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

胰腺癌被认为是一种极其致命的肿瘤，其主要原因是胰腺癌本身具有极高的侵袭和转移潜能。从N-亚硝基(2-氧丙基)胺(BGP)诱发的叙利亚金黄地鼠胰腺导管癌模型中，建立了高侵袭转移细胞系PC-1.0和低侵袭转移细胞系PC-1。高侵袭性和高转移性的PC-1.0细胞产生解离因子(Df)，诱导癌细胞解离。对PC-1.0细胞在无血清条件培养液中的癌细胞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。两种细胞株在体外表现出不同的生长形态，弱侵袭性细胞系PC-1形成岛状集落，高侵袭性细胞系PC-1.0以单细胞生长为主。条件培养液…PC-1.0细胞出现较多的岛状集落解体，PC-1细胞形态改变为细长细胞，伪足形成频率较高。解离活性不与肝素柱结合，而与羟基磷灰石结合。从凝胶过滤中推断得到。在免疫印迹分析中，用无气旋封闭抗体检测到主要的免疫反应蛋白条带，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，而且增强了细胞的运动性、细胞与纤维连接蛋白的黏附和化学侵袭。DF对细胞运动的诱导作用与c-fos基因的表达密切相关。环磷酸腺苷拮抗剂、PKC抑制剂(星形孢子素)和反义c-fos可显著抑制DF诱导的PC-1细胞运动。代表性差异分析(RDA)显示MAPK2与DF诱导的细胞解离密切相关，提示MEK2、依赖于cAMP的PKC和c-fos的激活可能参与了细胞解离和细胞运动的诱导机制。对该因子的分析可以为阐明胰腺癌的侵袭和转移机制以及开发新的治疗方法提供重要信息。较少

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