ALPHA-1-6 FUCOSYLTRANSFERASE ANTISENSE-OLIGONUCLEOTIDE THERAPY FOR HEPATOCELLULAR CARCINOMA.

肝细胞癌（HCC）是人类最常见的恶性肿瘤之一，尽管有多种治疗方法，但其肝内和肝外转移的预防仍然是一个难题。我们已经调查了血清学特征表明肝癌的恶性潜力。虽然，在一般情况下，甲胎蛋白（AFP）的血清浓度已被用于诊断肝癌，我们已经表明，相对量的透镜culinaris凝集素（LCA）反应种类的AFP是显着更大的肝癌比非肿瘤性肝病。此外，我们已经阐明，较高比例的（LCA）-反应种类的AFP与肿瘤的侵袭性显着相关。AFP的LCA反应性物质的分子基础是AFP的双触角糖链的最内侧N-乙酰葡糖胺残基的岩藻糖基化。此外，据报道，N-连接的β1-6支链寡糖可能直接促进了细胞的生长。 关于我们 e恶性表型，包括几种类型癌症（包括HCC）的转移潜力。据推测，这些表型变化必须由GDP-L-Fuc：N-乙酰基-β-D-氨基葡萄糖苷提供：β1-6岩藻糖基转移酶（αFT）和N-乙酰葡糖胺基转移酶V（GnT V）分别催化β1 - 6分支，αFT催化GDP-岩藻糖通过α 1 -6键加成到糖蛋白N-连接寡糖的N-乙酰葡糖胺残基的还原末端，GnT V催化β1-6分支。在本研究中，我们首先确定了各肝组织中αFT和GnT V的信息量是否真的与组织和血清中的各酶活性相关。因此，定量逆转录-聚合酶链反应显示，肝癌中每种mRNA的数量与肝癌和相应血清中每种酶的活性显著相关。基于这些观察结果，接下来我们试图研究针对αFT和GnT V的反义RNA的作用。为了在体内评价该作用，我们采用了基于四环素反应元件（TRE）的高水平基因表达系统。尽管该系统具有根据四环素的存在或不存在来严格调节体内基因表达的能力的巨大优势，但是建立稳定的细胞系相对困难。因为它需要转染两个关键成分，四环素控制的反式激活因子（TA）和TRE融合到感兴趣的基因。此外，没有用于选择的基因整合到由TRE和目的基因组成的质粒中。最后，必须转化三个组分，包括选择性标记。我们成功地克隆了αFT和GnT V基因，并建立了稳定的TA细胞系。在不久的将来，我们将能够开发双稳定细胞系，并在分子水平上阐明岩藻糖基化和葡糖胺化对癌症侵袭的生物学意义。少

Hepatocellular carcinoma (HCC) is one of the most prevalent human cancers, and the prevention of intra and extrahepatic metastasis of HCC is still difficult even with the development of several therapeutic modalities. We have investigated serological features indicating a malignant potential of HCC. Although, in general, the serum concentration of alpha-fetoprotein (AFP) has been used for the diagnosis of HCC, we have shown that the relative amount of the Lens culinaris agglutinin (LCA)-reactive species of AFP is significantly greater in HCC than in non-neoplastic liver diseases. In addition, we have elucidated that the higher proportion of (LCA)-reactive species of AFP is significantly associated with the tumor invasiveness. The molecular basis of the LCA-reactive species of AFP is the fucosylation at the innermost N-acetylglucosamine residue of the biantennary sugar chain of AFP. Furthermore, it was reported that N-linked β1-6 branched oligosaccharides might contribute directly to th … More e malignant phenotype including metastatic potential of several types of cancers including HCC. It is supposed that these phenotypic changes must be provided by GDP-L-Fuc : N-acethyl-β-D-glucosaminide : β1-6 fucosyltransferase (αFT), which catalyzes the addition of fucose from GDP-fucose through an αl-6 linkage to the reducing end of N-acetylglucosamine residue of N-linked oligosaccharides of glycoproteins, and N-acetylglucosaminyltransferase V (GnT V), which catalyzes β1-6 branching, respectively.Therefore, in this research term, at first we determined weather the amount of message of αFT and GnT V in each liver tissue were really associated with each enzyme activity in the tissues and the serum. Consequently, quantitative reverse transcription coupled polymerase chain reaction revealed that the quantity of each mRNA in HCC was significantly associated with each enzyme activity in HCC and the corresponding serum. From the point of these observations, next we tried to investigate effects of antisense RNAs against αFT and GnT V. In order to evaluate the effects in vivo, we employed the high-level gene expression system based on tetracycline-responsive element (TRE). Although this system has a great advantage of capability for strict regulation of gene expression in vivo according to the presence or absence of tetracycline, it is relatively difficult to establish stable cell lines. Because it requires transfection of two critical components, the tetracycline-controlled transactivator (TA) and TRE fused to the gene of interest. Furthermore, none of genes for the selection is integrated into the plasmid consisting of TRE and the gene of interest. Finally, three components have to be transformed including a selectable marker. We have finished to clone αFT and GnT V genes in an appropriate construct, and create a stable cell line of TA. In a near future, we will be able to develop double-stable cell lines, and elucidate a biological significance of fucosylation and glucosaminylation at a molecular level on cancer invasiveness. Less