title: Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro.
abstract: OBJECTIVE
  To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma.
  
  
  METHODS
  Human p16 cDNA was ligated to the downstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells.
  
  
  RESULTS
  Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16 expression in cells transfected with pcDNA3.1-Egr.1p-p16 induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak (87.00 ng/L), and was significantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone.
  
  
  CONCLUSIONS
  X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells. The detection of dose and time provides an experimental basis for in vivo study in future.
authors: Department of Oncology, Second Hospital, Medical college of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China. mahongbingxa@126.com
authors: Hong-Bing
authors: HB
authors: Ma
authors: Xi-Jing
authors: XJ
authors: Wang
authors: Zheng-Li
authors: ZL
authors: Di
authors: Hui
authors: H
authors: Xia
authors: Zheng
authors: Z
authors: Li
authors: Jie
authors: J
authors: Liu
authors: Jie
authors: J
authors: Ma
authors: Hua-Fen
authors: HF
authors: Kang
authors: Cong-Mei
authors: CM
authors: Wu
authors: Ming-Hua
authors: MH
authors: Bai
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger
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