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Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions
© Shiraishi et al; licensee BioMed Central Ltd. 2010
Received: 8 February 2010
Accepted: 30 June 2010
Published: 30 June 2010
Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells.
We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT).
We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406) targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512) targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping) and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone.
We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.
Antisense molecules with significantly modified backbones such as peptide nucleic acids (PNA), methoxyethoxy (MOE) and locked nucleic acids (LNA), or morpholino oligos, rendering them RNase H inactive, are able to modulate mRNA splicing when targeting intron-exon junctions in pre-mRNA [1–7]. For instance correction of aberrant splicing by blocking cryptic 5'- or 3'- splice sites, induction of exon skipping [8–10] and force selection of an alternative splice site by targeting antisense molecules to original splice sites  have been demonstrated. Thus antisense targeting of splice junctions have the potential of inducing shifts in the ratio between biologically functional splice variants or even induce non-natural splice variants with novel biological function of the resulting protein. Therefore, splicing targeting technology may open a range of opportunities for gene targeting in drug discovery and molecular biology contexts [5, 12, 13].
The mdm2 oncogene is amplified and/or over expressed in several cancer types . This oncogene encodes a protein that negatively controls the functions of the p53 tumor suppressor protein by blocking the transactivation domain and by stimulating the degradation of p53. Down regulation of MDM2 has been recognized as a potential mechanism for cancer therapy [15, 16] because down-regulation of MDM2 in tumors exhibiting MDM2 over-expression should induce p53 stability and thus sensitization to DNA-damaging treatments via p53-dependent pathways [17–20]. Accordingly, recent studies have shown down regulation of full-length MDM2 protein through a traditional RnaseH dependent antisense approach. In addition, more than 40 different splice variants of mdm2 mRNA have been detected in tumors and normal cells [17, 21], but the potential functions or oncogenic properties of the different MDM2 isoforms are far from fully understood [22–24]. Therefore, targeting of mdm2 mRNA splicing could be an effective way of controlling and studying overall MDM2 expression and function.
It has recently been shown that PNA oligomers targeted to exon-intron splice junctions are potent inhibitors/modulators of mRNA splicing [6, 25]. By targeting a 3'- splice site, at least two outcomes have been found although no systematic studies have yet been published. The spliceosome will either skip the exon and thus produce a truncated mRNA missing the exon, or skip the intron excision and thereby produce a larger mRNA still containing the intron. Therefore, PNA molecules designed to down-regulate full length mdm2 mRNA or shift relative populations of splice variants by splicing modulation may be a useful approach for both future therapy as recently indicated for PNA targeting of CD40 pre-mRNA (7), as well as for investigating the functions of mdm2 splice variants . However, limited information is available concerning the optimum design of antisense PNA oligomers, including target location, for the desired splicing modulation [6, 7, 26]. Therefore we decided to perform a more systematic study of PNA oligomers targeting the 5'- or 3'-splice sites in the human MDM2 gene.
In this study, we have tested ten PNAs (15-mer) targeting intron2 (seven PNAs targeting the 3'-splice site and three PNAs are targeting to the 5'-splice site) as well as a PNAs targeting the 3'-splice site of intron3. Depending on the target site intron and/or exon skipping was observed, and most interestingly targeting of intron2 also affected the splicing of intron3. Finally, treatment of JAR cells with the PNA targeting intron3 resulted in a significant reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53
Synthesis of PNA
Nomenclatures and sequences of PNAs
H-Acr-eg1- TCG GTG CTT ACC TGG-NH2
H-Acr-eg1-TGC TTA CCT GGA TCA-NH2
H-Acr-eg1-TTA CCT GGA TCA GCA-NH2
H-Acr-eg1-TGT TGG TAT TGC ACA-NH2
H-Acr-eg1-TGG TAT TGC ACA TTT-NH2
H-Acr-eg1-TAT TGC ACA TTT GCC-NH2
H-Acr-eg1-TGC ACA TTT GCC TAC-NH2
H-Acr-eg1-ACA TTT GCC TAC AAG-NH2
H-Acr-eg1-TTT GCC TAC AAG GAA-NH2
H-Acr-eg1-GCC TAC AAG GAA AAA-NH2
H-Acr-eg1-TTT GGT CTA ACC TAT -NH2
H-(D-Arg)8-TTT GGT CTA ACC TAT-NH2
H-(D-Arg)8-Lys(Deca)-TTT GGT CTA ACC TAT-NH2
H-Acr-eg1-TGC AGA TTT CCC TAC-NH2
H-Acr-eg1-TTT AGT CTA GCC TAT -NH2
JAR cells were provided by Dr. Peter Ebbesen (The Stem Cell Laboratory, Aalborg University, Denmark). The cells were cultured in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 1.5% glutamax (Gibco) and streptomycin/penicillin (100 U/ml each)) at 37°C in a humidified atmosphere of 95% air and 5% CO2.
Nomenclatures of primer sets and sequences of RT-PCR primers.
Product size (bp)
intron2 5'-splice site
intron2 3'-splice site
intron3 5'-splice site
intron3 3'-splice site
intron4 5'-splice site
intron5 3'-splice site
Western blot analysis
Cells were transfected with PNA as described above and subjected to protein sample preparation: The cells were lysed in boiling lysis buffer (1% SDS, 10 mM Tris-HCl, pH 7.2, 1X protease inhibitor cocktail tablet (Roche, Germany)), and 40 μg of the protein was fractionated by SDS-polyacrylamide gel electrophoresis (SDS-10% PAGE) and transferred to a PVDF-membrane (ADVANTEC MFS). Blocking, detection and re-probing were performed with the ECL-Plus system (Amersham) following the manufacture's instruction. Briefly, the PVDF-membrane was blocked with 5% skim milk (in 20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.1% Tween 20) over night at 4°C and incubated with a primary antibody (anti-MDM2, SMP14 (Sigma); anti-p53, ab6 (Santa Cruz); anti-β-actin, AC-15, (Sigma)) overnight at 4°C. The membrane was washed with washing buffer (20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.01% Tween 20) three times for a minimum of 15 min each at room temperature, and then incubated with 1:20000 diluted anti-mouse IgG-horse radish peroxidase conjugated secondary antibody (DAKO, P0161) for 1 h at room temperature. After washing as described above, the protein of interest was detected with ECL-Plus (Amersham).
JAR cells, plated in a 96 well plate the day before transfection, were subjected to PNA transfection in combination with camptothecin (CPT) treatment. CPT was added to the PNA transfection solution at the desired concentration and incubated for 48 h. Subsequently, cell viability was determined by the MTS-assay using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer's instructions. The absorbance is presented as relative cellular viability (absorbance from non-PNA treated cells was set as 100%).
Results and Discussion
Ten PNAs targeting intron2 at the 5' or 3' splice sites were tested for their ability to inhibit splicing of mdm2 pre-mRNA. As the first in frame AUG translation start codon is located in exon3, we focused on splice interference that might result in exon3 skipping, thereby prohibiting any translation initiation from the mRNA. In order to facilitate cellular uptake, we used 9-aminoacridine conjugated PNAs in combination with cationic lipid transfection reagent as previously described .
In summary, we have identified several PNAs targeting the 5' or 3' splice sites in intron2 of mdm2 pre-mRNA which can inhibit splicing. One of the most efficient PNAs (PNA2406) is targeting the 3' splice site and is complementary to 4 bases in intron2 and 11 bases in exon3. Interestingly, PNA2512, which is targeting the 3' splice site of intron3 and is complementary to 4 bases in intron3 and 11 bases in exon4, showed both splicing inhibition as well as exon skipping. Furthermore, PNA2406 inhibited splicing of both intron2 as well as the downstream intron (intron3), whereas PNA2512 only inhibited the splicing of intron3 (and not downstream intron4). These results reflect the complexity of the splicing mechanism and thus the splicing modulation by antisense PNAs (and presumably other antisense agents as well). Further studies are required to obtain more generally applicable guidelines for designing antisense PNAs for a desired splicing modulation. Treatment of JAR cells in culture with the most potent PNA (2512) for pre-mRNA splicing modulation also affected protein expression, causing a reduction of the level of MDM2 protein and (consequently) an increase in the level of p53. Finally, this PNA increased the cytotoxicity of the anticancer camptothecin presumably caused by the PNA mediated up-regulation of p53. In conclusion these results add to the accumulating evidence that antisense mediated mRNA splice modulation have interesting prospects both within drug discovery but also as a molecular biology tool. Once more potent in vivo PNA delivery methods are available, the present results provide the basis for further drug discovery studies.
This study was supported by the Danish Cancer Society, The Lundbeck Foundation (Senior research fellowship to TS) and the Danish Medical Research Council.
- Kole R, Vacek M, Williams T: Modification of alternative splicing by antisense therapeutics. Oligonucleotides. 2004, 14: 65-74. 10.1089/154545704322988067.View ArticlePubMedGoogle Scholar
- Lu QL, Rabinowitz A, Chen YC, Yokota T, Yin H, Alter J, Jadoon A, Bou-Gharios G, Partridge T: Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc Natl Acad Sci USA. 2005, 102: 198-203. 10.1073/pnas.0406700102.View ArticlePubMedGoogle Scholar
- Suwanmanee T, Sierakowska H, Lacerra G, Svasti S, Kirby S, Walsh CE, Fucharoen S, Kole R: Restoration of human beta-globin gene expression in murine and human IVS2-654 thalassemic erythroid cells by free uptake of antisense oligonucleotides. Mol Pharmacol. 2002, 62: 545-553. 10.1124/mol.62.3.545.View ArticlePubMedGoogle Scholar
- Giles RV, Spiller DG, Clark RE, Tidd DM: Antisense morpholino oligonucleotide analog induces missplicing of C-myc mRNA. Antisense Nucleic Acid Drug Dev. 1999, 9: 213-220.View ArticlePubMedGoogle Scholar
- Gebski BL, Mann CJ, Fletcher S, Wilton SD: Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet. 2003, 12: 1801-1811. 10.1093/hmg/ddg196.View ArticlePubMedGoogle Scholar
- Karras JG, Maier MA, Lu T, Watt A, Manoharan M: Peptide nucleic acids are potent modulators of endogenous pre-mRNA splicing of the murine interleukin-5 receptor-alpha chain. Biochemistry. 2001, 40: 7853-7859. 10.1021/bi010263l.View ArticlePubMedGoogle Scholar
- Siwkowski AM, Malik L, Esau CC, Maier MA, Wancewicz EV, Albertshofer K, Monia BP, Bennett CF, Eldrup AB: Identification and functional validation of PNAs that inhibit murine CD40 expression by redirection of splicing. Nucleic Acids Res. 2004, 32: 2695-2706. 10.1093/nar/gkh584.View ArticlePubMedPubMed CentralGoogle Scholar
- van Deutekom JC, Bremmer-Bout M, Janson AA, Ginjaar IB, Baas F, den Dunnen JT, van Ommen GJ: Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells. Hum Mol Genet. 2001, 10: 1547-1554. 10.1093/hmg/10.15.1547.View ArticlePubMedGoogle Scholar
- Pramono ZA, Takeshima Y, Alimsardjono H, Ishii A, Takeda S, Matsuo M: Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence. Biochem Biophys Res Commun. 1996, 226: 445-449. 10.1006/bbrc.1996.1375.View ArticlePubMedGoogle Scholar
- Mann CJ, Honeyman K, Cheng AJ, Ly T, Lloyd F, Fletcher S, Morgan JE, Partridge TA, Wilton SD: Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci USA. 2001, 98: 42-47. 10.1073/pnas.011408598.View ArticlePubMedGoogle Scholar
- Taylor JK, Zhang QQ, Wyatt JR, Dean NM: Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides. Nat Biotechnol. 1999, 17: 1097-1100. 10.1038/15079.View ArticlePubMedGoogle Scholar
- Mercatante DR, Kole R: Control of alternative splicing by antisense oligonucleotides as a potential chemotherapy: effects on gene expression. Biochim Biophys Acta. 2002, 1587: 126-132.View ArticlePubMedGoogle Scholar
- Mercatante D, Kole R: Modification of alternative splicing pathways as a potential approach to chemotherapy. Pharmacol Ther. 2000, 85: 237-243. 10.1016/S0163-7258(99)00067-4.View ArticlePubMedGoogle Scholar
- Momand J, Jung D, Wilczynski S, Niland J: The MDM2 gene amplification database. Nucleic Acids Res. 1998, 26: 3453-3459. 10.1093/nar/26.15.3453.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang H, Nan L, Yu D, Agrawal S, Zhang R: Antisense anti-MDM2 oligonucleotides as a novel therapeutic approach to human breast cancer: in vitro and in vivo activities and mechanisms. Clin Cancer Res. 2001, 7: 3613-3624.PubMedGoogle Scholar
- Tortora G, Caputo R, Damiano V, Bianco R, Chen J, Agrawal S, Bianco AR, Ciardiello F: A novel MDM2 anti-sense oligonucleotide has anti-tumor activity and potentiates cytotoxic drugs acting by different mechanisms in human colon cancer. Int J Cancer. 2000, 88: 804-809. 10.1002/1097-0215(20001201)88:5<804::AID-IJC19>3.0.CO;2-Z.View ArticlePubMedGoogle Scholar
- Bartel F, Harris LC, Wurl P, Taubert H: MDM2 and its splice variant messenger RNAs: expression in tumors and down-regulation using antisense oligonucleotides. Mol Cancer Res. 2004, 2: 29-35.PubMedGoogle Scholar
- Chen L, Lu W, Agrawal S, Zhou W, Zhang R, Chen J: Ubiquitous induction of p53 in tumor cells by antisense inhibition of MDM2 expression. Mol Med. 1999, 5: 21-34.PubMedPubMed CentralGoogle Scholar
- Chen L, Agrawal S, Zhou W, Zhang R, Chen J: Synergistic activation of p53 by inhibition of MDM2 expression and DNA damage. Proc Natl Acad Sci USA. 1998, 95: 195-200. 10.1073/pnas.95.1.195.View ArticlePubMedPubMed CentralGoogle Scholar
- Shiraishi T, Nielsen PE: Down-regulation of MDM2 and activation of p53 in human cancer cells by antisense 9-aminoacridine-PNA (peptide nucleic acid) conjugates. Nucleic Acids Res. 2004, 32: 4893-4902. 10.1093/nar/gkh820.View ArticlePubMedPubMed CentralGoogle Scholar
- Bartel F, Taubert H, Harris LC: Alternative and aberrant splicing of MDM2 mRNA in human cancer. Cancer Cell. 2002, 2: 9-15. 10.1016/S1535-6108(02)00091-0.View ArticlePubMedGoogle Scholar
- Perry ME, Mendrysa SM, Saucedo LJ, Tannous P, Holubar M: p76(MDM2) inhibits the ability of p90(MDM2) to destabilize p53. J Biol Chem. 2000, 275: 5733-5738. 10.1074/jbc.275.8.5733.View ArticlePubMedGoogle Scholar
- Bartel F, Meye A, Wurl P, Kappler M, Bache M, Lautenschlager C, Grunbaum U, Schmidt H, Taubert H: Amplification of the MDM2 gene, but not expression of splice variants of MDM2 MRNA, is associated with prognosis in soft tissue sarcoma. Int J Cancer. 2001, 95: 168-175. 10.1002/1097-0215(20010520)95:3<168::AID-IJC1029>3.0.CO;2-A.View ArticlePubMedGoogle Scholar
- Fridman JS, Hernando E, Hemann MT, de Stanchina E, Cordon-Cardo C, Lowe SW: Tumor promotion by Mdm2 splice variants unable to bind p53. Cancer Res. 2003, 63: 5703-5706.PubMedGoogle Scholar
- Sazani P, Kang SH, Maier MA, Wei C, Dillman J, Summerton J, Manoharan M, Kole R: Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res. 2001, 29: 3965-3974.PubMedPubMed CentralGoogle Scholar
- Sazani P, Gemignani F, Kang SH, Maier MA, Manoharan M, Persmark M, Bortner D, Kole R: Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat Biotechnol. 2002, 20: 1228-1233. 10.1038/nbt759.View ArticlePubMedGoogle Scholar
- Christensen L, Fitzpatrick R, Gildea B, Petersen KH, Hansen HF, Koch T, Egholm M, Buchardt O, Nielsen PE, Coull J, et al: Solid-phase synthesis of peptide nucleic acids. J Pept Sci. 1995, 1: 175-183. 10.1002/psc.310010304.View ArticlePubMedGoogle Scholar
- Koppelhus U, Shiraishi T, Zachar V, Pankratova S, Nielsen PE: Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain. Bioconjug Chem. 2008, 19: 1526-1534. 10.1021/bc800068h.View ArticlePubMedGoogle Scholar
- Shiraishi T, Nielsen PE: Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption. Nat Protoc. 2006, 1: 633-636. 10.1038/nprot.2006.92.View ArticlePubMedGoogle Scholar
- Mae M, Langel U: Cell-penetrating peptides as vectors for peptide, protein and oligonucleotide delivery. Curr Opin Pharmacol. 2006, 6: 509-514. 10.1016/j.coph.2006.04.004.View ArticlePubMedGoogle Scholar
- Ivanova GD, Arzumanov A, Abes R, Yin H, Wood MJ, Lebleu B, Gait MJ: Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle. Nucleic Acids Res. 2008, 36: 6418-6428. 10.1093/nar/gkn671.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://0-www.biomedcentral.com.brum.beds.ac.uk/1471-2407/10/342/prepub
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