Sharma S D, Jiang J, Hadley M E, Bentley D L, Hruby V J
Department of Chemistry, Arizona Research Laboratories, University of Arizona, Tucson 85721, USA.
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13715-20. doi: 10.1073/pnas.93.24.13715.
We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone (MSH) receptors of melanoma cells. Multiple copies of [Nle-4,D-Phe-7]-alpha-MSH, a potent analog of alpha-MSH, were conjugated to microspheres (latex beads) or macrospheres (polyamide beads) through a thioether or disulfide bond. Binding between the beads and mouse and human melanoma cells was examined by scanning electron microscopy and by light microscopy. Each mouse and human melanoma cell (of all cell lines) evinced binding to the beads. Binding of the melanotropin conjugates was not restricted to any one phase of the cell cycle. Specificity of binding was demonstrated by several studies. Negative controls included cell types of nonmelanocyte origin (e.g., mammary cancer cells) and beads that lacked the melanotropic ligand or had other attached ligands. Beads with a disulfide-linked melanotropin analog served as a direct control. Treatment of these beads with DTT during or before incubation of the beads with melanoma cells (resulting in release of the MSH analog from the beads) eliminated binding of the beads to melanoma cells. Binding interactions between melanoma cells and melanotropin-bound beads also could be abolished by prior incubation with unconjugated MSH analog. During these experiments, certain membrane receptor-hormone associated phenomena, such as capping (aggregation) of the receptor-ligand complex, also were observed. These results provide visual evidence that MSH receptors are a property common to melanoma cells. Normal human epidermal melanocytes and keratinocytes were also shown to express melanotropin receptors by the same criteria established for melanoma cells.
我们开发了两种固相试剂系统，用于黑色素瘤细胞促黑素（MSH）受体的显微镜可视化和表征。强效α-MSH类似物[Nle-4,D-Phe-7]-α-MSH的多个拷贝通过硫醚或二硫键与微球（乳胶珠）或大球（聚酰胺珠）偶联。通过扫描电子显微镜和光学显微镜检查珠子与小鼠和人类黑色素瘤细胞之间的结合。每个小鼠和人类黑色素瘤细胞（所有细胞系）均显示出与珠子的结合。促黑素缀合物的结合不限于细胞周期的任何一个阶段。多项研究证明了结合的特异性。阴性对照包括非黑素细胞来源的细胞类型（例如乳腺癌细胞）以及缺乏促黑素配体或带有其他附着配体的珠子。带有二硫键连接的促黑素类似物的珠子用作直接对照。在珠子与黑色素瘤细胞孵育期间或之前用二硫苏糖醇处理这些珠子（导致MSH类似物从珠子上释放）消除了珠子与黑色素瘤细胞的结合。黑色素瘤细胞与促黑素结合的珠子之间的结合相互作用也可以通过预先与未偶联的MSH类似物孵育来消除。在这些实验中，还观察到了某些膜受体-激素相关现象，例如受体-配体复合物的封帽（聚集）。这些结果提供了视觉证据，表明MSH受体是黑色素瘤细胞共有的特性。通过为黑色素瘤细胞建立的相同标准，还表明正常人表皮黑素细胞和角质形成细胞表达促黑素受体。
