Ungefroren H, Kruse M L, Trauzold A, Roeschmann S, Roeder C, Arlt A, Henne-Bruns D, Kalthoff H
Clinic for General Surgery and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany.
J Cell Sci. 2001 Aug;114(Pt 15):2735-46. doi: 10.1242/jcs.114.15.2735.
In this study we investigated the functional role of FAP-1 as a potential inhibitor of CD95 (Fas, APO-1)-mediated apoptosis in pancreatic cancer cells. Stable transfection of the CD95-sensitive, FAP-1-negative cell line Capan-1 with an FAP-1 cDNA resulted in a strongly decreased sensitivity to CD95-induced apoptosis, as measured by DNA fragmentation and caspase-3 activity. Inhibition of cellular protein tyrosine phosphatases with orthovanadate dose-dependently increased CD95-induced apoptosis in CD95-resistant FAP-1-positive Panc89 and Capan-1-FAP-1 cells almost to the level seen in wild-type Capan-1 cells. Blocking the CD95/FAP-1 interaction in Panc89 cells by cytoplasmic microinjection of a synthetic tripeptide mimicking the C terminus of CD95 resulted in a mean 5.5-fold increase in apoptosis compared to cells that received a control peptide. Using confocal laser scanning microscopy we show that in Panc89 cells FAP-1 is mainly associated with the Golgi complex and with peripheral vesicles. FAP-1 displayed enhanced colocalization with CD95 upon CD95 stimulation in the Golgi complex but not in surface-associated vesicles. This correlated with a decrease in plasma membrane staining for CD95 as determined by FACS analysis. Inhibition of Golgi anterograde transport by brefeldin A abolished the anti-CD95-induced colocalization of FAP-1 and CD95 as well as the decrease in cell-surface-associated CD95. Finally, we demonstrate by immunohistochemistry that FAP-1 is strongly expressed in tumor cells from pancreatic carcinoma tissues. Taken together, these results show that FAP-1 can protect pancreatic carcinoma cells from CD95-mediated apoptosis, probably by preventing anti-CD95-induced translocation of CD95 from intracellular stores to the cell surface.
在本研究中，我们调查了FAP-1作为胰腺癌细胞中CD95（Fas，APO-1）介导的细胞凋亡潜在抑制剂的功能作用。用FAP-1 cDNA稳定转染对CD95敏感、FAP-1阴性的细胞系Capan-1，导致对CD95诱导的细胞凋亡敏感性显著降低，这通过DNA片段化和caspase-3活性来测定。用原钒酸盐抑制细胞蛋白酪氨酸磷酸酶可剂量依赖性地增加CD95抗性FAP-1阳性的Panc89和Capan-1-FAP-1细胞中CD95诱导的细胞凋亡，几乎达到野生型Capan-1细胞中的水平。通过细胞质显微注射模拟CD95 C末端的合成三肽来阻断Panc89细胞中的CD95/FAP-1相互作用，与接受对照肽的细胞相比，细胞凋亡平均增加了5.5倍。使用共聚焦激光扫描显微镜，我们显示在Panc89细胞中，FAP-1主要与高尔基体复合体和外周囊泡相关。在高尔基体复合体中，CD95刺激后FAP-1与CD95的共定位增强，但在表面相关囊泡中没有。这与通过流式细胞术分析确定的CD95细胞膜染色减少相关。用布雷菲德菌素A抑制高尔基体顺向转运消除了FAP-1和CD95的抗CD95诱导的共定位以及细胞表面相关CD95的减少。最后，我们通过免疫组织化学证明FAP-1在胰腺癌组织的肿瘤细胞中强烈表达。综上所述，这些结果表明，FAP-1可能通过阻止抗CD95诱导的CD95从细胞内储存库转运到细胞表面，从而保护胰腺癌细胞免受CD95介导的细胞凋亡。
