Downregulation and forced expression of EWS-Fli1 fusion gene results in changes in the expression of G1regulatory genes

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

Alteration of cyclin D1 transcript elongation by a mutated transcription factor up-regulates the oncogenic D1b splice isoform in cancer

“…As also expected from previous studies (27), Dox treatment resulted in an Ϸ2-fold decrease in cyclin D1a mRNA levels specifically in A673-shEF1 cells but not in A673-Ctrl cells (Fig. 1B).…”

Rapamycin induces the fusion-type independent downregulation of the EWS/FLI-1 proteins and inhibits Ewing's sarcoma cell proliferation

“…Thus, it appears that, in ES cells, downregulation of EWS/FLI-1 expression may be an important contributor to the observed G 1 cell cycle arrest. This interpretation is in agreement with the previous observation that inhibition of the expression of the fusion protein with specific antisense oligodeoxynucleotides induced G 1 cell cycle arrest (Matsumoto et al, 2001). This does not exclude the possibility that abnormalities in G 1 checkpoint regulators known to occur in ES cells (Deneen and Denny, 2001) may also contribute to, and most likely increase their sensitivity to rapamycin.…”

“…; (*), Pp0.02 for test group versus untreated control group Downregulation of EWS/FLI-1 by rapamycin S Mateo-Lozano et al Several studies reported that the induction of cell cycle arrest by rapamycin resulted from a decrease in the levels of cyclins (Seufferlein and Rozengurt, 1996;Decker et al, 2003). Moreover, it has been recently proposed that cyclin-dependent kinase inhibitors and other G 1 regulators are targets of the EWS/FLI-1 transcriptional activity (Matsumoto et al, 2001;Nakatani et al, 2003). Thus, it appears that, in ES cells, downregulation of EWS/FLI-1 expression may be an important contributor to the observed G 1 cell cycle arrest.…”

Upregulation of Id2, an oncogenic helix-loop-helix protein, is mediated by the chimeric EWS/ets protein in Ewing sarcoma

“…EWS/Fli-1 is a strong transactivator of the c-myc promoter (Bailly et al, 1994). Several genes such as EAT-2 (Thompson et al, 1996), stromelysin , and fringe (May et al, 1997) are reported to be upregulated, while TGF-b type II receptor (TGFbIIR) (Im et al, 2000), p21 (Matsumoto et al, 2001), and p57KIP2 (Dauphinot et al, 2001) are downregulated. Furthermore, EWS/Fli-1 interacts with hsRPB7, a subunit of human RNA polymerase II (Petermann et al, 1998), and the EWS/ Fli-1 fusion protein alters the IGF-IR signaling pathway (Toretsky et al, 1997).…”