Lee Kyung-Han, Byun Sang Sung, Choi Joon Hun, Paik Jin-Young, Choe Yearn Seong, Kim Byung-Tae
Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Kangnamgu, Seoul, Korea.
Eur J Nucl Med Mol Imaging. 2004 Mar;31(3):433-8. doi: 10.1007/s00259-003-1395-7. Epub 2004 Jan 27.
There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest. The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene. We synthesised and purified radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli beta-galactosidase. We showed that [(125)I]iodo-PETG specifically binds to beta-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein. We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against beta-galactosidase activity. COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in [(125)I]iodo-PETG uptake ( r(2)=0.897; P=0.001), which reached up to 642.5%+/-16.7% of control levels ( P<0.00001). Moreover, the level of uptake was highly correlated to luminescent measurements of beta-galactosidase activity ( r(2)=0.878; P<0.0001). These results confirm that radioiodine-labelled PETG specifically targets beta-galactosidase and that its uptake rates faithfully reflect levels of expression of the lacZ reporter gene. Further investigations were performed in nude mice bearing human neuroblastoma tumours transferred with the lacZ gene. Compared with control tumours, lacZ-expressing tumours were slightly better visualised on [(123)I]iodo-PETG images and had a modest increase in tumour to muscle count ratio (2.6+/-0.2 vs 1.9+/-0.1, P<0.05). The present results provide proof-of-principle for the potential of radiolabelled inhibitors as promising radiotracers to monitor lacZ gene expression levels. Future modifications to improve cell permeability should enhance in vivo contrast levels and may allow the use of radiolabelled beta-galactosidase inhibitors for non-invasive monitoring of lacZ gene expression.
最近，人们对开发特异性靶向从感兴趣的报告基因表达转录而来的蛋白质的放射性探针的兴趣日益浓厚。本研究的目的是开发一种靶向最广泛使用的报告基因之一——细菌lacZ基因的放射性探针。我们合成并纯化了放射性碘标记的苯乙基-β-D-硫代半乳糖苷（PETG），它是一种对大肠杆菌β-半乳糖苷酶具有特异性的竞争性抑制剂。我们发现，放射性示踪剂与该蛋白质孵育后，经柱色谱和聚丙烯酰胺凝胶电泳验证，[¹²⁵I]碘代PETG能特异性结合β-半乳糖苷酶。我们还通过酶动力学研究表明，碘代PETG对β-半乳糖苷酶活性保留抑制作用。感染表达lacZ基因的重组腺病毒的COS-7细胞，其[¹²⁵I]碘代PETG摄取量有病毒滴度依赖性增强（r² = 0.897；P = 0.001），达到对照水平的642.5%±16.7%（P < 0.00001）。此外，摄取水平与β-半乳糖苷酶活性的发光测量高度相关（r² = 0.878；P < 0.0001）。这些结果证实，放射性碘标记的PETG特异性靶向β-半乳糖苷酶，其摄取率忠实地反映了lacZ报告基因的表达水平。对携带转染了lacZ基因的人神经母细胞瘤肿瘤的裸鼠进行了进一步研究。与对照肿瘤相比，表达lacZ的肿瘤在[¹²³I]碘代PETG图像上的显影略好，肿瘤与肌肉计数比有适度增加（2.6±0.2对1.9±0.1，P < 0.05）。目前的结果为放射性标记抑制剂作为监测lacZ基因表达水平的有前景的放射性示踪剂的潜力提供了原理证明。未来为提高细胞通透性所做的改进应能提高体内对比度，并可能允许使用放射性标记的β-半乳糖苷酶抑制剂对lacZ基因表达进行非侵入性监测。
