Published online Feb 15, 1999. doi: 10.3748/wjg.v5.i1.18
Revised: September 14, 1998
Accepted: October 6, 1998
Published online: February 15, 1999
AIM 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-promo-ters, with recombinant DNA technology of mole-cular biology. So, it is easy to generate digoxi-genin (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 im-munohistochemistry, 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%), increased 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 (18.9 × 104 to 4.8 × 105 and 4.8 × 105), 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 anti-sense RNA to cyclin D1 can reverse the trans-formed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.