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Transformation induced by Ewing's sarcoma associated EWS/FLI-1 is suppressed by KRAB/FLI-1.

Chan D, Wilson TJ, Xu D, Cowdery HE, Sanij E, Hertzog PJ, Kola I - Br. J. Cancer (2003)

Bottom Line: Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1.The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor.This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Centre for Functional Genomics and Human Diseases, Monash Institute of Reproduction and Development, Monash University, Melbourne, Australia.

ABSTRACT
Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1. To elucidate the mechanisms by which EWS/FLI-1 mediates transformation in mouse models, we have generated a murine Ews/Fli-1 fusion protein. We demonstrate that this protein transforms fibroblast cells in vitro similar to human EWS/FLI-1 as demonstrated by serum and anchorage-independent growth, the formation of tumours in nude mice and elevation of the oncogenic marker c-myc. Furthermore, transformation of these cells was inhibited by a specific repressor, KRAB/FLI-1. The KRAB/FLI-1 repressor also suppressed the tumorigenic phenotype of a human Ewing's sarcoma cell line. These findings suggest that the transformed phenotype of Ewing's sarcoma cells can be reversed by using the sequence-specific FLI-1-DNA-binding domain to target a gene repressor domain. The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor. This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.

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Western blot showing the expression of EWS/FLI-1 (68 kDa), KRAB/FLI-1 or mutant KRAB/FLI-1 (45 kDa), and alteration of c-myc protein levels in transfected NIH3T3 cells. (A) Cells transfected with empty construct (M=mock control) or murine EWS/Fli-1 (#1) and subclones of #1 cotransfected with KRAB/FLI-1 (K1, K22) or mutant KRAB/FLI-1 (mK12). (B) Similar human EWS/FLI-1-transformed clones transfected with KRAB/FLI-1 (K13, K19) or mutant KRAB/FLI-1 (mK10).
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fig3: Western blot showing the expression of EWS/FLI-1 (68 kDa), KRAB/FLI-1 or mutant KRAB/FLI-1 (45 kDa), and alteration of c-myc protein levels in transfected NIH3T3 cells. (A) Cells transfected with empty construct (M=mock control) or murine EWS/Fli-1 (#1) and subclones of #1 cotransfected with KRAB/FLI-1 (K1, K22) or mutant KRAB/FLI-1 (mK12). (B) Similar human EWS/FLI-1-transformed clones transfected with KRAB/FLI-1 (K13, K19) or mutant KRAB/FLI-1 (mK10).

Mentions: Murine and human EWS/FLI-1-transformed clones were used to test the effects of the KRAB/FLI-1 hybrid protein on the transformed phenotype. KRAB/FLI-1 and mutant KRAB/FLI-1 vectors were generated such that they contained the equivalent region of the FLI-1 DBD as that found in human type I EWS/FLI-1 translocations and our human and mouse EWS/FLI-1 constructs. The mutant KRAB domain, which was used as a control, contains two amino-acid substitutions which abolishes KRAB binding to the corepressor KAP-1 and thus repressor function (Margolin et al, 1994; Friedman et al, 1996). Both KRAB/FLI-1 and mutant KRAB/FLI-1 fusion genes were placed under the control of the human EF-1α promoter (Figure 2). These genes were followed by a promoterless IRES/neomycin cassette to ensure that all G418-resistant clones expressed the KRAB/FLI-1 or mutant KRAB/FLI-1 fusion genes. Protein expression in G418-resistant clones was demonstrated by Western blot with the Fli-1 polyclonal antibody that detected both the 68 kDa EWS/FLI-1 protein band and the 45 kDa KRAB/FLI-1 or mutant KRAB/FLI-1 protein bands. Three murine and human EWS/FLI-1-transformed clones were each transfected with KRAB/FLI-1 and data from two representative clones, mEF#1 and HuEF#16, are shown in Figure 3AFigure 3


Transformation induced by Ewing's sarcoma associated EWS/FLI-1 is suppressed by KRAB/FLI-1.

Chan D, Wilson TJ, Xu D, Cowdery HE, Sanij E, Hertzog PJ, Kola I - Br. J. Cancer (2003)

Western blot showing the expression of EWS/FLI-1 (68 kDa), KRAB/FLI-1 or mutant KRAB/FLI-1 (45 kDa), and alteration of c-myc protein levels in transfected NIH3T3 cells. (A) Cells transfected with empty construct (M=mock control) or murine EWS/Fli-1 (#1) and subclones of #1 cotransfected with KRAB/FLI-1 (K1, K22) or mutant KRAB/FLI-1 (mK12). (B) Similar human EWS/FLI-1-transformed clones transfected with KRAB/FLI-1 (K13, K19) or mutant KRAB/FLI-1 (mK10).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2376791&req=5

fig3: Western blot showing the expression of EWS/FLI-1 (68 kDa), KRAB/FLI-1 or mutant KRAB/FLI-1 (45 kDa), and alteration of c-myc protein levels in transfected NIH3T3 cells. (A) Cells transfected with empty construct (M=mock control) or murine EWS/Fli-1 (#1) and subclones of #1 cotransfected with KRAB/FLI-1 (K1, K22) or mutant KRAB/FLI-1 (mK12). (B) Similar human EWS/FLI-1-transformed clones transfected with KRAB/FLI-1 (K13, K19) or mutant KRAB/FLI-1 (mK10).
Mentions: Murine and human EWS/FLI-1-transformed clones were used to test the effects of the KRAB/FLI-1 hybrid protein on the transformed phenotype. KRAB/FLI-1 and mutant KRAB/FLI-1 vectors were generated such that they contained the equivalent region of the FLI-1 DBD as that found in human type I EWS/FLI-1 translocations and our human and mouse EWS/FLI-1 constructs. The mutant KRAB domain, which was used as a control, contains two amino-acid substitutions which abolishes KRAB binding to the corepressor KAP-1 and thus repressor function (Margolin et al, 1994; Friedman et al, 1996). Both KRAB/FLI-1 and mutant KRAB/FLI-1 fusion genes were placed under the control of the human EF-1α promoter (Figure 2). These genes were followed by a promoterless IRES/neomycin cassette to ensure that all G418-resistant clones expressed the KRAB/FLI-1 or mutant KRAB/FLI-1 fusion genes. Protein expression in G418-resistant clones was demonstrated by Western blot with the Fli-1 polyclonal antibody that detected both the 68 kDa EWS/FLI-1 protein band and the 45 kDa KRAB/FLI-1 or mutant KRAB/FLI-1 protein bands. Three murine and human EWS/FLI-1-transformed clones were each transfected with KRAB/FLI-1 and data from two representative clones, mEF#1 and HuEF#16, are shown in Figure 3AFigure 3

Bottom Line: Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1.The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor.This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Centre for Functional Genomics and Human Diseases, Monash Institute of Reproduction and Development, Monash University, Melbourne, Australia.

ABSTRACT
Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1. To elucidate the mechanisms by which EWS/FLI-1 mediates transformation in mouse models, we have generated a murine Ews/Fli-1 fusion protein. We demonstrate that this protein transforms fibroblast cells in vitro similar to human EWS/FLI-1 as demonstrated by serum and anchorage-independent growth, the formation of tumours in nude mice and elevation of the oncogenic marker c-myc. Furthermore, transformation of these cells was inhibited by a specific repressor, KRAB/FLI-1. The KRAB/FLI-1 repressor also suppressed the tumorigenic phenotype of a human Ewing's sarcoma cell line. These findings suggest that the transformed phenotype of Ewing's sarcoma cells can be reversed by using the sequence-specific FLI-1-DNA-binding domain to target a gene repressor domain. The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor. This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus