Muscarinic cholinergic receptors in the human melanoma cell line SK-Mel 28: modulation of chemotaxis
Muscarinic cholinergic receptors in the human melanoma cell line SK-Mel 28: modulation of chemotaxis
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DOI：
10.1111/j.1365-2230.2005.01865.x
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发表时间：
2005-09-01
影响因子：
4.1
通讯作者：
Drews, U
中科院分区：
文献类型：
--
作者：
Boss, A;Oppitz, M;Drews, U
原发性和转移性黑色素瘤表达毒蕈碱受体。在胚胎组织中，毒蕈碱受体的表达与形态发生相关。已经提出的假设是，毒蕈碱受体参与胚胎的形态发生运动，并在黑色素瘤细胞在侵袭性生长过程中的细胞运动。本研究的目的是在人黑色素瘤细胞系SK-Mel 28中表征毒蕈碱受体，并在Boyden室测定中测试对纤连蛋白的趋化活性是否可以受到毒蕈碱刺激的影响。在Western印迹与单克隆抗体M35，毒蕈碱受体定位在66 kDa的强带，并在63 kDa的弱带。用M3亚型特异性抗体的Western印迹在66 kDa处再现了该线。RT-PCR显示M3和M5亚型的mRNA。这些发现表明SK-Mel 28细胞表达大量的M3亚型和少量的M5亚型受体。显微镜观察毒蕈碱刺激后的钙动员表明，所有细胞携带功能性毒蕈碱受体。以纤连蛋白为趋化剂，在改良的Boyden小室中建立了标准化的趋化试验。加入卡巴胆碱后的上室，纤连蛋白诱导的趋化性增加类似于30%，观察到的效果废除阿托品。这些结果表明，毒蕈碱胆碱能治疗具有调节作用的SK-梅尔28黑色素瘤细胞中的纤连蛋白诱导的趋化性，表明毒蕈碱系统参与调节细胞运动。
Primary and metastatic human melanomas express muscarinic receptors. In embryonic tissues, expression of muscarinic receptors is correlated with morphogenesis. The hypothesis has been put forward that muscarinic receptors are involved in morphogenetic movements in the embryo, and in cellular movements in melanoma cells during invasive growth. The purpose of the present study was to characterize the muscarinic receptors in the human melanoma cell line SK-Mel 28 and to test in a Boyden chamber assay whether the chemotactic activity towards fibronectin can be influenced by muscarinic stimulation. In Western blots with the monoclonal antibody M35, muscarinic receptors were localized in a strong band at 66 kDa, and in a weak band at 63 kDa. Western blot with M3 subtype specific antibodies reproduced the line at 66 kDa. RT-PCR revealed mRNA for subtypes M3 and M5. These findings suggest that SK-Mel 28 cells express a large number of subtype M3 and a small number of subtype M5 receptors. Microscopic observation of calcium mobilization after muscarinic stimulation indicated that all cells carried functional muscarinic receptors. A standardized chemotaxis assay was established in modified Boyden chambers using fibronectin as chemotactic agent. After addition of carbachol to the upper compartment, an increase of fibronectin induced chemotaxis of similar to 30% was observed, an effect abrogated by atropine. These results demonstrate that muscarinic cholinergic treatment has a modulatory effect on fibronectin-induced chemotaxis in SK-Mel 28 melanoma cells, indicating that the muscarinic system is involved in regulation of cell movement.