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

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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