SHERP

Development of a dual membrane protein reporter system using sodium iodide symporter and mutant dopamine D2 receptor transgenes

Cited 0 time in webofscience Cited 0 time in scopus
Authors
Hwang, Do Won; Kang, Joo Hyun; Chang, Young Soo; Jeong, Jae Min; Chung, June-Key; Lee, Myung Chul; Kim, Soonhag; Lee, Dong Soo
Issue Date
2007-04-03
Publisher
The Society of Nuclear Medicine Inc
Citation
J Nucl Med. 2007 Apr;48(4):588-95.
Keywords
AnimalsAutoradiography/methodsCell Line, TumorCell Membrane/*metabolismHumansMaleMiceMice, Inbred BALB CMice, NudeModels, GeneticNeoplasm TransplantationProtein BindingReceptors, Dopamine D2/*geneticsSymporters/*metabolism*Transgenes
Abstract
For noninvasive monitoring of cellular status by dual reporters, a dual membrane protein reporter system was developed and its in vivo applicability was examined. Human sodium iodide symporter (hNIS) and mutant dopamine D(2) receptor (D(2)R) transgenes were chosen considering their complementarity. METHODS: pIRES-hNIS/D(2)R containing NIS and D(2)R linked with an internal ribosomal entry site (IRES) was constructed and transfected into human hepatoma SK-Hep1 and rat glioma C6 cells. The cell lines stably expressing hNIS and D(2)R (named SK-ND and C6-ND) were produced, which was confirmed by messenger RNA expression of reporter genes. The functional activities of hNIS and D(2)R were measured by (125)I uptake assay and (3)H-spiperone receptor-binding assays. A biodistribution study was performed on SK-ND tumor-bearing mice using (99m)Tc-pertechnetate and (3)H-spiperone. In vivo hNIS expression was examined using (99m)Tc-pertechnetate gamma-camera imaging and, D(2)R expression was examined using a (3)H-spiperone autoradiographic study. RESULTS: (125)I uptake of SK-ND and C6-ND cell lines showed a maximum 97-fold and 43-fold increase, respectively, which were completely inhibited by KClO(4). Specific (3)H-spiperone binding to SK-ND and C6-ND cell homogenates was observed, which were completely inhibited by (+)-butaclamol. Among the dual reporter gene-expressing cell lines, the activities of both reporters were inversely correlated with each other. Competition assay of hNIS-expressing cells by D(2)R vector transfection and D(2)R-expressing cells by hNIS vector transfection showed a dose-dependent decrease of hNIS and D(2)R activities, respectively. In the biodistribution study, (99m)Tc-pertechnetate accumulated 10-fold and (3)H-spiperone accumulated 4-fold more in SK-ND tumors than that in parental SK tumors. In vivo imaging of (99m)Tc-pertechnetate persisted until 5 wk after the cell graft in SK-ND tumors. Autoradiographic study of brain tissues from these mice also revealed an accumulation of (3)H-spiperone in SK-ND tumors. CONCLUSION: We developed a dual membrane-bound positron and gamma-imaging reporter system of hNIS and D(2)R. We observed its reporting capability in vitro and in vivo and elucidated that these 2 membrane protein reporters competed with each other in their expression. Although we expect that hNIS and D(2)R transgenes can complement each other as a dual reporter system, we suggest that one needs to validate the ratio of expression of the 2 membrane protein reporter transgenes for cellular status tracking.
ISSN
0161-5505 (Print)
Language
English
URI
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17401096

http://hdl.handle.net/10371/29708
Files in This Item:
There are no files associated with this item.
Appears in Collections:
College of Medicine/School of Medicine (의과대학/대학원)Nuclear Medicine (핵의학전공)Journal Papers (저널논문_핵의학전공)
  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse