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We here determine the influence of mesenchymal stem cell (MSC) therapy on the progression of solid tumors. The influence of MSCs was investigated in human colorectal cancer cells as well as in an immunocompetent rat model of colorectal carcinogenesis representative of the human pathology. Treatment with bone marrow (BM)‐derived MSCs significantly reduced both cancer initiation and cancer progression by increasing the number of tumor‐free animals as well as decreasing the number and the size of the tumors by half, thereby extending their lifespan. The attenuation of cancer progression was mediated by the capacity of the MSCs to modulate the immune component. Specifically, in the adenocarcinomas (ADKs) of MSC‐treated rats, the infiltration of CD68+ monocytes/macrophages was 50% less while the presence of CD3+ lymphocytes increased almost twofold. The MSCs reprogrammed the macrophages to become regulatory cells involved in phagocytosis thereby inhibiting the production of proinflammatory cytokines. Furthermore, the MSCs decreased NK (Natural Killer) and rTh17 cell activities, Treg recruitment, the presence of CD8+ lymphocytes and endothelial cells while restoring Th17 cell activity. The expression of miR‐150 and miR‐7 increased up to fivefold indicating a likely role for these miRNAs in the modulation of tumor growth. Importantly, MSC administration limited the damage of healthy tissues and attenuated tumor growth following radiotherapy. Taken together, we here show that that MSCs have durable action on colon cancer development by modulating the immune component of the tumor microenvironment. In addition, we identify two miRNAs associated with the capacity of MSCs to attenuate cancer growth. stem cells translational medicine 2019;8:285&300

Travo A, Piot O, Wolthuis R, Gobinet C, Manfait M, Bara J, Forgue-Lafitte M-E & Jeannesson P (2010) Histopathology 56, 921–931 IR spectral imaging of secreted mucus: a promising new tool for the histopathological recognition of human colonic adenocarcinomas Aims: During colonic carcinogenesis, mucin-type glycoproteins are known to undergo quantitative and qualitative alterations. The aim of this study was to determine the value of infrared (IR) spectral histology for the histopathological recognition of colonic adenocarcinomas based on mucin-associated IR spectral markers. Methods and results: Paraffin-embedded tissue sections of normal human colon and adenocarcinomas were analysed directly by IR-microspectroscopy (IR-MSP), without prior chemical dewaxing. IR-MSP imaging combined with multivariate analysis permitted the construction of IR colour-coded images of the tissue sections providing spatially resolved biochemical information. This allowed localization of mucin-rich areas and provided label-free spectral-based staining of secreted mucus related to the biochemical heterogeneity of its mucin content. IR images of secreted mucus display the same spectral clusters in both normal and adenocarcinomatous colonic tissues, but with significant differences in surface percentages. Such differences allow a distinction between these two tissue types. Spectral variations associated with changes of mucin secondary structure were the most accurate mucus spectral marker for discriminating between normal colon and adenocarcinomas in the sample set. Conclusions: IR-MSP imaging provides a new type of histology, independent of visual morphology, presenting tremendous possibilities for discovery and clinical monitoring of cancer markers.

Summary The secreted thiol-activated cytolysin listeriolysin O (LLO) was responsible for L. monocytogenes -induced high-molecular glycoproteins (HMGs) exocytosis in cultured human mucosecreting HT29-MTX cells. By biochemical analysis we demonstrate that the majority of secreted HMGs in LLO-stimulated cells are of mucin origin. In parallel, analysis of the expression of MUCs genes showed that the transcription of the MUC3 , MUC4 and MUC12 genes encoding for membrane-bound mucins was increased in LLOstimulated cells. Upregulation of the MUC3 gene correlates with an increased expression of the membrane-bound MUC3 mucin. In contrast, increase in secretion of the gel-forming MUC5AC mucin develops without upregulation of the MUC5AC gene. Finally, results showed that NF- κ κ κ B and AP-1 transcription factors were not involved in LLO-induced upregulation of MUCs genes in HT29-MTX cells, whereas L. monocytogenes infection was able to promote the degradation of I κ κ κ B proteins in the cells.

Mucinous cystic tumors of the pancreas must be distinguished from other cystic lesions because of their potential malignancy. Our purpose was to assess the reliability of gastric M1 mucin analysis in the fluid of cystic lesions of the pancreas in comparison or association with carcinoembryonic antigen. M1 mucin and carcinoembryonic antigen were measured in cyst fluid obtained preoperatively by fine-needle aspiration. The lesions consisted of 12 serous cystadenomas, 9 mucinous cystadenomas, 8 cystadenocarcinomas and 6 intraductal mucinous hypersecreting neoplasms. Thirty pancreatic pseudocysts complicating well-documented chronic pancreatitis were also examined. In addition, M1 mucins were localized by immunoperoxidase staining in fetal and normal adult pancreas and in mucinous and serous tumors. Carcinoembryonic values of G20 ng/ml and M1 mucin values of G50 U M1/ml represented 82 and 78% sensitivity, respectively, as well as 100% specificity for distinguishing mucinous lesions from serous cystadenomas; the sensitivity for this purpose was 100% using these criteria in combination. Carcinoembryonic antigen values of G300 ng/ml and M1 mucin values of G1,200 U M1/ml represented 56 and 30% sensitivity, respectively, as well as 100% specificity for distinguishing mucinous lesions from pseudocysts; the sensitivity for this purpose was 60% using these criteria in combination. By immunohistology, M1 mucins were detected in the wall of mucinous lesions but not in fetal and normal adult pancreas and in serous cystadenomas. Measurement of M1 mucin antigen in cyst fluid could thus improve the diagnosis of mucinous cystic lesions of the pancreas.Int. J. Cancer74:286‐290, 1997. r 1997 Wiley-Liss, Inc.

The arotinoid Ro 40-8757 was previously shown to inhibit the growth of a variety of human cancer cell lines derived from breast, lung and uterus. In view of the high incidence of human digestive cancers, and the slow progress in the development of new therapy, we examined in this paper several combinations between the new arotinoid Ro 40-8757, 5-fluorouracil (5FU) and interferon alpha-2a on the growth of nine human cancer cell lines derived from the gastrointestinal and pancreatic system. Half-maximal inhibition of cell proliferation by Ro 40-8757 was observed at concentrations ranging between 0.18 and 0.57 microM, and increased up to 4.7 microM in retinoid-resistant CAPAN 620 pancreatic cells. All-trans-retinoic acid was 70 times less potent. The sensitivity of HT29-5FU-resistant colonic cells was similar to that observed in the parental cells, suggesting an action independent of pyrimidine metabolism. Ro 40-8757 did not induce any differentiation on HT29 cells, as suggested by ultrastructural analysis. The arotinoid did not interact with receptor signal transduction pathways under the control of serum components, such as growth factors as half-maximal inhibiton of growth was similar in HT29-S-B6 cells cultured in the absence or presence of serum. Cell cycle analysis showed that Ro 40-8757 was not acting at a phase-specific transition in HT29 cells and, accordingly, did not induce overexpression of the protein kinase C (PKC)alpha isoform, or conversion of hyperphosphorylated p105 Rb into hypophosphorylated forms. However, the arotinoid induced significant accumulation of the dephosphorylated, active form of the tumour-suppressor protein. Combinations of Ro 40-8757 with 5FU and interferon alpha 2a resulted in an additive but not synergistic antiproliferative action in HT29 cells. Our data support the interest in Ro 40-8757 as a potent anti-cancer drug, especially in combination therapy with 5FU and interferon, in gastrointestinal and pancreatic cancers, where new active therapeutic modalities are urgently needed. Images Figure 3

During colonic carcinogenesis, mucin-type glycoproteins are known to undergo quantitative and qualitative alterations. The aim of this study was to determine the value of infrared (IR) spectral histology for the histopathological recognition of colonic adenocarcinomas based on mucin-associated IR spectral markers.Paraffin-embedded tissue sections of normal human colon and adenocarcinomas were analysed directly by IR-microspectroscopy (IR-MSP), without prior chemical dewaxing. IR-MSP imaging combined with multivariate analysis permitted the construction of IR colour-coded images of the tissue sections providing spatially resolved biochemical information. This allowed localization of mucin-rich areas and provided label-free spectral-based staining of secreted mucus related to the biochemical heterogeneity of its mucin content. IR images of secreted mucus display the same spectral clusters in both normal and adenocarcinomatous colonic tissues, but with significant differences in surface percentages. Such differences allow a distinction between these two tissue types. Spectral variations associated with changes of mucin secondary structure were the most accurate mucus spectral marker for discriminating between normal colon and adenocarcinomas in the sample set.IR-MSP imaging provides a new type of histology, independent of visual morphology, presenting tremendous possibilities for discovery and clinical monitoring of cancer markers.

Objectives Biomarker detection in pancreatic cyst fluids is of importance to improve the diagnosis of mucinous cystadenoma, a precancerous lesion. However, assay protocols are generally established for serum testing. Methods Immunoradiometric assay of gastric M1/MUC5AC mucin was performed on pancreatic cyst fluids with well-characterized monoclonal antibodies. Results Among 1466 pancreatic cyst fluids tested, about 10% to 15% of samples presented abnormal behaviors: (i) radioactivity measured after immunoradiometric assay much lower than the blank of the assay and (ii) increasing dilution of the fluids leading to apparent increase of M1/MUC5AC concentration. In contrast, none of the 109 hepatic cyst fluids tested presented interference.We demonstrate that some (n = 54) interfering fluids cause mucin degradation as well as antibody degradation. Western blot analysis showed that the C-terminal part of the M1/MUC5AC apomucin is most sensitive to degradation. Conclusions The presence of proteases that degrade antibodies as well as mucin may explain the pitfalls observed in 3.6% of the samples. To detect this interference, each fluid has to be systematically tested at 1:100 dilution in the presence of a saturating concentration of M1/MUC5AC mucin standard and in the absence of antiprotease reagents. Detection of interference could prevent false results caused by mucin degradation in situ.

Background: Resistin and resistin-like molecule (RELM)β comprise a novel class of cysteine-rich proteins secreted into the circulation implicated in hepatic insulin resistance and inflammation. RELMβ is specifically produced by intestinal goblet cells but regulation of its expression and much of its local function are not elucidated. RELMβ has been suggested to regulate colonic inflammation susceptibility, which is dependent on the mucosal barrier integrity. Methods: In this work we explored the physiopathological role of RELMβ in the colon. Among agents tested, carbachol and gastrin were strong inhibitors of RELMβ mRNA accumulation. We examined the effect of recombinant RELMβ on mucin secretion by human mucus-secreting HT29-Cl.16E cells in culture and by mouse colonic goblet cells in vivo. Results: RELMβ upregulated MUC2 and M1/MUC5AC gene expression in HT29-Cl.16E cells. RELMβ enhanced M1/MUC5AC secretion by human colonic HT29-Cl.16E cells and MUC2 secretion by murine intestinal goblet cells. RELMβ exerted its action exclusively on the apical side of HT29-Cl.16E cells, in agreement with its luminal mucosecretagogue effect in mice. Its action required calcium, protein kinase C, tyrosine kinases, and extracellular-regulated protein kinase activities and was synergized by carbachol. An intracolonic RELMβ challenge was performed in the trinitrobenzene sulfonic acid (TNBS)-murine model of colitis and macroscopic and histological scores were monitored. The macroscopic and histopathological severity of TNBS-induced colitis was significantly attenuated by RELMβ pretreatment. Conclusions: A direct participation in maintaining the mucosal defense barrier can be ascribed to RELMβ in line with a regulatory role in intestinal inflammation. (Inflamm Bowel Dis 2008)

International audience; The abnormal expression of gastric M1/MUC5AC mucin in precancerous lesions and colon cancer evidenced by immunohistochemistry led us to check for its presence in the mucus obtained directly from patients undergoing surgery for cancerous (adenocarcinoma) or inflammatory (diverticulitis or ulcerative colitis) diseases. In parallel, the authors quantified aberrant crypt foci (ACF) and their immunolabelling by M1/MUC5AC in mucosae of cancer and diverticulitis patients. Immuno-Radio-Metric Assay of M1/MUC5AC mucin developed by the authors was used to detect M1/MUC5AC mucin in the colonic mucus scraped from surgical specimens. M1/MUC5AC mucin was detected in the mucus of 51/69 (74%) patients with colon adenocarcinoma, versus 7/27 (26%) patients with diverticulitis (threshold: 30 units of M1 mucin per mg protein, area under ROC curve: 0.80). M1/MUC5AC was present in significantly (p < 0.001) larger amounts in the mucus of cancer versus diverticulitis patients. All (10/10) patients with ulcerative colitis tested showed levels above the threshold and their mucosae were strongly labelled with the anti-M1/MUC5AC antibody by immunohistochemistry. Patients with cancer exhibited 3 fold more ACF than those with diverticulitis, but no significant difference was observed in the mean size and M1/MUC5AC expression pattern of ACF between these two groups. The expression of M1/MUC5AC was in correlation with their size. In macroscopically normal mucosa, ACF were the most important source of M1/MUC5AC mucin. Testing of M1/MUC5AC can enhance the detection of precancerous lesions and colon cancer.

International audience; Aberrant crypt foci (ACF) are microscopic lesions which have been postulated to precede the development of adenomas, precursors of colon cancer. The gastric M1/MUC5AC mucin has also been described as an early marker of colon carcinogenesis in the human and in the rat. To study changes in mucin expression associated with the genesis of tumors, Wistar rats were treated by intrarectal instillations of MNNG, twice a week for 2 weeks, and were sacrificed 10 (n = 20), 14 (n = 20), 22 (n = 20), 30 (n = 10) and 66 (n = 16) weeks after the beginning of the treatment. In the treated rats, the MUC5AC mucin was mainly expressed in ACF compared with the histologically normal mucosae, which showed few isolated MUC5AC-positive normal crypts. During carcinogenesis, the percentage of large ACF [> or =10 aberrant crypts] increased and the number of MUC5AC-positive (NCs) decreased. At Week 30, small tumors were observed arising from large ACF, both types of lesions expressing MUC5AC. At Week 66, large tumors showed remnants of MUC5AC-positive ACF in their adjacent mucosae. This observation suggests that the expression of MUC5AC is associated with the ACF/adenoma sequence and supports the notion of large ACF as precursors of adenomas/adenocarcinomas. Moreover, the expression of MUC5AC in the transitional mucosa adjacent to both rat and human colon tumors suggests that some human tumors could arise from large ACF, and reinforces the concept of the premalignant potential of these lesions.

We have developed 11 monoclonal antibodies (MAbs) against human gastric mucin, (1-13M1, 2-11M1, 2-12M1, 9-13M1, 58M1, 19M1, 21M1, 45M1, 463M, 589M, 62M1), which specifically stained by immunohistochemisty both the human gastric surface mucosa and colon adenoma. Among them, five (19M1, 21M1, 463M, 589M, 62M1) immunoreacted with the peptide encoded by the 3' region of the MUC5AC gene (Nollet et al: Int J Cancer 2002;99:336-343). In this study, we identified in the 5' region of this gene the nucleotide fragments encoding peptides immunoreacting with three other anti-M1 MAbs (1-13M1, 2-11M1 and 9-13M1), as well as the SOMU1 MAb (Sotozono et al: J Immunol Methods 1996;192:187-196). 1-13M1 MAb immunoreacts with peptides, including the Cys 2 and Cys 4 domains. The SOMU1 MAb recognized the Cys 5 domain, and the MAbs 2-11M1 and 9-13M1 the globular D1/D2 and D3 domains, respectively. Using serial sections of the mucosae adjacent to colon adenocarcinomas and colon adenomas, we observed that the anti-M1 and anti-SOMU1 MAbs displayed the same immunostaining patterns. The three anti-M1 MAbs (2-12M1, 58M1, and 45M1) did not react with the products of the MUC5AC gene tested until now. The MUC5AC apomucin is now well characterized by MAbs immunoreacting against seven different epitopes belonging to the different main cystein globular domains of this macromolecule. Such antibodies are useful tools for studying the biosynthesis, polymerization, and degradation of mucin.

Three hybridomas secreting monoclonal antibodies (MAbs) against human (62M MAb) or rat (463M and 589M MAbs) gastric mucins were isolated. These MAbs immunoreacted against a human recombinant protein encoded by the 3′ region of the MUC5AC gene. We have mapped 2 new gastric mucin epitopes and the M1-f epitope previously characterized by the 19/21M1 MAbs on MUC5AC-encoded apomucin. The M1-f, 463/589M and 62M epitopes are located in the MUC11p15/von Willebrand factor (vWF)-A3uD4 domain, in the D4-(vWF)-like domain and in the C- and CK-vWF-like domains of MUC5AC, respectively. The 463/589M and 62M MAbs stained the surface epithelium of human gastric mucosae, but not the normal colon mucosae (except 463/589M MAbs, which immunoreacted with 5 of 49 cases). All hyperplastic polyps are stained strongly with the 463/589M MAbs and faintly with the 62M MAb. In addition, 463/589M epitope was detected in 64% of the adenomas and in 93% of the mucosae adjacent to adenocarcinomas; in contrast, only 9% of the adenomas and 29% of the mucosae adjacent to adenocarcinomas expressed the 62M epitope. The expression pattern of the 463/589M epitope in colonic carcinogenesis is different from that of the 19/21M1 epitope, although the 2 epitopes are encoded by MUC5AC gene. © 2002 Wiley-Liss, Inc.

Publisher Summary This chapter describes expression of gastric MUC5AC mucin during colon carcinogenesis. The originality of these monoclonal antibodies (MAbs) is the unusual method of the selection of hybridomas secreting anti-M1 antibodies. Using immunohistology, ones selected the clones secreting antibodies that showed on serial sections the same immunostaining pattern as the polyclonal anti-M1 antibodies: strong staining of colonic adenomatous glands and normal surface gastric epithelium but negative on the normal colon. When the complete sequence of the MUC5AC gene became available, it was possible to show that 8 of these 11 anti-gastric M1 MAbs immunoreacted with the product of this MUC5AC gene. The epitopes recognized by the three other MAbs were precipitated by each of eight anti-M1/MUC5AC MAbs that immunoreacted with the product of MUC5AC gene. Consequently, the link between gastric M1 mucin and the MUC5AC gene was clearly established. Secretory mucins may be regarded also as specific differentiation markers for each of the mucous cells of the digestive epithelium. This last characteristic is of importance to study the modifications of cell differentiation during the carcinogenesis processes. Thus, these mucins can also be of interest as potential tumor markers.

To the Editors: In a recent issue of Clinical Cancer Research , Dr. Gold describes MUC1 as a biomarker of pancreatic intraepithelial neoplasm of low grade (PanIN-1) using PAM4 monoclonal antibodies (mAb; ref. [1][1]). This claim is questionable. PAM4 mAb does not react in normal pancreas, but

Our previous investigations clearly indicate that the transfer of SV40LT in human and murine intestinal epithelial cells induced alterations of the ultrastructural organization and polarization of the resulting immortalized cell lines. We now demonstrate that the functional expression of the SV40LT oncogene in Caco-2 cells (C2) did not modify the enterocytic phenotype, including expression of villin, sucrase-isomaltase, brush border and dome formation. As compared to C2 cells, the transfected C2LT9 cells exhibited similar growth curves and no change in invasive properties in vitro, reduced latency times necessary for manifestation of the tumors in vivo, and increased proportion of cells in the S phase of the cell cycle. The LT antigen-RB complexes are clearly identified at the different phases of the growth curve. Differentiation is characterized by the conversion of Rb into hypophosphorylated forms in mature enterocvtes. These results suggest that: (1) C2 cells and Rb exert a dominant control against LT; (2) the enterocytic differentiation is compatible with LT expression when this oncogene is introduced in proliferating cells already engaged in the differentiation process; (3) Rb and its partners may contribute to the genetic and molecular alterations observed during the progression of human colorectal cancer; (4) LT requires p53 for the expression of his oncogenic activity.

An homogeneous cell population isolated from the inguinal tissue of 3-day-old rats is able to proliferate in primary culture. In the presence of a physiological concentration of insulin (1.5 nM) it converts into cells exhibiting the morphology and the biochemical characteristics of adipocytes. Insulin and epidermal growth factor (EGF) receptors were studied during both the exponential growth and the adipose conversion phases of these cells. Binding experiments with 125 I-labelled peptides were performed directly in the culture dishes. The number of high affinity insulin binding sites increased, during the entire culture period studied, reaching 18 days after plating the value of 10 600 × 2360. Control cells (cultured in the presence of anti-insulin antibody) exhibited an increase of the concentration of insulin binding sites from no more than 500 sites/cell to 6880 ± 1710 sites/cell between dat 0 and 9 (corresponding to the exponential growth phase); this increase was followed by a rapid reduction in insulin receptors during the stationary phase. The density of EGF binding sites increased between day 0 and 4 (one cell cycle), whether the cells were maintained or not with insulin, and plateaued therafter. Mature adipocytes freshly isolated from the inguinal tissue of 3-day-old rats had no detectable EGF binding sites, but their content in high affinity binding sites for insulin was similar to that of cells complete adipocyte conversion in primary culture.

Transfer of the SV40 large-T (LT) oncogene into isolated human and murine intestinal epithelial cells induced alterations of the ultrastructural organization and polarization of the resulting immortalized cell lines. We now demonstrate that the functional expression of the SV40 LT antigen in Caco-2 cells did not alter phenotypic markers of differentiation, including expression of villin, sucrase–isomaltase, brush border and dome formation. As compared to parental cells, the transfected Caco-2 LT9 cells exhibited similar growth curves and no invasive properties in vitro. The major oncogenic function of the SV40 LT antigen in transfected Caco-2 cells is associated with reduced latency times necessary for the manifestation of tumors in athymic nude mice. The Caco-2 cell line contained deleted and mutant p53 alleles (stop codon in position 204) and has no detectable truncated p53 protein by Western blot. Molecular complexes between the SV40 LT antigen and the retinoblastoma-related proteins pRb1 and Rb2 were clearly identified at the different phases of the growth curve. When compared to normal human colonic crypts, Caco-2 cell differentiation is related to partial redistribution of pRb1 into hypophosphorylated, antiproliferative forms. The pRb2 protein is found elevated in a subset of human colorectal tumors and their corresponding liver metastases. We conclude that: (1) Caco-2 cells exert a dominant control against the oncogenic functions of the LT antigen; (2) loss of p53 function is not restrictive for the establishment of polarity and differentiation of the enterocyte lineage; (3) the levels and phosphorylation status of the Rb1 and Rb2 proteins may play important roles in the proliferation and differentiation of normal and neoplastic human colonic mucosa.

Receptors of regulatory peptides such as Vasoactive Intestinal Peptide (VIP) and insulin are present in transformed gut epithelial cell lines from human and coupled to a biological activity, the magnitude of which varies according to the cell line.

Insulin receptors have been demonstrated in isolated rat intestinal epithelial cells. The specific binding of 125I-insulin was time--and temperature--dependent, the optimal temperature of study being 15 degrees. Dissociation of bound 125I-insulin by an excess of unlabelled hormone was rapid and attained 66 +/- 2% in 2 h. When initiated by dilution, the dissociation attained 35 +/- 4% in 2 h, and 72 +/- 1% in 2 h when 10(-7) mol/l unlabelled insulin was added. The pH optimum for the binding process was between 7.5 and 8, and the binding increased proportionally to cell protein concentration up to 1.5 mg/ml. Under standard conditions (2 h at 15 degrees) the degradation of the labelled hormone in the medium accounted for 20--50% of total tracer, depending on the concentration of cells. At apparent equilibrium (2 h at 15 degrees), unlabelled insulin in the range of 10(-10) to 10(-7) mol/l inhibited competitively the binding of 4.3--7 X 10(-11) mol/l 125I-insulin; fifty per cent inhibition was obtained with 3 X 10(-9) mol/l native insulin. Scatchard analysis, after correction for degradation, gave curvilinear plots, that may be explained by two orders of binding sites, with 2,000 +/- 200 sites/cell of high affinity (Ka = 2.2 +/- 0.2 X 10(9) l/mol) and 39,000 +/- 3,000 sites/cell of low affinity (Ka = 5.6 +/- 1.6 X 10(7) l/mol). The potency of proinsulin to compete with 125I-insulin for the binding site was 3% that of insulin, unrelated peptides were inactive. Such results give a molecular basis to different reports suggesting that the intestine could be a target-tissue for insulin.

Physiological studies indicate that epidermal growth factor-urogastrone (EGF) acts on stomach epithelium as mitogen and modulator of acid secretion. Here, we studied the binding of 125I-EGF to gastric glands isolated from the guinea-pig fundus (acid-secreting part) and antrum. At 20 degrees C, the association of 125I-EGF to gastric glands was time-dependent (plateau at 90 min) and reversible (75-85% dissociation in 1 h). No degradation of the peptide was detected, but a time-dependent loss of binding capacity was observed. At apparent equilibrium (90 min, 20 degrees C) unlabelled EGF (80 pM to 80 nM) competed with 125I-EGF-binding in the same manner in antrum and fundus (50% inhibition, with 0.6 nM EGF). Whereas kinetics properties were similar in antrum and fundus, the binding capacity was 40-55% lower in fundus than in antrum in young animals (6-8 weeks). By contrast, in adult animals (20-30 weeks), binding was the same in both parts of stomach. Scatchard analysis showed that two orders of binding sites were present in all cases (Ki 0.34-0.47 nM, Ki 2.2-3.4 nM), and that the differences observed were only accounted for by number of binding sites. These results show that EGF possess high affinity binding sites on gastric epithelium. These sites, dependent upon the age of the animals, may be related to the modulations by EGF of gastric trophism and secretions.