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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 of tumours in nude mice. Cells (1×106) were inoculated into the flanks of BALB/c nu/nu mice and tumours measured with calipers at 3-day intervals. (A) The parental murine Ews/Fli-1-transformed NIH3T3 cells (mEF#1) and KRAB/FLI-1 (K1) or mutant KRAB/FLI-1 (mK12) cotransfected subclones. (B) The parental human EWS/FLI-1-transformed NIH3T3 cells (HuEF#16) and KRAB/FLI-1 (K13) or mutant KRAB/FLI-1 (mK10) cotransfected subclones. Mean tumour volumes (±s.e.m.) are shown and were calculated by the formula (mean diameter)3×π/6. The table shows the number of inoculation sites forming tumours at each time point.
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fig6: KRAB/FLI-1 inhibits the growth of tumours in nude mice. Cells (1×106) were inoculated into the flanks of BALB/c nu/nu mice and tumours measured with calipers at 3-day intervals. (A) The parental murine Ews/Fli-1-transformed NIH3T3 cells (mEF#1) and KRAB/FLI-1 (K1) or mutant KRAB/FLI-1 (mK12) cotransfected subclones. (B) The parental human EWS/FLI-1-transformed NIH3T3 cells (HuEF#16) and KRAB/FLI-1 (K13) or mutant KRAB/FLI-1 (mK10) cotransfected subclones. Mean tumour volumes (±s.e.m.) are shown and were calculated by the formula (mean diameter)3×π/6. The table shows the number of inoculation sites forming tumours at each time point.

Mentions: For each murine or human EWS/FLI-1-transformed clone, one KRAB/FLI-1 cotransfectant and one mutant KRAB/Fli-1 cotransfectant were inoculated into BALB/c nu/nu mice. Control cells were not observed to form any tumours (data not shown). The HuEF#16-transformed clone formed tumours of 222±53 mm3 by 30 days after inoculation, whereas the mEF#1 transformed clone had formed tumours of only 128±26 mm3 at the same stage (Figure 6AFigure 6


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 of tumours in nude mice. Cells (1×106) were inoculated into the flanks of BALB/c nu/nu mice and tumours measured with calipers at 3-day intervals. (A) The parental murine Ews/Fli-1-transformed NIH3T3 cells (mEF#1) and KRAB/FLI-1 (K1) or mutant KRAB/FLI-1 (mK12) cotransfected subclones. (B) The parental human EWS/FLI-1-transformed NIH3T3 cells (HuEF#16) and KRAB/FLI-1 (K13) or mutant KRAB/FLI-1 (mK10) cotransfected subclones. Mean tumour volumes (±s.e.m.) are shown and were calculated by the formula (mean diameter)3×π/6. The table shows the number of inoculation sites forming tumours at each time point.
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Related In: Results  -  Collection

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fig6: KRAB/FLI-1 inhibits the growth of tumours in nude mice. Cells (1×106) were inoculated into the flanks of BALB/c nu/nu mice and tumours measured with calipers at 3-day intervals. (A) The parental murine Ews/Fli-1-transformed NIH3T3 cells (mEF#1) and KRAB/FLI-1 (K1) or mutant KRAB/FLI-1 (mK12) cotransfected subclones. (B) The parental human EWS/FLI-1-transformed NIH3T3 cells (HuEF#16) and KRAB/FLI-1 (K13) or mutant KRAB/FLI-1 (mK10) cotransfected subclones. Mean tumour volumes (±s.e.m.) are shown and were calculated by the formula (mean diameter)3×π/6. The table shows the number of inoculation sites forming tumours at each time point.
Mentions: For each murine or human EWS/FLI-1-transformed clone, one KRAB/FLI-1 cotransfectant and one mutant KRAB/Fli-1 cotransfectant were inoculated into BALB/c nu/nu mice. Control cells were not observed to form any tumours (data not shown). The HuEF#16-transformed clone formed tumours of 222±53 mm3 by 30 days after inoculation, whereas the mEF#1 transformed clone had formed tumours of only 128±26 mm3 at the same stage (Figure 6AFigure 6

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