Expression and function of DMT1 without IRE in C6 cells mediated by recombinant adenovirus

Abstract

Divalent metal transporter 1 (DMT1) is a ferrous iron import protein. The improper expression of DMT1 is involved in neurodegenerative diseases. In the present study, we constructed a recombinant adenovirus containing the gene of DMT1 without the iron response element (DMT1-IRE) and investigated its expression and function in the C6 glioma cell line. The DMT1-IRE gene, obtained by RT-PCR, was cloned into the shuttle plasmid pAdTrack-CMV containing green fluorescent protein (GFP) reporter gene. Linearized plasmid pAdTrack-CMV-DMT1-IRE was subsequently co-transformed into Escherichia coli (E. coli) BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1 after digestion with Pme I. Pac I-digested pAdEasy1-DMT1-IRE was then transfected into E1-transformed human embryonic kidney cells (HEK293 cells) , in which recombinant adenoviruses were generated within 7 to 10 days. The results demonstrated that we obtained the DMT1-IRE gene. pAdEasy1-DMT1-IRE yielded a large fragment, plus a smaller fragment of 4.5 kb after digestion with PacI. PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE, indicating the successful construction of recombinant adenovirus plasmid containing DMT1-IRE. GFP fluorescence further confirmed the generation of adenovirus. AdDMT1-IRE could efficiently infect C6 glioma cells. And cell viability decreased in Ad-DMT1-IRE infected cells after iron overload compared to the control. These results suggest that the over expressed DMT1-IRE can aggravate the iron induced cell death due to its iron influx function.

Keywords

divalent metal transporter 1 / recombinant adenovirus / homologous recombination / iron

References

[1] KarlinK D.Metalloenzymes, structural motifs, and inorganic models.Science,1993, 261(5122):701-708

[2] AisenP.Iron metabolism: An evolutionary perspective. In:Brock J H, Halliday J W, Powell L W,eds.Iron metabolism in health and disease. London: Saunders WB,1994,1-30

[3] JellingerK,PaulusW,Grundke-IqbalI,RiedererP,YoudimM B.Brain iron and ferritin in Parkinson’s and Alzheimer’s diseases.J Neural Transm Park Dis Dement Sect,1990,2(4): 327-340

[4] GrunheidS,CellierM,VidalS,GrosP.Identification and characterization of a second mouse Nramp gene.Genomics,1995,25(2):514-525

[5] GunshinH,MackenzieB,BergerU V,GunshinY,RomeroM F,BoronW F,NussbergerS, GollanJ L,HedigerM A.Cloning and characterization of a mammalian proton-coupled metal-ion transporter.Nature,1997,388(6641):482-488

[6] FlemingM D,TrenorC C 3rd,SuM A,FoernzlerD,BeierD R,DietrichW F,AndrewsN C. Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet, 1997,16(4):383-386

[7] CanonneH F,GruenheidS,PonkaP,GrosP.Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron.Blood,1999, 93(12):4406-4417

[8] HuangE,OngW Y,ConnorJ R.Distribution of divalent metal transporter-1 in the monkey basal ganglia.Neuroscience,2004,128(3):487-496

[9] AndrewsN C,FlemingM D,GunshinH.Iron transport across biological membranes.Nutr Rev,1999,57(4): 114-123

[10] JiangH,QianZ M,XieJ X.The increased DMT1 expression and iron content in MPTP-treated C57BL/6 mice.Acta Physiologica Sinica,2003,55(5): 571-576

[11] AtasoyH T,NuyanO,TuncT,YorubulutM,UnalA E,InanL E.T2-weighted MRI in Parkinson's disease; substantia nigra pars compacta hypointensity correlates with the clinical scores.Neurol India,2004,52(3):332-337

[12] GerlachM,DoubleK L,YoudimM B,RiedererP.Potential sources of increased iron in the substantia nigra of parkinsonian patients.J Neural Transm Suppl,2006, (70):133-142

[13] FlemingM D,RomanoM A,SuM A,GarrickL M,GarrickM D,AndrewsN C.Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci U S A,1998,95(3):1148-1153

[14] SuM A,TrenorC C,FlemingJ C,FlemingM D,AndrewsN C.The G185R mutation disrupts function of iron transporter Nramp2.Blood,1998,92(6):2157-2163

[15] BurdoJ R,MenziesS L,SimpsonI A,GarrickL M,GarrickM D,DolanK G,HaileD J, BeardJ L,ConnorJ R.Distribution of divalent metal transporter 1 and metal transport protein 1 in the normal and Belgrade rat.J Neurosci Res,2001,66(6):1198-1207

[16] WesleyR D,TangM,LagerK M.Protection of weaned pigs by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of H3N2 swine influenza virus.Vaccine,2004,22(25-26):3427-3434

[17] ChinsangaramJ,MoraesM P,KosterM,GrubmanM J.Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease.J Virol,2003, 77(2):1621-1625

[18] MascolaJ R,SamborA,BeaudryK,SantraS,WelcherB,LouderM K,VancottT C,HuangY,ChakrabartiB K,KongW P,YangZ Y,XuL,MontefioriD C,NabelG J,LetvinN L. Neutralizing antibodies elicited by immunization of monkeys with DNA plasmids and recombinant adenoviral vectors expressing human immunodeficiency virus type 1 proteins.J Virol, 2005,79(2):771-779

[19] MartinF S,HeikeO,HeinzS.Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: Crossing the species barrier.J Virol,2001,75(11):5108-5118

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