Establishment of highly metastasizing human salivary gland cancer cell lines and the analysis of mechanisms involved in the metastasis

用N-甲基-N-亚硝基脲（MNU）处理无转移能力的人涎腺腺癌细胞系HSGc。在暴露于MNU后，克隆具有改变的形态的细胞。皮下接种裸鼠后，MNU处理的HSGc克隆Gc 2 -100 cl-1在各种器官中形成转移灶，然后分离出5个转移克隆。检测组织型纤溶酶原激活物（tPA）、尿激酶型纤溶酶原激活物（uPA）、金属蛋白酶及其组织抑制剂1（TIMP-1）的表达。发现Gc 2 -100 cl-1和转移克隆分泌高水平的tPA，而HSGc产生检测不到的这种酶的水平。在任何细胞克隆中均未观察到uPA的表达。当明胶分解酶的分泌进行了检查，转移克隆产生更高水平的57-和32-kDa的，但不是92-或72-kDa的明胶酶，相比HSGc细胞。尽管TIMP-1在所有的细胞克隆中都被检测到，但转移性克隆表达TIMP-1。 ...更多信息 产生的TIMP-1少于HSGc细胞;此外，一个转移克隆产生的TIMP-1分子量不同于28-kDa TIMP-1。我们的研究结果表明，人类涎腺肿瘤细胞的转移能力的获得与几种金属蛋白酶的分泌增加以及TIMP-1表达的降低或改变密切相关。接下来，我们已经表明，来自转移克隆的条件培养基（CM）含有刺激牛主动脉内皮（BAE）细胞增殖和迁移的因子，并抑制BAE细胞产生胶原酶。为了进一步表征这一点，我们分析了EGF从细胞克隆的分泌以及EGF对BAE细胞生物学行为的影响。与HSGc相比，转移克隆释放大量EGF，然而，EGF受体的数量在所有细胞克隆中均以相似的水平一致地检测到。EGF不影响细胞克隆的胶原酶和TIMP-1的分泌。或者，EGF刺激BAE细胞的增殖和迁移，并抑制BAE细胞分泌胶原酶。因此，负责诱导BAE细胞生物学行为的含CM因子可归因于EGF。因此，这些观察结果表明，转移克隆分泌的EGF发挥其作为血管生成因子的生物学效应。少

A human salivary gland adenocarcinoma cell clone HSGc, with no metastatic ability was exposed to N-methyl-N-nitrosourea (MNU). Following exposure to MNU,cells with altered morphology were cloned. Upon s.c.inoculation into nude mice, an MNU-treated HSGc clone, Gc2-100 cl-1, formed metastatic foci in various organs, and then 5 metastasizing clones were isolated. Evaluation of expression of tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), metalloproteinases and tissue inhibitor of metalloproteinases-1 (TIMP-1) was performed. Gc2-100 cl-1 and metastasizing clones were found to secrete high levels of tPA,while HSGc produced undetectable levels of this enzyme. Expression of uPA was not observed in any of the cell clones. When the secretion of gelatinolytic enzymes was examined, metastasizing clones produced higher levels of 57- and 32-kDa, but not of 92- or 72-kDa gelatinases, as compared to HSGc cells. Although TIMP-1 was detected in all cell clones, metastasizing clones se … More creted less TIMP-1 than HSGc cells ; in addition, one metastasizing clone produced TIMP-1 with a molecular weight distinct from that of 28-kDa TIMP-1. Our results suggest that the aquisition of metastatic ability by human salivary gland tumor cells is closely associated with increased secretion of several metalloproteinases as well as decreased or altered TIMP-1 expression. Next, we have shown that conditioned medium (CM) from metastasizing clones contains factor (s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells. To further characterize this, we analyzed the secretion of EGF from cell clones as well as the effect of EGF on the biologic behaviors of BAE cells. Metastasizing clones released a large amount of EGF as compared with HSGc, however, the number of EGF receptors was detected consistently at a level that was similar in all cell clones. EGF did not affect the secretion of both collagenases and TIMP-1 from cell clones. Alternatively, EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF These observations, therefore, indicate that EGF secreted by metastasizing clones exerts its biologic effects as an angiogenic factor. Less