[HSV-TK gene therapy of lung adenocarcinoma xenografts using a hypoxia/radiation dual-sensitive promoter].
OBJECTIVE
Radio-genetic therapy is a novel strategy for cancer treatment; however, a limited success was shown due to lower sensitivity of tumor cells to radiation under hypoxia, which is a unique feature for solid tumors. In order to improve the efficacy of radiogenetic therapy for lung cancer, a hypoxia/radiation dual-sensitive promoter was constructed to enhance the expression of HSV-TK in transfected cells exposed to radiation under hypoxia.


METHODS
The chimeric promoter HRE-Egr was generated by insertion of hypoxia response elements (HREs) upstream of the Egr-1 (early growth response gene-1) promoter. HSV-TK expression vector was constructed by cloning HRE-Egr promoter upstream of HSV-TK gene, which was transfected into A549 cells via liposome. The expression of HSV-TK in transfected cells exposed to irradiation (6 Gy) and/or hypoxia (1% O2) were analyzed by Northern blot, and the relative survival rate of cells in presence of prodrug ganciclovir (GCV) was tested using MTT method. To examine the efficacy of this HRE-Egr-TK gene therapy in vivo, the A549 adenocarcinoma xenografts were planted in BALB/c nude mice. The tumor volumes and the suppression rates were assayed in nude mice bearing xenografts infected with plasmids and exposed to radiation.


RESULTS
HSV-TK gene expression in transfected cells was markedly increased in both radiation (227 U) and hypoxia (94 U) groups compared with control group (21 U). The HSV-TK expression (769 U) in transfected cells exposed to radiation under hypoxia is much more higher than the former groups. The survival rate of transfected cells exposed to radiation under hypoxia in the presence of GCV was obviously decreased with comparison of cells under normoxia (7.2%+/-1.8 % vs 32.7%+/-4.6 %). HRE-Egr promoter transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n=10), the tumor suppression rates was 91.2%.


CONCLUSIONS
The hypoxia/radiation dual-sensitive promoter HRE-Egr can enhance the HSV-TK gene expression in solid tumors under hypoxia. Enhanced tumor suppression effect was observed in A549 xenografts infected with HRE-Egr promoter exposed to radiation.
Wang
Wei-Dong
WD
Cancer Center of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing,400037, P.R.China. lirongwd@sohu.com
Chen
Zheng-Tang
ZT
Li
De-Zhi
DZ
Duan
Yu-Zhong
YZ
Wang
Zhi-Xin
ZX
Cao
Zheng-Huai
ZH
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D000818:Animals; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D003037:Cobalt Radioisotopes; D004268:DNA-Binding Proteins / Q000235:genetics*; D051766:Early Growth Response Protein 1; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011401:Promoter Regions, Genetic / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020218:Response Elements / Q000235:genetics*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D003037:Cobalt Radioisotopes; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D013937:Thymidine Kinase; D015774:Ganciclovir