Fernández-Salas E, Peracaula R, Frazier M L, de Llorens R
Unitat de Bioquímica, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Spain.
Eur J Biochem. 2000 Mar;267(5):1484-94. doi: 10.1046/j.1432-1327.2000.01148.x.
Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.
人核糖核酸酶被认为是胰腺癌可能的肿瘤标志物，许多作者报道了胰腺癌患者血清中核糖核酸酶活性升高。这种升高的原因尚不清楚。在本研究中，我们证明人胰腺腺癌细胞系合成并分泌不同的核糖核酸酶。我们从人胰腺腺癌细胞系Capan-1、MDAPanc-3、IBF-CP3和Panc-1以及壶腹腺癌细胞系MDAAmp-7的条件培养基中分离并鉴定了人胰腺或分泌型核糖核酸酶（RNase 1），这些细胞系代表了广泛的分化阶段。这些细胞系中只有Panc-1产生大量的非分泌型核糖核酸酶。然后，我们建立了分泌型和非分泌型核糖核酸酶的纯化程序，包括通过切向过滤浓缩上清液、阴离子交换和阳离子交换液相色谱以及C4反相高效液相色谱。使用分光光度法和负染酶谱技术监测核糖核酸酶活性级分。N端序列分析、动力学分析和内切糖苷酶消化研究结果表明，所有细胞系分泌的主要核糖核酸酶是分泌型核糖核酸酶，它由几种不同N-糖基化形式组成。Northern印迹分析证实，一些细胞系表达分泌型核糖核酸酶mRNA。Panc-1和MDAPanc-28产生的mRNA水平过低无法检测到。除Panc-1表达水平较高外，通过Northern印迹分析在所有细胞系中发现非分泌型核糖核酸酶的表达水平相似。在这里，我们首次描述了几种不同分化程度的人胰腺癌细胞系表达并分泌核糖核酸酶。这一事实表明，胰腺癌患者血清核糖核酸酶水平升高的一个来源是肿瘤细胞。对Capan-1分泌的RNase 1的寡糖部分分析表明，它高度糖基化，其N-聚糖链与正常胰腺产生的RNase 1有显著差异。这些结果再次表明了将人血清RNase 1测定用作肿瘤标志物的可能性。
