RESUMEN

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.

Asunto(s)

Colecistitis/metabolismo , Mucinas/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad Aguda , Línea Celular , Enfermedad Crónica , Fenómenos Fisiológicos del Sistema Digestivo , Células Epiteliales/fisiología , Epítopos/metabolismo , Vesícula Biliar/fisiología , Expresión Génica , Humanos , Inmunohistoquímica , Mucinas/genética , Mucinas/inmunología , ARN Mensajero/metabolismo , Valores de Referencia

RESUMEN

Secretory mucins play an important role in the cytoprotection of epithelial surfaces and are used as tumour markers in a variety of cancers. The MUC6 secretory mucin was originally isolated from a gastric cDNA library. The aim was to determine the specific type and location of MUC6 mucin gene expression in a wide range of human adult and fetal epithelial tissues. In situ hybridization, RNA analysis, and immunohistochemistry were used to quantify and localize mucin gene expression. The data obtained show that MUC6 is highly expressed in gastric mucosa, duodenal Brunner's glands, gall bladder, seminal vesicle, pancreatic centroacinar cells and ducts, and periductal glands of the common bile duct; focal expression is seen in basal endometrial and endocervical glands. MUC6 epitopes were also highly expressed in 7/10 pancreatic cancers and 7/10 cholangiocarcinomas and focally expressed in 4/10 endocervical adenocarcinomas. Expression of MUC6 occurs early in fetal development and was observed in Brunner's glands and pancreatic ducts at 18-19 weeks and in gastric glands at 20 weeks' gestation. The tissue distribution of the MUC6 secretory mucin indicates that it may function to protect epithelial tissues from a wide range of substances. Expression of MUC6 is frequently preserved in pancreatic and bile duct adenocarcinomas, but it is only sparsely expressed in endocervical carcinomas.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias del Sistema Digestivo/metabolismo , Sistema Digestivo/metabolismo , Mucinas/metabolismo , Proteínas de Neoplasias/metabolismo , Northern Blotting , Epitelio/metabolismo , Femenino , Feto/metabolismo , Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Masculino , Mucina 6 , Mucinas/genética , ARN/genética

RESUMEN

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.

Asunto(s)

Mucinas/química , Secuencia de Aminoácidos , Secuencia de Bases , Dimerización , Humanos , Datos de Secuencia Molecular , Factor de von Willebrand/química

RESUMEN

Recent characterizations of mucins at the molecular level indicate that at least eight mucin genes are expressed by epithelia of mucosal surfaces. The purpose of this study was to determine whether these cloned mucins, designated MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7, are expressed by epithelia of the female reproductive tract. Northern blot analysis, in situ hybridization, and immunohistochemistry were performed using RNA and tissue from surgically removed human reproductive tract specimens including endocervix, ectocervix, vagina, endometrium, and fallopian tube. Complementary DNA to the tandem repeat regions of MUCs 1, 2, 3, 5AC, 5B, and 6; oligonucleotides to the tandem repeat regions of MUCs 4, 6, and 7; and antibodies that recognize unique mucin tandem repeats were used. The data demonstrate that the endocervical epithelium expresses five mucin genes: MUCs 1, 4, 5AC, 5B, and 6. The ectocervical and vaginal epithelia express MUCs 1 and 4, although vaginal expression of MUC4 appears patchy. Endometrial epithelium expresses MUC1 and low amounts of MUC6. MUC6 immunoreactivity was detected only is scattered endometrial glands located in the basalis region in specimens from pre- and postmenopausal women. The only mucin detected in the fallopian tube was MUC1. These data indicate that the endocervical epithelium expresses multiple mucin genes and that the stratified epithelia of the ectocervix and vagina also produce mucins that may function in reproductive processes and protection of the reproductive tract tissues.

Asunto(s)

Genitales Femeninos/metabolismo , Mucinas/biosíntesis , Transcripción Genética , Secuencia de Bases , Northern Blotting , Cuello del Útero/metabolismo , Clonación Molecular , Endometrio/metabolismo , Epitelio/metabolismo , Trompas Uterinas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Mucinas/análisis , Mucinas/genética , Especificidad de Órganos , ARN Mensajero/biosíntesis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Secuencias Repetitivas de Ácidos Nucleicos , Vagina/metabolismo

RESUMEN

Previous studies from our laboratory have shown that HT29 cells selected by adaptation to methotrexate (HT29-MTX) express mature mucins that differ in their immunoreactivity to antibodies against gastric mucin and in the level of one of two major gastric mucin MUC5AC (MUC5) mRNA compared with parental HT29 cells. In this study, we examined the expression of another major gastric mucin, MUC6 mRNA, as well as that of MUC2, -3 and -5 mRNAs in HT29-MTX cells. We also examined their relationship to mucin-related antigen expression and biological properties of the cells such as adhesion to matrigel and E-selectin and in vitro invasiveness, liver colonising activity and degree of differentiation of nude mouse xenograft. Slot blot and Northern analysis revealed markedly increased levels of MUC5 mRNA but no change in MUC6 mRNA level in HT29-MTX cells compared with parental HT29 cells which express barely detectable levels of MUC6 mRNA. A nuclear run-on study showed that MUC5 mRNA was up-regulated at the transcriptional level. The marked increase in MUC5 mRNA was associated with a significant increase in the expression of human gastric mucin and apomucin antigens in HT29-MTX cells. When the adhesive capacity of two cell lines was compared, HT29-MTX cells showed significantly lower adhesion to E-selectin consistent with their lower expression of sialyl Le(x) and sialyl Le(a) antigens compared with HT29 cells. HT29-MTX cells also showed lower adhesive capacity to matrigel than HT29 cells. Interestingly, HT29-MTX cells exhibited significantly decreased liver colonisation capacity in nude mice following splenic vein injection. Furthermore, nude mouse xenograft tumours produced by HT29-MTX cells exhibited a significantly greater degree of differentiation, consisting of mucin-secreting glands than those produced by HT29 cells. In conclusion, these results indicate a shift of predominantly colonic-type mucins to the gastric type, specifically the surface epithelial cell type (MUC5) but not the mucous neck cell or antral gland type (MUC6) in HT29-MTX cells and strongly suggest that altered regulation of mucin genes and the degree of differentiation in cancer cells may be responsible for the altered biological behaviour of these cells.

Asunto(s)

Mucinas/genética , Animales , Northern Blotting , Colágeno/metabolismo , Combinación de Medicamentos , Resistencia a Antineoplásicos , Selectina E/metabolismo , Células HT29 , Humanos , Immunoblotting , Laminina/metabolismo , Metotrexato/farmacología , Ratones , Ratones Desnudos , Mucinas/metabolismo , Trasplante de Neoplasias , Proteoglicanos/metabolismo , ARN Mensajero/genética , ARN Neoplásico/genética , Transcripción Genética , Trasplante Heterólogo

RESUMEN

BACKGROUND & AIMS: Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS: Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS: Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunner's glands of the duodenum. CONCLUSIONS: MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.

Asunto(s)

ADN Complementario/genética , Mucosa Gástrica/metabolismo , Expresión Génica , Mucinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Epitelio/metabolismo , Biblioteca Genómica , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Mucina 5AC , Mucina 5B , Mucinas/química , Mucinas/metabolismo , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos

RESUMEN

Mucins synthesized by malignant cells may contribute (via decreased cellular adhesion and immune recognition) to cancer invasion and metastases. Human mucins are derived from a heterogeneous family of genes, labeled MUC1-6. Our aim was to determine the pattern of mucin gene expression in normal, preneoplastic (intestinal metaplasia), and malignant gastric specimens. Probes and antibodies for specific mucin tandem repeat sequences were used for RNA and immunohistochemical analysis. Normal stomach mucosa was characterized by expression of MUC1, MUC5, and MUC6 mRNA and immunoreactive protein, without MUC2, MUC3, and MUC4 gene expression. In contrast, high levels of MUC2 and MUC3 mucin mRNA and immunoreactive protein were found in specimens with intestinal metaplasia. Gastric cancers exhibited markedly altered secretory mucin mRNA levels compared with adjacent normal mucosa, with decreased levels of MUC5 and MUC6 mRNA and increased levels of MUC3 and MUC4 mRNA. Overall, immunoreactive MUC1 mucin was detected in 72% of 33 gastric cancers, and secretory mucin core peptides were expressed in 34% (MUC2), 45% (MUC3), 19% (MUC5), and 57% (MUC6) of these specimens. Coexpression of multiple (three or more) mucin core proteins occurred in 15 of 25 (60%) advanced (stages III and IV) cancers compared with 1 of 8 (12.5%) early (stages I and II) cancers (P < 0.048). We conclude that human gastric epithelium has a unique mucin gene pattern, which becomes markedly altered in preneoplastic and neoplastic specimens. Increased mucin gene heterogeneity in gastric adenocarcinomas is associated with advanced cancer stage.

Asunto(s)

Adenocarcinoma/metabolismo , Mucosa Gástrica/metabolismo , Expresión Génica , Mucinas/biosíntesis , Mucinas/genética , Familia de Multigenes , Lesiones Precancerosas/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Células Epiteliales , Epitelio/metabolismo , Epitelio/patología , Mucosa Gástrica/citología , Mucosa Gástrica/patología , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Lesiones Precancerosas/cirugía , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Valores de Referencia , Secuencias Repetitivas de Ácidos Nucleicos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/cirugía

RESUMEN

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.

Asunto(s)

Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/economía , Neoplasias Colorrectales/epidemiología , Neoplasias Colorrectales/genética , Análisis Costo-Beneficio , Genes APC , Mutación de Línea Germinal , Humanos , Sangre Oculta , Sigmoidoscopía/economía

RESUMEN

This discussion was selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from a transcription, it has been edited by Nathan M. Bass, MD, PhD, Associate Professor of Medicine, under the direction of Lloyd H. Smith Jr. MD, Professor of Medicine and Associate Dean in the School of Medicine.

Asunto(s)

Neoplasias Colorrectales , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/genética , Humanos , Pronóstico

RESUMEN

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.

Asunto(s)

Neoplasias del Colon/química , Mucinas Gástricas , Intestino Delgado/química , Mucinas/análisis , Mucinas/genética , Péptidos/análisis , Péptidos/genética , ARN Mensajero/análisis , Colon/química , Colon/citología , Neoplasias del Colon/patología , ADN/análisis , ADN/genética , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Intestino Delgado/citología , Mucinas/fisiología , Péptidos/fisiología , ARN Mensajero/genética

RESUMEN

Secretory mucins consist of a protein backbone that is catenated by disulfide bonds, heavily O-glycosylated, and packaged into storage granules prior to release from cells. In this paper, we identify and sequence cDNAs that encode the amino terminus of the MUC2 protein, a major form of mucin found in human intestines and airways. The protein sequence was found to contain a repetitive element of approximately 350 amino acids with considerable sequence similarity to the D domains of prepro-von Willebrand factor. A total of four of these D domains were found to be present in the MUC2 protein, three in the amino-terminal region, and one in the carboxyl-terminal region. Prepro-von Willebrand factor contains four D domains itself, and the overall positioning of the D domains in the two proteins is similar. Prepro-von Willebrand factor contains a 741 residue pro-protein that has been implicated in both the disulfide-linked oligomerization of von Willebrand factor and its packaging into secretory vacuoles. A similar region is present in the MUC2 amino terminus sequence, suggesting that the mechanisms involved in the polymerization and packaging of MUC2 may parallel those already described for von Willebrand factor.

Asunto(s)

Secuencia de Consenso , Mucinas/genética , Proteínas de Neoplasias/genética , Precursores de Proteínas/genética , Factor de von Willebrand/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Colon/metabolismo , Cartilla de ADN , ADN Complementario/metabolismo , Humanos , Datos de Secuencia Molecular , Mucina 2 , Mucinas/biosíntesis , Proteínas de Neoplasias/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transcripción Genética

RESUMEN

Gastric mucin is a large glycoprotein which is thought to play a major role in the protection of the gastrointestinal tract from acid, proteases, pathogenic microorganisms, and mechanical trauma. In this paper we describe the isolation by expression cloning and characterization of cDNAs which code for human gastric mucin. The cDNA sequence is characterized by a tandem repeat region whose individual repeat unit is 507 base pairs (169 amino acids) long. The translated sequence is rich in threonine, serine, and proline (31, 18, and 15%, respectively) and contains a relatively large amount of histidine (7.1%) and alanine (5.6%). RNA blot analysis shows a polydisperse pattern which is characteristic of mucins. Expression of this gene is highest in the stomach and gall bladder, with weaker expression in the terminal ileum and right colon. This expression pattern is different from other human mucins and indicates that this gene codes for a unique mucin. Fluorescence in situ hybridization techniques have localized this gene to chromosome 11p15.4-11p15.5. This is the third mucin to be localized to the 11p15 region and suggests a clustering of secretory mucin genes. We propose that this gene for human gastric mucin be called MUC6.

Asunto(s)

Mucinas Gástricas/genética , Mucinas/genética , Estómago/química , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Clonación Molecular , ADN/aislamiento & purificación , Mucinas Gástricas/análisis , Humanos , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Mucinas/análisis

RESUMEN

The human MUC2 mucin is a large secretory glycoconjugate that coats the epithelia of the intestines, airways, and other mucus membrane-containing organs. Previous work has shown that this mucin contains an extended tandem repeat-containing domain rich in Thr and Pro. In the present work we describe two additional regions of this mucin located both upstream and downstream of the tandem repeat array. The carboxyl-terminal domain contains 984 residues and can be divided into mucin-like (139 residues) and cysteine-rich (845 residues) subdomains. This latter subdomain exhibits varying degrees of sequence similarity to a wide range of mucins and mucin-like proteins including those isolated from rats, pigs, cows, and frogs. We also report here the sequence of 1270 residues lying immediately upstream of the tandem repeats. This region contains a repetitive, mucin-like subdomain and a second cysteine-rich stretch of more than 700 residues. Both cysteine-rich subdomains of this mucin have sequence similarity with von Willebrand factor, a serum protein that exists as a disulfide-linked polymer. This suggests that these cysteine-rich subdomains are important in the catenation of mucin monomers into oligomers, the structures that confer viscoelasticity upon mucus.

Asunto(s)

Colon/química , Cisteína/química , Mucinas/química , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN , Humanos , Datos de Secuencia Molecular , Mucinas/genética , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido

RESUMEN

We have prepared antisera to deglycosylated rat intestinal mucin and used it to obtain immunoreactive clones from a rat jejunum cDNA library. Four of these clones were sequenced, and all were found to be partial cDNAs that contained 18-base pair tandem repeats characteristic of a mucin. These cDNAs encoded a repetitive peptide with a consensus sequence of TTTPDV. Thus, they bear little resemblance to either of the two human intestinal mucin cDNAs isolated previously (Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 and Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. E., Byrd, J. C., Lamport, D. T. A., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415). One of these rat mucin clones, designated RMUC 176, was chosen for further analysis. This clone recognized a band of approximately 9 kilobases when used to probe RNA blots. A strong hybridization band was present using rat small intestine and colon RNA but was not detectable when RNA isolated from heart, liver, or kidney was tested. The RMUC 176 clone and the two previously isolated human intestinal mucin cDNA clones were used to probe blots prepared from BamHI-digested DNA of various species. Here, the human probes detected fragments present only in human and chimpanzee DNA, whereas the RMUC 176 clone recognized fragments only in rat and mouse DNA. Thus, the repetitive portions of intestinal mucin genes are apparently not well conserved between phylogenetically distant species.

Asunto(s)

Evolución Biológica , ADN/aislamiento & purificación , Intestino Delgado/fisiología , Yeyuno/fisiología , Mucinas/genética , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Secuencia de Bases , Carbohidratos/análisis , Cromatografía de Afinidad , Clonación Molecular/métodos , ADN/genética , Ensayo de Inmunoadsorción Enzimática , Biblioteca de Genes , Masculino , Datos de Secuencia Molecular , Mucinas/aislamiento & purificación , Ratas , Ratas Endogámicas , Secuencias Repetitivas de Ácidos Nucleicos

RESUMEN

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.

Asunto(s)

Intestino Delgado/química , Mucinas/genética , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Aminoácidos , Secuencia de Bases , Colon/química , Humanos , Datos de Secuencia Molecular

RESUMEN

Human intestinal mucins are large glycoconjugates (greater than 1,000,000 D) that coat the epithelium, serving to lubricate and protect. Apart from this physiologic function, mucins are important in that they are frequently altered in cancer; thus, they have potential usefulness as tumor markers. We have isolated mucins from human LS174T colon cancer cells and small intestine, deglycosylated these highly purified glycoconjugates, produced polyclonal antibodies to the apomucins, and used these antibodies to isolate two different types of cDNA clones that encode different apomucins. The first class of cDNA clones was isolated using antibodies to deglycosylated LS174T mucin. These cDNA, designated SMUC or MUC2, contain 69 nucleotide tandem repeats that encode a repetitive peptide that is extremely rich in threonine and proline. Northern blots using MUC2 cDNA as probes exhibit large (7,600 bases) and polydisperse hybridization bands. This gene is polymorphic within the human population and is located on chromosome 11. The second class of cDNA was isolated using antibodies to deglycosylated small intestinal mucin. These cDNA, designated SIB or MUC3, have 51 nucleotide tandem repeats that encode a threonine- and serine-rich repetitive peptide. This mucin also is encoded by a large, polydisperse message, but it is clearly distinct from MUC2 as it is located on chromosome 7. Both the MUC2 and MUC3 mucins are expressed in colonic tumors; however, the level of their expression is quite variable. Thus, at least two mucins are expressed by the human gastrointestinal tract. Elucidation of the regulation of these two genes will be important in understanding the physiology and pathophysiology of the human intestine.

Asunto(s)

Mucinas Gástricas , Intestinos/química , Mucinas/análisis , Péptidos/análisis , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Mucinas/genética , Péptidos/genética

RESUMEN

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.

Asunto(s)

Carcinoma/metabolismo , Carcinoma/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Mucinas/biosíntesis , Metástasis de la Neoplasia , Animales , Northern Blotting , Glicosilación , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Ratones , Ratones Desnudos , Mucinas/genética , Trasplante de Neoplasias , ARN Mensajero/genética , Trasplante Heterólogo

RESUMEN

Sodium butyrate induces morphological and biochemical changes consistent with a more differentiated phenotype in some colon cancer cell lines. These changes include increased expression of carcinoembryonic antigen (CEA) and other oncodevelopmental markers. We utilized domain-specific probes and polyclonal antibodies against CEA-related antigens to study sodium butyrate-induced expression of the CEA gene family in a villous adenoma-derived cell line, which is nontumorigenic in nude mice (VACO 235), and two colonic carcinoma cell lines known to respond to sodium butyrate exposure by phenotypic differentiation (HT-29 and LS 174T). The induction begins as quickly as 24 h after exposure and occurs primarily at a transcriptional level, although some translational control is also evident. No evidence was found for gene amplification, rearrangement, or methylation to account for the mechanism of this transcriptional control. [35S]Cysteine pulse-labeled cell lysate immunoblots and polyadenylated RNA blot hybridization suggest that increases in mRNA transcript and CEA-related glycoprotein levels are primarily due to increased synthesis rather than decreased degradation. A considerable amount of heterogeneity is seen in the biosynthesis of the CEA-related glycoproteins, with each cell line showing a distinct pattern of CEA-related antigen expression from a limited number of mRNA transcripts.

Asunto(s)

Adenocarcinoma/inmunología , Antígeno Carcinoembrionario/genética , Neoplasias del Colon/inmunología , Northern Blotting , Southern Blotting , Western Blotting , Butiratos/farmacología , Ácido Butírico , Antígeno Carcinoembrionario/aislamiento & purificación , Línea Celular , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Humanos , Peso Molecular , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación

RESUMEN

A human small intestine lambda gt11 cDNA library was screened using antisera prepared against the deglycosylated protein backbone of human colon cancer xenograft mucin. Three cDNAs were isolated from this screening, designated SMUC 40-42. These cDNAs were all found to contain tandem repeats of 69 nucleotides which encoded a threonine- and proline-rich protein consensus sequence of PTTTPITTTTTVTPTPTPTGTQT. RNA blots probed with one of these cDNAs, SMUC 41, exhibited large, polydisperse hybridization bands at approximately 7,600 bases. Band intensities were strongest when human small intestine, colon, and colon cancer poly(A)+ RNA was used. In vitro translation of poly(A)+ RNA from human small intestine, colon, and colon cancer cells produced a 162,000-dalton peptide that was immunoprecipitated with antibodies to deglycosylated mucin. SMUC 41 was also used to probe DNA blots, which indicated the presence of restriction fragment length polymorphisms in the intestinal mucin gene. These findings may be important in assessing the abnormal mucins found associated with several human diseases.

Asunto(s)

Mucinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Clonación Molecular , ADN/genética , Humanos , Intestinos , Datos de Secuencia Molecular , Mucinas/inmunología , Polimorfismo Genético , Biosíntesis de Proteínas

RESUMEN

Advances in the understanding of biochemical and molecular processes associated with cellular growth and differentiation, as well as colonic carcinogenesis hold promise for the development of new diagnostic and therapeutic modalities for this disease. Altered glycosylation of cell surface and secreted glycoconjugates appear to be useful markers in differentiating normal from malignant colonic tissue. New information regarding deletion and inappropriate expression of several blood group-related carbohydrate antigens as well as the synthesis of unique cancer-related carbohydrate structures has been derived from the use of monoclonal antibody technology, and may lead to more sensitive and specific screening tests and targeted therapies. Several glycoprotein markers for colon cancer have been studied whose diagnostic accuracy may surpass the limited sensitivity and specificity of traditional markers such as CEA. Colorectal cancers contain numerous quantitative and qualitative differences in metabolic and synthetic enzyme activities compared with normal colonic mucosa, which may be of potential importance in designing chemotherapeutic regimens or for following disease activity. Other cancer-associated markers, such as increases in orthinine decarboxylase activity and crypt cell labeling reflect abnormal proliferative activity and may be correlated with premalignant states. Studies of protooncogene expression and certain chromosomal deletions will provide insight into mechanisms of carcinogenesis and may also serve to define high-risk individuals. It is likely that as the biochemical and molecular mechanisms underlying malignancy are further delineated, cancer-associated markers will be defined that will improve diagnostic and more importantly, therapeutic efficacy.

Asunto(s)

Biomarcadores de Tumor , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/terapia , Humanos , Tamizaje Masivo