Construction Of Ewing's Sarcoma Specific Expression Vector Of Diphtheria Toxin And Observation Of Its Killing Effect On Cultured Ewing's Sarcoma Cells

Aims Previous studies have shown that EWS-FLI-1 fusion protein, formed by chromosomal translocation of t (11; 22)(q 24; q 12) in 95 percent of Ewing's sarcomas, can bind with special gene sequence (ACCGGAAGT), and has transcription activity. In this study, we constructed an expression vector, which contained the binding sequence and the diphtheria toxin A chain (DTA) gene sequence, and investigated the expression and killing effect of DTA, after transfected into Ewing's sarcoma cell lines in vitro. We hope this study can help to find a new way of gene therapy to cancer.Methods 1. The transfection of luciferase reporter plasmid (pS2), which contains the EWS-FLI-1 binding sequence, into cultured Ewing's sarcoma cell lines (RD-ES, SK-ES) and control cell lines (Hela, NIH3T3) was performed by liposome method. The luciferase activity in the cellular lysate was measured by spectrofluorometer. 2. To construct the DTA expression vector (pS2-DTA), we replaced the luciferase gene in pS2 with DTA gene at the points of Ncoâ…  and Xbaâ…  by molecular technique. The new plasmid was verified by restriction enzyme digest and nucleotide sequence determine. 3. To investigate the synthetic inhibition to luciferase, we co-transfected the pS2 and pS2-DTA and tested the activity of luciferase. 4. After transfected the pS2-DTA into the cells, the expression of DTA was detected by RT-PCR and immunohistochemistry; in situ cell death was detected by TUNEL method, the survival rate was counted by trypan blue exclusion test, the killing effect was detected by observing morphologic change and drawing growth curve.Results 1. The result of restriction enzyme digest and gene sequence showed that the EWS-FLI-1 binding sequence (ACCGGAAGT) lay at the upper stream of diphtheria toxin A fragment. 2. The result of luciferase activity showed: (1) The luciferase activity in Ewing's sarcoma cells was significantly higher than that in control cells (P<0.01), when pS2 was transfected. (2) The pS2-DTA inhibited the expression of luciferase especially in Ewing's sarcoma cells, when the pS2 and pS2-DTA were co-transfected. 3. The expressionand killing effection of DTA, when pS2-DTA was transfected showed: (1) The growth and survival rate of Ewing's sarcoma cells was lower than that of controls. The apoptosis rate of Ewing's sarcoma cells was higher than that of controls(P<0.01). (2) The expression of DTA in Ewing's sarcoma cells was much higher at the levels of mRNA and protein than that in controls(P<0.01).Conclusions 1. The expression vector pS2-DTA contains the EWS-FLI-1 binding sequence and the DTA gene sequence. 2. EWS-FLI-1 can regulate the expression of luciferase because of its powerful transcription activity. 3. The pS2-DTA can inhibite the synthesis of luciferase, when it is co-transfected into the Ewing's sarcoma cells with the pS2. 4. The pS2-DTA can kill and restrain Ewing's sarcoma cells but this function is lower than that of control cells.