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Development of Recombinant Adenovirus Encoding Antisense Cyclin D1 and Its Effect on Human Lung Adenocarcinoma Cells
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Abstract:
To construct the recombinant adenovirus encoding antisense cyclin D1 fragment, and to investigate its effect on tumor cell proliferation and apoptosis. Methods: The shuttle plasmid encoding antisense cyclin D1 was constructed by cloning cyclin D1 cDNA fragment in the reverse direction into the pAdCMV. Then the plasmid pJM17 and the shuttle plasmid were co-transferred into 293 cells with liposome for homologous recombination to acquire recombinant adenovirus. The cell cycle, apoptosis, and the expression of the related genes of the in vitro transferred human adenocarcinoma cell lines GLC-82 and SPC-A-1, as well as the human embryonic diploid lung cell line 2BS were studied. The effect on colony formation was also studied. Results: The recombinant adenovirus encoding antisense cyclin D1 fragment Ad-AS cyclin D1 was obtained with the titer of 1×10 10 pfu/ml. RT-PCR assay showed that the expression of cyclin D1 of GLC-82 and SPC-A-1 was reduced after Ad-AS cyclin D1 infection. Simultaneously, obvious G1 arrest and apoptosis, bcl-2 and bax gene expression changes were observed in the infected tumor cells. The infected 2BS cells showed no such changes. The colony formation ability in vitro was also reduced.]Conclusion: The reduced expression of cyclin D1 gene could induce human lung adenocarcinoma cells specific G1 arrest and apoptosis, while has no effect on normal cell.
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