title: [Effect of recombinant adenovirus Ad-DT-A in targeted therapy for malignant cancer cell lines with loss of IGF2 imprinting].
abstract: OBJECTIVE
  To explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.
  
  
  METHODS
  The mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.
  
  
  RESULTS
  The expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.
  
  
  CONCLUSIONS
  The recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.
authors.affiliation: Nanjing Medical University, Nanjing, China.
authors.forename: Yu-qin
authors.initials: YQ
authors.lastname: Pan
authors.forename: Bang-shun
authors.initials: BS
authors.lastname: He
authors.forename: Chan
authors.initials: C
authors.lastname: Zhu
authors.forename: Li-li
authors.initials: LL
authors.lastname: Qu
authors.forename: Xiong-fei
authors.initials: XF
authors.lastname: Xu
authors.forename: Shu-kui
authors.initials: SK
authors.lastname: Wang
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D004167:Diphtheria Toxin / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D018392:Genomic Imprinting; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D061986:MCF-7 Cells; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D011897:Random Allocation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D004167:Diphtheria Toxin; D010446:Peptide Fragments; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C029795:diphtheria toxin fragment A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007335:Insulin-Like Growth Factor II