Analysis of the mechanism of liver metastasis in pancreatic cancer -purification and gene cloning of cancer cell dissociation factor-

胰腺癌是一种非常致命的肿瘤，主要原因是胰腺癌本身具有极高的侵袭和转移潜力。利用n -亚硝基双（2-氧丙基）胺（BGP）诱导的叙利亚金仓鼠胰腺导管癌，建立了高侵袭性、转移性胰腺癌细胞系PC-1.0和弱侵袭性、低转移性胰腺癌细胞系PC-1。高侵袭性、高转移性PC-1.0细胞可产生诱导癌细胞分离的解离因子（DF）。对PC-1.0细胞在无血清条件培养基中的解离活性进行了部分纯化，并对部分纯化活性的几种生物学特性进行了评价。两种细胞系体外生长形态不同，弱侵袭性细胞系PC-1形成岛状菌落，高侵袭性细胞系PC-1.0主要以单细胞形态生长。在条件培养基中，PC-1.0细胞更多地诱导岛状菌落分离，PC-1细胞形态变化为细长型细胞，形成伪足的频率较高。解离活性不与肝素柱结合，而与羟基磷灰石培养体结合。并从凝胶分离中推断出来。采用多克隆阻断抗体，免疫印迹分析检测到主要的免疫反应蛋白带，其表观分子质量为> 55 kDa。部分纯化的DF不仅增强了细胞的解离，还增强了细胞的运动性、细胞对纤维连接蛋白的粘附和化学侵袭。检测到DF诱导细胞运动与c-fos mRNA表达有显著关系。环AMP拮抗剂、PKC抑制剂（staurosporine）和反蛋白c-fos显著抑制DF对PC-1细胞的诱导作用。此外，代表性差异分析（RDA）显示MAP激酶激酶2 （MEK2）与DF诱导的细胞解离密切相关。这些结果表明，MEK2、环AMP依赖性PKC和c-fos的激活可能参与了诱导细胞解离和细胞运动的机制。分析该因子可为阐明胰腺癌的侵袭转移机制和开发新的治疗方法提供重要信息。少

Pancreatic cancer is known to be an extremely lethal neoplasm, main reason being that pancreatic cancer itself has an extremely high potential of invasion and metastasis. Two pancreatic cancer cell lines, the highly invasive and metastatic cell line, PC-1.0, and weakly invasive and rarely metastatic cell line, PC-1, were established from a pancreatic ductal carcinoma induced by N-nitrosobis (2-oxopropyl) amine (BGP) in a Syrian golden hamster. And highly invasive and metastatic PC-1.0 cells has been found to produce dissociation factor (DF) which induces cancer cell dissociation. The cancer cell dissociation activity in serum-free conditioned medium of PC-1.0 cells was partially purified and several biological properties of the partially purified activity were evaluated.Two cell lines exhibited different growth morphology in vitro, the weakly invasive cell line PC-1 formed island-like colonies and the highly invasive cell line PC-1.0 grew mainly as single cells. The conditioned medium … More of PC-1.0 cells induced dissociation of island-like colonies, and morphological changes of PC-1 cells to elongated cells, with a high frequency of pseudopodia formation. The dissociation activity did not bind to the heparin column but bind to hydroxylapatite culum. and had as deduced from gel fitration. The major immunoreactive proteinous band with an apparent molecular mass of > 55 kDa was detected in immunoblotting analysis, using apolyclonal blocking antibody. The partially purified DF enhanced not only cell dissociation but also cell motility, cell adhesion to fibronectin, and chemoinvasion. The significant relationship between the induction of cell motility by DF and the c-fos mRNA expression was detected. The induction of cell motility of PC-1 cells by DF was significantly inhibited by cyclic AMP antagonist, PKC inhibitor (staurosporine), and antisence c-fos. Moreover, the representational difference analysis (RDA) revealed that MAP kinase kinase 2 (MEK2) was closely related to cell dissociation induced by DF.These results indicate that the activation of MEK2, cyclic AMP dependent PKC and c-fos are possibly involved in the mechanism of the induction of cell dissociation and cell motility. Analysis of this factor could provide the important information to clarify the mechanism of invasion and metastasis and to develop newly therapeutic method for panreatic cancer. Less