Invasion of melanoma cells into the dermal connective tissue is a major characteristic in the complex process of metastasis. Proteases play an important role in tumor cell invasion as these enzymes are able to degrade most components of the extracellular matrix (ECM), and thus enable cells to penetrate interstitial connective tissues and basement membranes. We developed an improved culture model that allows the detailed study of melanoma cell invasion in vitro. In this model, high (BLM) or low (530) invasive melanoma cells were seeded on the dermal side of dead deepidermized dermis (DDD) and cultured for 14 days at the air/liquid interface. The high invasive cells invaded the tissue, leading to dermal tumor formation, whereas the low invasive cells did not. Analysis of the enzymatic activity of gelatinases by in situ gelatin zymography at neutral pH revealed proteolysis only in those composites cultured with high invasive melanoma cells. Interestingly, in situ zymograms performed at more acidic conditions, favoring the activity of cysteine proteases, exhibited markedly enhanced and widespread gelatinolysis compared to neutral pH. Cysteine protease inhibitors (E-64 and leupeptin) significantly reduced invasion of melanoma cells into these composites. These results indicate an important role of cysteine proteases for tumor invasion. Key words: tumor invasion; extracellular matrix, proteases, in situ zymographyOne of the life threatening features of malignant melanoma is its capacity for early metastatic spread. A prerequisite for melanoma cells to metastasize via the lymphatic or the hematogenous system is that they have to penetrate several barriers, including the basement membranes and the interstitial dermal connective tissue. 1 Tumor cells have been shown to use different mechanisms, consisting of proteolytic alteration of the extracellular matrix (ECM), adhesion and de-adhesion to matrix constituents and cell shape changes leading to migration through these tissues. 2 Different in vitro systems have been developed to study in more detail the role of proteases during tumor cell invasion. However, most of these systems are rather artificial, e.g., analysis of invasion through matrigel (formed from extracts of the basement membrane proteins produced by EHS-tumor), or the use of tissues that do not adequately resemble the in vivo situation of human skin, such as chick heart fragments, 3 chicken chorioallantoic membrane 4 or human amnion membrane. 5 Additionally, several lines of investigation have demonstrated that the interaction of tumor cells with their surrounding ECM controls the phenotype of the cells, including cell growth, differentiation and protease activity. 6,7 Protease activity is tightly regulated at different levels, including gene expression, processing of latent zymogen forms, inhibition of enzyme activity by specific inhibitors and the control of enzyme activity by environmental factors such as ionic strength and pH. 8 Different ECM degrading proteases, including serine proteases, the matrix me...
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