title: [Anti-tumor effect of suicide gene therapy using chimeric promoter plus radiotherapy on cancer cell lines].
abstract: OBJECTIVE
  To explore the synergistic anti-tumor effect of radiotherapy and horseradish peroxidase/prodrug indole-3-acetic acid (HRP/IAA) gene therapy system using chimeric hTERT promoter responsive to ionizing radiation.
  
  
  METHODS
  The synthetic hTERT promoters containing four tandem-repeat copies of radio-inducible CArG elements, and the chimeric promoter containing cytomegalovirus (CMV) early promoter were both constructed. The activities of the chimeric promoters in cancer cell lines (HeLa, A549, and MHCC97) and normal cell line (MRC-5) were detected using luciferase reporter gene expression analysis after a (60)Co γ-irradiation treatment at a series of doses(a single dose of 0 to 10 Gy). The anti-tumor effect of combining irradiation with HRP/IAA gene-directed enzyme prodrug therapy system controlled by the chimeric promoter was tested by colony formation assay, cell counting and apoptosis analysis.
  
  
  RESULTS
  The chimeric promoters were ineffective in normal human cells, even after irradiation, but the expression of luciferase gene in tumor cells was significantly higher. The activity of the chimeric promoter in MRC-5 cells was 22.3%, 12.9% and 13.6% of that in HeLa, A549 and MHCC97 cells, respectively. After irradiation, the ratios were 11.7%, 8.7% and 8.8%, respectively. Furthermore, the chimeric promoters could successfully induce the expression of luciferase gene following different doses of radiation, with maximal inducible activity seen after 6 Gy irradiation. The chimeric promoter containing four tandem-repeat copies of radio-inducible CArG elements and CMV early promoter showed the highest activity with 6 Gy irradiation. The relative luciferase activities in HeLa, A549 and MHCC97 cells were 1.7 ± 0.2, 2.3 ± 0.2 and 2.3 ± 0.1, respectively. The chimeric promoter mediated suicide gene therapy system could increase radio-sensitivity in different cancer cells. Compared with the control system, it plus irradiation showed stronger cell proliferation inhibition, 67.3% vs. 26.1% in HeLa, 69.0% vs. 28.3% in A549, 64.6% vs. 20.8% in MHCC97 cells, and also higher apoptosis-inducing effect, 39.6% vs. 14.2% in HeLa, 33.0% vs. 12.4% in A549, and 33.2% vs. 14.2% in MHCC97 cells.
  
  
  CONCLUSIONS
  Chimeric promoter containing hTERT promoter, CArG elements and CMV promoter preserve the tumor-specificity in telomerase-positive tumor cells, and irradiation-responsive to low dose of radiation. The suicide gene therapy using this promoter plus radiotherapy show a strong anti-tumor effect in vitro. It is expected to have a good potential for future application in gene radiotherapy.
authors.affiliation: Department of Radiation and Medical Oncology, Zhongnan Hospital, Hubei Key Laboratory of Tumor Biological Behaviors, Wuhan University, Wuhan 430071, China.
authors.forename: Wen-jie
authors.initials: WJ
authors.lastname: Sun
authors.forename: Jie
authors.initials: J
authors.lastname: Xiong
authors.forename: Wei-feng
authors.initials: WF
authors.lastname: Wang
authors.forename: Zheng-kai
authors.initials: ZK
authors.lastname: Liao
authors.forename: Fu-xiang
authors.initials: FX
authors.lastname: Zhou
authors.forename: Yun-feng
authors.initials: YF
authors.lastname: Zhou
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003131:Combined Modality Therapy; D003587:Cytomegalovirus / Q000235:genetics; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006735:Horseradish Peroxidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007210:Indoleacetic Acids / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011355:Prodrugs; D011401:Promoter Regions, Genetic / Q000528:radiation effects; D011878:Radiotherapy / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D007210:Indoleacetic Acids; D011355:Prodrugs; D011994:Recombinant Proteins; C030737:indoleacetic acid; D006735:Horseradish Peroxidase; D008156:Luciferases; C509186:TERT protein, human; D019098:Telomerase