Evidence of antisense tumor targeting in mice
Evidence of antisense tumor targeting in mice
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DOI：
10.1021/bc0499073
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发表时间：
2004-11-01
影响因子：
4.7
通讯作者：
Hnatowich, DJ
中科院分区：
文献类型：
--
作者：
Nakamura, K;Fan, C;Hnatowich, DJ
尽管与对照DNA相比，放射性标记的反义DNA的积累增加正在成为培养的肿瘤细胞中的常规观察结果，但通过反义机制体内靶向肿瘤的可靠证据仍然难以捉摸。本研究的目的是通过使用两种不同的肿瘤和两种肿瘤内（i.t.）和静脉内（i. v.）施用放射性标记的反义和对照正义DNA。本研究使用MDR++细胞系KB-G2及其亲本MDR+细胞系KB-31。反义（AS）DNA针对MDR 1 mRNA的AUG起始密码子，并且与有义（S）对照DNA一起沿着，是裸施用的均匀硫代磷酸酯。在我们实验室以前的细胞培养研究中，这种AS DNA在KB-G2细胞中的积累非常高，而在KB-31细胞中仅为平均水平，我们将这一事实归因于在前一细胞系中通过RT-PCR测定的MDR 1 mRNA表达高出1000倍。在本研究中，两种DNA均通过MAG 3用Tc-99 m放射性标记，并i.t.或在处死和解剖前24小时静脉注射1 μ g（100 μ Ci）/动物。在i.t.给药后，除了在KB-G2小鼠中，其中肌肉水平低于S对照，在正常组织中AS和S DNA生物分布之间没有观察到统计学显著差异（Student’s t检验，p < 0.05，N = 4）。相比之下，AS DNA与S DNA相比，YB-G2动物中的肿瘤水平显著更高（14.7 vs 8.5%ID/g），而KB-31动物中的这种差异（8.6 vs 4.3%ID/g）不显著。处死前获得的全身图像清楚地显示KB-G2动物中AS DNA相对于S DNA的靶向改善，但KB-31动物中没有。基于这些结果的计算表明，在i.t.局当静脉注射而不是静脉注射时，与KB-31相比，在KB-G2动物中没有观察到更高的肿瘤水平，这很可能是因为到达肿瘤的剂量较低。当KB-G2和KB-31结果相结合时，除了血液中S DNA水平较高和脾脏中S DNA水平较低外，正常组织中AS和S DNA生物分布之间没有统计学显著差异。相比之下，AS DNA的肿瘤水平显著高于S DNA（0.100 vs 0.063%ID/g）。基于这些结果的计算表明，在静脉内给药后，每个肿瘤细胞特异性地积累了约400个AS DNA。因此，已经获得了通过反义机制体内靶向肿瘤的证据，因为与对照Tc-99 m-S DNA相比，在i.t.和静脉注射给药。AS DNA在肿瘤中的成功定位表明，体内AS靶向肿瘤是可行的，尽管实际的反义成像需要改进肿瘤递送和正常组织清除。
Even though increased accumulations of radiolabeled antisense DNAs compared to control DNAs are becoming a routine observation in cultured tumor cells, trustworthy evidence of tumor targeting in vivo by an antisense mechanism remains elusive. The goal of this study was to obtain convincing evidence of antisense tumor targeting in nude mice by using two different tumors and both intratumoral (i.t.) and intravenous (i.v.) administration of radiolabeled antisense and control sense DNAs. Both the MDR++ cell line KB-G2 and its parent MDR+ cell line KB-31 were used in this study. The antisense (AS) DNA was directed against the AUG start codon of the MDR1 mRNA and, along with the sense (S) control DNA, was a uniform phosphorothioate administered naked. In previous cell culture studies from our laboratories, the accumulation of this AS DNA was strikingly high in KB-G2 cells and only average in KB-31 cells, a fact we attribute to the 1000-fold higher expression by RT-PCR of MDR1 mRNA in the former cell line. In this study, both DNAs were radiolabeled with Tc-99m via MAG3 and administered i.t. or i.v. at 1 mug (100 muCi) per animal 24 h prior to sacrifice and dissection in mice bearing thigh tumors of about 1 g. Following i.t. administration, no statistically significant differences (Student's t test, p < 0.05, N = 4) between the AS and S DNA biodistributions in normal tissues were observed except in the KB-G2 mice in which muscle levels were lower for the S control. In contrast, tumor levels in the YB-G2 animals were significantly higher for the AS DNA vs S DNA (14.7 vs 8.5% ID/g) while this difference (8.6 vs 4.3% ID/g) was insignificant in the KB-31 animals. The whole body images obtained just prior to sacrifice clearly show improved targeting of AS DNA vs S DNA in the KB-G2 but not the KB-31 animals. Calculations based on these results show that about 60 000 AS DNAs accumulated specifically (i.e. AS DNA - S DNA) per KB-G2 tumor cell following i.t. administration. When administered i.v. rather than i.t., higher tumor levels in KB-G2 animals compared to KB-31 were not observed, most likely because of the lower dosage reaching the tumors. When the KB-G2 and KB-31 results are combined, no statistically significant differences between the AS and S DNA biodistributions in normal tissues were observed except in blood in which S DNA levels were higher and in spleen in which they were lower. In contrast, tumor levels were significantly higher for the AS DNA vs S DNA (0.100 vs 0.063% ID/g). Calculations based on these results show that about 400 AS DNAs accumulated specifically per tumor cell following i.v. administration. Therefore evidence for tumor targeting in vivo by an antisense mechanism has been obtained in that statistically higher tumor accumulations of the Tc-99m-AS DNA were observed compared to the control Tc-99m-S DNA both following i.t. and i.v. administrations. The successful localization of AS DNA in tumor demonstrates that in vivo AS targeting of tumor is feasible although improvements in tumor delivery and normal tissue clearance are needed for practical antisense imaging.