Hughes S J, Nambu Y, Soldes O S, Hamstra D, Rehemtulla A, Iannettoni M D, Orringer M B, Beer D G
Department of Surgery, Section of Thoracic Surgery, University of Michigan Medical School, Ann Arbor 48109, USA.
Cancer Res. 1997 Dec 15;57(24):5571-8.
This study describes Fas (CD95) expression in Barrett's esophagus, adenocarcinomas of the esophagus, and three esophageal adenocarcinoma cell lines. Immunohistochemical analysis of Barrett's esophagus demonstrated cell surface expression of Fas protein. In contrast, 30.5% of esophageal adenocarcinomas examined by immunohistochemical analysis demonstrated faint cytoplasmic staining, and 69.5% were negative for Fas. Similar levels of Fas mRNA were identified in tumors compared to mRNA levels in esophageal squamous mucosa or Barrett's esophagus. An approximately Mr 48,000 Fas protein was identified by Western blot analysis in tumors that were negative for Fas expression by immunohistochemical analysis. The esophageal adenocarcinoma cell line Seg-1 was negative for Fas expression by immunohistochemical analysis, but Western blot analysis demonstrated abundant, appropriately sized Fas protein. In agreement with the immunohistochemical analysis, flow cytometry of Seg-1 showed minimal amounts of Fas on the cell surface, which correlated with resistance to Fas-mediated apoptosis. No mutations in the Seg-1 Fas coding sequence or exon 1 were identified by sequence analysis. This was confirmed by transient transfection of COS cells with expression vectors generated from the Seg-1 Fas cDNA, which resulted in cell surface expression of the Fas protein. Stable transfection of Seg-1 with a Fas expression vector did not result in efficient Fas expression on the cell surface. Seg-1 cells, transiently transfected with a Fas-FLAG expression vector and examined for protein expression using confocal microscopy and an anti-FLAG antibody, showed that the Fas-FLAG protein was not present on the cell surface but was present in the cytoplasm. Taken together, these results indicate that expression of Fas on the cell surface by esophageal adenocarcinoma is reduced. In an esophageal adenocarcinoma cell line, wild-type Fas protein is retained in the cytoplasm, and this correlates with resistance to Fas-mediated apoptosis. The retention of wild-type Fas protein within the cytoplasm may represent a mechanism by which malignant cells evade Fas-mediated apoptosis.
本研究描述了Fas（CD95）在巴雷特食管、食管腺癌及三种食管腺癌细胞系中的表达情况。对巴雷特食管进行免疫组织化学分析显示Fas蛋白在细胞表面表达。相比之下，通过免疫组织化学分析检测的食管腺癌中，30.5%表现为微弱的细胞质染色，69.5%的Fas呈阴性。与食管鳞状黏膜或巴雷特食管中的mRNA水平相比，肿瘤中Fas mRNA的水平相似。通过蛋白质印迹分析在免疫组织化学分析中Fas表达阴性的肿瘤中鉴定出一种分子量约为48,000的Fas蛋白。食管腺癌细胞系Seg-1通过免疫组织化学分析Fas表达呈阴性，但蛋白质印迹分析显示有大量大小合适的Fas蛋白。与免疫组织化学分析结果一致，Seg-1的流式细胞术显示细胞表面Fas含量极少，这与对Fas介导的凋亡的抗性相关。通过序列分析未在Seg-1 Fas编码序列或外显子1中鉴定到突变。用从Seg-1 Fas cDNA生成的表达载体对COS细胞进行瞬时转染证实了这一点，这导致Fas蛋白在细胞表面表达。用Fas表达载体对Seg-1进行稳定转染未导致Fas在细胞表面有效表达。用Fas-FLAG表达载体对Seg-1进行瞬时转染，并使用共聚焦显微镜和抗FLAG抗体检测蛋白质表达，结果显示Fas-FLAG蛋白不在细胞表面，而是存在于细胞质中。综上所述，这些结果表明食管腺癌细胞表面Fas的表达减少。在一种食管腺癌细胞系中，野生型Fas蛋白保留在细胞质中，这与对Fas介导的凋亡的抗性相关。野生型Fas蛋白在细胞质中的保留可能代表恶性细胞逃避Fas介导的凋亡的一种机制。
