Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals

AIM:To establish a luciferase reporter cell line that respondsdioxin-like chemicals (DLCs) and on this basis to evaluateits characteristics and application in the determination ofDLCs.METHODS:A recombinant luciferase reporter plasmid wasconstructed by inserting dioxin-responsive element (DREs)and MMTV promoter segments into the pGL3-promoterplasmid immediately upstream of the luciferase gene,whichwas structurally demonstrated by fragment mapping analysisin gel electrophoresis and transfected into the humanhepatoma cell line HepG2,both transiently and stably,toidentify the inducible expression of luciferase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).The time course,responsive period,sensitivity,structure-inducibility and dose-effect relationships of inducible luciferase expression to DLCswas dynamically observed in HepG2cells stably transfectedby the recombinant vector (HepG2-Luc) and compared withthat assayed by ethoxyresorufin-O-deethylase (EROD) innon-transfected HepG2cells (HepG2-wt).RESULTS:The inducible luciferase expression of HepG2-Luc cells was noted in a time-,dose-,and AhR-dependentmanner,which peaked at 4 h and then decreased to a stablelevel at 14 h after TCDD treatment.The responsiveness ofHepG2-Luc cells to TCDD induction was decreased withculture time and became undetectable at 10thmonth ofHepG2-Luc cell formation.The fact that luciferase activityinduced by 3,3’,4,4’-PCB in HepG2-Luc cells was muchless than that induced by TCDD suggests a structure-inducibility relationship existing among DLCs.Within theconcentrations from 3.5×10-12to 5×10-9mol/L,significantcorrelations between TCDD doses and EROD activities wereobserved in both HepG2-luc and HepG2-wt cells.The correlationbetween TCDD doses from 1.1×10-13to 1×10-8mol/L andluciferase activities was also found to be significant in HepG2-luc cells (r=0.997,P<0.001),but not in their HepG2-wtcounterparts.For the comparison of the enzyme responsivenessbetween cell lines to TCDD,the luciferase sensitivity andreproducibility in HepG2-luc cells were both better than thatof EROD in HepG2-wt cells,the former was at 1.1×10-13mol/L and 3.5×10-12mol/L,and the coefficients of variation(CV) of the latter was 15-30% and 22-38%,respectively.CONCLUSION:The luciferase expression of HepG2-luc cellsestablished in the present study could sensitively respondto the DLCs stimulation and might be a prospective tool forthe determination of DLCs.