Optimization of radiation controlled gene expression by adenoviral vectors in vitro.
The radiation-inducible EGR-1-promoter has been used in different gene therapy approaches in order to enhance and locally restrict therapeutic efficacy. The aim of this study was to reduce nonspecific gene expression in the absence of irradiation (IR) in an adenoviral vector. Rat rhabdomyosarcoma R1H tumor cells were infected with adenoviral vectors expressing either EGFP or HSV-TK under control of the murine EGR-1 promoter/enhancer. Cells were irradiated at 0-6 Gy. Gene expression was determined by FACS-analysis (EGFP), or crystal violet staining (HSV-TK). The bovine growth hormone polyadenylation signal (BGH pA) was used as insulating sequence and was introduced upstream or upstream and downstream of the expression cassette. Infected R1H cells displayed IR dose-dependent EGFP expression. Cells treated with IR, AdEGR.TK and ganciclovir displayed a survival of 17.3% (6 Gy). However, significant gene expression was observed in the absence of IR with EGR.TK and EGR.EGFP constructs. Introduction of BGHpA upstream or upstream and downstream of expression cassette resulted in decreased nonspecific cytotoxicity by a factor of 1.6-2.3 with minor influence on the induced level of cytotoxicity. Introduction of insulating sequences in adenoviral vectors might allow tighter temporospatial control of gene expression by the radiation-inducible EGR-1 promoter.
Anton
Martina
M
Institut für Experimentelle Onkologie & Therapieforschung, München, Germany. m.anton@lrz.tu-muenchen.de
Gomaa
Iman E O
IE
von Lukowicz
Tobias
T
Molls
Michael
M
Gansbacher
Bernd
B
Würschmidt
Florian
F
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D003131:Combined Modality Therapy; D004268:DNA-Binding Proteins / Q000235:genetics*; D051766:Early Growth Response Protein 1; D005720:Gamma Rays; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000627:therapeutic use*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D013006:Growth Hormone / Q000235:genetics; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D066298:In Vitro Techniques; D011061:Poly A / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000528:radiation effects*; D051381:Rats; D012208:Rhabdomyosarcoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D014161:Transduction, Genetic; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011061:Poly A; D013006:Growth Hormone; D013937:Thymidine Kinase