Immunoluminescent Detection of Intercellular Adhesion Molecule-1 and Aminopeptidase N on Human Melanoma Cells

Abstract

Background: During the complex process of melanoma cell detachment from the primary tumor and subsequent metastasis, different cell surface proteins are involved in tumor cell interactions with extracellular matrix (ECM) components and surrounding cells. Melanoma cells are able to vary their expression patterns of adhesion proteins and proteases or peptidases in different stages of tumor development. Materials and Methods: This paper describes the detection of intercellular adhesion molecule-1 (ICAM-1) and aminopeptidase N (APN) on melanoma cells by immunoluminescence. The marker enzyme horseradish peroxidase (HRP) of the secondary antibody (Ab) was used to release luminescence. In addition, APN was detected on intact cells with an activity assay using alanine-p-nitroanilide (Ala-pNA) as substrate including inhibition experiments. Results: The cell surface expression of ICAM-1 and APN on melanoma cells was shown by indirect labelling using two different antibodies for each antigen. The activity and inhibition assays confirmed the immunoluminescent result for APN. Conclusion: Both cell surface proteins have a strong impact on tumor cell behaviour and are typical cell markers indicating a high metastatic tumor stage in melanoma. The extensive characterization of melanoma cells offers the possibility for therapeutic approaches with more than one target molecule. Moreover, increasing insights into the components involved in signal transduction could help to develop specific reagents that inhibit tumor-specific pathways.