Use of the norepinephrine transporter as a reporter gene for non-invasive imaging of genetically modified cells.
BACKGROUND
The norepinephrine transporter (NET) is a high-affinity transporter for catecholamines. Its expression is almost exclusively restricted to the sympathetic nervous system. In this study we evaluated whether the NET can be used as a reporter gene for non-invasive imaging of genetically modified cells with radiolabeled probes.


METHODS
Human A431, HT1080 and murine CMS-5 cells were retrovirally transduced with bovine NET cDNA. Transduced and parental cells were incubated in vitro with [(131)I]meta-iodobenzylguanidine ([(131)I]MIBG). The specificity of tracer uptake was determined by adding the NET inhibitor imipramine. Rat PC12 cells served as positive controls. Parental and A431NET cells were xenotransplanted into nude mice and tumor uptake of [(123)I]MIBG in vivo was determined after tracer administration.


RESULTS
In vitro stably transduced cells showed a 66- to 120-fold higher [(131)I]MIBG uptake than parental cells. Incubation with imipramine reduced [(131)I]MIBG uptake of transduced cells to the level found in parental cells. More than 70% of the initial radioactivity was retained in all transduced cell lines after 2 h incubation with tracer-free medium. [(131)I]MIBG uptake in PC12 cells, which express the NET endogenously, was 20- to 28-fold lower than in transduced cells. In vivo, A431NET tumors demonstrated a 33-fold higher [(123)I]MIBG uptake than parental tumors. Gamma camera images 24 h after tracer injection showed no tracer uptake in parental A431 tumors, but clear images of A431NET tumors.


CONCLUSIONS
Transduction of tumor cells with NET cDNA causes highly specific uptake and significant retention of catecholamine analogs in vitro and in vivo. These characteristics make the NET suitable as a reporter gene for non-invasive monitoring of gene transfer.
Anton
Martina
M
Institute of Experimental Oncology, Technische Universität München, Germany. m.anton@lrz.tum.de
Wagner
Bettina
B
Haubner
Roland
R
Bodenstein
Claudia
C
Essien
Bryan E
BE
Bönisch
Heinz
H
Schwaiger
Markus
M
Gansbacher
Bernd
B
Weber
Wolfgang A
WA
D019797:3-Iodobenzylguanidine / Q000493:pharmacokinetics; D000818:Animals; D002277:Carcinoma / Q000473:pathology; D018076:DNA, Complementary / Q000235:genetics*; D005354:Fibrosarcoma / Q000473:pathology; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008819:Mice, Nude; D050484:Norepinephrine Plasma Membrane Transport Proteins; D016716:PC12 Cells; D011877:Radionuclide Imaging / Q000379:methods; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D051381:Rats; D012190:Retroviridae; D027981:Symporters / Q000096:biosynthesis / Q000235:genetics*; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D050484:Norepinephrine Plasma Membrane Transport Proteins; D019275:Radiopharmaceuticals; C493228:Slc6a2 protein, mouse; C493226:Slc6a2 protein, rat; D027981:Symporters; D019797:3-Iodobenzylguanidine