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Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified, synthetic ribozyme

Cancer Gene Therapy volume 10, pages 201–208 (2003)Cite this article

Abstract

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.

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References

  • Birikh KR, Heaton PA, Eckstein F . The structure, function and application of the hammerhead ribozymes. Eur J Biochem. 1997;245:1–16.

    Article  CAS  Google Scholar 

  • Bramlage B, Luzi E, Eckstein F . Designing ribozymes for the inhibition of gene expression. Trends Biotechnol. 1998;16:434–438.

    Article  CAS  Google Scholar 

  • Cleary ML, Smith SD, Sklar J . Cloning and structural analysis of cDNA for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell. 1986;47:19–28.

    Article  CAS  Google Scholar 

  • Chen-Levy Z, Nourse J, Cleary ML . The bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation. Mol Cell Biol. 1989;9:701–710.

    Article  CAS  Google Scholar 

  • Vaux D, Cory S, Adams JM . Bcl-2 gene promoters haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988;335:440–442.

    Article  CAS  Google Scholar 

  • Reed JC . Bcl-2 family proteins:regulators od apoptosis and chemioresistance in hematologic malignancies. Semin Hematol. 1997;34:9–19.

    CAS  PubMed  Google Scholar 

  • Waters JS, Webb A, Cunningham D, et al. Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma. J Clin Oncol. 2000;18:1812–1823.

    Article  CAS  Google Scholar 

  • Gewirtz AM, Sokol DL, Ratajczak MZ . Nucleic acid therapeutic:state of the art and future prospects. Blood. 1998;92:712–736.

    CAS  PubMed  Google Scholar 

  • Schiavone N, Rosini P, Quattrone A, et al. A conserved AU-rich element in the 3′ untraslated region of bcl-2 mRNA is endowed with destabilizing function that is involved in bcl-2 down-regulation during apoptosis. FASEB J. 2000;14:174–184.

    Article  CAS  Google Scholar 

  • Wilson GM, Brewer G . Identification and characterization of proteins binding A+U-rich elements. Methods Enzymol. 1999;17:74–83.

    Article  CAS  Google Scholar 

  • Donnini M, Lapucci A, Papucci L, et al. Apoptosis is associated with modifications of bcl-2 mRNA AU-binding proteins. Biochem Biophys Res Comm. 2001;287:1063–1069.

    Article  CAS  Google Scholar 

  • Yang J-H, Usman N, Chartrand P, et al. Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain. Biochemistry. 1992;31:5005–5009.

    Article  CAS  Google Scholar 

  • Goodchild J . Enhancement of ribozyme catalytic activity by a contiguous oligodeoxynucleotide (facilitator) and by 2′-O-methylation. Nucleic Acids Res. 1992;20:4607–4612.

    Article  CAS  Google Scholar 

  • Heidenreich O, Benseler F, Fahrenholz A, et al. High activity and stability of hammerhead ribozymes containing 2′-modified pyrimidine nucleosides and phosphorothioate. J Biol Chem. 1994;269:2131–2138.

    CAS  PubMed  Google Scholar 

  • Bramlage B, Luzi E, Eckstein F . HIV-1 LTR as a target for synthetic ribozyme-mediated inhibition og gene expression: site selection and inhibition in cell culture. Nucleic Acids Res. 2000;28:4059–4067.

    Article  CAS  Google Scholar 

  • Shimayama T, Nishikawa F, Nishikawa S, et al. Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages. Nucleic Acids Res. 1993;21:2605–2611.

    Article  CAS  Google Scholar 

  • Bevilacqua A, Ceriani MC, Schiavone N, et al. BCL-2 regulation targeting the AU-rich domain. Nucleosides, Nucleotides Nucleic Acids. 2000;19:1273–1279.

    Article  CAS  Google Scholar 

  • Quattrone A, Papucci L, Santini V, et al. Quantitation of bcl-2 oncogene in cultured lymphoma/leukemia cell lines and in primary leukemia b-cells by a highly sensitive RT-PCR method. Haematologica. 1995;80:495–504.

    CAS  PubMed  Google Scholar 

  • Zuker M, Stiegler P . Optimal computer folding of large RNA sequences using thermodynamic and auxiliary information. Nucleic Acids Res. 1981;9:133–148.

    Article  CAS  Google Scholar 

  • Julien T, Frankel B, Longo S, et al. Antisense-mediated inhibition of the Bcl-2 gene induces apoptosis in human malignant glioma. Surg Neurol. 2000;53:360–368.

    Article  CAS  Google Scholar 

  • Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitisation of malignant melanoma by BCL2 antisense theraoy. Lancet. 2000;356:1728–1733.

    Article  CAS  Google Scholar 

  • Uchida T, Gao JP, Wang C, et al. Antitumor effect of bcl-2 antisense phosphorothioate oligodeoxynucleotides on human renal-cell carcinoma cells in vitro and in mice. Mol Urol. 2001;5:71–78.

    Article  CAS  Google Scholar 

  • Tolcher AW . Preliminary phase I results of G3139 (bcl-2 antisense oligonucleotide) therapy in combination with docetaxel in hormone-refractory prostate cancer. Semin Oncol. 2001;28:67–70.

    Article  CAS  Google Scholar 

  • Konopleva M, Tari AM, Estrov Z, et al. Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins. Blood. 2000;95:3929–3938.

    CAS  PubMed  Google Scholar 

  • Gibson S, Pellenz C, Hutchison RE, et al. Induction of apoptosis in oral cancer cells by an anti-bcl-2 ribozyme delivered by an adenovirus vector. Clin Cancer Res. 2000;6:213–222.

    CAS  PubMed  Google Scholar 

  • Potter PM, McKenzie PP, Hussain N, et al. Construction of adenovirus for high level expression of small RNAs in mammalian cells. Mol Biotechnol. 2000;15:105–114.

    Article  CAS  Google Scholar 

  • Dorai T, Perlman H, Walsh K, et al. A recombinant defective adenoviralagent expressing anti-bcl-2 ribozyme promotes apoptosis of bcl-2 expressing human prostate cancer cells. Int J Cancer. 1999;82:846–852.

    Article  CAS  Google Scholar 

  • Dorai T, Olson CA, Katz AE, et al. Development of a hammerhead ribozyme against bcl-2I. Preliminary evalutation of a potential gene therapeutic agent for hormone-refractory human prostate cancer. Prostate. 1997;32:246–258.

    Article  CAS  Google Scholar 

  • Scheid S, Heinzinger M, Waller CF, et al. Bcl-2 mRNA-targeted ribozymes:effects on programmed cell death in chronic myelogenous leukemia cell lines. Ann Hematol. 1998;76:117–125.

    Article  CAS  Google Scholar 

  • Webb A, Cunningham D, Cother F, et al. BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet. 1997;349:1137–1141.

    Article  CAS  Google Scholar 

  • Wilson GM, Sutphen K, Chuang K, et al. Folding of A+U-rich RNA elements modulates AUF1 binding. J Biol Chem. 2001;276:8695–8704.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

E Luzi thanks his wife Carla for her support and help. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano), Ministero dell'Istruzione, dell'Universita' e della Ricerca, Ministero della Salute (MIUR), Consiglio Nazionale Ricerche (CNR) and Ente Cassa di Risparmio di Firenze. Andrea Lapucci is supported by a fellowship from Federazione Italiana per la Ricerca sul Cancro (FIRC, Milano).

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  1. Ettore Luzi

    Present address: Biosensor Laboratory, Department of Chemistry, Polo Scientifico, Via della Lastruccia 3, Sesto Fiorentino-Firenze, 50019, Italy

Authors and Affiliations

  1. Department of Experimental Pathology and Oncology, University of Florence, Viale Morgagni 50, Firenze, 50134, Italy

    Ettore Luzi, Laura Papucci, Nicola Schiavone, Martino Donnini, Andrea Lapucci, Alessio Tempestini, Ewa Witort & Sergio Capaccioli

  2. Department of Pharmacology, University of Milan, Milan, 20129, Italy

    Angelo Nicolin

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Correspondence to Ettore Luzi.

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Luzi, E., Papucci, L., Schiavone, N. et al. Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified, synthetic ribozyme. Cancer Gene Ther 10, 201–208 (2003). https://doi.org/10.1038/sj.cgt.7700556

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