Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro.
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.
Ma
Hong-Bing
HB
Department of Oncology, Second Hospital, Medical college of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China. mahongbingxa@126.com
Wang
Xi-Jing
XJ
Di
Zheng-Li
ZL
Xia
Hui
H
Li
Zheng
Z
Liu
Jie
J
Ma
Jie
J
Kang
Hua-Fen
HF
Wu
Cong-Mei
CM
Bai
Ming-Hua
MH
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
D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger
Acta Oncol. 2001;40(6):735-8
11765068
Gene Ther. 2000 Jul;7(13):1121-5
10918478
Cancer Res. 1994 Aug 15;54(16):4266-9
8044769
Biochem Biophys Res Commun. 2003 May 2;304(2):241-7
12711305
Ann Surg. 2002 Jan;235(1):51-9
11753042
Nucleic Acids Res. 1988 Sep 26;16(18):8835-46
3140220
Anticancer Res. 2003 May-Jun;23(3B):2723-8
12894565
Yonsei Med J. 2005 Oct 31;46(5):597-613
16259056
Zhonghua Yi Xue Za Zhi. 2004 Feb 2;84(3):214-8
15059537
Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2419-22
8384722
Zhonghua Nei Ke Za Zhi. 2005 Jul;44(7):509-13
16080841
Am J Surg. 2003 Sep;186(3):279-86
12946833
J Natl Cancer Inst. 1995 Feb 1;87(3):175-82
7707404
CA Cancer J Clin. 2000 Jan-Feb;50(1):7-33
10735013
J Cell Physiol. 2002 Dec;193(3):287-92
12384981
Int J Cancer. 1999 Dec 10;83(6):870-873
10602059
Pancreas. 2005 Jul;31(1):13-22
15968242
Acta Oncol. 2006;45(2):124-35
16546857
World J Gastroenterol. 2004 Oct 15;10(20):2927-30
15378766
Pancreas. 2007 Jan;34(1):92-5
17198189
Hum Gene Ther. 1998 Jul 1;9(10):1409-17
9681412