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. 2001 Mar;84(6):768–775. doi: 10.1054/bjoc.2000.1652

Downregulation and forced expression of EWS-Fli1 fusion gene results in changes in the expression of G 1 regulatory genes

Y Matsumoto 1, K Tanaka 1, F Nakatani 1, T Matsunobu 1, S Matsuda 1, Y Iwamoto 1
PMCID: PMC2363806  PMID: 11259090

Abstract

Chromosomal translocation t(11;22)(q24:q12) is detected in approximately 90% of tumours of the Ewing family (ET). This translocation results in EWS-Fli1 gene fusion which produces a EWS-Fli1 fusion protein acting as an aberrant transcriptional activator. We previously reported that the inhibition of EWS-Fli1 expression caused the G 0/G 1 arrest of ET cells. We, therefore, hypothesized that EWS-Fli1 may affect the expression of G 1 regulatory genes. Downregulation of EWS-Fli1 fusion proteins was observed 48 hours after the treatment with EWS-Fli1 antisense oligonucleotides. The expressions of G 1 cyclins, cyclin D1 and cyclin E, were markedly decreased in parallel with the reduction of EWS-Fli1 fusion protein. On the other hand, the expression of p21 and p27, which are important cyclin-dependent kinase inhibitors (CKIs) for G 1–S transition, was dramatically increased after the treatment with EWS-Fli1 antisense oligonucleotides. RT-PCR analysis showed that alteration of the expressions of the cyclins and CKIs occurred at the mRNA level. Furthermore, transfection of EWS-Fli1 cDNA to NIH3T3 caused transformation of the cells and induction of the expression of cyclin D1 and E. Clinical samples of ET also showed a high level of expression of cyclin D1 mRNA, whereas mRNAs for p21 and p27 were not detected in the samples. These findings strongly suggest that the G 1–S regulatory genes may be involved in downstream of EWS-Fli1 transcription factor, and that the unbalanced expression of G 1–S regulatory factors caused by EWS-Fli1 may lead to the tumorigenesis of ET. © 2001 Cancer Research Campaign http://www. bjcancer.com

Keywords: EWS-Fli1, cell-cycle, G 0/G 1 arrest, antisense oligonucleotides, clinical samples, transfection

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Selected References

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  1. Albanese C., Johnson J., Watanabe G., Eklund N., Vu D., Arnold A., Pestell R. G. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem. 1995 Oct 6;270(40):23589–23597. doi: 10.1074/jbc.270.40.23589. [DOI] [PubMed] [Google Scholar]
  2. Arvand A., Bastians H., Welford S. M., Thompson A. D., Ruderman J. V., Denny C. T. EWS/FLI1 up regulates mE2-C, a cyclin-selective ubiquitin conjugating enzyme involved in cyclin B destruction. Oncogene. 1998 Oct 22;17(16):2039–2045. doi: 10.1038/sj.onc.1202129. [DOI] [PubMed] [Google Scholar]
  3. Bedi A., Zehnbauer B. A., Barber J. P., Sharkis S. J., Jones R. J. Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia. Blood. 1994 Apr 15;83(8):2038–2044. [PubMed] [Google Scholar]
  4. Botz J., Zerfass-Thome K., Spitkovsky D., Delius H., Vogt B., Eilers M., Hatzigeorgiou A., Jansen-Dürr P. Cell cycle regulation of the murine cyclin E gene depends on an E2F binding site in the promoter. Mol Cell Biol. 1996 Jul;16(7):3401–3409. doi: 10.1128/mcb.16.7.3401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chin Y. E., Kitagawa M., Su W. C., You Z. H., Iwamoto Y., Fu X. Y. Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1. Science. 1996 May 3;272(5262):719–722. doi: 10.1126/science.272.5262.719. [DOI] [PubMed] [Google Scholar]
  6. Funaoka K., Shindoh M., Yoshida K., Hanzawa M., Hida K., Nishikata S., Totsuka Y., Fujinaga K. Activation of the p21(Waf1/Cip1) promoter by the ets oncogene family transcription factor E1AF. Biochem Biophys Res Commun. 1997 Jul 9;236(1):79–82. doi: 10.1006/bbrc.1997.6909. [DOI] [PubMed] [Google Scholar]
  7. Geng Y., Whoriskey W., Park M. Y., Bronson R. T., Medema R. H., Li T., Weinberg R. A., Sicinski P. Rescue of cyclin D1 deficiency by knockin cyclin E. Cell. 1999 Jun 11;97(6):767–777. doi: 10.1016/s0092-8674(00)80788-6. [DOI] [PubMed] [Google Scholar]
  8. Giovannini M., Biegel J. A., Serra M., Wang J. Y., Wei Y. H., Nycum L., Emanuel B. S., Evans G. A. EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations. J Clin Invest. 1994 Aug;94(2):489–496. doi: 10.1172/JCI117360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gu Y., Turck C. W., Morgan D. O. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature. 1993 Dec 16;366(6456):707–710. doi: 10.1038/366707a0. [DOI] [PubMed] [Google Scholar]
  10. Keyomarsi K., Conte D., Jr, Toyofuku W., Fox M. P. Deregulation of cyclin E in breast cancer. Oncogene. 1995 Sep 7;11(5):941–950. [PubMed] [Google Scholar]
  11. Kovar H., Jug G., Aryee D. N., Zoubek A., Ambros P., Gruber B., Windhager R., Gadner H. Among genes involved in the RB dependent cell cycle regulatory cascade, the p16 tumor suppressor gene is frequently lost in the Ewing family of tumors. Oncogene. 1997 Oct;15(18):2225–2232. doi: 10.1038/sj.onc.1201397. [DOI] [PubMed] [Google Scholar]
  12. Lalli E., Sassone-Corsi P., Ceredig R. Block of T lymphocyte differentiation by activation of the cAMP-dependent signal transduction pathway. EMBO J. 1996 Feb 1;15(3):528–537. [PMC free article] [PubMed] [Google Scholar]
  13. Lee S. J., Ha M. J., Lee J., Nguyen P., Choi Y. H., Pirnia F., Kang W. K., Wang X. F., Kim S. J., Trepel J. B. Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21(WAF1/CIP1) in human prostate carcinoma cells. J Biol Chem. 1998 Apr 24;273(17):10618–10623. doi: 10.1074/jbc.273.17.10618. [DOI] [PubMed] [Google Scholar]
  14. Liu M., Lee M. H., Cohen M., Bommakanti M., Freedman L. P. Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937. Genes Dev. 1996 Jan 15;10(2):142–153. doi: 10.1101/gad.10.2.142. [DOI] [PubMed] [Google Scholar]
  15. Lukas J., Herzinger T., Hansen K., Moroni M. C., Resnitzky D., Helin K., Reed S. I., Bartek J. Cyclin E-induced S phase without activation of the pRb/E2F pathway. Genes Dev. 1997 Jun 1;11(11):1479–1492. doi: 10.1101/gad.11.11.1479. [DOI] [PubMed] [Google Scholar]
  16. May W. A., Gishizky M. L., Lessnick S. L., Lunsford L. B., Lewis B. C., Delattre O., Zucman J., Thomas G., Denny C. T. Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5752–5756. doi: 10.1073/pnas.90.12.5752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McGahon A., Bissonnette R., Schmitt M., Cotter K. M., Green D. R., Cotter T. G. BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood. 1994 Mar 1;83(5):1179–1187. [PubMed] [Google Scholar]
  18. 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]
  19. Pagano M., Tam S. W., Theodoras A. M., Beer-Romero P., Del Sal G., Chau V., Yew P. R., Draetta G. F., Rolfe M. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science. 1995 Aug 4;269(5224):682–685. doi: 10.1126/science.7624798. [DOI] [PubMed] [Google Scholar]
  20. Qian X., Kulig E., Jin L., Lloyd R. V. Expression of D-type cyclins in normal and neoplastic rat pituitary. Endocrinology. 1998 Apr;139(4):2058–2067. doi: 10.1210/endo.139.4.5955. [DOI] [PubMed] [Google Scholar]
  21. Sherr C. J., Roberts J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995 May 15;9(10):1149–1163. doi: 10.1101/gad.9.10.1149. [DOI] [PubMed] [Google Scholar]
  22. Srivastava R. K., Srivastava A. R., Cho-Chung Y. S. Synergistic effects of 8-chlorocyclic-AMP and retinoic acid on induction of apoptosis in Ewing's sarcoma CHP-100 cells. Clin Cancer Res. 1998 Mar;4(3):755–761. [PubMed] [Google Scholar]
  23. 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]
  24. Tanaka K., Iwamoto Y., Noguchi Y., Oda Y., Sugioka Y. The establishment and characterization of a peripheral neuroepithelioma cell line in soft tissue of extremity. Lab Invest. 1995 Feb;72(2):237–248. [PubMed] [Google Scholar]
  25. Waga S., Hannon G. J., Beach D., Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature. 1994 Jun 16;369(6481):574–578. doi: 10.1038/369574a0. [DOI] [PubMed] [Google Scholar]
  26. Wang T. C., Cardiff R. D., Zukerberg L., Lees E., Arnold A., Schmidt E. V. Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature. 1994 Jun 23;369(6482):669–671. doi: 10.1038/369669a0. [DOI] [PubMed] [Google Scholar]
  27. Wong H., Riabowol K. Differential CDK-inhibitor gene expression in aging human diploid fibroblasts. Exp Gerontol. 1996 Jan-Apr;31(1-2):311–325. doi: 10.1016/0531-5565(95)00025-9. [DOI] [PubMed] [Google Scholar]
  28. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

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