Identification and characterization of a Kunitz-type protease inhibitor in ascites fluid from patients with ovarian carcinoma.
Urinary trypsin inhibitor (UTI; Mr 40 kDa) is a Kunitz-type protease inhibitor that efficiently inhibits cell-associated trypsin and plasmin activities. The aim of this study is to examine the expression pattern of UTI in the human ovarian carcinoma ascites fluid by Western blotting, zymography, immunoprecipitation, immunohistochemistry, biochemical and gene analyses and animal experiments. We have identified and characterized the 40 kDa immunoreactive UTI (UTI(40)) and 8 kDa degradation fragment (UTI(8)) in ascites fluid. The levels of UTI(40) and UTI(8) are elevated in ascites fluid taken from patients with ovarian carcinoma relative to paired plasma samples. The UTI(40) and UTI(8) were identified immunologically by the reactivity with 2 different anti-UTI antibodies recognizing different epitopes of the UTI molecule, functionally by its ability to bind trypsin and structurally by its apparent molecular mass with and without deglycosylation treatment. The purified polypeptides have been sequenced and were identical with sequences obtained from UTI and the carboxyl-terminal domain of UTI, respectively. However, UTI mRNA was not detected in the ovarian carcinoma tissue and ovarian carcinoma cell lines examined. Based on extravasation experiments using intravenously injected biotinylated inter-alpha-trypsin inhibitor (IalphaI; a precursor of UTI), we conclude that UTI(40) and UTI(8) found in the ascites fluid may result from (i) the extravasation of plasma proteins such as IalphaI into the peritoneal cavity via hyperpermeable vessels and (ii) the subsequent degradation of IalphaI and UTI(40) by tumor cell-associated trypsin-like enzymes.
Kobayashi
H
H
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
Hirashima
Y
Y
Sun
G W
GW
Ohi
H
H
Fujie
M
M
Terao
T
T
D018262:Adenocarcinoma, Clear Cell / Q000378:metabolism; D002288:Adenocarcinoma, Mucinous / Q000378:metabolism; D000818:Animals; D001202:Ascitic Fluid / Q000378:metabolism*; D019921:Biotinylation; D015153:Blotting, Western; D018269:Carcinoma, Endometrioid / Q000378:metabolism; D018284:Cystadenocarcinoma, Serous / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008222:Lymphokines / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000378:metabolism*; D010455:Peptides; D010940:Plant Proteins; D011233:Precipitin Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014361:Trypsin Inhibitors / Q000096:biosynthesis* / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; C053865:Kunitz-type protease inhibitor, plant; D008222:Lymphokines; D010455:Peptides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors