Human trypsinogens in the pancreas and in cancer.

This study led to the development of monoclonal antibodies and time-resolved immunofluorometric methods recognizing human trypsinogen-1 and -2, respectively. Using these methods in normal sera the concentration of trypsinogen-1 was found to be higher than that of trypsinogen-2. However, in acute pancreatitis the concentration of serum trypsinogen-2 was 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentration was only 15-fold. Serum samples from patients who had undergone pancreatoduodenectomy contained trypsinogen-2, while trypsinogen-1 was detected in only one of nine samples. Furthermore, in human ovarian cyst fluids tumor-associated trypsinogen-2 (TAT-2) is the predominant isoenzyme and in mucinous cyst fluids the levels of TAT-2 were associated with malignancy. These results suggest that (i) trypsinogen-2 could be used as a diagnostic marker for acute pancreatitis, (ii) its expression is not restricted to the pancreas, and (iii) TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. In ion exchange chromatography, isoelectric variants of the trypsinogen isoenzymes were seen. Mass spectrometric analysis of these revealed that pancreatic trypsinogens are sulfated at tyrosine 154 (Tyr154), whereas TAT-2 from a colon carcinoma cell line is not. Tyr154 is located within the primary substrate binding pocket of trypsin. Thus, Tyr154 sulfation is likely to influence substrate binding. The previously known differences in charge and substrate binding between pancreatic and tumor-associated trypsinogens are suggested to be caused by sulfation of Tyr154 in pancreatic trypsinogens.

Tropical calcific pancreatitis and its association with CTRC and SPINK1 (p.N34S) variants.

BACKGROUND: Tropical calcific pancreatitis (TCP) is a relatively common form of chronic pancreatitis in parts of Asia and Africa. The SPINK1 variant p.N34S is strongly associated with TCP, but other genetic factors remain to be defined. Chymotrypsinogen C (CTRC) degrades trypsinogen and loss-of-function variants have been found in European patients with chronic pancreatitis. Preliminary data indicate that CTRC might increase the risk for TCP. MATERIALS AND METHODS: We selected 150 Indian TCP patients and 150 Indian controls to perform mutational screening of the complete coding region of CTRC and exon 3 of SPINK1. We performed in-silico analysis and functional studies of novel CTRC variants. RESULTS: We identified eight variants among this sample. Three were synonymous and c.180 C>T was significantly enriched in patients (odds ratio=2.09; 95% confidence interval=1.19-3.67; P=0.03). We identified a novel nonsynonymous CTRC (p.G61R) variant in one of 146 patients (0.7%), but absent from controls. In-silico analysis showed that this variant affected a conserved residue, and functional analysis showed that p.G61R results in a complete loss of CTRC secretion from transiently transfected human embryonic kidney 293T cells. SPINK1 p.N34S was present in 31.8% of patients compared with 4.7% in controls, there was no significant cosegregation with CTRC variants. CONCLUSION: The contribution of CTRC variants to TCP is relatively small, but the identification of novel loss-of-function variants (p.G61R) underscores the importance of the trypsinogen pathway in causing TCP.

Divergent roles of SPINK1 and PRSS2 variants in tropical calcific pancreatitis.

BACKGROUND/AIMS: Tropical calcific pancreatitis (TCP) refers to a type of idiopathic pancreatitis prevalent in Asia. The trypsin inhibitor (SPINK1) N34S variant partially explains the genetic susceptibility to TCP. As anionic trypsinogen (PRSS2) G191R protects against chronic pancreatitis in Europeans, we investigated whether this variant protects from TCP in Indians. METHODS: We enrolled 174 patients and 794 controls from two Indian tertiary care referral hospitals. We analyzed PRSS2 and SPINK1 variants by melting curve analysis, allele-specific discrimination assay, and sequencing. RESULTS: G191R was detected in 1 TCP patient (0.6%) compared to 13 controls (1.6%; OR 0.27, 95% CI 0.03-2.1; p = 0.33). SPINK1 N34S was enriched in the TCP population 67/174 (38.5%) compared to controls 10/234 (4.3%; OR 14, 95% CI 6.9-28.3; p < 0.001). CONCLUSION: G191R PRSS2 is a rare allele in the Indian population and the data suggest a nonsignificant trend towards a protective effect. N34S SPINK1 represents the major genetic risk factor in TCP.

The role of NF-kappaB activation in the pathogenesis of acute pancreatitis.

Acute pancreatitis is an inflammatory disease of the pancreas which, in its most severe form, is associated with multi-organ failure and death. Recently, signalling molecules and pathways which are responsible for the initiation and progression of this disease have been under intense scrutiny. One important signalling molecule, nuclear factor kappaB (NF-kappaB), has been shown to play a critical role in the development of acute pancreatitis. NF-kappaB is a nuclear transcription factor responsible for regulating the transcription of a wide variety of genes involved in immunity and inflammation. Many of these genes have been implicated as central players in the development and progression of acute pancreatitis. This review discusses recent advances in the investigation of pancreatic and extrapancreatic (lungs, liver, monocytes and macrophages, and endothelial cells) NF-kappaB activation as it relates to acute pancreatitis.

Germline mutations and gene polymorphism associated with human pancreatitis.

A wide range of mutations and polymorphisms in genes that relate to pancreatic function seem to be involved in the development of pancreatitis. Some of these genetic alterations lead to disease phenotypes with unequivocal mendelian inheritance patterns, whereas others seem to act as modifier genes in conjunction with environ-mental or, as yet unidentified, genetic cofactors. This article reviews germline changes in the genes for trypsin, pancreatic secretory trypsin inhibitor, the cystic fibrosis conductance regulator, lipid metabolism proteins, inflammatory mediators for cytokines, and cathepsin B.

Enhanced invasiveness of pancreatic adenocarcinoma cells stably transfected with cationic trypsinogen cDNA.

Various studies have described increased expression of cationic trypsinogen in malignant tumor cells. To explore the role of secreted cationic trypsinogen in invasion by cancer cells, we introduced cationic trypsinogen cDNA into Panc-1, a pancreatic adenocarcinoma-derived cell line that lacks expression of endogeneous trypsinogen. Four independent clones (designated Panc-1-Try-7, -9, -11 and -24) stably expressing cationic trypsinogen mRNA were isolated and processed for further study. In a zymographic analysis, gelatinolytic activity for cationic trypsinogen was detectable in serum-free conditioned media obtained from all 4 transfectants but not in media from mock-transfected or parental Panc-1 cells. A Matrigel invasion assay revealed that all trypsinogen-expressing transfectants acquired significantly greater invasive ability than that shown by mock-transfected and parental Panc-1 cells. In addition, enhanced invasiveness of the transfectants was suppressed by FUT-175, a serine protease inhibitor, to the level seen in parental cells. These results provide direct evidence that cationic trypsinogen can increase the invasive ability of carcinoma cells.

CCK independently activates intracellular trypsinogen and NF-kappaB in rat pancreatic acinar cells.

In the cholecystokinin (CCK) hyperstimulation model of acute pancreatitis, two early intracellular events, activation of trypsinogen and activation of nuclear factor-kappaB (NF-kappaB), are thought to be important in the development of the disease. In this study, the relationship between these two events was investigated. NF-kappaB activity was monitored by using a DNA binding assay and mob-1 chemokine gene expression. Intracellular trypsin activity was measured by using a fluorogenic substrate. Protease inhibitors including FUT-175, Pefabloc, and E-64d prevented CCK stimulation of intracellular trypsinogen and NF-kappaB activation. Likewise, the NF-kappaB inhibitors pyrrolidine dithiocarbamate and N-acetyl-L-cysteine inhibited CCK stimulation of NF-kappaB and intracellular trypsinogen activation. These results suggested a possible codependency of these two events. However, CCK stimulated NF-kappaB activation in Chinese hamster ovary-CCK(A) cells, which do not express trypsinogen, indicating that trypsin is not necessary for CCK activation of NF-kappaB. Furthermore, adenovirus-mediated expression in acinar cells of active p65 subunits to stimulate NF-kappaB, or of inhibitory kappaB-alpha molecules to inhibit NF-kappaB, did not affect either basal or CCK-mediated trypsinogen activation. Thus trypsinogen and NF-kappaB activation are independent events stimulated by CCK.

Expression and characterization of trypsinogen produced in the human male genital tract.

Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M(r) of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.

Human colon carcinoma, fibrosarcoma and leukemia cell lines produce tumor-associated trypsinogen.

Previous studies have indicated that cyst fluid of ovarian tumors contains 2 trypsinogen isoenzymes, called tumor-associated trypsinogen-I (TAT-I) and trypsinogen-2 (TAT-2), the levels of which correlate with the degree of malignancy of the tumors. In addition, these cyst fluids contain large amounts of tumor-associated trypsin inhibitor (TATI), which is also expressed in many other human tumors. In the present study we examined the production of TAT-I, TAT-2 and TATI in 9 established tumor-cell lines. TAT-2 was produced by 5 cell lines. Its concentration in the conditioned medium of COLO 205 colon adenocarcinoma cells, K-562 erythroleukemia cells and fibrosarcoma cell lines HT 1080, 8387 and A 9733 was 460 micrograms/l, 9.8 micrograms/l, 21 micrograms/l, 8.8 micrograms/l and 0.24 micrograms/l, respectively. TAT-I was detectable in the conditioned medium of COLO 205 and HT 1080 cells at concentrations of 64 micrograms/l and 0.5 micrograms/l, respectively. TATI was detected only in the media of COLO 205 cells at a concentration of 23 micrograms/l. TAT-2 zymogen was purified from the conditioned medium of COLO 205 and HT 1080 cells by immunoaffinity chromatography. According to its aminoterminal amino acid sequence, a molecular mass of 28 kDa by SDS-PAGE, elution pattern in ion-exchange chromatography and ability to be activated by enteropeptidase, the zymogen is identical to that previously isolated from cyst fluid of ovarian tumors. In addition, we found that TAT-2 secretion could be down-regulated by dexamethasone in HT 1080 cells but not in COLO 205 cells. The abundant production of TAT-2 isoenzyme in different cancer cell lines suggests that it could contribute to the increased proteolytic activity of many human tumors.