Lotan R, Lotan D, Deutsch V
Cancer Res. 1987 Jun 15;47(12):3152-8.
Previous studies have shown that treatment of S91-C2 murine melanoma cells with beta-all-trans-retinoic acid (RA) results in growth inhibition, enhanced activity of sialyltransferase, and increased glycosylation of a Mr 160,000 cell surface sialoglycoprotein (gp160). None of these effects could be detected in mutant clones (e.g., S91-C154) selected from the S91-C2 cells for resistance to RA-induced growth inhibition. These findings suggest that modulation by RA of gp160 might be related causally to growth inhibition. In this study we examined the possible role of gp160 in growth regulation using specific antibodies to this glycoprotein. Metabolic labeling of S91-C2 cells with either [3H]glucosamine or [35S]methionine revealed that the cells shed into the growth medium a gp160-like glycoprotein, in addition to several other macromolecules. The gp160-like glycoprotein was isolated from concentrated conditioned medium after preparative polyacrylamide slab gel electrophoresis in the presence of sodium dodecylsulfate by excision of the corresponding protein band. Rabbits were immunized with this material and immunoblotting revealed that their sera contained antibodies that bound specifically to gp160 in extracts of untreated or RA-treated S91-C2 cells. Indirect immunofluorescence staining followed by fluorescence-activated cell sorter analysis demonstrated that the anti-gp160 antibodies bound to the surface of both untreated and RA-treated S91-C2 cells and that the treated cells bound more of the antibodies than untreated ones. In contrast, these antibodies bound to the same extent to untreated and RA-treated resistant S91-C154 cells. The growth of S91-C2 cells in the presence of anti-gp160 antibodies in semisolid medium as well as in monolayer cultures was inhibited in a dose-dependent fashion. Fifty % growth inhibition was obtained at an immunoglobulin concentration of 10 micrograms/ml. The growth of cells exposed concurrently to RA and anti-gp160 antibodies was also inhibited strongly in semisolid medium, but the antibodies caused only a small increase in the inhibitory effect of RA in monolayer cultures. No inhibition by the antibodies of either anchorage-independent growth or anchorage-dependent growth of S91-C154 cells, grown in the absence or presence of RA, was observed. These results support the suggestion that cell surface gp160 might be involved in growth regulation in the S91-C2 cells.
先前的研究表明，用β-全反式维甲酸（RA）处理S91-C2小鼠黑色素瘤细胞会导致生长抑制、唾液酸转移酶活性增强以及一种分子量为160,000的细胞表面唾液酸糖蛋白（gp160）的糖基化增加。在从S91-C2细胞中筛选出的对RA诱导的生长抑制具有抗性的突变克隆（例如S91-C154）中，未检测到这些效应中的任何一种。这些发现表明，RA对gp160的调节可能与生长抑制存在因果关系。在本研究中，我们使用针对这种糖蛋白的特异性抗体来研究gp160在生长调节中的可能作用。用[3H]葡萄糖胺或[35S]甲硫氨酸对S91-C2细胞进行代谢标记显示，除了其他几种大分子外，细胞还向生长培养基中释放出一种gp160样糖蛋白。在十二烷基硫酸钠存在的情况下，通过制备性聚丙烯酰胺平板凝胶电泳从浓缩的条件培养基中分离出gp160样糖蛋白，方法是切除相应的蛋白条带。用这种材料免疫兔子，免疫印迹显示它们的血清中含有能与未处理或经RA处理的S91-C2细胞提取物中的gp160特异性结合的抗体。间接免疫荧光染色后进行荧光激活细胞分选分析表明，抗gp160抗体与未处理和经RA处理的S91-C2细胞表面均有结合，且处理后的细胞比未处理的细胞结合更多的抗体。相比之下，这些抗体与未处理和经RA处理的抗性S91-C154细胞的结合程度相同。在半固体培养基以及单层培养中，抗gp160抗体存在时S91-C2细胞的生长受到剂量依赖性抑制。在免疫球蛋白浓度为10微克/毫升时可获得50%的生长抑制。在半固体培养基中，同时暴露于RA和抗gp160抗体的细胞生长也受到强烈抑制，但在单层培养中，抗体仅使RA的抑制作用略有增加。未观察到抗体对在有无RA情况下生长的S91-C154细胞的非锚定依赖性生长或锚定依赖性生长有抑制作用。这些结果支持了细胞表面gp160可能参与S91-C2细胞生长调节的观点。
