title: [Effects of human telomerase reverse transcriptase promoter and survivin promoter in targeted tumor gene therapy].
abstract: OBJECTIVE
  To investigate the effects of human telomerase reverse transcriptase (hTERT) promoter and survivin promoter in tumor-specific gene therapy.
  
  
  METHODS
  hTERT promoter and survivin promoter were obtained by PCR using Jurkat genomic DNA. Recombinant adeno-associated virus (AAV) vectors containing exogenous TRAIL gene and hTERT promoter or survivin promoter were constructed and designated as rAAV-hTERT-TRAIL (h/TRAIL) or rAAV-survivin-TRAIL (s/TRAIL). rAAV particles were obtained after packing and purification and the virus titer was calculated by real-time PCR. Human hepatocellular carcinoma (HCC) cells of the lines SMMC-7721, BEL-7402, HepG2, and Hep3B, and primary human hepatocytes (PHHs) were transfected with h/TRAIL or s/TRAIL. Flow cytometry was used to detect the expression of the reporter gene enhanced green fluorescent protein (EGFP), so as to examine the activity of the two promoters. MTT method was used to detect the activity of the cells. Fifteen BalB/C mice underwent subcutaneous injection of SMMC-7721 cells so as to establish tumor models and then were randomly divided into 3 groups to undergo intra-tumor injection of h/TRAIL, s/TRAIL, or PBS. The growth of tumor was observed for 5 weeks, and then peripheral blood samples were collected to examine the serum AST and ALT levels. TUNEL was used to detect the apoptosis if the tumor cells.
  
  
  RESULTS
  All the SMMC-7721, BEL-7402, HepG2, and Hep3B cells driven by both h/TRAIL and s/TRAIL showed EGFP expression, however, no fluorescence could be seen in the PHHs transfected with h/TRAIL and s/ TRAIL. MTT method showed that 72 hours after the transfection of h/TRAIL and s/TRAIL the survival rates of the SMMC-7721, BEL-7402, and HepG2 cells all decreased, however, the survival rate of the Hep3B cells and PHHs did not changed significantly. The size of the subcutaneous tumor of the mice of the h/ TRAIL group was 625 mm3, significantly smaller than that of the PBS group (1500 mm3, P <0.05), however, the tumor size of the s/TRAIL group was 1117 mm3, not significantly different from that of the PBS group (P >0.05). The AST and ALT levels of all mice did not change significantly 5 weeks after the intratumor injection.
  
  
  CONCLUSIONS
  Tumor-specific promoters are promising candidates in targeted tumor gene therapy.
authors.affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hong
authors.initials: H
authors.lastname: Ma
authors.forename: Shi-Lian
authors.initials: SL
authors.lastname: Liu
authors.forename: Yan-Xin
authors.initials: YX
authors.lastname: Liu
authors.forename: De-Xian
authors.initials: DX
authors.lastname: Zheng
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000628:therapy; D002460:Cell Line; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D019169:Jurkat Cells; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000077022:Survivin; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000235:genetics*; D014162:Transfection; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D000077022:Survivin; D053221:TNF-Related Apoptosis-Inducing Ligand; D049452:Green Fluorescent Proteins; C509186:TERT protein, human; D019098:Telomerase