Loss of adhesion to basement membrane components but not to keratinocytes in proliferating melanocytes.

We studied the adhesive characteristics of melanocytes, cultured either in the presence of the mitogen phorbol 12-myristate 13-acetate (PMA) that keeps them in a proliferative state, or in the absence of PMA allowing them to differentiate. On proliferating melanocytes, several integrins, ICAM-1, E-cadherin, and CD44 were expressed. In the absence of PMA, proliferation was arrested, melanin synthesis increased, and the morphology of the melanocytes became more spreaded. Under these conditions, expression of integrins alpha 3 beta 1 and alpha 5 beta 1 decreased, whereas expression of alpha 2 beta 1, alpha 4 beta 1, and alpha 6 beta 1 increased. No changes were observed for any of the other adhesion molecules. Immunoprecipitations from metabolically labeled cells confirmed the shift in integrin expression at the level of biosynthesis. The increased surface expression of alpha 2 beta 1 and alpha 6 beta 1 in the absence of PMA was accompanied by an induction of adhesion to basement membrane components collagen and laminin through these integrins. Integrin alpha 5 beta 1/alpha v beta 3-mediated adhesion to fibronectin, CD44-mediated adhesion to hyaluronate, and E-cadherin/beta 1-integrin-mediated adhesion to keratinocytes were not affected by PMA. These findings indicate that by selective modulation of the expression of adhesion molecules, adhesion to components of the basement membrane is reduced in proliferating melanocytes, whereas adhesion to keratinocytes is maintained. Similar events may be involved in melanocyte proliferation and migration during wound healing and initial steps of melanocytic tumor progression.

Alpha v-integrins in human melanoma: gain of alpha v beta 3 and loss of alpha v beta 5 are related to tumor progression in situ but not to metastatic capacity of cell lines in nude mice.

We investigated the expression of alpha v-integrins in different stages of human cutaneous melanocytic tumor progression. We observed that alpha v beta 5 was the alpha v-integrin expressed in all common nevocellular nevi, in 78% of dysplastic nevi, in 63% of early primary melanomas, in 43% of advanced primary melanomas, and in 33% of melanoma metastases. Hence, loss of alpha v beta 5 expression was related to melanocytic tumor progression. In line with earlier reports, alpha v beta 3 was exclusively detected in advanced primary melanomas and metastases (24% and 50% respectively). Staining with anti-alpha v monoclonal antibodies (MAbs) in lesions where both alpha v beta 3 and alpha v beta 5 were absent showed that alternative alpha v-integrins were expressed in advanced primary melanomas and metastases. By FACS analysis, we determined expression of alpha v beta 5 and alpha v beta 3 in 4 human melanoma cell lines with different metastatic capacities after s.c. inoculation into nude mice. One of the non-metastatic and both highly metastatic cell lines expressed alpha v beta 5 at their surface. Surprisingly, alpha v beta 3 was detected exclusively in the non-metastatic cell lines. Absence of alpha v beta 3 in the highly metastatic cell lines was confirmed by lack of immunoprecipitation from 35S-methionine-labeled cells and by absence of immunohistochemical staining on primary and metastatic xenograft lesions. Our findings indicate that alpha v beta 5 expression is often lost in advanced stages of melanocytic tumor progression in situ, while alpha v beta 3 is acquired, but that a decrease in alpha v beta 5 and an increase in alpha v beta 3 expression are not necessarily related to the metastatic behavior of human melanoma cells in nude mice.

Regulation of integrin-mediated adhesion to laminin and collagen in human melanocytes and in non-metastatic and highly metastatic human melanoma cells.

We compared integrin-mediated adhesion to extracellular matrix (ECM) components of cultured human melanocytes and 6 human melanoma cell lines with different metastatic capacities in nude mice. Cultured melanocytes and most melanoma cell lines adhered strongly to fibronectin (FN), whereas only highly metastatic cell lines adhered to laminin (LM), collagen type I (COI) and type IV (COIV). Adhesion to LM and CO could be blocked by anti-alpha 6 and anti-alpha 2 monoclonal antibodies (MAbs) respectively. This observation is consistent with the finding that expression of LM receptor alpha 6 beta 1 and LM/CO receptor alpha 2 beta 1 was low on melanocytes and non- or poorly metastatic cell lines, whereas these integrins were strongly expressed on highly metastatic cell lines. In addition, immunoprecipitation from [35S]-methionine-labeled cells demonstrated increased synthesis of alpha 6, alpha 2 and beta 1 in highly metastatic cell lines and immunohistochemistry showed expression of alpha 6 beta 1 and alpha 2 beta 1 only in xenograft lesions from highly metastatic cell lines. Furthermore, the observation that adhesion of melanocytes and non- or poorly metastatic cell lines could be stimulated with anti beta 1 MAbs demonstrates that these receptors, on these cells, are expressed in an inactive state. Our results suggest that alpha 2 beta 1 and alpha 6 beta 1 play a role in human melanoma metastasis in nude mice and demonstrate that interactions of these integrins with their ligands can be regulated at the level of surface expression and activation state of the receptor.

Establishment and characterization of a human melanoma cell line (MV3) which is highly metastatic in nude mice.

To select human melanoma cells that are highly tumorigenic and metastatic in nude mice we have implanted fragments of a fresh human melanoma metastasis subcutaneously (s.c.) into a nude mouse. After 3 passages in nude mice, part of the xenograft was cultured and a new melanoma cell line, MV3, was established. After intravenous (i.v.) inoculation of 2 x 10(6) MV3 cells, 95% of the nude mice (n = 20) developed lung colonies within 6 weeks. S.c. inoculation of 2 x 10(6) MV3 cells resulted in 95% tumor take, while 90% of the mice (n = 20) showed spontaneous metastases in the lungs within 7 weeks. Histological and immunohistological features of the original tumor of the patient were largely retained in the tumors of the mice and in the cell line in vitro. As shown by Alcian blue staining, MV3 cells contain large quantities of glycosaminoglycans (GAGs) and/or proteoglycanes (PGs), both in vivo and in vitro. The cells showed a marked expression of transferrin receptor, ICAM-1, EGF-receptor, and VLA-2 integrin. As only few human melanoma cell lines are available that frequently show metastasis in nude mice, the highly metastatic MV3 cell line represents a useful tool for studying the expression and regulation of molecules on human melanoma cells involved in the process of metastasis.