Original Articles
Copyright ©The Author(s) 1999. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 15, 1999; 5(1): 18-21
Published online Feb 15, 1999. doi: 10.3748/wjg.v5.i1.18
Antisense to cyclin D1 reverses the transformed phenotype of human gastric cancer cells
Bing Chen, Xue-Yong Zhang, Yu-Jing Zhang, Ping Zhou, Yan Gu, Dai-Ming Fan
Bing Chen, Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Xue-Yong Zhang, Dai-Ming Fan, Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medi-cal University, Xi’an 710033, Shaanxi Province, China
Yu-Jing Zhang, Ping Zhou, Columbia-Presbyterian Cancer Center, Columbia University, New York 10032, USA
Yan Gu, Department of Gastroenterology, Jiuxianqiao Hospital, Beijing 100016, China
Bing Chen, male, born on 1960-11-27 in Nanjing City, China, asso-ciate professor of internal medicine, Ph.D. & MD in gastroentero-logy, post doctor of Third Military Medical University, China, having 24 papers published.
Author contributions: All authors contributed equally to the work.
Supported by the National Outstanding Youth Science Foundation of China, No.3952520.
Correspondence to: Dr Bing Chen, Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. bingchen@cq.col.com.cn
Telephone: +86-23-68754259
Received: July 30, 1998
Revised: September 14, 1998
Accepted: October 6, 1998
Published online: February 15, 1999
Abstract

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.

Keywords: stomach neoplasms; cyclin D1; RNA, antisense; gene therapy