Antisense to cyclin D1 reverses the transformed phenotype of human gastric cancer cells.
AIM:To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor.METHODS:A cyclin D1 subcloning plasmid termed BKSD1 was constructed by subcloning the human cyclin D1 cDNA into Bluescript-KS, a plasmid vector 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 successfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine-mediated introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpression of antisense RNA to cyclin D1, was obtained by selection in G418. The subline, control subline transfected pDOR-neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morphology and cell biology.RESULTS:Compared with control cell lines, SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%), increased cell size and cytoplasm to nucleus ratio, increased doubling time (42.2h to 26.8h and 26.4h), decreased saturation density (18.9X10(4) to 4.8X10(5) and 4.8X10(5)), increased percentage of cells in the G(1)/G(0) phase (80.9%-64.6% and 63.8%), reacquired serum dependence, and a loss of tumorigenicity 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.
Chen
Bing
B
Zhang
Xue-Yong
XY
Zhang
Yu-Jing
YJ
Zhou
Ping
P
Gu
Yan
Y
Fan
Dai-Ming
DM