FEBS Lett. 461 (1999) 136– 140. [28] W.Y. Gao, F.S. Han, C. Storm, W. Egan, Y.C. Cheng, Phosphorothioate oligodeoxynucleotides are inhibitors of human DNA polymerases and Rnase H: implications for antisense technology, Mol. Pharmacol. 41 (1992) 223–229. [29] D.A. Brown, S.H. Kang, S.M. Gryaznov, L. DeDionisio, O. Heidenreich, S. Sullivan, X. Xu, M.I. Nerenberg, Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding, J. Biol. Chem. 269 (1994) 26801–26805. [30] T.L. Burgess, E.F. Fisher, S.L. Ross, J.V. Bready, Y.X. Qian, L.A. Bayewitch, A.M. Cohen, C.J. Herrera, S.S. Hu, T.B. Kramer, The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism, Proc. Natl. Acad. Sci. USA 92 (1995) 4051–4055. [31] N. Dyson, The regulation of E2F by pRB-family proteins, Genes Dev. 12 (1998) 2245–2262. [32] K.M. Hsiao, S.L. McMahon, P.J. Farnham, Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter, Genes Dev. 8 (1994) 1526–1537. [33] E. Neuman, E.K. Flemington, W.R. Sellers, W.G. Kaelin Jr., Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites with its promoter, Mol. Cell. Biol. 14 (1994) 6607–6615. [34] D.G. Johnson, K. Ohtani, J.R. Nevins, Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression, Genes Dev. 8 (1994) 1514–1525.
E2F decoy oligodeoxynucleotides effectively inhibit growth of human tumor cells Jong Deok Ahn,a,b Cheorl-Ho Kim,c Junji Magae,d Young Ho Kim,a Hye Jin Kim,a,b Kwan-Kyu Park,e SaHyun Hong,e Keun-Gyu Park,b In Kyu Lee,b,* and Young-Chae Change,* a Department of Microbiology, Kyungpook National University, Daegu 701-702, Republic of Korea Department of Internal Medicine, Keimyung University School of Medicine, Daegu 700-712, Republic of Korea National Research Laboratory for Glycobiology, Korean Ministry of Science and Technology, College of Oriental Medicine, Dongguk University, Kyungju City, Kyungbuk 780-714, Republic of Korea d Department of Biotechnology, Institute of Research and Innovation, 1201 Takada, Kashiwa 277-0861, Japan e Kidney Institute, Keimyung University School of Medicine, Daegu 700-712, South Korea b
c
Received 9 September 2003
Abstract Abnormal cell proliferation, largely dependent upon deregulation of cell-cycle regulatory proteins, is an important feature of several forms of human cancer. The transcription factor, E2F, plays a critical role in the trans-activation of several genes involved in cell-cycle regulation, thereby regulating cell growth. We have demonstrated that E2F decoy oligodeoxynucleotides (ODNs) with a circular dumbbell structure (CD-E2F decoy) corresponding to E2F binding sites effectively inhibit cell proliferation of primary cultured cells. Here we found that the E2F decoy ODNs inhibited serum-induced promoter activity of E2F-dependent genes in a sequence-specific manner in a RB-positive human osteosarcoma, U2OS, as well as in a RB-negative human cervical carcinoma, C33A. This E2F decoy ODN strongly inhibited gene expression of endogenous E2F1 and PCNA and proliferation of these cancer cells. Our results suggest that this decoy ODN strategy could represent a powerful investigative and potentially therapeutic strategy in the prevention and treatment of cancer. Ó 2003 Elsevier Inc. All rights reserved. Keywords: E2F; RB; Decoy; Dumbbell; Tumor growth inhibition; Osteosarcoma; Cervical carcinoma
It is now widely accepted that cancer is a disease characterized by defects in cell cycle regulation. Transcription factor E2F plays an important role in the regulation of cell cycle progression and entry into S phase [1–3]. E2F has been implicated in the periodic regulation of cellular genes required for transition through G1 and entry into the S phase including dihydrofolate reductase (DHFR), c-myc, DNA polymerase, cdc2, and proliferating cell nuclear antigen (PCNA) [4– 7]. E2F activity is regulated by interactions with RB family members. As cells progress toward the S phase, * Corresponding authors. Fax: +82-53-250-7892 (I.K. Lee), +82-53250-7095 (Y.-C. Chang). E-mail addresses: [email protected] (I.K. Lee), ycchang@ dsmc.or.kr (Y.-C. Chang).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.124
RB family proteins are phosphorylated by G1 cyclin– cdk complexes, resulting in the release of transcriptionally active E2F which leads to the activation of genes required for cell cycle progression [8–10]. Over-expression of various E2F proteins can induce cell cycle progression [11–14]. Several previous studies have shown that over-expression of various E2F proteins can induce cell cycle progression, and several intervention trials have been used to block the action of E2F proteins, including antisense methods, ectopic expression of dominant-negative mutants, and inhibition of upstream kinases [15,16]. An alternative approach to repress the action of transcription factors involves the use of double-stranded “decoy” oligonucleotides (ODNs) which inhibit the expression of genes by providing a competitor for the
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053
binding element of specific transcription factors such as E2F. Several studies have reported that synthetic double-stranded ODNs act as decoys against their target transcription factor when introduced into cells, and the decoys down-regulate the expression of several genes in a sequence-specific manner [17–20]. Therefore, decoy oligonucleotides have been proposed as a potential approach to block the action of transcription factors on gene expression. Moreover, we have demonstrated previously that treatment of E2F decoy ODN inhibits cell proliferation in vascular smooth muscle cells and mesangial cells [21,22]. Together with these findings led to us to investigate the inhibitory effect of E2F ODN decoys on cell cycle progression as a possible candidate for therapeutic intervention in proliferation of cancer cells. In this study, we developed and optimized E2F decoy ODN targeting activated E2F and found that circular dumbbell E2F decoy (CD-E2F decoy) ODN effectively inhibited transactivation of essential E2F-dependent cell-cycle regulatory genes (E2F1, PCNA) and proliferation of cancer cells. This study demonstrates the feasibility of E2F decoy ODN as a therapeutic strategy for human cancers. Materials and methods Cell culture. A human osteosarcoma, U2OS, and a human cervical carcinoma, C33A, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT) and 1% penicillin–streptomycin (Sigma, St. Louis, MO) at 37 °C in a 5% CO2 atmosphere. Construction of the circular dumbbell E2F decoy ODN. The sequences of circular dumbbell double-stranded ODN against the E2F binding site and mutated ODNs were as follows: CD-E2F decoy ODN (note: consensus sequences are underlined), 50 -ATGCGCGAA ACGCGTTTTCGCGTTTCGCGCATAGTTTTCT-30 ; mutated E2F decoy (M-E2F decoy) ODN, 50 -ATAATCTAAACGCGTTTTCGCG TTTAGATTATAGTTTTCT-30 . CD-E2F decoy ODN was predicted to form a stem-loop structure. Following the addition of T4 DNA ligase, the mixture was incubated for 24 h at 16 °C to generate a covalently ligated circular dumbbell decoy molecule. CD-E2F decoy ODN comprises two loops and one stem containing an E2F consensus sequence. In vitro gene transfer. Cells were fed with fresh culture medium the day before decoy was added and washed twice with Opti-MEM (Gibco-BRL), prior to the experiment. Cells were transfected with decoy ODN (100 nM) or plasmids combined with LipofectAMINE 2000 (molar ratio; DNA:lipid ¼ 1:3) (Gibco-BRL). The mixture was added dropwise to cells, according to manufacturer’s instructions. Transfection of plasmid expressing wild type RB protein (pCMV-RB) was carried out in a similar manner. Cells were incubated at 37 °C for 5 h. After the addition of fresh medium containing 10% FBS, cells were maintained in a CO2 incubator until use. The pSV-b-gal (Promega, Madison, WI, USA) was co-transfected to monitor the transfection efficiencies. Luciferase assay. [E2F]x4 luciferase reporter and a series of E2F1 reporter constructs were used in transient transfection assays as described previously [23]. Briefly, the [E2F]x4 luciferase reporter, which contains four E2F sites with the sequence TTTCGCGC and the TATA
1049
box from the adenovirus E1B gene, was generated by subcloning the promoter from [E2F]x4 chloramphenicol acetyltransferase into the reporter plasmid pGL3-Basic. For E2F1 promoter assays, E2F1-luc construct was supplied as a gift from N. Dyson of MGH Cancer Center [24]. The [E2F]x4-luc and E2F1 reporter gene plasmid were used to investigate the effects of E2F decoy ODN on promoter activity. To analyze luciferase expression, cells were washed twice with PBS and lysed with 200 ll of 1 Reporter lysis buffer (Promega, Madison, WI, USA). Each lysate (50 ll) was examined for luciferase activity and b-galactosidase activity as described previously [22]. Northern blot analysis. The radiolabeled probes used for Northern blot analysis were prepared by random primer labeling method with [a-32 P]dCTP using a kit (Amersham, Arlington Heights, IL, USA). After the labeling reaction, the radiolabeled probes were purified on a NAP-5 column (Pharmacia, Uppsala, Sweden). Total RNA for Northern blot analysis was extracted by RNeasy RNA extraction kit (Qiagen, Hilden, Germany). Ten micrograms of total RNA was applied to a 1% formaldehyde–agarose gel and transferred to a nylon membrane. The nylon membrane was hybridized in Express Hyb solution at 65 °C for 2 h with a radiolabeled E2F1 and PCNA cDNA probe and washed according to the manufacturer’s instruction. The membrane was exposed to X-ray film for 24–48 h and the mRNA expression was quantified with densitometric analysis. Loading differences were normalized using an 18s rRNA cDNA probe. Western blot analysis. Cells were washed twice with PBS and suspended in IPH lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 100 mM PMSF, leupeptin 1 mg/ml, aprotinin 1 mg/ml, and 1 M DTT]. Cells were extracted at 4 °C for 30 min. After centrifugation at 12,000 rpm for 20 min, the supernatant was obtained as cell lysate. Protein quantification was performed using a Bio-Rad protein assay system (Bio-Rad Laboratories). Cell lysates (30 lg/lane) were electrophoresed on 10% SDS–PAGE and transferred to Immobilon-P-membrane (Millipore, USA). The membrane was allowed to react with anti-E2F1 polyclonal antibody and anti-PCNA polyclonal antibody (Santa Cruz Biotechnology). Antibodies were detected by horseradish peroxidase-linked secondary antibody using the ECL Western blotting detection system, as specified by the manufacturer. Effect of decoy ODN on cell proliferation. Cells were seeded onto 96-well tissue culture plates. At 30% confluence, cells were rendered quiescent by incubation for 24 h in defined serum-free medium. LipofectAMINE 2000: decoy ODN (100 nM of decoy ODN) complex was added to the wells and cells were incubated at 37 °C for a further 5 h. After 48 h, an index of cell proliferation was determined with a WST cell counting kit (Wako, Osaka, Japan). Statistical analyses. Results are expressed as means SE. Analysis of variance was performed with Duncan’s test and used to determine significant differences in multiple comparisons. Values of p < 0:05 were taken as statistically significant. All experiments were performed at least three times.
Results Effect of E2F decoy ODNs on promoter activities E2F responsible promoter constructs were co-transfected with E2F decoy ODN alone or combined with pCMV-RB plasmids to investigate the effect on serumstimulated E2F responsible promoter activity. At first, we investigated the inhibitory effect of E2F decoy ODNs on the promoter activity of reporter gene plasmids [E2F]x4-luc, which contains four E2F binding sites in the promoter region (Fig. 1A), and E2F1 reporter gene plasmids (Fig. 1B) in U2OS and C33A cancer cell lines.
1050
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053
Effect of the E2F decoy ODNs on cellular gene expression Given the finding that E2F decoy ODN inhibits serum-induced promoter activity in a sequence-specific manner, we reasoned that the decoys would also reduce expression of endogenous cell cycle regulatory genes. As shown in Figs. 2 and 3, serum treatment stimulated the mRNA and protein expression levels of E2F1 and PCNA genes, representative downstream targets for E2F, in U2OS cells (p < 0:01). Transfection of the CDE2F decoy ODN, but not the M-E2F decoy ODN, resulted in the significant attenuation of serum-induced gene expression of E2F1 and PCNA (p < 0:01). In contrast, 18S rRNA expression was not affected by E2F decoy ODNs. In C33A cells, the basal E2F1 and PCNA expression levels were much higher than those of U2OS cells and the expression was slightly increased by treatment of serum (p < 0:001). Transfection of the CD-E2F decoy ODN or overexpression of RB protein significantly attenuated both E2F1 and PCNA gene expression (p < 0:01) and that gene expression was blocked much more efficiently by the combined treatment with CD-E2F decoy ODN (p < 0:001).
Fig. 1. Effect of E2F decoy ODN on serum-induced promoter activities in cancer cells. Cells (3 105 ) were cultured for 24 h in the presence of 10% serum, co-transfected with E2F decoy ODN (100 nM) and [E2F]x4-luc (A) and E2F1-luc (B) reporter plasmids, and further incubated in the presence of 0.1% or 10% serum. Expression of luciferase and b-galactosidase was determined 36 h after the transfection. Data are presented as means SE of five independent experiments after normalization of b-galactosidase activity. Statistical significance was determined as *p < 0:01 compared to U2OS control, # p < 0:001 compared to U2OS control, **p < 0:01 compared to U2OS control + 10% serum, ## p < 0:001 compared to C33A + 10% serum, and þ p < 0:01 compared to C33A + 10% serum. Abbreviations: Decoy, cells transfected with E2F decoy ODNs; CD, CD-E2F decoy ODN; and M, M-E2F decoy ODN.
Treatment with 10% serum resulted in a significant increase in the luciferase gene expression, compared to control (Fig. 1A, p < 0:01) in U2OS cells. As expected, transfection of CD-E2F decoy ODN significantly attenuated the promoter activity induced by serum (p < 0:01), whereas M-E2F decoy ODN did not affect the activity induced by serum. In C33A cells, the basal E2F activity was much higher than that of U2OS cell and the activity was not affected by treatment of serum. It might be due to the deficiency of RB function in C33A cells, because overexpression of RB protein significantly suppressed the promoter activity (p < 0:01). CD-E2F decoy significantly attenuated E2F1 promoter activity and the activity was blocked almost completely by the treatment of CD-E2F decoy ODN in conjunction with RB protein overexpression (p < 0:001).
Fig. 2. Effect of E2F decoy ODN on serum-induced mRNA expression in cancer cells. Cells (1 106 ) were cultured for 24 h in the presence of 10% serum and further incubated in the presence of 0.1% or 10% serum for 48 h. (A) Typical Northern blot results for E2F1 and PCNA. (B) Gene expression of E2F1 and PCNA in cancer cells was quantified by densitometric analysis. Values represent means SE of five independent experiments. Statistical significance was determined as *p < 0:01 compared to U2OS control, # p < 0:001 compared to U2OS control, **p < 0:01 compared to U2OS control + 10% serum, ## p < 0:001 compared to C33A + 10% serum, and þ p < 0:01 compared to C33A + 10% serum. Abbreviations: Decoy, cells transfected with E2F decoy ODNs; CD, CD-E2F decoy ODN; and M, M-E2F decoy ODN.
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053
1051
stimulated the growth of both U2OS (p < 0:01) and C33A (increase slightly but not significant) as compared to control cultures. Transfection of the CD-E2F decoy ODNs resulted in significant inhibition of cell growth stimulated by serum treatment (p < 0:01), while the ME2F decoy ODNs did not abolish this observed increase in cell proliferation. Combined treatment with CD-E2F decoy ODNs together with RB overexpression blocked the proliferation of C33A cells more efficiently (p < 0:001).
Discussion
Fig. 3. Effect of E2F decoy ODN on serum-induced protein expression in cancer cells. Cells (1 106 ) were cultured for 24 h in the presence of 10% serum and further incubated in the presence of 0.1% or 10% serum for 48 h. (A) Typical Western blot results for E2F1 and PCNA. (B) Protein expression of E2F1 and PCNA in cancer cell line U2OS and C33A was quantified by densitometric analysis. Values represent means SE of five independent experiments. Statistical significance was determined as *p < 0:01 compared to U2OS control, # p < 0:001 compared to U2OS control, **p < 0:01 compared to U2OS control + 10% serum, ## p < 0:001 compared to C33A + 10% serum, and þ p < 0:01 compared to C33A + 10% serum. Abbreviations: Decoy, cells transfected with E2F decoy ODNs; CD, CD-E2F decoy ODN; and M, M-E2F decoy ODN.
Effects of E2F decoy ODN on the inhibition of cell proliferation Since one of the major phenomena of cancer cell is abnormal growth and dividing in an unregulated fashion, E2F decoy ODNs were tested for their ability to inhibit cell proliferation (Fig. 4). Treatment of serum
Fig. 4. Effect of E2F decoy ODN on serum-induced cell proliferation in cancer cells. Cell proliferation was determined as described in Materials and methods. Values are presented as means SE of five independent experiments. Statistical significance was determined as *p < 0:01 compared to U2OS control, # p < 0:05 compared to C33A + 10% serum, ## p < 0:001 compared to C33A + 10% serum, and **p < 0:01 compared to 10% serum treated group in each cell line. Abbreviations: Decoy, cells transfected with E2F decoy ODNs; CD, CD-E2F decoy ODN; and M, M-E2F decoy ODN.
Recently, several new technologies have been introduced in the inhibition of target gene expression in a sequence-specific manner, such as antisense oligonucleotides, ribozymes, and RNA interference. A more successful oligonucleotide-based approach has been the use of synthetic double-stranded oligonucleotides containing a cis-transcription element (called as “decoy ODN”) that can penetrate cells, can bind sequence-specific DNA-binding proteins, and can interfere with eukaryotic transcription [17–22]. Decoy strategy targeting E2F was recently reported to be quite effective in inhibiting proliferation in gene therapy studies in vitro, in vivo or ex vivo [21,25,26]. To test the clinical potential of E2F decoy in cancer therapy, we introduced E2F decoy ODN to two human cancer cell lines, U2OS and C33A. Our E2F decoy effectively suppressed E2F-dependent promoter activity in reporter plasmids and gene expression of downstream targets for E2F, E2F1, and PCNA. Concomitant with the repression of E2F-dependent transcription, it suppressed the proliferative response of human cancer cell lines. We also examined the effect of inhibition on E2F decoy in the other cancer cell lines, HeLa and COS-1 cells. Transfection of both HeLa and COS-1 by the E2F decoy also suppressed the proliferation of these cells (data not shown). Our results demonstrated that E2F decoy strategy is clinically effective in cancer therapy. The therapeutic effectiveness of synthetic doublestranded ODNs in modulation of specific gene expression largely depends on several factors, including the stability, specificity, and the efficiency of ODN in tissue and cellular delivery. The main limitation of unmodified ODN is rapid degradation by nucleases prevalent in sera, cell, and tissue. To rectify this problem, ODNs have been chemically modified with sulfur ion, methyl group, or other foreign materials to enhance the stability against nuclease activity. Although the stability of ODNs is enhanced by these chemical modifications, other problems have been encountered that are attributed to foreign materials [27–30]. In this study, we employed CD decoy ODN instead of phosphorothioate modified decoy ODN to overcome the disadvantages of
1052
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053
the chemically modified form. We demonstrated previously that CD decoy ODN is structurally more stable and effective than chemically modified decoy ODN during in vitro as well as in vivo neointimal formation following vascular injury and mesangial cell proliferation [21,22]. The present study demonstrated that unmodified CD decoy ODN is also sufficiently stable and effective in cancer cells. In this study, we examined the effect of serum on trans-activity of E2F in cancer cells and its downstream genes. In U2OS cells, serum treatment stimulated promoter activities of the [E2F]x4 and E2F1 reporters as well as expression of endogenous downstream targets of E2F, E2F1, and PCNA. In contrast, basal E2F activity was significantly higher than that of U2OS cells and the stimulatory effect of serum was hardly observed in C33A cells. This difference could be due to the functional defect of a tumor suppressor, pRB, in C33A cells, because ectopic expression of pRB through transient transfection resulted in significant reduction of E2F activity and expression of E2F1 and PCNA as well as proliferation. CD-E2F decoy, but not M-E2F decoy, markedly attenuated proliferation and gene expression induced by the serum treatment in both cancer cell lines. These data suggest that CD-E2F decoy is effective even in the cancer cells deficient in pRB. Although E2F activates transcription of target genes, it also represses transcription of specific targets by recruiting pRB family proteins to their promoter [31]. When E2F serves as a transcription repressor, deprivation of E2F from the target gene should enhance the transcription. In our study, abrogation of E2F binding by E2F decoy suppressed the growth and expression of E2F1 and PCNA, and the co-transfection with pRB further enhanced the suppressive effect in C33A cells. These results suggest that E2F activates transcription of these target genes and pRB suppresses their transcription through the inactivation of E2F in the physiological condition. On the other hand, promoter analysis with reporter experiments suggests that E2F in E2F1 promoter serves as a transcription repressor [32–34]. Results of artificial reporter experiments are sometimes inconsistent with endogenous gene expression. Decoy strategy that removes the interaction of an endogenous transcription factor and an authentic promoter may provide important information about functions of the endogenous transcription factor in physiological relevance. In conclusion, the present study demonstrates that inhibition of trans-activity of E2F proteins using circular dumbbell E2F decoy ODN significantly decreased cell cycle regulatory gene expression and cell proliferation in cancer cell lines, U2OS and C33A. This new molecular strategy, using CD-E2F decoy ODN, could represent a powerful investigative and potentially therapeutic strategy in the prevention and treatment of cancer.
Acknowledgments We are grateful to H. Heintz (University of Vermont, USA), N. Dyson (MGH Cancer center, Boston, USA), and K. Helin (European Institute of Oncology) for plasmids and other reagents. This study was supported by a grant from Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (00-PJ3-PG6-GN07-001).
References [1] S.J. Weintraub, C.A. Prater, D.C. Dean, Retinoblastoma protein switches the E2F site from positive to negative element, Nature 358 (1992) 259–261. [2] R.A. Weinberg, The retinoblastoma protein and cell cycle control, Cell 81 (1995) 323–330. [3] J. DeGregori, T. Kowalik, J.R. Nevins, Cellular targets for activation by the E2F1 transcription factor includes DNA synthesis- and G1/S-regulatory genes, Mol. Cell. Biol. 15 (1995) 4215–4224. [4] S. Dalton, Cell cycle regulation of the human cdc2 gene, EMBO J. 11 (1992) 1797–1804. [5] K. Thalmeier, H. Synovzik, R. Mertz, E.L. Winnacker, M. Lipp, Nuclear factor E2F mediates basic transcription and transactivation by E1a of the human MYC promoter, Genes Dev. 3 (1989) 527–536. [6] S. Wagner, M.R. Green, A transcriptional tryst, Nature 352 (1991) 189–190. [7] R.J. Watson, P.J. Dyson, J. McMahon, Multiple c-myb transcript cap sites are variously utilized in cells of mouse haematopoietic origin, EMBO J. 6 (1987) 1643–1651. [8] A.B. Pardee, A restriction point for control of normal animal cell proliferation, Proc. Natl. Acad. Sci. USA 71 (1974) 1286–1290. [9] S.J. Weintraub, C.A. Prater, D.C. Dean, Retinoblastoma protein switches the E2F site from positive to negative element, Nature 358 (1992) 259–261. [10] M.G. Pagano, G. Draetta, J. Durr, Association of cdk2 kinase with the transcription factor E2F during S phase, Science 255 (1992) 1144–1147. [11] D.K. Strom, J.L. Cleveland, S. Chellappan, J. Nip, S.W. Hiebert, E2F-1 and E2F-3 are functionally distinct in their ability to promote myeloid cell cycle progression and block granulocyte differentiation, Cell Growth Differ. 9 (1998) 59–69. [12] R.J. Duronio, P.H. O’Farrell, J.E. Xie, A. Brook, N. Dyson, The transcription factor E2F is required for S phase during Drosophila embryogenesis, Genes Dev. 9 (1995) 1445–1455. [13] X.H. Yang, T.L. Sladek, Overexpression of the E2F-1 transcription factor gene mediates cell transformation, Gene Expr. 4 (1995) 195–204. [14] P. Singh, S.H. Wong, W. Hong, Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation, EMBO J. 13 (1994) 3329–3338. [15] R.J. Lee, C. Albanese, M. Fu, M. D’Amico, B. Lin, G. Watanabe, G.K. Haines 3rd, P.M. Siegel, M.C. Hung, Y. Yarden, J.M. Horowitz, W.J. Muller, R.G. Pestell, Cyclin D1 is required for transformation by activated Neu and is induced through an E2Fdependent signaling pathway, Mol. Cell. Biol. 20 (2000) 672–683. [16] A. Sala, N.C. Nicolaides, A. Engelhard, T. Bellon, D.C. Lawe, A. Arnold, X. Grana, A. Giordano, B. Calabretta, Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells, Cancer Res. 54 (1994) 1402–1406. [17] A. Bielinska, R.A. Shivdasani, L. Zhang, G.J. Nabel, Regulation of gene expression with double-stranded phosphorothioate oligonucleotides, Science 250 (1990) 997–1000.
J.D. Ahn et al. / Biochemical and Biophysical Research Communications 310 (2003) 1048–1053 [18] R. Morishita, G.H. Gibbons, M. Horiuchi, Y. Kaneda, T. Ogihara, V.J. Dzau, Role of AP-1 complex in angiotensin IImediated transforming growth factor-b expression and growth of smooth muscle cells: Using decoy approach against AP-1 binding site, Biochem. Biophys. Res. Commun. 243 (1998) 361–367. [19] J.D. Ahn, R. Morishita, Y. Kaneda, K.U. Lee, J.Y. Park, Y.J. Jeon, H.S. Song, I.K. Lee, Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose- and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells, Diabetologia 44 (2001) 713–720. [20] Y. Sawa, R. Morishita, K. Suzuki, A novel strategy for myocardial protection using in vivo transfection of cis element ‘decoy’ against NFjB binding site, Circulation 96 (1997) II-280–II-285. [21] J.D. Ahn, R. Morishita, Y. Kaneda, H.S. Kim, Y.C. Chang, K.U. Lee, J.Y. Park, H.W. Lee, Y.H. Kim, I.K. Lee, Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia, Gene Ther. 9 (2002) 1682–1692. [22] K.K Park, J.D. Ahn, I.K. Lee, J.J. Magae, N.H. Heintz, J.Y. Kwak, Y.C. Lee, Y.S. Cho, H.C. Kim, Y.M. Chae, Y.H. Kim, C.H. Kim, Y.C. Chang, Inhibitory effects of novel E2F decoy oligodeoxynucleotides on mesangial cell proliferation by coexpression of E2F/DP, Biochem. Biophys. Res. Commun. 308 (2003) 689–697. [23] Y.C. Chang, S. Illenye, N.H. Heintz, Cooperation of E2F-p130 and Sp1-pRb complexes in repression of the Chinese hamster dhfr gene, Mol. Cell. Biol. 21 (2001) 1121–1131. [24] F.A. Dick, E. Sailhamer, N.J. Dyson, Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins, Mol. Cell. Biol. 20 (2000) 3715–3727. [25] R. Morishita, G.H. Gibbons, M. Horiuchi, K.E. Ellison, M. Nakama, L. Zhang, Y. Kaneda, T. Ogihara, V.J. Dzau, A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo, Proc. Natl. Acad. Sci. USA (1995) 5855–5859.
1053
[26] M.J. Mann, A.D. Whittemore, M.C. Donaldson, M. Belkin, M.S. Conte, J.F. Polak, E.J. Orav, A. Ehsan, G. Dell’Acqua, V.J. Dzau, Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomized, controlled trial, Lancet 354 (1999) 1493–1498. [27] T. Hosoya, H. Takeuchi, Y. Kanesaka, H. Yamakawa, N. Miyano-Kurosaki, K. Takai, N. Yamamoto, H. Takaku, Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides, FEBS Lett. 461 (1999) 136– 140. [28] W.Y. Gao, F.S. Han, C. Storm, W. Egan, Y.C. Cheng, Phosphorothioate oligodeoxynucleotides are inhibitors of human DNA polymerases and Rnase H: implications for antisense technology, Mol. Pharmacol. 41 (1992) 223–229. [29] D.A. Brown, S.H. Kang, S.M. Gryaznov, L. DeDionisio, O. Heidenreich, S. Sullivan, X. Xu, M.I. Nerenberg, Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding, J. Biol. Chem. 269 (1994) 26801–26805. [30] T.L. Burgess, E.F. Fisher, S.L. Ross, J.V. Bready, Y.X. Qian, L.A. Bayewitch, A.M. Cohen, C.J. Herrera, S.S. Hu, T.B. Kramer, The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism, Proc. Natl. Acad. Sci. USA 92 (1995) 4051–4055. [31] N. Dyson, The regulation of E2F by pRB-family proteins, Genes Dev. 12 (1998) 2245–2262. [32] K.M. Hsiao, S.L. McMahon, P.J. Farnham, Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter, Genes Dev. 8 (1994) 1526–1537. [33] E. Neuman, E.K. Flemington, W.R. Sellers, W.G. Kaelin Jr., Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites with its promoter, Mol. Cell. Biol. 14 (1994) 6607–6615. [34] D.G. Johnson, K. Ohtani, J.R. Nevins, Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression, Genes Dev. 8 (1994) 1514–1525.