Li Hua, Zhao Songji, Jin Yongnan, Nishijima Ken-Ichi, Akizawa Hiromichi, Ohkura Kazue, Tamaki Nagara, Kuge Yuji
Department of Tracer Kinetics & Bioanalysis, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
Nucl Med Commun. 2011 Dec;32(12):1211-5. doi: 10.1097/MNM.0b013e32834b7ea7.
We have developed a radiolabeled uracil derivative, 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) as a novel single photon emission computed tomography probe for thymidine phosphorylase (TP). This radioiodinated IIMU has a high affinity for TP and highly accumulates in the TP-expressing tumor cell line A431 (human epidermoid carcinoma). To evaluate the specificity of the cellular uptake of IIMU to TP expression, we examined the effects of TP knockdown on the uptake of ¹²⁵I-labeled IIMU (¹²⁵I-IIMU) in the tumor cells.
TP-specific small interfering RNA (siRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific siRNA (positive control), and negative control siRNA were transfected into A431 cells, respectively. Target-mRNA and protein expression levels of TP and GAPDH were examined 48 and 72 h after transfection, respectively. The cellular uptake level of ¹²⁵I-IIMU was also evaluated 72 h after transfection. The results were compared after normalization with the corresponding negative controls.
After TP-specific and GAPDH-specific siRNA transfection, the expression levels of TP and GAPDH mRNA decreased significantly to 41 and 29%, respectively, compared with the negative control (P<0.001 for both). The expression levels of TP and GAPDH protein also significantly decreased to 34 and 30%, respectively (P<0.001 for both). After TP-specific siRNA transfection, the cellular uptake level of ¹²⁵I-IIMU decreased significantly to 66% (P<0.001). In contrast, GAPDH siRNA transfection did not significantly affect the cellular uptake level of ¹²⁵I-IIMU.
siRNA-mediated TP knockdown significantly decreased the cellular uptake level of ¹²⁵I-IIMU. This finding indicates that the uptake of IIMU in tumor cells is TP specific and directly corresponds to TP expression levels.
我们已研发出一种放射性标记的尿嘧啶衍生物，即5-碘-6-[(2-亚氨基咪唑烷基)甲基]尿嘧啶（IIMU），作为一种用于胸苷磷酸化酶（TP）的新型单光子发射计算机断层扫描探针。这种放射性碘化的IIMU对TP具有高亲和力，并在表达TP的肿瘤细胞系A431（人表皮样癌）中高度积聚。为了评估IIMU细胞摄取对TP表达的特异性，我们检测了TP基因敲低对肿瘤细胞中¹²⁵I标记的IIMU（¹²⁵I-IIMU）摄取的影响。
分别将TP特异性小干扰RNA（siRNA）、甘油醛-3-磷酸脱氢酶（GAPDH）特异性siRNA（阳性对照）和阴性对照siRNA转染至A431细胞中。分别在转染后48小时和72小时检测TP和GAPDH的靶mRNA及蛋白表达水平。转染后72小时也评估¹²⁵I-IIMU的细胞摄取水平。将结果与相应的阴性对照进行标准化比较。
与阴性对照相比，在转染TP特异性和GAPDH特异性siRNA后，TP和GAPDH mRNA的表达水平分别显著降低至41%和29%（两者均P<0.001）。TP和GAPDH蛋白的表达水平也分别显著降低至34%和30%（两者均P<0.001）。在转染TP特异性siRNA后，¹²⁵I-IIMU的细胞摄取水平显著降低至66%（P<0.001）。相反，转染GAPDH siRNA对¹²⁵I-IIMU的细胞摄取水平没有显著影响。
siRNA介导的TP基因敲低显著降低了¹²⁵I-IIMU的细胞摄取水平。这一发现表明，肿瘤细胞中IIMU的摄取具有TP特异性，且与TP表达水平直接相关。
