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Quantitation of the Protein MIA as a Marker for,,,Chondrocytes in Research Samples

MIA (Melanoma Inhibitory Activity) was cloned as a secreted protein from human melanoma cell lines [1]. MIA is expressed in and secreted from malignant melanomas and chondrocytes [2, 3]. As MIA expression in chondrocytes is dependent on the differentiation status of the cells it was also designated CD-RAP (cartilage-derived retinoic acid sensitive protein). Retinoic acid is known as a regulator of chondrocytic cell differentiation in vitro.

Recent results indicate an important role in tumor progression and metastasis as MIA mediates detachment of melanoma cells from extracellular matrix molecules such as fibronectin [4]. MIA expression levels closely parallel the capability of melanoma cells to form metastases in syngeneic animals. Recently, MIA-deficient mice were shown to have structural abnormalities in cartilage [5].

Materials and Methods

Cell culture

Human primary chondrocytes derived from articular cartilage were cultivated in DMEM supplemented with penicillin (100 U/ml), streptomycin (10 g/ml) (both Sigma), and 10 % fetal calf serum (Gibco) under a humidified atmosphere of 5% CO2 at 37 C, then split 1:2 at 80 % confluency. As chondrocytes spontaneously dedifferentiate in cell culture, redifferentiation of the cells was induced by treatment with transforming growth factor-b (TGF-b) for 4 days. Differentiation was controlled by determining collagen type II with RT-PCR. Human mesenchymal stem cells were obtained from BioWhittaker Europe (Poietics) and cultured as described by the manufacturer.

MIA-ELISA

MIA was measured by a commercially available ELISA
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