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Biochemical Journal logoLink to Biochemical Journal
. 1999 May 1;339(Pt 3):547–553.

Inhibition of gene expression by anti-sense C-5 propyne oligonucleotides detected by a reporter enzyme.

Y Hamel 1, J Lacoste 1, C Frayssinet 1, A Sarasin 1, T Garestier 1, J C François 1, C Hélène 1
PMCID: PMC1220189  PMID: 10215592

Abstract

Using a reporter plasmid containing the luciferase gene under the control of the insulin-like growth factor 1 (IGF-1) promoter region [including its 5' untranslated region (UTR)], we demonstrate that a 17-mer oligophosphorothioate containing C-5 propyne pyrimidines is able to inhibit luciferase gene expression in the nanomolar concentration range when the anti-sense oligonucleotide is targeted either to a coding sequence in the luciferase gene or to the 5' UTR of the gene for IGF-1. Inhibition was obtained independently of whether the plasmid and the anti-sense oligonucleotide were co-transfected or transfected separately into hepatocarcinoma cells. However, the efficiency of inhibition by the anti-sense oligonucleotides was 10-fold greater in the first case. The unmodified oligophosphorothioate targeted to the 5' UTR of IGF-1 did not inhibit luciferase gene expression at a 100-fold higher concentration unless its length was increased from 17 to 21 nt, in which case an inhibition of gene expression was obtained and an IC50 of 200 nM was observed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adamo M. L., Ben-Hur H., Roberts C. T., Jr, LeRoith D. Regulation of start site usage in the leader exons of the rat insulin-like growth factor-I gene by development, fasting, and diabetes. Mol Endocrinol. 1991 Nov;5(11):1677–1686. doi: 10.1210/mend-5-11-1677. [DOI] [PubMed] [Google Scholar]
  2. Alt M., Renz R., Hofschneider P. H., Paumgartner G., Caselmann W. H. Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides. Hepatology. 1995 Sep;22(3):707–717. [PubMed] [Google Scholar]
  3. Angelloz-Nicoud P., Binoux M. Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3). Endocrinology. 1995 Dec;136(12):5485–5492. doi: 10.1210/endo.136.12.7588299. [DOI] [PubMed] [Google Scholar]
  4. Baserga R., Hongo A., Rubini M., Prisco M., Valentinis B. The IGF-I receptor in cell growth, transformation and apoptosis. Biochim Biophys Acta. 1997 Jun 7;1332(3):F105–F126. doi: 10.1016/s0304-419x(97)00007-3. [DOI] [PubMed] [Google Scholar]
  5. Boussif O., Lezoualc'h F., Zanta M. A., Mergny M. D., Scherman D., Demeneix B., Behr J. P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7297–7301. doi: 10.1073/pnas.92.16.7297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brasier A. R., Tate J. E., Habener J. F. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques. 1989 Nov-Dec;7(10):1116–1122. [PubMed] [Google Scholar]
  7. Cantor C. R., Warshaw M. M., Shapiro H. Oligonucleotide interactions. 3. Circular dichroism studies of the conformation of deoxyoligonucleotides. Biopolymers. 1970;9(9):1059–1077. doi: 10.1002/bip.1970.360090909. [DOI] [PubMed] [Google Scholar]
  8. Chan P. P., Glazer P. M. Triplex DNA: fundamentals, advances, and potential applications for gene therapy. J Mol Med (Berl) 1997 Apr;75(4):267–282. doi: 10.1007/s001090050112. [DOI] [PubMed] [Google Scholar]
  9. Coats S., Flanagan W. M., Nourse J., Roberts J. M. Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science. 1996 May 10;272(5263):877–880. doi: 10.1126/science.272.5263.877. [DOI] [PubMed] [Google Scholar]
  10. Fenster S. D., Wagner R. W., Froehler B. C., Chin D. J. Inhibition of human immunodeficiency virus type-1 env expression by C-5 propyne oligonucleotides specific for Rev-response element stem-loop V. Biochemistry. 1994 Jul 19;33(28):8391–8398. doi: 10.1021/bi00194a002. [DOI] [PubMed] [Google Scholar]
  11. Flanagan W. M., Kothavale A., Wagner R. W. Effects of oligonucleotide length, mismatches and mRNA levels on C-5 propyne-modified antisense potency. Nucleic Acids Res. 1996 Aug 1;24(15):2936–2941. doi: 10.1093/nar/24.15.2936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flanagan W. M., Su L. L., Wagner R. W. Elucidation of gene function using C-5 propyne antisense oligonucleotides. Nat Biotechnol. 1996 Sep;14(9):1139–1145. doi: 10.1038/nbt0996-1139. [DOI] [PubMed] [Google Scholar]
  13. Giles R. V., Spiller D. G., Grzybowski J., Clark R. E., Nicklin P., Tidd D. M. Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells. Nucleic Acids Res. 1998 Apr 1;26(7):1567–1575. doi: 10.1093/nar/26.7.1567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gutierrez A. J., Matteucci M. D., Grant D., Matsumura S., Wagner R. W., Froehler B. C. Antisense gene inhibition by C-5-substituted deoxyuridine-containing oligodeoxynucleotides. Biochemistry. 1997 Jan 28;36(4):743–748. doi: 10.1021/bi9620971. [DOI] [PubMed] [Google Scholar]
  15. Hall L. J., Kajimoto Y., Bichell D., Kim S. W., James P. L., Counts D., Nixon L. J., Tobin G., Rotwein P. Functional analysis of the rat insulin-like growth factor I gene and identification of an IGF-I gene promoter. DNA Cell Biol. 1992 May;11(4):301–313. doi: 10.1089/dna.1992.11.301. [DOI] [PubMed] [Google Scholar]
  16. Hatzfeld A., Feldmann G., Guesnon J., Frayssinet C., Schapira F. Location of adult and fetal aldolases A, B, and C by immunoperoxidase technique in LF fast-growing rat hepatomas. Cancer Res. 1978 Jan;38(1):16–22. [PubMed] [Google Scholar]
  17. Hélène C., Toulmé J. J. Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim Biophys Acta. 1990 Jun 21;1049(2):99–125. doi: 10.1016/0167-4781(90)90031-v. [DOI] [PubMed] [Google Scholar]
  18. Kim H. G., Miller D. M. A novel triplex-forming oligonucleotide targeted to human cyclin D1 (bcl-1, proto-oncogene) promoter inhibits transcription in HeLa cells. Biochemistry. 1998 Feb 24;37(8):2666–2672. doi: 10.1021/bi972399i. [DOI] [PubMed] [Google Scholar]
  19. Kim H. G., Reddoch J. F., Mayfield C., Ebbinghaus S., Vigneswaran N., Thomas S., Jones D. E., Jr, Miller D. M. Inhibition of transcription of the human c-myc protooncogene by intermolecular triplex. Biochemistry. 1998 Feb 24;37(8):2299–2304. doi: 10.1021/bi9718191. [DOI] [PubMed] [Google Scholar]
  20. Kim S. W., Lajara R., Rotwein P. Structure and function of a human insulin-like growth factor-I gene promoter. Mol Endocrinol. 1991 Dec;5(12):1964–1972. doi: 10.1210/mend-5-12-1964. [DOI] [PubMed] [Google Scholar]
  21. Lacoste J., François J. C., Hélène C. Triple helix formation with purine-rich phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative. Nucleic Acids Res. 1997 May 15;25(10):1991–1998. doi: 10.1093/nar/25.10.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lafarge-Frayssinet C., Duc H. T., Frayssinet C., Sarasin A., Anthony D., Guo Y., Trojan J. Antisense insulin-like growth factor I transferred into a rat hepatoma cell line inhibits tumorigenesis by modulating major histocompatibility complex I cell surface expression. Cancer Gene Ther. 1997 Sep-Oct;4(5):276–285. [PubMed] [Google Scholar]
  23. LeRoith D., Baserga R., Helman L., Roberts C. T., Jr Insulin-like growth factors and cancer. Ann Intern Med. 1995 Jan 1;122(1):54–59. doi: 10.7326/0003-4819-122-1-199501010-00009. [DOI] [PubMed] [Google Scholar]
  24. Lewis J. G., Lin K. Y., Kothavale A., Flanagan W. M., Matteucci M. D., DePrince R. B., Mook R. A., Jr, Hendren R. W., Wagner R. W. A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3176–3181. doi: 10.1073/pnas.93.8.3176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lowe W. L., Jr, Roberts C. T., Jr, Lasky S. R., LeRoith D. Differential expression of alternative 5' untranslated regions in mRNAs encoding rat insulin-like growth factor I. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8946–8950. doi: 10.1073/pnas.84.24.8946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lund O. S., Nielsen J. O., Hansen J. E. Inhibition of HIV-1 in vitro by C-5 propyne phosphorothioate antisense to rev. Antiviral Res. 1995 Sep;28(1):81–91. doi: 10.1016/0166-3542(95)00041-j. [DOI] [PubMed] [Google Scholar]
  27. McCarthy T. L., Thomas M. J., Centrella M., Rotwein P. Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element. Endocrinology. 1995 Sep;136(9):3901–3908. doi: 10.1210/endo.136.9.7649098. [DOI] [PubMed] [Google Scholar]
  28. Moats-Staats B. M., Retsch-Bogart G. Z., Price W. A., Jarvis H. W., D'Ercole A. J., Stiles A. D. Insulin-like growth factor-I (IGF-I) antisense oligodeoxynucleotide mediated inhibition of DNA synthesis by WI-38 cells: evidence for autocrine actions of IGF-I. Mol Endocrinol. 1993 Feb;7(2):171–180. doi: 10.1210/mend.7.2.7682287. [DOI] [PubMed] [Google Scholar]
  29. Monia B. P., Johnston J. F., Ecker D. J., Zounes M. A., Lima W. F., Freier S. M. Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides. J Biol Chem. 1992 Oct 5;267(28):19954–19962. [PubMed] [Google Scholar]
  30. Monia B. P., Lesnik E. A., Gonzalez C., Lima W. F., McGee D., Guinosso C. J., Kawasaki A. M., Cook P. D., Freier S. M. Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. J Biol Chem. 1993 Jul 5;268(19):14514–14522. [PubMed] [Google Scholar]
  31. Moulds C., Lewis J. G., Froehler B. C., Grant D., Huang T., Milligan J. F., Matteucci M. D., Wagner R. W. Site and mechanism of antisense inhibition by C-5 propyne oligonucleotides. Biochemistry. 1995 Apr 18;34(15):5044–5053. doi: 10.1021/bi00015a015. [DOI] [PubMed] [Google Scholar]
  32. Neuenschwander S., Roberts C. T., Jr, LeRoith D. Growth inhibition of MCF-7 breast cancer cells by stable expression of an insulin-like growth factor I receptor antisense ribonucleic acid. Endocrinology. 1995 Oct;136(10):4298–4303. doi: 10.1210/endo.136.10.7664648. [DOI] [PubMed] [Google Scholar]
  33. Ojwang J. O., Mustain S. D., Marshall H. B., Rao T. S., Chaudhary N., Walker D. A., Hogan M. E., Akiyama T., Revankar G. R., Peyman A. Modified antisense oligonucleotides directed against tumor necrosis factor receptor type I inhibit tumor necrosis factor alpha-mediated functions. Biochemistry. 1997 May 20;36(20):6033–6045. doi: 10.1021/bi970124x. [DOI] [PubMed] [Google Scholar]
  34. Pierga J. Y., Magdelenat H. Applications of antisense oligonucleotides in oncology. Cell Mol Biol (Noisy-le-grand) 1994 May;40(3):237–261. [PubMed] [Google Scholar]
  35. Resnicoff M., Coppola D., Sell C., Rubin R., Ferrone S., Baserga R. Growth inhibition of human melanoma cells in nude mice by antisense strategies to the type 1 insulin-like growth factor receptor. Cancer Res. 1994 Sep 15;54(18):4848–4850. [PubMed] [Google Scholar]
  36. Resnicoff M., Sell C., Rubini M., Coppola D., Ambrose D., Baserga R., Rubin R. Rat glioblastoma cells expressing an antisense RNA to the insulin-like growth factor-1 (IGF-1) receptor are nontumorigenic and induce regression of wild-type tumors. Cancer Res. 1994 Apr 15;54(8):2218–2222. [PubMed] [Google Scholar]
  37. Rotwein P. Structure, evolution, expression and regulation of insulin-like growth factors I and II. Growth Factors. 1991;5(1):3–18. doi: 10.3109/08977199109000267. [DOI] [PubMed] [Google Scholar]
  38. St Croix B., Flørenes V. A., Rak J. W., Flanagan M., Bhattacharya N., Slingerland J. M., Kerbel R. S. Impact of the cyclin-dependent kinase inhibitor p27Kip1 on resistance of tumor cells to anticancer agents. Nat Med. 1996 Nov;2(11):1204–1210. doi: 10.1038/nm1196-1204. [DOI] [PubMed] [Google Scholar]
  39. Stein C. A. Exploiting the potential of antisense: beyond phosphorothioate oligodeoxynucleotides. Chem Biol. 1996 May;3(5):319–323. doi: 10.1016/s1074-5521(96)90113-1. [DOI] [PubMed] [Google Scholar]
  40. Stein C. A. How to design an antisense oligodeoxynucleotide experiment: a consensus approach. Antisense Nucleic Acid Drug Dev. 1998 Apr;8(2):129–132. doi: 10.1089/oli.1.1998.8.129. [DOI] [PubMed] [Google Scholar]
  41. Stein C. A., Subasinghe C., Shinozuka K., Cohen J. S. Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res. 1988 Apr 25;16(8):3209–3221. doi: 10.1093/nar/16.8.3209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Trojan J., Johnson T. R., Rudin S. D., Ilan J., Tykocinski M. L., Ilan J. Treatment and prevention of rat glioblastoma by immunogenic C6 cells expressing antisense insulin-like growth factor I RNA. Science. 1993 Jan 1;259(5091):94–97. doi: 10.1126/science.8418502. [DOI] [PubMed] [Google Scholar]
  43. Wagner R. W., Matteucci M. D., Lewis J. G., Gutierrez A. J., Moulds C., Froehler B. C. Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science. 1993 Jun 4;260(5113):1510–1513. doi: 10.1126/science.7684856. [DOI] [PubMed] [Google Scholar]
  44. Yang H., Adamo M. L., Koval A. P., McGuinness M. C., Ben-Hur H., Yang Y., LeRoith D., Roberts C. T., Jr Alternative leader sequences in insulin-like growth factor I mRNAs modulate translational efficiency and encode multiple signal peptides. Mol Endocrinol. 1995 Oct;9(10):1380–1395. doi: 10.1210/mend.9.10.8544846. [DOI] [PubMed] [Google Scholar]

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