Sodi S A, Chakraborty A K, Platt J T, Kolesnikova N, Rosemblat S, Keh-Yen A, Bolognia J L, Rachkovsky M L, Orlow S J, Pawelek J M
Department of Dermatology, Yale University School of Medicine, New Haven, CT 06520, USA.
Pigment Cell Res. 1998 Oct;11(5):299-309. doi: 10.1111/j.1600-0749.1998.tb00739.x.
We recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential. With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content and increased responsiveness to MSH compared to parental cells. Here we investigated the hybrid melanotic phenotype in more detail, comparing the pigmentary systems of hybrids and parental Cloudman S91 cells by several techniques. Cells were studied by electron microscopy, cell lysates were analyzed for tyrosinase (E.C.1.14.18.1) activity, and melanosomal proteins were analyzed by gel electrophoresis and immunoblotting. Melanosomes in parental Cloudman melanoma cells were few in number and relatively amorphous, whereas those in the hybrids were numerous and heavily pigmented, containing highly organized lattice structures. Both basal and MSH-inducible tyrosinase activities were elevated several fold in hybrids compared to parental cells. Tyrosinase, TRP-2, and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages, which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. By using 3H-glucosamine as a marker of N-glycosylation, its incorporation into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors, and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.
我们最近报道，在体外由低转移性小鼠Cloudman S91黑色素瘤细胞与人类或小鼠巨噬细胞生成的大多数杂交细胞显示出增强的转移潜力。除少数例外，与亲代细胞相比，具有增强转移潜力的杂交细胞还具有更高的基础黑色素含量以及对促黑素（MSH）的反应性增强。在此，我们通过几种技术比较杂交细胞和亲代Cloudman S91细胞的色素系统，更详细地研究了杂交细胞的黑素沉着表型。通过电子显微镜研究细胞，分析细胞裂解物中的酪氨酸酶（E.C.1.14.18.1）活性，并通过凝胶电泳和免疫印迹分析黑素小体蛋白。亲代Cloudman黑色素瘤细胞中的黑素小体数量少且相对无定形，而杂交细胞中的黑素小体数量众多且色素沉着严重，含有高度组织化的晶格结构。与亲代细胞相比，杂交细胞中基础和MSH诱导的酪氨酸酶活性均提高了几倍。与亲代黑色素瘤细胞中的相同蛋白质相比，杂交细胞中的酪氨酸酶、TRP-2和LAMP-1在凝胶上迁移得更慢，这与糖基化增加一致。杂交细胞中LAMP-1的迁移与腹膜巨噬细胞中的相似，腹膜巨噬细胞中的LAMP-1似乎也比Cloudman细胞中的LAMP-1糖基化程度更高。通过使用3H-葡糖胺作为N-糖基化的标志物，发现其掺入杂交细胞中的酪氨酸酶和LAMP-1的量增加，被N-糖基化抑制剂抑制，并且与亲代细胞相比，杂交细胞中受MSH刺激的程度更大。这些结果表明N-糖基化是MSH诱导的黑素生成的重要调节途径，并进一步表明N-连接糖基化的改变可能是巨噬细胞x黑色素瘤杂交细胞中黑素生成和转移调节的潜在机制。
