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Etoposide enhances transgene expression mediated by recombinant replication defective adenovirus in tumor cell lines
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Abstract:
To investigate the effects of chemotherapeutic agent Etoposide on transgene expression mediated by recombinant replication-defective adenovirus in tumor cell lines. Methods：Cultured tumor cells, including NCI-H446, NCI-H460, A549, SMMC-7721, SGC7901, SKBR-3, and BTT were infected by Ad-CMV-EGFP alone （MOI being 1 and 10）or in combination with Etoposide at different final concentrations (0.2, 2, 20, 40, 80, 100 and 200 μg/ml). GFP positive cell rates and the mean intensities of GFP fluorescence in tumor cells were detected by Fluorescence Activated Cell Sorting (FACS) after cultured with different strategies. EGFP protein expression in tumor cells was analyzed by Western blotting. Quantitative analysis of mRNA and DNA copies of EGFP in tumor cells were performed by RT-PCR and real-time PCR. Results: FACS results indicated that Etoposide efficiently enhanced the mean intensities of EGFP fluorescence to different degrees in all 7 cell lines but had no evident effects on the EGFP positive rate. Twenty-four hours after cultured at the presence of 10 MOI Ad5-CMV-EGFP and 40 μg/ml Etoposide, the mean intensities of EGFP fluorescence in NCI-H446, NCI-H460, A549, SMMC-7721, SGC7901, SKBR-3, and BTT cells were respectively 3.3, 3.5, 3.1, 6.2, 70, 5.4, and 3.4 folds that cultured with 10 MOI Ad5-CMV-EGFP alone. Western blotting showed that EGFP protein expression in cells co cultured with Ad5-CMV-EGFP and Etoposide was 2-5 times that cultured with 10 MOI Ad5-CMV-EGFP alone. EGFP mRNA expression had a similar tendency as EGFP protein, but the copies of EGFP DNA had no evident changes. Conclusion: Etoposide can enhance transgene expression mediated by recombination replication defective adenovirus in several tumor cell lines, which may play a role at the transcriptional level.
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Clc Number:
R730.3
