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Abstract
Malignant melanoma is the cancer with the most rapid increase in incidence in the United States. Ultraviolet light and deficiency of the p16ink4a gene are known factors that predispose one to the development of malignant melanoma. The signal transduction pathways that underlie the progression of melanoma from their precursors, atypical nevi, are not well understood. We examined activation of the MAP kinase pathway in atypical nevi and melanoma cells and found that this pathway is activated in melanomas. To determine the functional significance of this activation, we introduced constitutively active MAP kinase kinase (MAPKK) into immortalized melanocytes. The introduction of this gene into melanocytes leads to tumorigenesis in nude mice, activation of the angiogenic switch, and increased production of the proangiogenic factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs). Activation of MAP kinase signaling may be an important pathway involved in melanoma transformation. Inhibition of MAP kinase signaling may be useful in the prevention and treatment of melanoma.
Highlights
Introduction of Active MAP kinase kinase (MAPKK) into ImmortalizedMelanocytes—The L10BIOBR cell line is an immortalized murine melanocyte cell line that has been used to assess tumorigenesis
We demonstrate that MAP kinase activation functionally contributes to the development of melanoma, as the introduction of a constitutively active MAPKK into melanocytes leads to transformation in vivo
Early melanoma is curable through surgical excision, the prognosis of advanced melanoma is dismal
Summary
Introduction of Active MAPKK into ImmortalizedMelanocytes—The L10BIOBR cell line is an immortalized murine melanocyte cell line that has been used to assess tumorigenesis. Substrate gel electrophoresis using gelatin as the substrate indicated that the introduction of MAPKK into melanocytes resulted in an increase in the gelatinase activity of MMP-9 (ϳ92 kDa) and MMP-2 (ϳ65 kDa) in comparison to control cells (Fig. 4A) When this same conditioned media was tested in a radiometric enzyme assay for TIMP activity, we found that the MAPKK cells produced significantly higher levels of MMP inhibitory activity than did the control cells (Fig. 4C). Analysis of Tumor Dormancy and Vascularity—In situ hybridization of vector control and MAPKK-expressing tumors for the proliferation-associated marker histone H3 revealed little expression of this marker in vector control dormant tumors but diffuse expression in MAPKK-expressing L10BIOBR tumors (Fig. 5, A–D) These results are similar to what we have observed with immortalized and transformed endothelial cells in that both three-dimensional growth and increased angiogenesis are required for tumor growth in vivo (14). MAPKK-expressing tumors were notable for a high level of vascularity compared with GFP vector controls (Fig. 5, E and F)
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