Ponomarev Vladimir, Doubrovin Michael, Shavrin Aleksander, Serganova Inna, Beresten Tatiana, Ageyeva Ludmila, Cai Changde, Balatoni Julius, Alauddin Mian, Gelovani Juri
Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
J Nucl Med. 2007 May;48(5):819-26. doi: 10.2967/jnumed.106.036962. Epub 2007 Apr 27.
A human-derived intrinsically nonimmunogenic reporter gene was tested for PET imaging of different molecular-genetic processes for potential clinical use.
The human mitochondrial thymidine kinase type 2 (hTK2) reporter gene truncated at the N terminus (DeltahTK2), alone or fused with green fluorescent protein (GFP), was used for preclinical evaluation in a mouse model. The levels of enzymatic activity of DeltahTK2 and DeltahTK2 GFP proteins were assessed using radiotracer accumulation and prodrug activation assays in vitro and in subcutaneous tumors grown from the corresponding cell lines in nude mice. Kinetic analyses of (124)I-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-iodouracil (FIAU), (18)F-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-ethyluracil (FEAU), or (18)F-9-(4-(18)F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) uptake in tumors and biodistribution studies were performed.
DeltahTK2 was successfully expressed in the cytoplasm of transduced cells. A new anti-hTK2 monoclonal antibody 8G2 was developed. The levels of FIAU and FEAU accumulation in cells expressing DeltahTK2 and DeltahTK2 GFP were at least 10-fold higher than in wild-type cells in vitro and about 6 times higher in vivo. We determined that FEAU is a more specific reporter substrate for DeltahTK2 than FIAU, whereas FHBG is not phosphorylated by this enzyme. In addition, we showed that DeltahTK2 transduced cells can be eliminated by treatment with d-arabinofuranosyl-cytosine.
We have tested a human-derived reporter gene that is likely to be nonimmunogenic and potentially allows for long-term monitoring of different molecular-genetic processes by nuclear imaging techniques in humans. Using (124)I-FIAU, (18)F-FIAU, or (18)F-FEAU, it should be possible to image DeltahTK2 reporter gene expression with PET in preclinical and clinical studies.
对一种源自人类的本质上无免疫原性的报告基因进行了测试，用于不同分子遗传过程的正电子发射断层显像（PET）成像，以探索其潜在临床应用价值。
将在N端截短的人类线粒体胸苷激酶2（hTK2）报告基因（DeltahTK2）单独或与绿色荧光蛋白（GFP）融合，用于小鼠模型的临床前评估。使用放射性示踪剂积累和前体药物激活试验，在体外以及在裸鼠体内由相应细胞系生长的皮下肿瘤中，评估DeltahTK2和DeltahTK2-GFP蛋白的酶活性水平。对（124）I-2'-氟-2'-脱氧-1-β-D-β-阿拉伯呋喃糖基-5-碘尿嘧啶（FIAU）、（18）F-2'-氟-2'-脱氧-1-β-D-β-阿拉伯呋喃糖基-5-乙基尿嘧啶（FEAU）或（18）F-9-（4-（18）F-氟-3-羟甲基丁基）鸟嘌呤（FHBG）在肿瘤中的摄取进行动力学分析，并开展生物分布研究。
DeltahTK2在转导细胞的细胞质中成功表达。开发了一种新的抗hTK2单克隆抗体8G2。在体外，表达DeltahTK2和DeltahTK2-GFP的细胞中FIAU和FEAU的积累水平比野生型细胞至少高10倍，在体内则高约6倍。我们确定，与FIAU相比，FEAU是DeltahTK2更特异的报告底物，而FHBG不能被该酶磷酸化。此外，我们表明，用阿糖胞苷处理可以消除转导了DeltahTK2的细胞。
我们测试了一种源自人类的报告基因，它可能无免疫原性，并有可能通过核成像技术对人类不同分子遗传过程进行长期监测。使用（124）I-FIAU、（18）F-FIAU或（18）F-FEAU，应该能够在临床前和临床研究中用PET对DeltahTK2报告基因的表达进行成像。
