Abstract: We investigated the biochemical properties and cellular expression of the c.346C>T (p.R116C) human cationic trypsinogen (PRSS1) mutant, which we identified in a German family with autosomal dominant hereditary pancreatitis. This mutation leads to an unpaired Cys residue with the potential to interfere with protein folding via incorrect disulfide bond formation. Recombinantly expressed p.R116C trypsinogen exhibited a tendency for misfolding in vitro. Biochemical analysis of the correctly folded, purified p.R116C mutant revealed unchanged activation and degradation characteristics compared to wild type trypsinogen. Secretion of mutant p.R116C from transfected 293T cells was reduced to approximately 20% of wild type. A similar secretion defect was observed with another rare PRSS1 variant, p.C139S, whereas mutants p.A16V, p.N29I, p.N29T, p.E79K, p.R122C, and p.R122H were secreted normally. All mutants were detected in cell extracts at comparable levels but a large portion of mutant p.R116C was present in an insoluble, protease-sensitive form. Consistent with intracellular retention of misfolded trypsinogen, the endoplasmic reticulum (ER) stress markers immunoglobulin-binding protein (BiP) and the spliced form of the X-box binding protein-1 (XBP1s) were elevated in cells expressing mutant p.R116C. The results indicate that mutation-induced misfolding and intracellular retention of human cationic trypsinogen causes hereditary pancreatitis in carriers of the p.R116C mutation. ER stress triggered by trypsinogen misfolding represents a new potential disease mechanism for chronic pancreatitis.
Abstract: BACKGROUND: Aberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour. METHODS: We fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish. RESULTS: In the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature. CONCLUSION: Our results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.
Abstract: The mammalian pancreas originates from two developing buds on the dorsal and ventral side of the duodenum which fuse and convert into a single mixed gland, composed of exocrine and endocrine cells. In the adult organism, the exocrine pancreas consists of acinar and ductal cells which are organised in a lobular branched tissue architecture and secrete and transport digestive enzymes into the duodenum. Mature endocrine cells, which represent only 1-2% of the pancreatic organ volume, form aggregates of so called islets of Langerhans within the exocrine pancreatic tissue and control glucose homeostasis by secretion of glucagon, insulin and other hormones into the bloodstream. Pancreatitis is the most common and a potentially lethal disorder of the exocrine pancreas with limited therapeutic options. A major obstacle in the development of successful treatment strategies has, until today, been our limited knowledge of the disease pathophysiology. This review will summarise recent advances in our understanding of the physiological mechanisms involved in the early disease processes of the exocrine pancreas.
Abstract: OBJECTIVE: The R122H mutation represents the most common point mutation of the cationic trypsinogen gene (PRSS1) in patients with hereditary pancreatitis (HP; Online Mendelian inheritance in man [OMIM] 167800), a rare variety of chronic pancreatitis. We identified a large number of HP families carrying this mutation in a confined region of Northern Germany within a 100-km radius. This apparent clustering could be due to the inheritance from a common ancestor (founder effect). METHODS: To address this question, we genotyped SNPs in close vicinity of the PRSS1 locus and determined common haplotypes. RESULTS: In members from 10 unrelated HP families (all R122H-positive), we found 7 different haplotypes to segregate with the R122H mutation. CONCLUSIONS: This virtually excludes a founder effect and suggests the presence of a mutational hot spot in codon 122 of the PRSS1 gene. An ascertainment bias of a large-volume referral center may have contributed to the locally increased detection of HP cases.
Abstract: Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.
Abstract: 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.
Abstract: Keratin 8 (KRT8) is one of the major intermediate filament proteins expressed in single-layered epithelia of the gastrointestinal tract. Transgenic mice over-expressing human KRT8 display pancreatic mononuclear infiltration, interstitial fibrosis and dysplasia of acinar cells resulting in exocrine pancreatic insufficiency. These experimental data are in accordance with a recent report describing an association between KRT8 variations and chronic pancreatitis. This prompted us to investigate KRT8 polymorphisms in patients with pancreatic disorders. The KRT8 Y54H and G62C polymorphisms were assessed in a cohort of patients with acute and chronic pancreatitis of various aetiologies or pancreatic cancer originating from Austria (n=16), the Czech Republic (n=90), Germany (n=1698), Great Britain (n=36), India (n=60), Italy (n=143), the Netherlands (n=128), Romania (n=3), Spain (n=133), and Switzerland (n=129). We also studied 4,234 control subjects from these countries and 1,492 control subjects originating from Benin, Cameroon, Ethiopia, Ecuador, and Turkey. Polymorphisms were analysed by melting curve analysis with fluorescence resonance energy transfer probes. The frequency of G62C did not differ between patients with acute or chronic pancreatitis, pancreatic adenocarcinoma and control individuals. The frequency of G62C varied in European populations from 0.4 to 3.8%, showing a northwest to southeast decline. The Y54H alteration was not detected in any of the 2,436 patients. Only 3/4,580 (0.07%) European, Turkish and Indian control subjects were heterozygous for Y54H in contrast to 34/951 (3.6%) control subjects of African descent. Our data suggest that the KRT8 alterations, Y54H and G62C, do not predispose patients to the development of pancreatitis or pancreatic cancer.
Abstract: Hereditary pancreatitis should be assumed if other risk factors for the disease can not been identified and if the patient has a family history for recurrent pancreatitis, chronic pancreatitis or pancreatic cancer. Since patients with chronic pancreatitis due to mutations in the cationic trypsinogen-gene have a much higher lifetime risk of developing pancreatic cancer, specifically if they are smokers, an adequate long-term follow up in specialized centers is recommended. The most frequent genetic changes in patients with hereditary pancreatitis are mutations in the cationic trypsinogen gene. Mutations in the CFTR-gene or SPINK1-gene have been reported in patients with idiopathic pancreatitis. The clinical relevance and the therapeutic consequences of these mutations is still controversial. Genetic testing is recommended when a patient with idiopathic pancreatitis is under 25 years at diagnosis or when one or more family members have either pancreatitis or pancreatic cancer. Genetic analysis of asymptomatic family members should only be offered after adequate genetic counselling. Prenatal diagnostic is not recommended.
Abstract: BACKGROUND: We have previously shown that cell contacts between pancreatic acinar cells dissociate early in pancreatitis and that this is a prerequisite for the development of pancreatic oedema. Here we studied the underlying mechanism. METHODS: Employing experimental caerulein induced pancreatitis in vivo and isolated pancreatic acini ex vivo, in conjunction with protein chemistry, morphology, and electron microscopy, we determined whether cell contact regulation in the pancreas requires or involves: (1) changes in cadherin-catenin protein expression, (2) tyrosine phosphorylation of adhesion proteins, or (3) alterations in the actin cytoskeleton. RESULTS: During initial cell-cell contact dissociation at adherens junctions, expression of adhesion proteins remained stable. At time points of dissociated adherens junctions, the cadherin-catenin complex was found to be tyrosine phosphorylated and internalised. The receptor type protein tyrosine phosphatase (PTP)kappa was constitutively associated with the cadherin-catenin complex at intact cell contacts whereas following the dissociation of adherens junctions, the internalised components of the cadherin-catenin complex were tyrosine phosphorylated and associated with the cytosolic PTP SHP-1. In isolated acini, inhibition of endogenous protein tyrosine phosphatases alone was sufficient to induce dissociation of adherens junctions analogous to that found with supramaximal caerulein stimulation. Dissociation of actin microfilaments had no effect on adherens junction integrity. CONCLUSIONS: These data identify tyrosine phosphorylation as the key regulator for cell contacts at adherens junctions and suggest a definitive role for the protein tyrosine phosphatases PTPkappa and SHP-1 in the regulation, maintenance, and restitution of cell adhesions in a complex epithelial organ such as the pancreas.
Abstract: BACKGROUND: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene-many of which cause cystic fibrosis-have also been reported in patients with chronic pancreatitis. The authors examine whether mild or severe CFTR mutations, homozygous or compound heterozygous CFTR mutations, or even simple cystic fibrosis carrier status alone increases the risk of developing pancreatitis. METHODS: After exclusion of patients with trypsinogen (PRSS1) mutations, cystic fibrosis, or pulmonary disease, and with known risk factors for pancreatitis 67 patients with idiopathic chronic pancreatitis (ICP) from northwest Germany and 60 geographically and ethnically matched controls were recruited. The entire coding region of the CFTR gene was sequenced in all patients and controls. ICP patients were also analysed for serine protease inhibitor Kazal type 1 (SPINK1) gene mutations. RESULTS: Abnormal CFTR alleles were found to be twice as frequent in ICP patients as in controls (25/134 v 11/120; p<0.05). Three of four severe CFTR mutations detected in patients were compound heterozygous with another abnormal CFTR allele, whereas among controls three severe CFTR mutations were found in heterozygous cystic fibrosis carriers. In ICP patients 19 uncommon/mild mutations, including combinations of the 5T allele with 12TG repeats, were identified compared with only five in controls (p = 0.012). Heterozygous SPINK1 mutations were detected in eight ICP patients (15% v 1% in controls) but only one also carried an additional mild CFTR mutation. CONCLUSIONS: These data show that not only compound heterozygosity, but also cystic fibrosis carrier status for different types of CFTR mutations, including uncommon/mild mutations, significantly increase the risk of developing pancreatitis. Although 45% of the study's ICP patients carried predisposing genetic risk factors (for example, mutations in CFTR or SPINK1), the authors found no evidence that the risk conveyed by CFTR mutations depends on co-inherited SPINK1 mutations.
Abstract: BACKGROUND & AIMS: Cadherins play an important role in cell-cell contact formation at adherens junctions. During the course of acute pancreatitis, adherens junctions are known to dissociate-a requirement for the interstitial accumulation of fluid and inflammatory cells-but the underlying mechanism is unknown. METHODS: Acute pancreatitis was induced in rats by supramaximal cerulein infusion. The pancreas and lungs were either homogenized for protein analysis or fixed for morphology. Protein sequencing was used to identify proteolytic cleavage sites and freshly prepared acini for ex vivo studies with recombinant proteases. Results were confirmed in vivo by treating experimental pancreatitis animals with specific protease inhibitors. RESULTS: A 15-kilodalton smaller variant of E-cadherin was detected in the pancreas within 60 minutes of pancreatitis, was found to be the product of E-cadherin cleavage at amino acid 394 in the extracellular domain that controls cell-contact formation, and was consistent with E-cadherin cleavage by leukocyte elastase. Employing cell culture and ex vivo acini leukocyte elastase was confirmed to cleave E-cadherin at the identified position, followed by dissociation of cell contacts and the internalization of cleaved E-cadherin to the cytosol. Inhibition of leukocyte elastase in vivo prevented E-cadherin cleavage during pancreatitis and reduced leukocyte transmigration into the pancreas. CONCLUSIONS: These data provide evidence that polymorphonuclear leukocyte elastase is involved in, and required for, the dissociation of cell-cell contacts at adherens junctions, the extracellular cleavage of E-cadherin, and, ultimately, the transmigration of leukocytes into the epithelial tissue during the initial phase of experimental pancreatitis.
Abstract: A case of a 24-year-old male with fatal cerebro-renal oxalosis assumed to be due to infusions of the sugar surrogate xylitol after appendectomy is reported. The diagnosis was established only after intensive histological investigations following the autopsy. The clinical picture was characterized by an acute seizure, coma and renal failure 2 days after the first xylitol infusion. Death occurred due to cerebral dysregulation as a very rare complication after parenteral administration of xylitol. Subendothelial double refractive calcium oxalate crystals were found in the walls of cerebral blood vessels, in particular in the stem ganglion regions and in the cortical renal tubules. The most common type of primary oxalosis was excluded by sequencing analysis. The young age, the minor surgical intervention and the otherwise unremarkable history are special features of this case. Since the genetic background of xylitol intolerance is still unclear, it is suggested that it should be banned as a sugar surrogate in clinical practice.
Abstract: BACKGROUND & AIMS: Cell adhesion proteins have been implicated as tumor suppressors because they prevent malignant cells from dissociating their cell contacts. We have studied the role of p120(ctn), a recently discovered member of the cadherin/catenin family, in human pancreatic cancer. METHODS: In 32 resection specimens of pancreatic adenocarcinoma and 10 control samples the expression of p120(ctn) was studied by Northern blot, immunocytochemistry, and immunogold electron microscopy. Patient survival data, tumor grading, and staging were correlated to the experimental results. In PaTu 8889 T pancreatic cancer cells, p120(ctn) expression was suppressed with 21-nucleotide small interfering RNA (siRNA) duplexes and proliferation was determined by bromodeoxyuridine (BrdU) incorporation. RESULTS: In pancreatic cancer p120(ctn) messenger RNA (mRNA) was increased 3- to 4-fold. Although p120(ctn) was localized exclusively at cell contacts in controls it was found in the cytosol and nucleus of pancreatic cancer cells. This redistribution correlated to the degree of tumor dedifferentiation but was independent of tumor stage. The mean survival of patients with predominant membrane localization of p120(ctn) was 24 +/- 7 (SEM) months vs. 9 +/- 2 months for patients with predominant cytoplasmic p120(ctn) expression (P < 0.05). Silencing of p120(ctn) with siRNA duplexes reduced pancreatic cancer cell growth by 40%. CONCLUSIONS: Up-regulation, cytoplasmic redistribution, and nuclear import of p120(ctn) are associated with a more malignant phenotype of pancreatic cancer. This study further represents conclusive evidence for a direct involvement of p120(ctn) in malignant tumor cell proliferation. Both p120(ctn)-defective tumor cell contacts and p120(ctn)-mediated growth signals appear to contribute to the aggressive spread of pancreatic cancer.
Abstract: Hereditary pancreatitis has been found to be associated with germline mutations in the cationic trypsinogen (PRSS1) gene. Here we report a family with hereditary pancreatitis that carries a novel PRSS1 mutation (R122C). This mutation cannot be diagnosed with the conventional screening method using AflIII restriction enzyme digest. We therefore propose a new assay based on restriction enzyme digest with BstUI, a technique that permits detection of the novel R122C mutation in addition to the most common R122H mutation, and even in the presence of a recently reported neutral polymorphism that prevents its detection by the AflIII method. Recombinantly expressed R122C mutant human trypsinogen was found to undergo greatly reduced autoactivation and cathepsin B-induced activation, which is most likely caused by misfolding or disulfide mismatches of the mutant zymogen. The K(m) of R122C trypsin was found to be unchanged, but its k(cat) was reduced to 37% of the wild type. After correction for enterokinase activatable activity, and specifically in the absence of calcium, the R122C mutant was more resistant to autolysis than the wild type and autoactivated more rapidly at pH 8. Molecular modeling of the R122C mutant trypsin predicted an unimpaired active site but an altered stability of the calcium binding loop. This previously unknown trypsinogen mutation is associated with hereditary pancreatitis, requires a novel diagnostic screening method, and, for the first time, raises the question whether a gain or a loss of trypsin function participates in the onset of pancreatitis.
Abstract: Acute and chronic recurrent pancreatitis have been reported in patients with a variety of inborn errors of metabolism. Among these are hyperlipidaemias, various disorders of branched-chain amino acid degradation, homocystinuria, haemolytic disorders, acute intermittent porphyria and several amino acid transporter defects. Some of these disease entities are exceedingly rare. In most of these disorders, pancreatitis is not very common and, with the exception of lipoprotein lipase and apolipoprotein C-II deficiency, is neither the leading nor the clinically most distressing manifestation of the underlying metabolic defect. The majority of these syndromes are, however, inherited, and often entire kindreds are carriers of well-defined germline mutations that can, to varying degrees, be associated with pancreatitis. We have reviewed the clinical, biochemical and genetic characteristics of those inborn errors of metabolism because interesting information can be gained from the in regard to the pathophysiology of pancreatitis and because they need to be distinguished from other hereditary causes of the disease.
Abstract: Recent findings shed new light on the process of receptor tyrosine kinase (RTK) activation and signal definition. In extension to the established mechanism of ligand-induced homodimeric receptor complex formation, recent findings highlight heterodimeric receptor aggregation as a powerful means of signal diversification. Promiscuous receptor interactions involve different ligand binding kinetics and generate divergent receptor phosphorylation sites that could allow enhanced or modified signal generation. Besides activation by a specific ligand, a newly defined RTK function involves signal integration of a variety of stimuli, including calcium-dependent responses in neuronal cells, activation of G-protein-coupled receptors or cellular stress such as UV irradiation. On the basis of existing evidence for such crossactivation pathways, RTKs must be considered as representing critical foci and switch points for multiple environmental and internal stimuli.
Abstract: Members of the epidermal growth-factor-receptor tyrosine-kinase (EGFR) family play important roles both in normal growth regulation/cell differentiation and in the genesis and progression of human neoplasia. In the present study, we analysed distinct heregulin (HRG) signals mediated by the HRG receptors HER3 and HER4. In overexpression cell systems, we demonstrate that HRG-induced transformation by "kinase-impaired" HER3 is dependent on coexpression of kinase active HER2. In cells coexpressing HER2 and HER4, however, both kinases significantly contribute to the HRG-induced mitogenic stimulus. In addition, we show that HER3 is no substrate of HRG-activated HER4. Analysis of EGFR crosstalk in a panel of human carcinoma cell lines revealed mainly HRG-induced activation of HER2/HER3, whereas HER4 activation is also detectable to various extents. Evidence for HRG-induced activation of HER3 and/or HER4 indicates relevance of cell-specific expression patterns of these high- and low-affinity HRG receptors in the modulation of a ligand-induced stimulus. Specific signal modulation and definition can be demonstrated further by distinct time courses of mitogen-activated protein (MAP) kinase (MAPK) activation, which are induced by distinct HRG isotypes via differential binding to HER2/HER3 versus HER2/HER4. In concert, these mechanisms of signal modulation may be decisive for the diverse biological activities of HRG in different cell types.
Abstract: Transduction of a mitogenic signal from the cell membrane to the nucleus involves the adapter proteins SHC and Grb2, which mediate activation of the Ras/mitogen-activated protein (MAP) kinase pathway. In contrast to receptor tyrosine kinases (RTKs), the signalling steps leading to Ras/MAP kinase activation by G-protein-coupled receptors (GPCRs) are still poorly characterized but appear to include beta gamma subunits of heterotrimeric G-proteins and as-yet unidentified tyrosine kinases. We report here that the epidermal growth factor receptor (EGFR) and the neu oncoprotein become rapidly tyrosine-phosphorylated upon stimulation of Rat-1 cells with the GPCR agonists endothelin-1, lysophosphatic acid and thrombin, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by either the selective tyrphostin AG1478 or a dominant-negative EGFR mutant suppressed MAP kinase activation and strongly inhibited induction of fos gene expression and DNA synthesis. Our results demonstrate a role for RTKs as downstream mediators in GPCR mitogenic signalling and suggest a ligand-independent mechanism of RTK activation through intracellular signal crosstalk.
Abstract: The class I receptor tyrosine kinase (RTK) HER2 is an oncoprotein that is frequently involved in the pathogenesis of tumors of epithelial origin. Here we report mRNA expression in peripheral blood and bone marrow cells from healthy donors in hematopoietic cell lines and leukemic blasts from patients with acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphoblastic leukemia (CLL), and chronic myeloid leukemia (CML). However, cell surface expression of HER2 protein (p185HER2) was found exclusively on a subset of leukemic cells of the B-lymphoblastic lineage. p185HER2 expression was found on blasts in 2 of 15 samples from infants, 9 of 19 samples from adult patients with C-ALL (CD19+CD10+), and 1 of 2 samples from patients with pro-B ALL (CD19+CD10-), whereas none of the leukemic cells from patients with AML (0/30), T-ALL (0/7), CLL (0/5) (CD19+CD5+), or CML in chronic and accelerated phase (0/5) or in blast crisis with myeloid differentiation (0/14) were positive for p185HER2. However, cells from 3 of 4 patients with CML in B-lymphoid blast crisis (CD19+CD10+) expressed high levels of p185HER2, which was also found on the surface of the CML-derived B-cell lines BV-173 and Nalm-1. Our study shows p185HER2 expression on malignant cells of hematopoietic origin for the first time. Aberrant expression of this oncogenic receptor tyrosine kinase in hematopoietic cell types may be an oncogenic event contributing to the development of a subset of B-lymphoblastic leukemias.
Abstract: Amplification and/or overexpression of HER2/neu and HER3 genes have been implicated in the development of cancer in humans. The fact that these receptor tyrosine kinases (RTKs) are frequently coexpressed in tumor-derived cell lines and that heterodimers form high affinity binding sites for heregulin (HRG) suggests a novel mechanism for signal definition, diversification or amplification. In cells expressing HER2 and HER3, tyrosine phosphorylation of HER3 is markedly increased upon exposure to recombinant HRG. ATP binding site mutants of HER2 and HER3 demonstrate transphosphorylation of HER3 by HER2, but not vice versa. HRG-induced transphosphorylation of HER3 results in a substrate phosphorylation pattern distinct from HER2 cells and enhances association of the receptor with SHC and phosphoinositol 3-kinase in transfected 293 and mammary carcinoma-derived MCF-7 cells. The physiological relevance of HER2/HER3 heterodimerization is demonstrated by HRG-dependent transformation of NIH 3T3 cells coexpressing the two receptors. These findings demonstrate the acquisition of expanded signaling capacities for HER2 by HRG-induced heterodimerization with HER3 and provide a molecular basis for the involvement of receptor heteroactivation in the development of human malignancies.
