Neil W. Toribara

Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer.

BACKGROUND/AIMS Several studies have reported Northern blot data showing that mucin is expressed in a tissue-specific manner. To determine whether expression is limited to specific cell types within these tissues requires histological analysis. METHODS Both immunocytochemistry and in situ hybridization were used to identify cell types expressing the MUC2 and MUC3 mucins in the human small intestine, colon, and colon carcinoma. RESULTS In the normal small intestine and colon, an antibody recognizing the MUC2 apomucin stained goblet cells. In contrast, an antibody recognizing the MUC3 apomucin stained both goblet and absorptive cells. Consistent with this, in situ hybridization showed MUC2 messenger RNA (mRNA) only in goblet cells and MUC3 mRNA in both goblet and absorptive cells. In several samples of moderately well-differentiated colon cancer, MUC2 and MUC3 showed distinct patterns of expression, but the expression level of each was reduced compared with levels in normal tissue; there was considerable tumor-to-tumor and cell-to-cell variability using both mucin antibodies and complementary DNA probes. CONCLUSIONS Individual mucin genes have distinct patterns of expression within mucin-producing tissues, suggesting that the various mucin gene products play distinct functional roles.

Mucin production by human colonic carcinoma cells correlates with their metastatic potential in animal models of colon cancer metastasis.

Patients with mucinous colorectal cancers characteristically present with advanced disease, however, the relationship between mucin production by colon cancer cells and their metastatic potential remains unclear. We therefore sought to define the relationship between mucin production by human colon cancer cells and metastatic ability by employing animal models of colon cancer metastasis. LS LiM 6, a colon carcinoma cell line with high liver metastasizing ability during cecal growth in nude mice produced twofold more metabolically labeled intracellular mucin and secreted four- to fivefold more mucin into the culture medium compared to poorly metastatic parental line LS174T. This was accompanied by a similar elevation in poly(A)+ RNA detected by blot hybridization with a human intestinal mucin cDNA probe, and increases in mucin core carbohydrate antigens determined immunohistochemically. Variants of LS174T selected for high (HM 7) or low (LM 12) mucin synthesizing capacity also yielded metastases after cecal growth and colonized the liver after splenic-portal injection in proportion to their ability to produce mucin. Inhibition of mucin glycosylation by the arylglycoside benzyl-alpha-N-acetyl-galactosamine greatly reduced liver colonization after splenic-portal injection of the tumor cells. These data suggest that mucin production by human colon cancer cells correlates with their metastatic potential and affects their ability to colonize the liver in experimental model systems.

Screening for Colorectal Cancer

In the past few years, knowledge of the clinical, biologic, and molecular genetic characteristics of colorectal cancer has greatly increased. Although the most cost-effective approach remains to be identified, screening for colorectal cancer can decrease mortality due to this disease by detecting cancers at earlier stages and allowing the removal of adenomas, thus preventing the subsequent development of cancer. Molecular studies that have helped define the genetic basis for this disease hold great promise for the development of better and more powerful methods to identify populations at risk. Individually, these technological, clinical, and basic-science advances are exciting; together, they promise to move us closer to the goal of substantially reducing mortality due to colorectal cancer.

MUC-2 human small intestinal mucin gene structure. Repeated arrays and polymorphism.

MUC-2, the first described intestinal mucin gene, has become important as a prototype for secreted mucins in several organ systems. However, little is known about its protein backbone structure and hence its role in diseases such as colon cancer, ulcerative colitis, and cystic fibrosis, which are known to have mucin abnormalities. Studies in this manuscript show that MUC-2 contains two distinct regions with a high degree of internal homology, but the two regions bear no significant homology to each other. Region 1 consists mostly of 48-bp repeats which are interrupted in places by 21-24-bp segments. Several of these interrupting sequences show similarity to each other, creating larger composite repeat units. Region 1 has no length polymorphisms. Region 2 is composed of 69-bp tandem repeats arranged in an uninterrupted array of up to 115 individual units. Southern analysis of genomic DNA samples using TaqI and HinfI reveals both length and sequence polymorphisms which occur within region 2. The sequence polymorphisms have different ethnic distributions, while the length polymorphisms are due to variable numbers of tandem repeats.

The Carboxyl-terminal Sequence of the Human Secretory Mucin, MUC6 ANALYSIS OF THE PRIMARY AMINO ACID SEQUENCE

The distribution of MUC6 suggests that its primary function is protection of vulnerable epithelial surfaces from damaging effects of constant exposure to a wide range of endogenous caustic or proteolytic agents. A combination of genomic, cDNA. and 3′ rapid amplification of cDNA ends techniques was used to isolate the carboxyl-terminal end of MUC6. The 3′ nontandem repeat region contained 1083 base pairs of coding sequence (361 amino acids) followed by 632 base pairs of 3′-untranslated region. The coding sequence consists of two distinct regions; region 1 contained the initial 270 amino acids (62% Ser-Thr-Pro with no Cys residues), and region 2 contained the COOH-terminal 91 amino acids (22% Ser-Thr-Pro with 12% Cys). Although region 1 had no homology to any sequences in GenBank, region 2 had approximately 25% amino acid homology to the COOH-terminal regions of human mucins MUC2, -5, and -5B and von Willebrand factor. The shortness of region 2 would leave little of the peptide backbone exposed to a potentially hostile environment. Antibody studies suggest that MUC6 in its native form exists as a disulfide-bonded multimer. The conservation of the 11 cysteine positions in region 2 suggests the importance of this short region to mucin polymerization.

Variable number tandem repeat polymorphism of the mucin genes located in the complex on 11p15.5

Abstract A family of four genes that encode major secreted mucins (MUC6, MUC2, MUC5AC and MUC5B) map to within 400kb on chromosome 11p15.5. These genes contain long stretches of tandem repeats of sequence that encode serine- and threonine-rich domains but that otherwise show no similarity from gene to gene, and regions of unique sequence domains that do show evidence of sequence homology. We have previously reported the existence of polymorphism in three of these genes but the extent and nature of this allelic variation is now described here in detail. Variable number tandem repeat polymorphisms of MUC6, MUC2 and MUC5AC are predicted to encode mucin polypeptides that differ in length. In the case of MUC2 and MUC6 these length differences are substantial (up to twofold). MUC5B in contrast does not show common allele length variation. Three MUC2 mutations are reported, none of which are associated with the meiotic recombinations previously observed in this region of chromosome 11.

Altered mucin core peptide expression in acute and chronic cholecystitis

Human mucin genes include membrane-bound mucins (MUC1, MUC3, MUC4) and secretory mucins (MUC2, MUC5AC, MUC5B, MUC6). Our aim was to determine mucin gene expression in human gallbladder cell lines, normal gallbladder from liver donors (N = 7) and surgical specimens with mild chronic cholecystitis (N = 29), chronic cholecystitis (N = 48), and acute and chronic cholecystitis (N = 27). MUC1 mRNA was ubiquitous; however, only rare MUC1 immunoreactivity was detected. MUC3, MUC5AC, MUC5B, and MUC6 mRNA were present in all gallbladder specimens and cell lines examined. Prominent MUC3, MUC5AC, MUC5B, and MUC6 immunoreactivity was present in 86–100% of normal gallbladders. The frequency of MUC5AC reactivity was decreased in specimens with acute cholecystitis (P < 0.05). In contrast, MUC2-reactivity was absent in normal gallbladder and present in 53.8% of acute cholecystitis specimens (P < 0.05). Surface epithelium is characterized by MUC3, MUC5AC, and MUC5B, whereas deeper mucosal folds display MUC5B and MUC6 immunoreactivity. Gallbladder epithelium demonstrates a unique and diverse pattern of mucin core proteins that becomes altered with increasing degrees of inflammation.