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    ABSTRACT: Introduction: The technique of RNA interference is rapidly growing in popularity, as it is an effective tool among molecular biologists conducting gene-silencing experiments. Esophageal adenocarcinoma (EA) responds poorly to conventional clinical therapy. It has been previously noted by immunohistochemistry that the BCL-XL protein, an important determinant of apoptosis resistance, appears to be highly expressed in EA tumors. Currently there is a no in vitro data evaluating BCL-XL in EA. We sought to determine BCL-XL protein levels in EA cell lines and assess the effectiveness of bcl-xl RNA interference (RNAi) and subsequent protein down-regulation in this tumor type. Methods: Western blot analysis evaluated baseline BCL-XL protein levels in human EA cell lines BE3 and SK5. Preliminary studies of three 21 nucleotide, double-stranded BCL-XL specific siRNA identified the most potent construct. The cells were then transfected with this construct utilizing oligofectamine, with scrambled siRNA as a control. After 72 hours, protein and transcriptional down regulation were evaluated by Western blot and real-time PCR. Flow cytometry was used to determine apoptotic cell death (subG0/G1 phase). Results were compared using the student’s t-test. Results: Greater than 90% short interfering RNA construct (siRNA) transfection was established with a fluorescent labeled system. Both cell lines exhibited baseline BCL-XL over-expression. After treatment, a 60% reduction in mRNA levels and a corresponding 50% decrease in protein expression was noted. The percentage of apoptotic cells was as follows: 3.16% (BE3 control) vs. 27.02% (BE3 treated) and 8.02% (SK5 control) vs. 32.96% (SK5 treated) (p < .03 and p < .001). Conclusion: We have demonstrated increased expression of BCL-XL in cultured EA cells. The anti-apoptotic function of this gene can be disrupted in specific fashion by RNA interference. These findings establish BCL-XL as a valid target in this aggressive malignancy and encourage further studies involving BCL-XL expression inhibition by RNAi.
    Journal of Surgical Research - J SURG RES. 01/2003; 114(2):278-278.