Regular Article

British Journal of Cancer (2000) 82, 16–19. doi:10.1054/bjoc.1998.0870 www.bjcancer.com
Published online 10 December 1999

Preferential down-regulation of phospholipase C-bold beta in Ewing's sarcoma cells transfected with antisense EWS-Fli-1

T Dohjima1, T Ohno1, Y Banno2, Y Nozawa2, Y Wen-yi1 and K Shimizu1

  1. 1Departments of Orthopaedic Surgery, University School of Medicine, Tsukasamachi-40, Gifu, 500-8705, Japan
  2. 2Biochemistry Gifu University School of Medicine, Tsukasamachi-40, Gifu, 500-8705, Japan

Received 28 May 1998; Revised 5 July 1999; Accepted 8 July 1999.

Top

Abstract

EWS-Fli-1, a fusion gene found in Ewing's sarcoma and primitive neuro-ectodermal tumour (PNET), encodes a transcriptional activator and promotes cellular transformation. We have made stable Ewing's sarcoma cells expressing antisense EWS-Fli-1 transcripts by transfecting the antisense EWS-Fli-1 expression plasmid. These cells showed partial loss of endogenous EWS-Fli-1 proteins and suppression of the cell growth. To elucidate the molecular mechanisms underlying the growth inhibition, we examined the changes of signal transducing proteins by immunoblot analysis in Ewing's sarcoma cells stably expressing antisense EWS-Fli-1 transcripts. Western blotting of the cell proteins revealed that expressions of phospholipase Cbeta2 and beta3 (PLCbeta2, PLCbeta3), and also protein kinase C alpha and beta (PKCalpha, beta) were significantly reduced by transfecting with antisense EWS-Fli-1. The inositol phosphates production by bradykinin (BK), but not platelet-derived growth factor (PDGF), was suppressed in these cells. These results suggest that the PLCbeta2 and PLCbeta3 may play a role in tumour proliferation in Ewing's sarcoma cells. © 2000 Cancer Research Campaign

Keywords:

phospholipase C, signal transduction, Ewing's sarcoma, antisense EWS-Fli-1, growth inhibition

Top

References

  1. Bailly RA, Bosselut R, Zucman J, Cormier F, Delattre O, Roussel M, Thomas G and Ghysdael J (1994) DNA-binding and transcriptional activation properties of the EWS-Fli-1 fusion protein resulting from the t(11;22)translocation in Ewing sarcoma. Mol Cell Biol 14: 3230–3241 | PubMed | ISI | ChemPort |
  2. Banno Y, Okano Y and Nozawa Y (1994) Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta1. J Biol Chem 269: 15846–15852
  3. Beekman A, Helfrich B, Bunn PA and Heasley LE (1998) Expression of catalytically inactive phospholipase Cbeta disrupts phospholipase Cbeta and mitogen-activated protein kinase signaling and inhibits small cell lung cancer growth. Cancer Res 58: 910–913 | PubMed | ISI | ChemPort |
  4. Berridge MJ and Irvine RF (1984) Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312: 315–321 | Article | PubMed | ISI | ChemPort |
  5. Braun BS, Frieden R, Lessnick SL, May W and Denny CT (1995) Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis. Mol Cell Biol 15: 4623–4630 | PubMed | ISI | ChemPort |
  6. Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M, Kovar H, Joubert I, De Jong P, Rouleau G, Aurias A and Thomas G (1992) Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumors. Nature 359: 162–165 | Article | PubMed | ISI | ChemPort |
  7. Haeuptle MT, Frank R and Dobberstein B (1986) Translation arrest by oligodeoxynucleotide complementary mRNA coding sequences yields polypeptides of predetermined length. Nucleic Acid Res 14: 1427–1448
  8. Ji QS, Winnier GE, Niswender KD, Horstman D, Wisdom R, Magnuson MA and Carpenter G (1997) Essential role of the tyrosine kinase substrate phospholipase C-gamma1 in mammalian growth and development. Proc Natl Acad Sci USA 94: 2999–3003 | Article | PubMed | ChemPort |
  9. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227: 680–685 | Article | PubMed | ISI | ChemPort |
  10. Lee SB, Rao AK, Lee KH, Yang X, Bae YS and Rhee SG (1996) Decreased expression of phospholipase C-beta2 isozyme in human platelets with impaired function. Blood 88: 1684–1691 | PubMed | ISI | ChemPort |
  11. May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, Lunsford LB, Hromas R and Denny CT (1993) The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol 13: 7393–7398 | PubMed | ISI | ChemPort |
  12. May WA, Arvand A, Thompson AD, Braun BS, Wright M and Denny CT (1997) EWS/FLI-1-induced manic fringe renders NIH 3T3 cell tumorigenic. Nature Genetics 17: 495–497 | Article | PubMed |
  13. Mohammadi M, Dionne CA, Li W, Li N, Spivak T, Honegger AM, Jaye M and Schlessinger J (1992) Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Nature 358: 681–684 | Article | PubMed | ChemPort |
  14. Munroe SH (1988) Antisense RNA inhibits splicing of pre-mRNA in vitro. EMBO J 7: 2523–2532 | PubMed | ChemPort |
  15. Nishizuyka Y (1986) Studies and perspectives of protein kinase C. Science 233: 305–312 | PubMed | ISI | ChemPort |
  16. Ohno T, Rao VN and Reddy ESP (1993) EWS/Fli-1 chimeric protein is a transcriptional activator. Cancer Res 53: 5859–5863 | PubMed | ChemPort |
  17. Ohno T, Ouchida M, Lee L, Gatalica Z, Rao VN and Reddy ESP (1994) The EWS gene, involved in Ewing family of tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors, codes for an RNA binding protein with novel regulatory domains. Oncogne 9: 3087–3097
  18. Ouchida M, Ohno T, Fujimura Y, Rao VN and Reddy ESP (1995) Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. Oncogene 11: 1049–1054 | PubMed | ISI | ChemPort |
  19. Peters KG, Marie J, Wilson E, Ives HE, Escobedo J, Del Rosario M, Mirda D and Williams LT (1992) Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+flux but not mitogenesis. Nature 358: 678–681 | Article | PubMed | ChemPort |
  20. Rabbitts TH (1994) Chromosomal translocations in human cancer. Nature 372: 143–149 | Article | PubMed | ISI | ChemPort |
  21. Rao VN, Ohno T, Prasad DDK, Bhattacharya G and Reddy ESP (1993) Analysis of the DNA-binding and transcriptional activation functions of human Fli-1 protein. Oncogene 8: 2167–2173 | PubMed | ISI | ChemPort |
  22. Reddy ESP and Rao VN (1991) erg, an ets related gene, codes for sequence-specific transcriptional activators. Oncogens 6: 2285–2289
  23. Rhee SG (1994) Regulation of phosphoinositide-specific phospholipase C by G protein. Signal-Activated Phospholipases, Liscovitch M (ed) pp 1–30. RG Landes Company: Austin
  24. Rhee SG and Bae YS (1997) Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem 272: 15045–15048 | Article | PubMed | ISI | ChemPort |
  25. Singer WD, Brown HA and Sternweis PC (1997) Regulation of eukaryotic phosphatidylinositol-specific phospholipase C and phospholipase. D. Annu Rev Biochem 66: 475–509
  26. Sorensen PH, Lessmick SL, Lopez-Terrada D, Liu XF, Triche TJ and Denny CT (1994) A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nat Genet 6: 146–151 | Article | PubMed | ISI | ChemPort |
  27. Tanaka K, Iwakuma T, Harimay K, Sato H and Iwamoto Y (1997) EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest 99: 239–247 | Article | PubMed | ISI | ChemPort |
  28. Towbin H, Staehelin T and Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 | Article | PubMed | ChemPort |
  29. Walder RY and Walder JA (1988) Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci USA 85: 5011–5015 | Article | PubMed | ChemPort |
  30. Weber G, Friedman E, Grimmond S, Hayward NK, Phelan C, Skogseid B, Gogl A, Zedenius J, Sandelin K, Teh BT, Carson E, White I, Oberg K, Shepherd J, Nordenskjold M and Larsson C (1994) The phospholipase C beta3 gene located in the MEN1 region shows loss of expression in endocrine tumours. Hum Genet 3: 1775–1781
  31. Whang-Peng J, Triche J, Knutsen T, Miset J, Douglass EC and Israel MA (1984) Chromosome translocation in peripheral neuroepithelioma. N Engl J Med 311: 584–585 | PubMed | ChemPort |