Anton Martina, Wagner Bettina, Haubner Roland, Bodenstein Claudia, Essien Bryan E, Bönisch Heinz, Schwaiger Markus, Gansbacher Bernd, Weber Wolfgang A
Institute of Experimental Oncology, Technische Universität München, Germany.
J Gene Med. 2004 Jan;6(1):119-26. doi: 10.1002/jgm.472.
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.
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.
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.
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.
去甲肾上腺素转运体（NET）是一种对儿茶酚胺具有高亲和力的转运体。其表达几乎仅局限于交感神经系统。在本研究中，我们评估了NET是否可用作放射性标记探针非侵入性成像转基因细胞的报告基因。
用牛NET cDNA逆转录病毒转导人A431、HT1080和小鼠CMS-5细胞。将转导细胞和亲本细胞与[¹³¹I]间碘苄胍（[¹³¹I]MIBG）在体外孵育。通过添加NET抑制剂丙咪嗪来确定示踪剂摄取的特异性。大鼠PC12细胞用作阳性对照。将亲本细胞和A431NET细胞异种移植到裸鼠体内，并在给予示踪剂后测定体内[¹²³I]MIBG在肿瘤中的摄取情况。
体外稳定转导的细胞显示出比亲本细胞高66至120倍的[¹³¹I]MIBG摄取。与丙咪嗪孵育可将转导细胞的[¹³¹I]MIBG摄取降低至亲本细胞中的水平。在用无示踪剂培养基孵育2小时后，所有转导细胞系中超过70%的初始放射性被保留。内源性表达NET的PC12细胞中的[¹³¹I]MIBG摄取比转导细胞低20至28倍。在体内，A431NET肿瘤显示出比亲本肿瘤高33倍的[¹²³I]MIBG摄取。示踪剂注射后24小时的γ相机图像显示亲本A431肿瘤无示踪剂摄取，但A431NET肿瘤图像清晰。
用NET cDNA转导肿瘤细胞会导致儿茶酚胺类似物在体外和体内具有高度特异性摄取和显著保留。这些特性使NET适合作为基因转移非侵入性监测的报告基因。
