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 Cβ2 and β3 (PLCβ2, PLCβ3), and also protein kinase C α and β (PKCα, β) 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 PLCβ2 and PLCβ3 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
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- Bailly R. A., Bosselut R., Zucman J., Cormier F., Delattre O., Roussel M., Thomas G., Ghysdael J. 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. 1994 May;14(5):3230–3241. doi: 10.1128/mcb.14.5.3230. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Banno Y., Okano Y., Nozawa Y. Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta 1. J Biol Chem. 1994 Jun 3;269(22):15846–15852. [PubMed] [Google Scholar]
- Beekman A., Helfrich B., Bunn P. A., Jr, Heasley L. E. Expression of catalytically inactive phospholipase Cbeta disrupts phospholipase Cbeta and mitogen-activated protein kinase signaling and inhibits small cell lung cancer growth. Cancer Res. 1998 Mar 1;58(5):910–913. [PubMed] [Google Scholar]
- Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
- Braun B. S., Frieden R., Lessnick S. L., May W. A., Denny C. T. Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis. Mol Cell Biol. 1995 Aug;15(8):4623–4630. doi: 10.1128/mcb.15.8.4623. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Delattre O., Zucman J., Plougastel B., Desmaze C., Melot T., Peter M., Kovar H., Joubert I., de Jong P., Rouleau G. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature. 1992 Sep 10;359(6391):162–165. doi: 10.1038/359162a0. [DOI] [PubMed] [Google Scholar]
- Haeuptle M. T., Frank R., Dobberstein B. Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length. Nucleic Acids Res. 1986 Feb 11;14(3):1427–1448. doi: 10.1093/nar/14.3.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ji Q. S., Winnier G. E., Niswender K. D., Horstman D., Wisdom R., Magnuson M. A., Carpenter G. Essential role of the tyrosine kinase substrate phospholipase C-gamma1 in mammalian growth and development. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2999–3003. doi: 10.1073/pnas.94.7.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lee S. B., Rao A. K., Lee K. H., Yang X., Bae Y. S., Rhee S. G. Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function. Blood. 1996 Sep 1;88(5):1684–1691. [PubMed] [Google Scholar]
- May W. A., Arvand A., Thompson A. D., Braun B. S., Wright M., Denny C. T. EWS/FLI1-induced manic fringe renders NIH 3T3 cells tumorigenic. Nat Genet. 1997 Dec;17(4):495–497. doi: 10.1038/ng1297-495. [DOI] [PubMed] [Google Scholar]
- May W. A., Lessnick S. L., Braun B. S., Klemsz M., Lewis B. C., Lunsford L. B., Hromas R., Denny C. T. 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. 1993 Dec;13(12):7393–7398. doi: 10.1128/mcb.13.12.7393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mohammadi M., Dionne C. A., Li W., Li N., Spivak T., Honegger A. M., Jaye M., Schlessinger J. Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Nature. 1992 Aug 20;358(6388):681–684. doi: 10.1038/358681a0. [DOI] [PubMed] [Google Scholar]
- Munroe S. H. Antisense RNA inhibits splicing of pre-mRNA in vitro. EMBO J. 1988 Aug;7(8):2523–2532. doi: 10.1002/j.1460-2075.1988.tb03100.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
- Ohno T., Ouchida M., Lee L., Gatalica Z., Rao V. N., Reddy E. S. 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. Oncogene. 1994 Oct;9(10):3087–3097. [PubMed] [Google Scholar]
- Ohno T., Rao V. N., Reddy E. S. EWS/Fli-1 chimeric protein is a transcriptional activator. Cancer Res. 1993 Dec 15;53(24):5859–5863. [PubMed] [Google Scholar]
- Ouchida M., Ohno T., Fujimura Y., Rao V. N., Reddy E. S. Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. Oncogene. 1995 Sep 21;11(6):1049–1054. [PubMed] [Google Scholar]
- Peters K. G., Marie J., Wilson E., Ives H. E., Escobedo J., Del Rosario M., Mirda D., Williams L. T. Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis. Nature. 1992 Aug 20;358(6388):678–681. doi: 10.1038/358678a0. [DOI] [PubMed] [Google Scholar]
- Rabbitts T. H. Chromosomal translocations in human cancer. Nature. 1994 Nov 10;372(6502):143–149. doi: 10.1038/372143a0. [DOI] [PubMed] [Google Scholar]
- Rao V. N., Ohno T., Prasad D. D., Bhattacharya G., Reddy E. S. Analysis of the DNA-binding and transcriptional activation functions of human Fli-1 protein. Oncogene. 1993 Aug;8(8):2167–2173. [PubMed] [Google Scholar]
- Reddy E. S., Rao V. N. erg, an ets-related gene, codes for sequence-specific transcriptional activators. Oncogene. 1991 Dec;6(12):2285–2289. [PubMed] [Google Scholar]
- Rhee S. G., Bae Y. S. Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem. 1997 Jun 13;272(24):15045–15048. doi: 10.1074/jbc.272.24.15045. [DOI] [PubMed] [Google Scholar]
- Singer W. D., Brown H. A., Sternweis P. C. Regulation of eukaryotic phosphatidylinositol-specific phospholipase C and phospholipase D. Annu Rev Biochem. 1997;66:475–509. doi: 10.1146/annurev.biochem.66.1.475. [DOI] [PubMed] [Google Scholar]
- Sorensen P. H., Lessnick S. L., Lopez-Terrada D., Liu X. F., Triche T. J., Denny C. T. A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nat Genet. 1994 Feb;6(2):146–151. doi: 10.1038/ng0294-146. [DOI] [PubMed] [Google Scholar]
- Tanaka K., Iwakuma T., Harimaya K., Sato H., Iwamoto Y. EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest. 1997 Jan 15;99(2):239–247. doi: 10.1172/JCI119152. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walder R. Y., Walder J. A. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5011–5015. doi: 10.1073/pnas.85.14.5011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weber G., Friedman E., Grimmond S., Hayward N. K., Phelan C., Skogseid B., Gobl A., Zedenius J., Sandelin K., Teh B. T. The phospholipase C beta 3 gene located in the MEN1 region shows loss of expression in endocrine tumours. Hum Mol Genet. 1994 Oct;3(10):1775–1781. doi: 10.1093/hmg/3.10.1775. [DOI] [PubMed] [Google Scholar]
- Whang-Peng J., Triche T. J., Knutsen T., Miser J., Douglass E. C., Israel M. A. Chromosome translocation in peripheral neuroepithelioma. N Engl J Med. 1984 Aug 30;311(9):584–585. doi: 10.1056/NEJM198408303110907. [DOI] [PubMed] [Google Scholar]