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The proteolytic activities of the ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) protein families play important roles in normal and numerous pathological conditions. The ADAM family members have potential implications in the metastasis of human tumor cells via cell adhesion and protease activities. This family is characterized by the presence of both disintegrin and metalloproteinase domains, responsible for the adhesive and proteolytic properties, respectively. MMPs are a family of proteases responsible for the degradation of the extracellular matrix to allow cell growth and to facilitate remodeling. Under pathological conditions these proteases are involved in many diverse processes from tumor cell migration to cartilage destruction in rheumatoid arthritis.
The gene expression levels of eight ADAMs, previously described to possess proteolytic activities; and seven MMPs, previously described to have significant roles in the remodeling of the extracellular matrix during the metastatic process, were analyzed by Real Time PCR using an ABI SDS Prism 7000. RNA was isolated from multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic normal tissue and tumor samples. This method allowed for detected changes in mRNA expression of the individual metalloproteainase genes to be compared between normal and metastatic states, and also between tissue and cultured cells.
Based on the preliminary results, there are substantial differences in the level of ADAM and MMP mRNA expression between tissue and cell lines. In general, the level of expression is several fold higher in cultured cells compared to the isogenic tissue they are derived from. We are currently performing in situ fluorogenic enzyme assays on cultured cells to determine if the elevated expression in accompanied by enhanced proteolytic activity.