Association of downregulated glycotumor-markers and in vivo rejection

我们最初的胆管癌细胞系TK具有在培养上清液中分泌一些糖基化的肿瘤标志物的特性，如CA19-9、CA50或唾液酸化Lewis x(SLE^x)。将IL-2和/或B7-L基因导入TK细胞后，观察到免疫缺陷小鼠皮下移植瘤细胞的强烈细胞病变效应和体内排斥反应，这在野生型细胞系中是不存在的。根据宝贵的研究结果，我推测这些特征性的变化可能源于转基因强制表达导致糖基化抗原的异常表达。这些抗原被糖转移酶，特别是Fuc-T组酶糖基化。目前，已在人类中克隆了TII、TIV、TV、TVI和TVii 5个亚型，FucTIII和TVii被证实与CA19-9和SLE^x的合成以及Le^a或Le^b的合成有关。此外，我还研究了这些细胞是否存在糖转移酶基因关键元件的突变。当用高效液相色谱检测时，尽管CA19-9、CA50和SLeX在每个转染体中的表达不同，但这些差异与酶活性无关。RT-PCR法也证实Fuc-TVii基因转录没有变化。基因组DNA测序显示，在每个转染体中都没有Fuc-tiII基因特异区的突变。然而，由于我通过植入研究再次确认了显著的肿瘤排斥反应，还需要进一步的研究来解释这一现象。

Our original cholangiocarcinoma cell line, TK has a property to secrete some glycosylated tumor markers such as CA19-9, CA50 or sialyl Lewis x (sLe^x) in the culture medium. When IL-2 and/or B7-l gene was transfected into the TK, I observed strong cytopathic effects and in vivo rejections in subcutaneous implantation of the tumor cells in immunocompromised SCID mouse, which did not occur in the wild type cell line.From the precious results of studies, I reasoned that these characteristic changes might originate from aberrant expressions of gylcosylated antigens caused by forced expression of transfected genes. These antigens are glycosylated by glycotransferase, especially by Fuc-T group enzymes. Currently, 5 subtypes, TIII, TIV, TV, TVI, and TVII, has been cloned in human and FucTIII and TVII are proved to be frankly responsible for synthesis of CA19-9 and sLe^x as well as Le^a or Le^b. In this stance, I performed direct comparisons of glycotransferase activity and amounts of gylcosylated antigens in each transfectants. Furthermore, I studied if these cells have mutations in the key elements of glycotransferase gene. When determined by HPLC, although expressions of CA19-9, CA50, and sLex varied in each transfectant these variances were not correlated with enzyme activities. I also confirmed no changes in Fuc-TVII transcription by RT-PCR.In each transfectant, there were no mutations in Fuc-TIII gene specific region by genomic DNA sequences. However, since I reconfirmed significant tumor rejections by implantation study, further studies will be needed to explain the phenomenon.