title: Modification of the hTERT promoter by heat shock elements enhances the efficiency and specificity of cancer targeted gene therapy.
abstract: One of the current challenges facing cancer gene therapy is the tumour-specific targeting of therapeutic genes. Effective targeting in gene therapy requires accurate spatial and temporal control of gene expression. To develop a sufficient and accurate tumour-targeting method for cancer gene therapy, we have investigated the use of hyperthermia to control the expression of a transgene under the control of the human telomerase reverse transcriptase (hTERT) promoter and eight heat shock elements (8HSEs).
  
  
  
  Luciferase reporters were constructed by inserting eight HSEs and the hTERT promoter (8HSEs-hTERTp) upstream of the pGL4.20 vector luciferase gene. The luciferase activity of the hTERT promoter and 8HSEs-hTERT promoter were then compared in the presence and absence of heat. The differences in luciferase activity were analysed using dual luciferase assays in SW480 (high hTERT expression), MKN28 and MRC-5 cells (low hTERT expression). The luciferase activity of the Hsp70B promoter was also compared to the 8HSEs-hTERT promoter in the above listed cell lines. Lentiviral vector and heat-induced expression of EGFP expression under the control of the 8HSEs-hTERT promoter in cultured cells and mouse tumour xenografts was measured by reverse transcription polymerase (RT-PCR), Western blot and immunofluorescence assays.
  
  
  
  hTERT promoter activity was higher in SW480 cells than in MKN28 or MRC-5 cells. At 43 °C, the luciferase activity of the 8HSEs-hTERT promoter was significantly increased in SW480 cells, but not in MKN28 or MRC-5 cells. Importantly, the differences in luciferase activity were much more obvious in both high (SW480) and low (MKN28 and MRC-5) hTERT expressing cells when the activity of the 8HSEs-hTERT promoter was compared to the Hsp70B promoter. Moreover, under the control of 8HSEs-hTERT promoter in vitro and in vivo, EGFP expression was obviously increased by heat treatment in SW480 cells but not in MKN28 or MRC-5 cells, nor was expression increased under normal temperature conditions.
  
  
  
  The hTERT promoter is a potentially powerful tumour-specific promoter and gene therapy tool for cancer treatment. Incorporating heat-inducible therapeutic elements (8HSEs) into the hTERT promoter may enhance the efficiency and specificity of cancer targeting gene therapy under hyperthermic clinical conditions.
authors.affiliation: a Department of General Surgery , First Affiliated Hospital of Xi'an Jiaotong University , Xi'an , Shaanxi .
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Wang
authors.affiliation: a Department of General Surgery , First Affiliated Hospital of Xi'an Jiaotong University , Xi'an , Shaanxi .
authors.forename: PeiHua
authors.initials: P
authors.lastname: Zhou
authors.affiliation: a Department of General Surgery , First Affiliated Hospital of Xi'an Jiaotong University , Xi'an , Shaanxi .
authors.forename: XueJun
authors.initials: X
authors.lastname: Sun
authors.affiliation: a Department of General Surgery , First Affiliated Hospital of Xi'an Jiaotong University , Xi'an , Shaanxi .
authors.forename: GuangBing
authors.initials: G
authors.lastname: Wei
authors.affiliation: b Department of General Surgery , Second Affiliated Hospital of Xi'an Jiaotong University , Xi'an , Shaanxi .
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: c Shaanxi Provincial People's Hospital , Xi'an , Shaanxi , and.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: c Shaanxi Provincial People's Hospital , Xi'an , Shaanxi , and.
authors.forename: JianFeng
authors.initials: J
authors.lastname: Yao
authors.affiliation: d First People's Hospital of XianYang City , XianYang , Shaanxi , China.
authors.forename: PengBo
authors.initials: P
authors.lastname: Jia
authors.affiliation: a Department of General Surgery , First Affiliated Hospital of Xi'an Jiaotong University , Xi'an , Shaanxi .
authors.forename: JianBao
authors.initials: J
authors.lastname: Zheng
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D005260:Female; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D018869:Heat-Shock Response; D006358:Hot Temperature; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008807:Mice, Inbred BALB C; D009369:Neoplasms / Q000235:genetics / Q000628:therapy*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D019098:Telomerase / Q000235:genetics*; D023041:Xenograft Model Antitumor Assays
chemical_list: D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; C509186:TERT protein, human; D019098:Telomerase
keywords: Gene therapy; hTERT promoter; heat shock element (HSE); heat-inducible gene expression; hyperthermia