All issues > Volume 43(7); 2000

Original Article

J Korean Pediatr Soc. 2000;43(7):942-951. Published online July 15, 2000.

Induction of Apoptosis of Ewing`s Sarcoma Cells by Regulating Fusion Protein Expression

Ho Keun HK Yi¹, Ran Ju RJ Kim¹, Dae Yeol DY Lee¹, Jung Soo JS Kim¹, Pyoung Han PH Hwang¹

¹Department of Pediatrics, Institute for Medical Sciences, Chonbuk National University Medical School, Chonju, Chonbuk, Korea

Correspondence Pyoung Han PH Hwang ,Email: hwaph@moak.chonbuk.ac.kr

Abstract

Purpose
: Fusion genes(EWS-Fli-1 and EWS-erg ) function as transcription activators and are essential for maintaining tumorigenic properties in Ewing`s sarcoma cells. Several reports have noted that Ets family transcription factors bind with CBP(CREB binding protein) in vitro. To understand the interaction of fusion proteins and CBP, we studied the CBP protein in TC135 cells expressing the EWS-Fli-1 gene. We also studied the hypothesis that downregulation of fusion gene expression may induce susceptibility to apoptosis in Ewing`s sarcoma cells.
Methods
: For targeting fusion proteins, we reconstructed the antisense EWS-fli-1, EWS-erg and CBP genes in pcDNA3, and transfected these genes to Ewing`s sarcoma cells showing high levels of expression for Ve3 and 5838 genes. These vectors were transfected to cells by the calcium phosphate method, and transformed cells were selected using G418. We measured DNA fragments for apoptosis using FACScan. We used crystal violet staining and MTT assay to evaluate cell viability, and Western blot analysis was used to assess CBP gene expression. Results : Cells transfected with antisense fusion genes Ve3 and 5838 showed inhibition of fusion protein expression. These cells also showed decreased cell viability. Susceptibility to apoptosis was induced by treatment with chemotherapeutic agents at low concentrations. Antisense CBP -transfected cells showed loss of cell viability in 0.1% and 0.5% serum. This loss of cell viability was similar to the response by antisense fusion protein-transfected cells treated with chemotherapeutic agents at low concentrations.
Conclusion
: Our results suggest that fusion proteins and CBP co-regulate apoptosis in Ewing`s sarcoma cells. Antisense fusion gene therapy may be an useful adjunct in combining with chemotherapeutic regimens to downregulate the expression of fusion proteins in Ewing`s sarcoma.

Keywords :Ewing`s sarcoma, EWS-fusion protein, Antisense, Apoptosis