DOI:
Authors:
Bing Chen
Abstract:
To further investigate the effect of cyclin D1 on the bio logic behavior of cancer cells and its potential role in gene therapy of tumor. METHODS A cyclin D1 subcloning plasmid termed BKSD1 was const ructed by subcloning the human cyclin D1 cDNA into Bluescript-KS, a plasmid vec tor with a pair of T7 and T3-promoters, with recombinant DNA technology of molecular biology. So, it is easy to generate digoxigenin (DIG)-labeled RNA probes of antisense and sense to cyclin D1 using RKSD1 as a template vector. PDORD1AS, an eukaryotic expression vector containing the full-length human cyclin D1 cDNA in its antisense orientation cloned into the retroviral vector pDOR-neo, was succ essfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine-mediate d introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpr ession of antisense RNA to cyclin D1, was obtained by selection in G418. The sub line, control subline transfected pDOR-neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morpho logy and cell biology. RESULTS Compared with control cell lines, SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%), in-creased ce ll size and cytoplasm to nucleus ratio, increased doubling time (42.2 h to 26.8 h and 26.4 h), decreased saturation density (18.910 4 to 4.810 5 and 4.810 5 ), increased percentage of cells in the G1/G0 phase (80.9%-64.6% and 63.8%), reacquired serum dependence, and a loss of tumorigenici ty in nude mice (0/4 to 4/4 and 4/4). CONCLUSION Stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.