| Objective: To screen compounds that can induce readthrough of nonsense mutations and develop promising drug candidates for future treatment of tumour and inherited diseases caused by nonsense mutations.Methods: Cells which can stably express luciferase containing nonsense mutations were treated by 10 ?mol/L of each candidate compound obtained from Shanghai Institute of Biochemistry and Cell Biology,and then the luciferase activities were tested to screen positive compounds.The activities of these positive compounds were further confirmed using the cell model above and protein truncation test-ELISA(PTT-ELISA).Finally,the positive readthrough agents were used to treat HT-29 cells containing APC nonsense mutation.Then the expression levels of APC and downstream proteins ?-catenin and c-myc were determined;HT-29 cells motility was tested by Wound-Healing assay and Transwell assay;cell growth was estimated by MTT assay;the cytotoxicity was analyzed by flow cytometry and TUNEL assay.Results: Blasticidin(BSD)was found to have readthrough activity in luciferase cell model.Further results of cell model and PTT-ELISA confirm that BSD can significantly increase the m RNA level and activity of luciferase and shows better readthrough activity than G418.In HT-29 cells,BSD can also obviously induce readthrough of APC gene containing nonsense mutations and subsequently downregulate the protein expression level of ?-catenin and c-myc significantly.The efficiency of BSD is higher than G418 in HT-29 cells.Moreover,the migration and growth of HT-29 cells are both suppressed.The data of flow cytometry and TUNEL assay illustrate that the cytotoxicity of BSD is little less than G418.Conclusion: BSD was found to have the ability to induce readthrough of nonsense mutation using luciferase cell model and PTT-ELISA,and significantly alleviate the malignant phenotype of tumor cells.BSD may be a promising candidate for further study. |
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