Identification and characterization of a Kunitz-type protease inhibitor inascites fluid from patients with ovarian carcinoma

Urinary trypsin inhibitor (UTI; Mr 40 kDa) is a Kunitz-type protease inhibi tor that efficiently inhibits cell-associated trypsin and plasmin activitie s. The aim of this study is to examine the expression pattern of UTI in the human ovarian carcinoma ascites fluid by Western blotting, zymography, imm unoprecipitation, immunohistochemistry, biochemical and gene analyses and a nimal experiments. We have identified and characterized the 40 kDa immunore active UTI (UTI40) and 8 kDa degradation fragment (UTI8) in ascites fluid. The levels of UTI40 and UTI8 are elevated in ascites fluid taken from patie nts with ovarian carcinoma relative to paired plasma samples. The UTI40 and UTI8 were identified immunologically by the reactivity with 2 different: a nti-UTI antibodies recognizing different epitopes of the UTI molecule, func tionally by its ability to bind trypsin and structurally by its apparent mo lecular mass with and without deglycosylation treatment. The purified polyp eptides have been sequenced and were identical with sequences obtained from UTI and the carboxyl-terminal domain of UTI, respectively. However, UTI mR NA was not detected in the ovarian carcinoma tissue and ovarian carcinoma c ell lines examined. Based on extravasation experiments using intravenously injected biotinylated inter-alpha-trypsin inhibitor (I alpha I; a precursor of UTI), we conclude that UTI40 and UTI8 found in the ascites fluid may re sult from (i) the extravasation of plasma proteins such as I alpha I into t he peritoneal cavity via hyperpermeable vessels and (ii) the subsequent deg radation of I alpha I and UTI40 by tumor cell-associated trypsin-like enzym es. Int. J. Cancer 87:44-54, 2000. (C) 2000 Wiley-Liss. Inc.