Liver Cancer
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 15, 2004; 10(10): 1402-1408
Published online May 15, 2004. doi: 10.3748/wjg.v10.i10.1402
Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes
Xing Zhong, Yu-Liang Ran, Jin-Ning Lou, Dong Hu, Long Yu, Yu-Shan Zhang, Zhuan Zhou, Zhi-Hua Yang
Xing Zhong, Yu-Liang Ran, Dong Hu, Long Yu, Yu-Shan Zhang, Zhuan Zhou, Zhi-Hua Yang, Department of Cell and Molecular Biology, Cancer Institute (Hospital), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing , 100021, China
Jin-Ning Lou, Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, China
Author contributions: All authors contributed equally to the work.
Supported by the National 863 Program, No.2001AA221251 and the National Natural Science Foundation of China, No.30230150
Correspondence to: Professor Zhi-Hua Yang, Department of Cell and Molecular Laboratory, Cancer Institute (Hospital), Chinese Academy of Medical Sciences and Peking Union Medical College, Panjiayuan, Chaoyang Qu PO Box, Beijing, 100021, China.
Telephone: +86-10-87771740 Fax: +86-10-67713359
Received: October 15, 2003
Revised: November 25, 2003
Accepted: December 2, 2003
Published online: May 15, 2004

AIM: To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs) cDNA expression library, so as to find some new possible targets for the diagnosis and therapy of liver tumor.

METHODS: HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample, and were cultured and proliferated in vitro. A cDNA expression library was constructed with the mRNA extracted from HLCVECs. Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs, and were then tested for their specificity against HLCVECs and angiogenic effects in vitro, such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells, using immunocytochemistry, immunofluorescence, cell cycle analysis and MTT assays, etc. The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX). The positive clones were sequenced and analyzed by bio-informatics.

RESULTS: The primary cDNA library consisted of 2 × 106 recombinants. Thirty-six positive clones were obtained from 6 × 105 independent clones by immunoscreening. Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes. Among them, 3 were new genes previously unreported, 2 of which were hypothetical genes. The other 15 were already known ones. Series analysis of gene expression (SAGE) database showed that ERP70, GRP58, GAPDH, SSB, S100A6, BMP-6, DVS27, HSP70 and NAC alpha in these genes were associated with endothelium and angiogenesis, but their effects on HLCVECs were still unclear. GAPDH, S100A6, BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries.

CONCLUSION: By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs, the functional genes associated with tumor endothelium or angiogenesis were identified. The modified SEREX, xenogeneic functional serum screening, was demonstrated to be effective for isolation and identification of antigen genes of tumor endothelium, and also for other tumor cell antigen genes. These antigen genes obtained in this study could be a valuable resource for basic and clinical studies of tumor angiogenesis, thus facilitating the development of anti- angiogenesis targeting therapy of tumors.

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