Abstract

PURPOSE
High levels and aberrant patterns of Bcl-2 gene expression have been reported in a variety of human cancers, including prostate, colorectal, lung and gastric cancers, neuroblastoma, non-Hodgkins lymphoma, and both acute and chronic leukemia. The Ewings sarcoma is a common malignant bone tumor in children and adolescents. Nearly all Ewings sarcomas have a t(11;22) chromosomal translocations which involves EWS gene and Fli-1 of ETS family transcription factors. The patterns of Bcl-2 gene expression in Ewings sarcoma is less well known. The inhibition of Bcl-2 gene expression leads to increase a apoptosis in several cancer cells. This study was undertaken to characterize the patterns of Bcl-2 gene expression in Ewings sarcoma cell (TC135) expressing fusion protein, EWS-Fli-l, and to induce the apoptosis of Ewings sarcoma cell by targeting Bcl-2 gene expression using antisense strategy.
MATERIALS AND METHODS
We used two types of antisense EWS-Fli-1 and Bcl-2 expression vectors. These vectors were transfected to TC135 cells by calcium phosphate method, and transformed cells were selected using G418. The transformed cells were stimulated with apoptosis-inducing reagents, and changes of Bcl-2 expression were analyzed by Western and Northern blot analyses. To confirm the increased apoptosis, we checked DNA fragmentation, cell viability assay by MTT and ICE (interleuldn converting enzyme) activity.
RESULTS
The TC135 cells transfected with antisense EWS-Fli-1 expression vector showed negatively regulated Bcl-2 protein and mRNA expression, but those transfected with control vector (pcDNA3) revealed no change of Bcl-2 gene expression. These results strongly suggested that the EWS-Fli-1 fusion protein directly regulate Bcl-2 gene expression on the Bcl-2 gene promotor region. And the TC135 cells transfected with antisense Bcl-2 expression vector showed increased apoptosis. These results suggested that the apoptosis pathway of Ewings sarcoma is regulated by EWS-Fli-1 fusion protein and following Bcl-2 gene. Finally, TC135 cells transfected with antisense Bcl-2 expression vector did not form colonies in soft agar, which may infer the loss of tumorigenicity.
CONCLUSION
The targeting of Bcl-2 gene in the TC135 cells using antisense strategy lead to an increased apoptosis in .Ewings sarcoma cells. This approach can be considered as an efficient candidate strategy of cancer gene therapy.