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Cloning and characterization of mouse intestinal MUC3 mucin: 3' sequence contains epidermal-growth-factor-like domains.

ABSTRACT: Mucin glycoproteins are a heterogeneous family of high-molecular-mass, heavily glycosylated proteins differentially expressed in epithelial tissue of the gastrointestinal, reproductive and respiratory tracts. We report here the cloning of a mouse caecal mucin (MCM). Amino acid analysis of purified MCM revealed a high content of serine (10.8%) and threonine (25.1%). Antibodies against deglycosylated MCM were prepared for immunohistochemical analysis and for screening a mouse caecal cDNA library. Immunohistochemical analysis showed strong staining of goblet cells and patchy staining of surface columnar cells in the duodenum, small intestine, caecum, colon and rectum. Screening of a mouse caecal cDNA library yielded clones containing tandem repeats of 18 bp with two predominant peptide sequences of TTTADV and TTTVVV. The tandem repeat domain is followed by 1137 bp of non-repetitive sequence and 521 bp of 3' untranslated sequence prior to the poly(A) tail. Two cysteine-rich regions lie within the 3' non-repetitive domain. The arrangement of the cysteines within these regions corresponds to epidermal growth factor-like domains. Following the second cysteine-rich region is a stretch of 19 hydrophobic amino acids which may act as a transmembrane domain or allow for interaction with hydrophobic molecules. Northern blot analysis indicates the mRNA is approximately 13.5 kb with greatest expression in the caecum and lesser amounts in the colon and small intestine. No MCM message is found in mouse stomach, trachea, lung, kidney, oesophagus or pancreas. In situ hybridization studies show that MCM message is expressed at the tips of villi in the intestine and in the upper crypts and surface cells of the caecum and colon. Chromosomal analysis assigns this gene to mouse chromosome 5 in a region of conserved linkage with human chromosome 7, the location of the human MUC3 gene. We conclude that we have identified a mouse caecal mucin which represents the mouse homologue of human MUC3. The mouse MUC3 cDNA sequence suggests that it is a novel non-polymerizing mucin which may participate in membrane or intermolecular interactions through its 3' non-repetitive region.

SUBMITTER: Shekels LL

PROVIDER: S-EPMC1219276 | biostudies-other | 1998 Mar

REPOSITORIES: biostudies-other

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Project description:BackgroundIdentification of early molecular pathway changes may be useful as biomarkers for tumour response/resistance prediction, and here we provide direct in vivo proof of this concept. The type 1 insulin-like growth factor receptor (IGF1R) has been implicated in various aspects of adenoma development and metastasis. We show here that, in murine intestinal adenomas acutely exposed to a small molecular inhibitor of EGFR (gefitinib), there is concurrent suppression of EGFR downstream signalling and induction of IGF signalling. We therefore tested the hypothesis that blockade of EGFR signalling was being tempered by compensatory activation of the IGF pathway by examining the effect of chronic suppression of IGF1R using AZ12253801, a small molecular tyrosine kinase inhibitor of IGF1R.MethodsMale Apc(min/+) mice with an intestinal tumour burden were exposed to a single dose of an inhibitor against EGFR (gefitinib), IGF1R (AZ12253801), 0.5% Tween 80 or combined EGFR/IGF1R inhibitor and culled 4 h post dosing. Tumour tissue was analysed to detect the early molecular pathways induced and anti-tumour phenotypic changes. Cohorts of male Apc(min/+) mice (n=15-17) were subsequently treated with gefitinib for a period of 8 weeks and subsequently exposed to single (either gefitinib or AZ12253801) or combined (gefitinib and AZ12253801) therapy. We also included a vehicle-treated cohort, which was never exposed to gefitinib and became symptomatic of the disease by day 150.ResultsBoth single treatments delayed the onset of disease symptoms. Combined dosing with gefitinib and AZ12253801 similarly delayed the onset of symptoms, and at 200 days suppressed small intestinal tumourigenesis more effectively than either treatment alone (median small intestinal adenoma volume (47 mm(3) (comb) vs 248 mm(3) (AZ12253801), P=0.0003 and 47 mm(3) (comb) vs 123 mm(3) (gefitinib), P=0.0042, Mann-Whitney (two-sided) test).ConclusionOur data provide evidence in support of the use of combinatorial therapy, and establishes the need to further define the precise benefit in vivo.

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Cloning and characterization of mouse intestinal MUC3 mucin: 3' sequence contains epidermal-growth-factor-like domains.

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