title: Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.
abstract: 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.
authors.affiliation: Unitat de Bioquímica, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Fernández-Salas
authors.forename: R
authors.initials: R
authors.lastname: Peracaula
authors.forename: M L
authors.initials: ML
authors.lastname: Frazier
authors.forename: R
authors.initials: R
authors.lastname: de Llorens
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000473:pathology; D001483:Base Sequence; D017077:Culture Media, Conditioned; D017931:DNA Primers; D006801:Humans; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D012260:Ribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D017077:Culture Media, Conditioned; D017931:DNA Primers; D012333:RNA, Messenger; D012260:Ribonucleases; D014357:Trypsin