Germann C, Shields A F, Grierson J R, Morr I, Haberkorn U
Department of Oncological Diagnostics and Therapy, German Cancer Research Center, Heidelberg.
J Nucl Med. 1998 Aug;39(8):1418-23.
The planning and individualization of gene therapy with suicide genes such as herpes simplex virus thymidine kinase (HSV-tk) necessitates the assessment of the enzyme activity expressed in the tumor. This can be done by uptake measurements of specific substrates for HSV-tk. Due to the molecular structure of 5-fluoro-1-(2'-deoxy-fluoro-beta-D-ribofuranosyl)uracil (FFUdR), it may be a substrate for both the mammalian thymidine kinase and HSV-tk.
Using a HSV-tk-expressing rat hepatoma cell line and a control cell line (bearing the empty vector) the uptake of 3H-FFUdR was determined with increasing incubation periods. Furthermore, measurements with graded mixtures of HSV-tk-expressing cells and control cells were made. To elucidate the mechanism of FFUdR transport into cells, a series of inhibition/competition experiments was performed with challenge inhibitors of the nucleoside and the nucleobase transport systems.
The uptake studies with tritiated FFUdR revealed a 14- to 19-fold higher accumulation in the HSV-tk-expressing cell line compared to the control cell line. While the 3H-FFUdR uptake was 3- to 4-fold higher than the 3H-ganciclovir uptake in the HSV-tk-expressing cells, it was also higher in control cells (5-fold). Furthermore, FFUdR accumulation was linearly correlated with the amount of HSV-tk-expressing cells. FFUdR uptake and growth inhibition by therapeutic doses of ganciclovir were highly correlated, with r = 0.96. Inhibition/competition experiments showed that FFUdR is transported mainly by the equilibrative and the concentrative nucleoside transporter but not by the nucleobase transport systems.
The FFUdR uptake is an indicator of the HSV-tk activity in tumor cells and can be used as a prognostic marker during gene therapy with HSV-tk. The relative merits of ganciclovir and FFUdR as specific substrates for HSV-tk will need to be further explored in vivo.
使用单纯疱疹病毒胸苷激酶（HSV-tk）等自杀基因进行基因治疗的规划和个体化需要评估肿瘤中表达的酶活性。这可以通过测量HSV-tk特异性底物的摄取来完成。由于5-氟-1-（2'-脱氧-氟-β-D-呋喃核糖基）尿嘧啶（FFUdR）的分子结构，它可能是哺乳动物胸苷激酶和HSV-tk的底物。
使用表达HSV-tk的大鼠肝癌细胞系和对照细胞系（携带空载体），随着孵育时间的增加测定3H-FFUdR的摄取。此外，对表达HSV-tk的细胞和对照细胞的分级混合物进行了测量。为了阐明FFUdR转运到细胞中的机制，用核苷和核碱基转运系统的挑战抑制剂进行了一系列抑制/竞争实验。
用氚标记的FFUdR进行的摄取研究表明，与对照细胞系相比，表达HSV-tk的细胞系中的积累高14至19倍。虽然在表达HSV-tk的细胞中3H-FFUdR摄取比3H-更昔洛韦摄取高3至4倍，但在对照细胞中也更高（5倍）。此外，FFUdR积累与表达HSV-tk的细胞数量呈线性相关。治疗剂量的更昔洛韦对FFUdR摄取和生长抑制高度相关，r = 0.96。抑制/竞争实验表明，FFUdR主要通过平衡型和浓缩型核苷转运体转运，而不是通过核碱基转运系统。
FFUdR摄取是肿瘤细胞中HSV-tk活性的指标，可作为HSV-tk基因治疗期间的预后标志物。更昔洛韦和FFUdR作为HSV-tk特异性底物的相对优点需要在体内进一步探索。
