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Introduction: Trefoil factor family member 2 (Tff2) is a small gut peptide, mainly known for its protective and healing functions. As previously demonstrated, high-fat (HF) feeding can rapidly and specifically modulate Tff2 transcription in key tissues of mice, including the duodenum and mesenteric adipose tissue, therefore suggesting a novel role for this gene in energy balance., Design and Methods: To explore whether and how Tff2 can influence feeding behavior and energy metabolism, Tff2 knock-out (KO) mice were challenged with HF diet for 12 weeks, hence food and energy intakes, body composition, as well as energy excretion and serum lipid and hormonal levels were analyzed. Finally, energy efficiency was estimated., Results: Tff2 KO mice showed a greater appetite and higher energy intake compared to wild-type (WT). Consistently, they presented lower levels of serum leptin, and increased transcription of agouti-related protein (Agrp) in the hypothalamus. Though energy and triglyceride fecal excretion were augmented in Tff2 KO mice, digestible energy intake was superior. However, KO mice were finally protected from HF diet-induced obesity, and accumulated less weight and fat depots than WT animals, while keeping a normal lean mass. Energy efficiency was lower in HF-KO mice, while energy expenditure and locomotor activity were globally increased., Conclusions: The present work demonstrates previously unsuspected roles for Tff2 and suggests it to be a mastermind in the control of energy balance and a promising therapeutic target for obesity., (Copyright © 2013 The Obesity Society.)

The trefoil peptide family, consisting in mammals of three members namely TFF1, 2 and 3, plays a cytoprotective role in epithelial cells of various tissues, mainly in the digestive tract. Tff1, Tff2 or Tff3 knock-out mouse models developed various kinds of gastrointestinal impairment. microRNAs are known to be novel gene regulators. We aimed to investigate the physiological role of such miRNAs in Tff2 knock-out mice. Whole miRNome profiling and in silico analysis were performed for Tff2-KO and WT mice. Our latest data explored the role of miRNAs in the regulatory cascades and molecular processes of Tff2-/- mice. As much as 6% of the Tff2-KO mice miRNome was significantly dys-regulated. Further in silico analysis suggests that the respective dys-regulated part of the miRNome is involved in human pathological processes, including pancreatic, colorectal and basal cell cancer. Additionally, the dys-regulated miRNome targets pathways involved in carbohydrate metabolism and adipocytokine signaling. The latter links deficient caloric maintenance in Tff2 and previous observation in Tff3-KO mice with miRNAs. In summary, our proof-of-concept study indicates that miRNAs may play an important role in the regulatory processes of the trefoil peptide family, especially in the regulation of cancer-related cascades.

TFF2 is one of the members of the trefoil factor family, known for its role in protection of gastrointestinal epithelia upon injury; however, recent studies suggest that TFF2 could also play an important role in the immune system. In the present study Tff2 deficient and wild type mice were infected by Y. enterocolitica which resulted in a lethal outcome in all Tff2 deficient mice, but not in WT animals. Yersinia invaded Peyer's patches more efficiently as shown by high bacterial titers in the KO mice while wild type mice displayed lower titers and a visible bacterial accumulation in the intestine. Bacterial accumulation in Peyer's patches of Tff2 deficient mice was accompanied by increased recruitment of macrophages. While an increased level of MAC-1 positive cells was observed in the spleens of both Tff2 deficient and WT mice at third day post infection, bacterial dissemination to liver, lung and kidneys was observed only in Tff2 knock-out mice. Analysis of the cellular composition of spleen did not reveal any substantial alteration to WT animals, suggesting possible disregulation of hemopoietic cells involved in immune response to Y. enterocolitica. These new data indicate that Tff2 plays an important role in immune response by protecting the organism from consequences of infection and that Tff2 knock-out mice react adversely to bacterial infections, in this case specifically to Y. enterocolitica., (Copyright © 2012 S. Karger AG, Basel.)

The 3 members of the mammalian trefoil factor family (TFF) are expressed and secreted as cytoprotective peptides along the entire length of the normal gastrointestinal tract. More recently, they were shown to display multifunctional properties. Goblet cells of the small and large intestine constitute a major source for the synthesis of the third family member, TFF3 (formerly intestinal trefoil factor, ITF). TFF3, like the other family members, is rapidly up-regulated in response to physical wounding of the digestive tract. In addition, Tff3 was also detected in the posterior pituitary gland. Apart from this Tff3 function as a neuropeptide, also presence of Tff3 in the mouse cochlea was noted and Tff3-deficient animals display hearing impairment and accelerated presbyacusis. To elucidate Tff3's mode of function and its unexpected contribution to the hearing process, we strived to determine Tff3's interacting partners and to establish the functional network. To this end, we used a protein-protein binding assay based on a specific transcriptional regulation in yeast cells (the yeast-two-hybrid assay). We looked for interacting partners of Tff3 in a mouse cochlea cDNA library (from donors aged 3-15 days, P3-P15). Our data show that several binding candidates exist and that they could contribute to the known involvement of the trefoil peptides to apoptosis.

Trefoil factor family (TFF) peptides provide protective and reparative effects by enhancing epithelial integrity and promoting mucosal restitution. TFF peptide expression is induced after mucosal damage. These processes are of central physiological relevance during the postnatal intestinal development and are strongly influenced during the weaning period. In piglets, weaning at early maturation stages frequently causes mucosal inflammation. The aim of this study was to evaluate postnatal intestinal TFF expression in a piglet probiotic trial. Low intestinal TFF2 expression was measured at early maturation stages. Weaning, however, was associated with a distinct response of increased TFF2 expression, indicating an important role in enhancing mucosal integrity. In the distal jejunum and ileum weaning could as well be associated with increased TFF3 mRNA levels. Differential TFF1 expression was not detected. Furthermore, TFF2 localization studies in different intestinal loci were performed by means of immunohistochemistry. Expression of selected genes (TGFA, EGFR, Cox-2) known to promote TFF signaling showed differential expression pattern as well, thereby providing further functional background. Furthermore, the expression patterns of EGFR observed in this study contribute to an advanced view of previous findings of EGFR regulation mainly obtained in rodents. An upregulated EGFR expression during early postnatal development suggests a local relevance to porcine intestinal maturation. However, a feed supplementation with the probiotic strain Enterococcus faecium did not influence TFF expression., (2009 S. Karger AG, Basel.)

The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.

Background and Aims: The gastrointestinal trefoil factor family (TFF1, TFF2, TFF3) peptides are considered to play an important role in maintaining the integrity of the mucosa. The physiological role of TFF2 in the protection of the GI tract was investigated in TFF2 deficiency., Methods: TFF2-/- mice were generated and differential expression of various genes was assessed by using a mouse expression microarray, quantitative real time PCR, Northern blots or immunohistochemistry., Results: On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach., (Copyright (c) 2005 S. Karger AG, Basel.)

Background: Damage to the gastrointestinal mucosa results in the acute up-regulation of the trefoil factor family peptides TFF1, TFF2, and TFF3. They possess protective, healing, and tumour suppressive functions. Little is known about the regulation of TFF gene expression. The promoters of all three TFF genes contain binding sites (E box) for upstream stimulating factor (USF) and Myc/Max/Mad network proteins., Aims: To determine the nature and function of transcription factors that bind to these E boxes and to understand their role for TFF gene expression., Methods: TFF promoter activities were determined by reporter gene assays. DNA binding was monitored by electromobility shift assays and by chromatin immunoprecipitation analyses. Expression of endogenous TFF was determined by multiplex RT-PCR., Results: It was observed that the TFF2 promoter is specifically and efficiently activated by USF transcription factors but not by c-Myc. USF displayed comparable binding to a high affinity Myc/Max binding site compared with the three TFF E boxes, while c-Myc exhibited lower affinity to the TFF E boxes. In contrast, pronounced binding differences were observed in cells with a strong preference for USF to interact specifically with the TFF2 E box, while Myc was not above background. Exogenous expression of USF was sufficient to activate the chromosomal TFF2 and to a lesser extent, the TFF1 gene., Conclusion: These findings define USF factors as regulators of the TFF2 gene and suggest that promoter specific effects are important for a pronounced gene activation of this cytoprotective peptide.

It has been advanced that the trefoil factor (TFF) 1 gene is a candidate tumor-suppressor gene and may be involved in the development and/or progression of human gastric cancer. We aimed to clarify the putative role of TFF1 in gastric carcinogenesis. Ninety gastric carcinomas and eight gastric carcinoma-derived cell lines were screened for TFF1 mutations; subsets of the primary tumors and of the cell lines were subjected to loss of heterozygosity (LOH), immunohistochemistry, and promoter methylation analyses. TFF1 mutations were not detected in any of 90 gastric carcinomas. Eight (28%) of 28 informative cases displayed LOH at the TFF1 locus and absence of TFF1 staining by immunohistochemistry. These results indicate a frequent loss of TFF1 expression in gastric carcinomas through a mutation-independent mechanism. Extensive TFF1 promoter methylation was observed in nonexpressing gastric carcinoma-derived cell lines and tissues. Expressing cell lines, as well as normal gastric mucosa, presented little or no methylation of the promoter. Gastric carcinoma DNA presented de novo methylation of the promoter. These results point to the involvement of promoter methylation in the shutting down of TFF1. We conclude that TFF1 point mutations seem to be a rare event in gastric carcinogenesis. The loss of expression of TFF1 in a proportion of gastric carcinomas may be explained by LOH and methylation of the TFF1 promoter region. Our results further support the role of TFF1 inactivation in gastric carcinogenesis, in agreement with the results obtained in the Tff1-knockout mice model.

Antimicrobial peptides are an important component of the innate response in many species. Here we describe the isolation of the gene Dermcidin, which encodes an antimicrobial peptide that has a broad spectrum of activity and no homology to other known antimicrobial peptides. This protein was specifically and constitutively expressed in the sweat glands, secreted into the sweat and transported to the epidermal surface. In sweat, a proteolytically processed 47-amino acid peptide was generated that showed antimicrobial activity in response to a variety of pathogenic microorganisms. The activity of the peptide was maintained over a broad pH range and in high salt concentrations that resembled the conditions in human sweat. This indicated that sweat plays a role in the regulation of human skin flora through the presence of an antimicrobial peptide. This peptide may help limit infection by potential pathogens in the first few hours following bacterial colonization.

One of the early events in inflammation and epithelial restitution of the gastrointestinal tract is the up-regulation of secretory peptides belonging to the trefoil factor family (TFF) that promote cell migration, protect and heal the mucosa. Their major expression site is stomach (TFF1, TFF2) and intestine (TFF3). Located in the 5'-flanking region of the genes are several consensus sites for members of the GATA transcription factors known to control gut-specific gene expression. By reverse transcription-PCR (RT-PCR), GATA-6 was shown to be expressed in a variety of tumor cell lines of gastric, intestinal and pancreatic origin. In MKN45, KATOIII and LS174T, cotransfection with TFF reporter genes and GATA-6 expression vectors revealed that GATA-6 activates TFF1 and TFF2 4-6-fold, without an effect on TFF3. The functional contribution of GATA binding sequences in the reverse orientation was further characterized by reporter gene assays using TFF2 deletion constructs and by gel shift experiments.

Mammalian trefoil factor family (TFF)-domain peptides promote gastrointestinal protection, healing and cell migration and may act as tumour suppressors. TFF-like domains also constitute modules of composite proteins like mucin glycoproteins and zona pellucida proteins. Database searches with a modified, less stringent consensus sequence - C-x(5,6)-[ST]-x(3)-C-x(4,5)-C-C-[FYWH]-x(2, 24)-C-[FY] - revealed that ancestors of the TFF-domain arose before amphibian evolution. Eggshell proteins and a zona pellucida-like protein in teleost species, an epidermis-specific protein in a tunicate as well as an open reading frame in a nematode exhibited TFF-like motifs suggesting that they most likely originated in some multicellular organism.

The winged helix transcription factors HNF-3/FKH (forkhead homologs) activate endodermal-derived and acute-phase gene expression and control gut development in Drosophila. Trefoil factor family (TFFs) peptides are vertebrate products secreted by mucin-producing epithelial cells of the gastrointestinal tract involved in restitution and repair of the mucosa. They are positively regulated in ulcerative and neoplastic conditions. We describe a consensus sequence in human and rodent TFF promoters close to the TATAA box showing striking similarity to the binding site of the HNF-3/FKH family. In gel retardation assays, HNF-3 alpha and beta bound predominantly to the site in TFF1 (formerly pS2) and, to a lesser extent, to the sites in TFF2 or TFF3. Mutations generated in this motif severely impaired transcription of TFF1 reporter genes. Cotransfection with expression vectors of HNF-3alpha and beta, but not the related HFH 11A and B, specifically activated the wild-type TFF1 reporter genes. Activation of endogenous expression of TFF1 by HNF-3 alpha and beta gene products was more than 1000 fold in the pancreatic cell line Capan-2 and fivefold in the gastric cell line MKN-45, whereas the intestinal cell lines HUTU 80 and HT-29 displayed no effect. Thus, HNF-3/FKH factors contribute causally to cell-specific regulation of TFF genes and may explain the acute-phase response of TFF peptides.

Background and Aims: In humans, trefoil peptides (TFF peptides) and some mucins have been reported to be expressed in a cell-specific manner at mucosal surfaces of normal gastrointestinal tissues. Neoplastic conditions cause characteristic changes of these expression patterns. To study such patterns in Barrett's metaplasia and squamous cell carcinoma of the oesophagus (SCC), the distribution of MUC1, MUC2, MUC5AC and the three TFF peptides (TFF1, TFF2 and TFF3) was investigated., Methods: In 40 archival samples of SCC and in 21 samples of Barret's metaplasia, expression of the three mucins and two TFF peptides (TFF1 and TFF2) was assessed by specific antibodies. Reverse transcriptase/polymerase chain reaction amplification (RT-PCR) was performed on frozen tissue samples from the 11 biopsies of SCC for the three TFF peptides., Results: Immunohistochemical tests for MUC2 and TFF2 were negative both in samples of Barret's metaplasia and in SCC. MUC1 expression was detected in 57.5% of the tumour samples, while TFF1 and MUC5AC were found in 10% and 7.5% of the cases respectively. In Barrett's metaplasia MUC1 was detected in 90.5% of the cases and TFF1 and MUC5AC in all of them. RT-PCR analysis revealed a more complex pattern: TFF1 and TFF3 expressed the corresponding mRNA in all samples investigated; the third member, TFF2, was active in 45.5% of the carcinoma biopsies and not in the corresponding native tissue., Conclusions: This finding in oesophageal carcinoma contrasts with the situation found in normal and neoplastic stomach epithelium where TFF1 and TFF2 are found co-expressed and TFF3 remains silent. Interestingly, MUC1 is expressed in a significant proportion of SCC. Both in Barett's metaplasia and in SCC the expression of MUC5AC mirrors the TFF1 synthesis in intensity and spatial distribution.

Peptides belonging to the trefoil factor family (TFF) protect the gastrointestinal epithelia. Overexpression of TFFs was observed in pathological conditions such as gastritis, ulceration, metaplasia and neoplasia of the gastrointestinal tract. The aims of this work were to investigate the recently described TFF2 gene polymorphism in different European populations. DNA samples from blood of healthy individuals and gastric cancer patients were genotyped using the polymerase chain reaction. They were compared to a gastric cancer population. The results do not show any significant difference in allelic frequencies between gastric cancer patients and healthy individuals from Portugal. However, the frequency of the two alleles found varies considerably among Europeans.

The gastrointestinal tract is exposed to environmental insult as a result of food intake or in pathological conditions such as diarrhoea, and is therefore protected by the mucus layer. As part of it, trefoil peptides (TFFs) are able to modify the visco-elastic properties of the mucus, protect against experimental ulceration, and promote repair of the epithelia. We investigated, using transient reporter gene assays and RT-PCR in the gastric carcinoma cell line MKN45 and colon carcinoma cell lines LS174T and HT29, whether ethanol and osmotic changes can modify transcriptional activity of TFFs. In a mild hypotonic environment (200 mosmol/l) all three TFF genes were up-regulated by at least a factor of 2. In hypertonic medium (400 mosmol/ll), TFF1 and TFF3 were down-regulated, whereas TFF2 was up-regulated by elevated concentrations of sodium or chloride in MKN45. Raising the osmolality by ethanol resulted in an up-regulation of TFF3 in both colon cell lines but not in the gastric cell line. We conclude that alteration in TFF gene expression is a response of gut epithelia to deal with osmotic forces and ethanol.

A group of secreted peptides (trefoil factor family; TFF) is abundantly expressed at mucosal surfaces of the gastrointestinal tract and promote epithelial restitution. They are upregulated around areas of epithelial damage, ulceration and neoplasia. The transcriptional regulation of the three human TFF genes was assayed by multiplex RT-PCR and reporter gene analysis in 8 gastrointestinal carcinoma cell lines. The level of endogenous mRNA matched well the reporter gene activity of all TFFs, indicating that the cis-acting elements located less than 1,000 bp upstream of the TATAA box account for cell-specific gene expression. In HT-29, the endogenous TFF expression profile changed in relation to cell growth conditions. Deletion and mutation analysis of TFF promoter constructs revealed enhancing elements shared within the three TFF promoters that were shown to bind nuclear proteins. Thus such specific DNA-protein interaction may explain the TFF peptides' cell specific expression pattern and altered levels in pathological conditions.

A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110-250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals.

Trefoil peptides are small secretory proteins characterized by three intrachain disulfide bonds forming the trefoil motif or P-domain. They are abundantly expressed on mucosal surfaces, especially of the gastrointestinal tract. In pathological conditions such as ulcers, metaplasia and neoplasia, their expression is upregulated. Three human trefoil peptides have been described: the estrogen-inducible pS2 protein, the spasmolytic protein and the intestinal trefoil factor. Recently, their role in the maintenance of surface integrity and ulcer healing was discussed. We already mapped the corresponding three genes (BCEI), SML1, TFF3) to the same genomic region (21q22.3). In this paper, we show that the three genes are clustered in a tandemly orientated fashion within 50 kb on a bacterial artificial chromosome (BAC) recombinant. This cluster is located adjacent to D21S19 and the locus order is cen-D21S212-TFF3-SML1-BCEI-D21S19-tel, whereas transcription of all three genes is directed towards the centromere. The gene structure of SML1 exhibits four exons, two of which encode the two separate trefoil motifs. TFF3 and BCEI, both containing one trefoil motif, are composed of three exons each, suggesting gene duplication and exon-shuffling events during evolution. The 5'-flanking region of SML1 was compared to the corresponding region of other trefoil genes. Two motifs with identical sequence and positions are shared between SML1 and BCEI, thus presenting possible targets for stomach-specific gene regulation. Two other motifs are shared within all known human and rat trefoil genes, suggesting a coordinated regulation and/or a common locus-controlling region. Using RT-PCR, a change in the pattern of trefoil gene expression is detected in tissue samples from normal gastric mucosa, hyperplastic polyps, gastric cancer, and gastric cancer cell lines, respectively.

Small peptides displaying a cysteine-rich module (termed P-domain or trefoil motif) form a recently increasing group of peptides abundantly expressed at mucosal surfaces of specific tissues and are associated with the maintenance of surface integrity. The estrogen-inducible pS2 gene (BCEI) and the human homolog to the porcine spasmolytic peptide (hsP) gene (SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be located at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene (TFF3) were described recently. By PCR analysis of a somatic cell hybrid panel and FISH using two large genomic recombinants (110 kb, 210 kb) cloned in the Bacterial Artificial Chromosome (BAC) system, we show that this gene coding for the new member of human P-domain/trefoil peptides also maps to chromosome region 21q22.3 suggesting a physical linkage of all three trefoil peptide genes.

A human cDNA corresponding to the porcine pancreatic spasmolytic protein (PSP) was isolated, and the recombinant clone was originally termed hSP for human spasmolytic protein. Later, the term SML1 for spasmolysin was suggested for the human gene. This protein shows a remarkable sequence homology to pS2, a protein coded by an estrogen-induced gene isolated from the breast carcinoma cell line MCF-7. Although, at the DNA level, the gene sequences pS2 and hSP/SML1 display insufficient homology for cross-hybridization, their expression in tumor cells occurs with remarkable coordination. The human pS2 gene sequence has been assigned to chromosome 21, and we have therefore attempted to map the hSP/SML1 gene by using cDNA and Southern blotting of genomic DNAs from a panel of human-rodent somatic cell hybrids carrying different complements of human chromosomes. Interestingly, the hSP/SML1 gene is also localized on chromosome 21.

The human pS2 gene, isolated from the breast carcinoma cell line MCF-7 and shown to be under estrogen transcriptional control in a subclass of breast cancer cells was reported to be secreted in normal stomach surface epithelial cells, whereas additional gastrointestinal tissues like pancreas and colon do not secrete pS2 at all. In porcine pancreas, a spasmolytic polypeptide (sharing domains of homology with pS2) was observed; a corresponding human gene (hSP) was shown to be active in normal stomach mucosa. hSP and pS2 gene activity in normal and neoplastic pancreas tissues was then compared. Whereas both genes are inactive in normal pancreatic cells, activation of the pS2 sequence in a primary pancreatic carcinoma cell culture and in 23 tumor tissues was noted when investigated by immunostaining. In all cases when pS2 showed a regular 0.6 kb transcript, hSP displayed a transcript of 0.7 kb. Six of these tumors showed a reduced pS2 immunoreactivity and, at the same time, aberrant pS2 mRNA bands and a complete shut-down of the hSP gene were noted. In one case, whereas normal pancreas remained negative, the corresponding tumor and its metastasis displayed regular transcripts of pS2 and hSP. This remarkably high correlation suggests that pS2 and hSP expression in the pancreatic tumors, but not in their corresponding healthy tissue is significantly linked to molecular steps leading to tumorigenesis.

Expression of pS2 (an estrogen-induced gene isolated from breast carcinoma MCF7 cells) and of the human spasmolytic peptide (hSP) gene was analyzed in 21 human biliary tract and gallbladder carcinomas and 16 non-neoplastic gallbladders at the protein level (immunochemistry) and, in a series of these cases, at the RNA level (Northern blots). Eighteen carcinomas and 14 non-neoplastic mucosae with inflammatory alterations showed pS2 activity by immunostaining or Northern blotting. In addition, in samples with pS2 expression, hSP RNA was demonstrated. In the corresponding non-neoplastic and healthy mucosae, pS2 was negative, as judged by immunostaining. Northern blots showed weak (basal) level of activity for pS2 and hSP. The genes' increased expression in correlation to inflammatory and neoplastic processes, by now observed in several carcinomas and idiopathic inflammatory bowel disease, hints to their essential role in such diseases.

Expression of the pancreatic spasmolytic peptide (hSP) gene and pS2 (a gene isolated from oestrogen-induced breast carcinoma cells) were analysed in 36 samples of human stomach carcinoma. 17 tumours were investigated at the RNA level (by northern blots) as well as at the gene product level (by immunochemistry). Since pS2 had been shown to be expressed in normal stomach mucosa its activity in carcinoma samples was expected. Surprisingly, strong pS2 immunoreactivity was noted in the diffuse carcinoma type, whereas the intestinal type displayed weak reactivity. The tumour samples showing strong immunostaining expressed the regular 0.6 kb pS2 RNA band and weak staining was paralleled by aberrant transcripts. Additionally, only in tumour samples with regular pS2 transcription was the typical 0.7 kb hSP RNA band seen; samples with aberrant pS2 bands did not express hSP at all. This is the first demonstration of hSP gene activity in a human tumour.

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