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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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KRAB/FLI-1 inhibits the growth rate of EWS/FLI-1-transformed cells in low serum medium. In all, 5000 cells were grown in 1% FCS and cells were counted in triplicate at 3-day intervals and mean±s.d. is shown. (A) The parental murine Ews/Fli-1-transformed NIH3T3 cells (mEF#1) and KRAB/FLI-1 (K1, K7) or mutant KRAB/FLI-1 (mKF12) cotransfected subclones. (B) The parental human EWS/FLI-1-transformed NIH3T3 cells (HuEF#16) and KRAB/FLI-1 (K13, K19) or mutant KRAB/FLI-1 (mK11) cotransfected subclones. NIH3T3 cells transfected with empty vector were used as controls. Data are representative of three separate experiments (mean±s.d.).
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fig4: KRAB/FLI-1 inhibits the growth rate of EWS/FLI-1-transformed cells in low serum medium. In all, 5000 cells were grown in 1% FCS and cells were counted in triplicate at 3-day intervals and mean±s.d. is shown. (A) The parental murine Ews/Fli-1-transformed NIH3T3 cells (mEF#1) and KRAB/FLI-1 (K1, K7) or mutant KRAB/FLI-1 (mKF12) cotransfected subclones. (B) The parental human EWS/FLI-1-transformed NIH3T3 cells (HuEF#16) and KRAB/FLI-1 (K13, K19) or mutant KRAB/FLI-1 (mK11) cotransfected subclones. NIH3T3 cells transfected with empty vector were used as controls. Data are representative of three separate experiments (mean±s.d.).

Mentions: Under normal culture conditions with 10% FCS there was no difference in the growth rate of clones expressing EWS/Fli-1 and/or KRAB/FLI-1 or mutant KRAB/FLI-1 fusion genes (data not shown). KRAB/FLI-1 also had no effect on the growth of wild-type NIH3T3 cells. However, under low serum culture conditions (1% FCS), the EWS/FLI-1-transformed cell lines and clones coexpressing mutant KRAB/FLI-1 grew rapidly (Figure 4AFigure 4


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)

KRAB/FLI-1 inhibits the growth rate of EWS/FLI-1-transformed cells in low serum medium. In all, 5000 cells were grown in 1% FCS and cells were counted in triplicate at 3-day intervals and mean±s.d. is shown. (A) The parental murine Ews/Fli-1-transformed NIH3T3 cells (mEF#1) and KRAB/FLI-1 (K1, K7) or mutant KRAB/FLI-1 (mKF12) cotransfected subclones. (B) The parental human EWS/FLI-1-transformed NIH3T3 cells (HuEF#16) and KRAB/FLI-1 (K13, K19) or mutant KRAB/FLI-1 (mK11) cotransfected subclones. NIH3T3 cells transfected with empty vector were used as controls. Data are representative of three separate experiments (mean±s.d.).
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Related In: Results  -  Collection

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fig4: KRAB/FLI-1 inhibits the growth rate of EWS/FLI-1-transformed cells in low serum medium. In all, 5000 cells were grown in 1% FCS and cells were counted in triplicate at 3-day intervals and mean±s.d. is shown. (A) The parental murine Ews/Fli-1-transformed NIH3T3 cells (mEF#1) and KRAB/FLI-1 (K1, K7) or mutant KRAB/FLI-1 (mKF12) cotransfected subclones. (B) The parental human EWS/FLI-1-transformed NIH3T3 cells (HuEF#16) and KRAB/FLI-1 (K13, K19) or mutant KRAB/FLI-1 (mK11) cotransfected subclones. NIH3T3 cells transfected with empty vector were used as controls. Data are representative of three separate experiments (mean±s.d.).
Mentions: Under normal culture conditions with 10% FCS there was no difference in the growth rate of clones expressing EWS/Fli-1 and/or KRAB/FLI-1 or mutant KRAB/FLI-1 fusion genes (data not shown). KRAB/FLI-1 also had no effect on the growth of wild-type NIH3T3 cells. However, under low serum culture conditions (1% FCS), the EWS/FLI-1-transformed cell lines and clones coexpressing mutant KRAB/FLI-1 grew rapidly (Figure 4AFigure 4

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