Killing Effect Of Expression Of The Diphtheria Toxin A Chain Gene Regulated By The EWS-FLI 1 Fusion Gene On Ewings Sarcoma

Objective Diphtheria toxin A chain (DTA) is known to kill cells by ribosylating the EF2 translation-initiation factor and inhibiting protein synthesis. Genetic recombination immunotoxin conjugated by the DTA and different vectors can kill cells that express special protein. Previous studies have shown that EWS-FLI1 fusion protein, formed by chromosomal translocation of t (11; 22)(q24; q 12) in 95 percent of Ewing's sarcomas, can combine to special gene sequence and has transcriptional activity. In this study, the expression and therapeutic effect of DTA was investigated by used of an special expression vector (pS2-DTA) which contains the binding sequence of the EWS-FLI1 fusion gene and the DTA gene sequence. Methods 1. To construct the nude mouse modles, the SK-ES-1 cell line was injected into the subcutaneousness of the nude mouse ridge.2.The mixture of the pS2-DTA and DOTAP was injected into the nude mouse through the tail vein. 3. The tumor volume was measured to evaluate the therapeutic effect of the DTA on the Ewing's sarcoma 4. Morphologic change of the transplanted tumor and the heart ,liver was observed by use of the pathologic slices .5. RT-PCR and immunohistochemistry were used to detect the expression of DTA; Apoptosis in situ was detected by TUNEL method.Results 1. The Ewing's sarcoma nude mouse model was constructed successfully. 2. The transplanted Ewing's sarcoma of the therapeutic group grew slower than that of the control groups, and the tumor volume of therapeutic group has a significant reduction after therapy (P<0.01). 3.The expression of DTA can be detected in the Ewing's sarcoma tissue and the liver by the RT-PCR, but the expresstion of the DTA in the liver tissue was significantly weaker than it in the tumor tissue (P<0.05). Expresstion of the DTA can be detected in the tumor tissue by immunohistochemistry, but not in heart and liver tissue. As apoptosis in situ, there was a significantly difference between the therapeutic group and the control groups(P<0.01). Conclusions 1.The expression vector pS2-DTA, which contains the EWS-FLI1 binding sequence and the DTA sequence, can express in the Ewing's sarcoma specially. 2. The expression vector pS2-DTA have a restraining and killing effect on Ewing's sarcoma.