Taylor D D, Taylor C G, Jiang C G, Black P H
Department of Microbiology, Hubert H. Humphrey Cancer Research Center, Boston, MA 02118.
Int J Cancer. 1988 Apr 15;41(4):629-35. doi: 10.1002/ijc.2910410425.
Tumor cells release intact portions of their plasma membranes in the process of membrane fragment shedding. This released material has been shown to inhibit various synthetic functions of normal cells, which may play an important role in certain patho-physiological events occurring in advanced-stage cancer patients. Our studies on metastatic variants of the murine B16 melanoma, B16-F1 (low incidence of lung colonization) and B16-F10 (high incidence of lung colonization) indicate that the shed membrane fragment material is composed predominantly of vesicles, ranging in size from 20 to 100 nm in diameter. The release of membrane fragments represents a small percentage (approximately 16%) of the total shedding of plasma membrane components. Membrane fragments were shed at a higher rate from the highly "metastatic" (colonizing) B16-F10 cells than from poorly metastatic B16-F1 cells, resulting in a 2-fold greater accumulation of membrane fragment material by cultures of B16-F10 cells than by B16-F1 cultures during the 48-hr assay period. The study of various intracellu ar metabolic processes (protein and RNA synthesis, glycosylation, and generation of ATP) required for the shedding of membrane fragments indicated that the shedding event is only dependent on energy when inhibitors of the above processes are present for 2 hr. Treatment of cells with these inhibitors for 8 hr results in cessation of the shedding process, indicating both a limited pool of components to be shed and the requirement for further synthesis of the shed material. Glycoprotein components of the shed membrane fragments were analyzed by SDS-polyacrylamide gel electrophoresis. In addition to quantitative differences, 2 additional bands were present in fluorographs from SDS-PAGE gels from the B16-F10 membrane fragment material which were not present in fluorographs from B16-F1 fragments. The glycoprotein components of shed membrane fragments were shown to represent selected domains of the cell's plasma membranes, in that only certain plasma membrane glycoproteins are shed as part of membrane fragments. The glycoproteins released as non-particulate molecules into the extracellular environment failed to exhibit these quantitative and qualitative differences.
肿瘤细胞在细胞膜片段脱落过程中释放其质膜的完整部分。已证明这种释放的物质可抑制正常细胞的各种合成功能，这可能在晚期癌症患者发生的某些病理生理事件中起重要作用。我们对小鼠B16黑色素瘤的转移变体B16-F1（肺定植发生率低）和B16-F10（肺定植发生率高）的研究表明，脱落的膜片段物质主要由直径为20至100nm的囊泡组成。膜片段的释放占质膜成分总脱落量的一小部分（约16%）。膜片段从高度“转移性”（定植）的B16-F10细胞中的脱落速率高于低转移性的B16-F1细胞，导致在48小时测定期内，B16-F10细胞培养物中膜片段物质的积累量比B16-F1培养物多2倍。对膜片段脱落所需的各种细胞内代谢过程（蛋白质和RNA合成、糖基化以及ATP生成）的研究表明，只有当上述过程的抑制剂存在2小时时，脱落事件才依赖于能量。用这些抑制剂处理细胞8小时会导致脱落过程停止，这表明待脱落的成分库有限，并且需要进一步合成脱落物质。通过SDS-聚丙烯酰胺凝胶电泳分析了脱落膜片段的糖蛋白成分。除了定量差异外，来自B16-F10膜片段物质的SDS-PAGE凝胶荧光图中还出现了另外两条带，而B16-F1片段的荧光图中没有这些带。脱落膜片段的糖蛋白成分被证明代表细胞质膜的特定区域，因为只有某些质膜糖蛋白作为膜片段的一部分脱落。作为非颗粒分子释放到细胞外环境中的糖蛋白没有表现出这些定量和定性差异。
