title: Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes.
abstract: OBJECTIVE
  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 x 10(6) recombinants. Thirty-six positive clones were obtained from 6 x 10(5) 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhong
authors.forename: Yu-Liang
authors.initials: YL
authors.lastname: Ran
authors.forename: Jin-Ning
authors.initials: JN
authors.lastname: Lou
authors.forename: Dong
authors.initials: D
authors.lastname: Hu
authors.forename: Long
authors.initials: L
authors.lastname: Yu
authors.forename: Yu-Shan
authors.initials: YS
authors.lastname: Zhang
authors.forename: Zhuan
authors.initials: Z
authors.lastname: Zhou
authors.forename: Zhi-Hua
authors.initials: ZH
authors.lastname: Yang
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis / Q000235:genetics / Q000473:pathology / Q000628:therapy; D002453:Cell Cycle / Q000502:physiology; D049109:Cell Proliferation; D004730:Endothelium, Vascular / Q000473:pathology* / Q000502:physiology*; D005260:Female; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000473:pathology / Q000628:therapy; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA