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J R Gum

Publications and source records attributed to J R Gum.

At least 19 recordsLinked to original sources
PubMed2001

Mouse intestinal goblet cells expressing SV40 T antigen directed by the MUC2 mucin gene promoter undergo apoptosis upon migration to the villi.

Mucinous colorectal cancers exhibit a characteristic set of molecular genetic alterations and may be derived from progenitor cells committed to the goblet cell lineage. Previously, we demonstrated that the MUC2 mucin gene promoter drives transgene reporter expression with high specificity in small intestinal goblet cells of transgenic mice. On the basis of these experiments, we reasoned that the MUC2 promoter could be used to drive SV40 T antigen (Tag) expression in the same cell type, decoupling them from their normal antiproliferative controls. A line of mice was established (MUCTag6) that expressed Tag in intestinal goblet cells as determined by RNA blot and immunohistochemical analysis. These goblet cells were markedly involuted however, most notably in the villi. Endogenous intestinal MUC2 message levels were reduced to about one third the normal level in these mice. However, absorptive cell lineage markers were comparable with nontransgenics. Bromodeoxyuridine-positive S-phase cells are limited to crypts in nontransgenic intestine but are present in both crypts and villi in MUCTag6. In contrast, mitotic cells were not present in the villi, indicating that MUCTag6 villi goblet cells do not progress into M phase. Apoptotic cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling were increased more than fourfold in MUCTag6 villi (P < 0.0001), and apoptotic goblet cells were evident. Electron microscopic examination of MUCTag6 intestinal villi revealed the presence of degraded cell remnants containing mucin goblets together with other cell debris, further indicating apoptosis of the goblet cell lineage. Thus, the expression of Tag in intestinal goblet cells releases them from normal antiproliferative controls, causing their inappropriate entry into S phase even after they transverse the crypt/villus junction. They do not, however, progress to M phase. Instead, they undergo apoptosis with a high degree of efficiency in S or G(2) phase. These experiments demonstrate that apoptosis effectively blocks inappropriate goblet cell proliferation in the intestine, supporting its proposed role as an antineoplastic mechanism.

Animals↗
PubMed2001

Efficient detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor microsatellite instability before germline genetic testing.

BACKGROUND & AIMS: The optimal strategy for the detection of hereditary nonpolyposis colorectal cancer (HNPCC) gene carriers remains uncertain. We evaluated whether microsatellite instability (MSI) analysis or MSH2 and MLH1 protein immunostaining of tumors will screen individuals efficiently for germline MSH2 and MLH1 testing. METHODS: We performed a case-series study of 114 eligible families enrolled in our high-risk colorectal cancer (CRC) registry. Medical history data were collected on probands and relatives. MSI analysis was performed on proband tumors, and MSH2 and MLH1 protein immunostaining was assessed. Denaturing gradient gel electrophoresis was used to identify germline MSH2 or MLH1 mutations in probands found to have tumors with high-frequency MSI. RESULTS: Tumor tissue and adequate clinical data were available in 109 of the 114 families. Amsterdam criteria and Bethesda guidelines were met by 23% and 70% of the families, respectively. High-frequency MSI was identified in the proband tumors in 47 of the 109 families (43%). Germline MSH2 and MLH1 gene testing was carried out in the probands of 32 of 47 families with MSI-H tumors. Mutations were detected in 16 families (9 in MSH2 and 7 in MLH1) and sequence variants of uncertain significance in 5 families (1 in MSH2 and 4 in MLH1). Germline mutations or sequence variants of uncertain significance were detected in 15 of 19 (79%) of our Amsterdam families and in 6 of 13 (46%) of our non-Amsterdam families with MSI-H tumors. MSH2 and MLH1 protein immunostaining was assessed in 38 of the 47 MSI-H tumors. Unequivocal loss of hMLH1 expression was found in 20 tumors and loss of MSH2 expression in 9 tumors. Corresponding loss of protein expression was seen in 17 of 18 (94%) of tumors from probands with germline mutations or variants. CONCLUSIONS: The detection of high-frequency MSI or the loss of MSH2 or MLH1 immunostaining in CRCs are both useful criteria for selecting high-risk patients who should be tested for germline mutations in MSH2 or MLH1.

Colorectal Neoplasms, Hereditary Nonpolyposis↗
PubMed2000

Analysis of dipeptidyl peptidase IV gene regulation in transgenic mice: DNA elements sufficient for promoter activity in the kidney, but not the intestine, reside on the proximal portion of the gene 5'-flanking region.

The dipeptidyl peptidase IV (DPPIV) gene encodes a brush border membrane exopeptidase that is expressed in a tissue-restricted fashion. To examine the regulation of DPPIV transcription in various tissues in vivo, we examined the expression of DPPIV 5'-flanking region (promoter)-human growth hormone reporter constructs in transgenic mice. These mice exhibited cell-type specific reporter expression in kidney. Surprisingly, however, only very low to non-detectable levels of reporter were found in small intestine. These results indicate that DNA elements sufficient for DPPIV expression in kidney, but not intestine, reside in the 5'-flanking region of the gene.

5' Untranslated Regions↗
PubMed2000

Multiple transcripts of MUC3: evidence for two genes, MUC3A and MUC3B.

The MUC3 gene encodes a transmembrane mucin-type glycoprotein. A number of consistent single nucleotide changes were observed in different MUC3 cDNAs from a single individual, suggesting the presence of at least three different transcripts per individual. This transcript heterogeneity is due both to the existence of a second copy of the MUC3 gene and to allelic changes. Sequencing of the second MUC3 shows that it has the same C-terminal domain and intron/exon structure as the previously described MUC3. The tandem repeat domain has the same amino acid consensus sequence but shows more substitutions. The unique exonic sequences range from 94 to 100% identity at the nucleotide level and correspondingly few amino acid changes have been identified. The introns show around 95% identity. We propose to name this second gene MUC3B, MUC3A being reserved for the first MUC3 gene. MUC3B, like MUC3A, is expressed in intestine and Caco-2 cells.

Alleles↗
PubMed2000

Polymorphism of human mucin genes in chest disease: possible significance of MUC2.

Most of the genes that encode epithelial mucins are highly polymorphic due to variations in the length of domains of tandemly repeated (TR) coding sequence, the part of the apomucin that is heavily glycosylated. We report here for the first time a difference in the distribution of MUC TR length alleles in chest disease. We examined the distribution of the length alleles of those MUC genes whose expression we have confirmed in the bronchial tree in an age- and sex-matched series of 50 pairs of atopic patients with and without asthma. There was no significant difference in the distribution of alleles of MUC1, MUC4, MUC5AC, and MUC5B. MUC2, however, showed a highly significant difference in distribution. The atopic, nonasthmatic individuals showed an allele distribution that was very different from all our other patient and control groups, this group showing a longer mean allele length. The observations suggest that longer MUC2 alleles may help protect atopic individuals from developing asthma, though the effect may be due to a linked gene. The biological significance of this variation with respect to susceptibility to asthma will merit further investigation, and it will also be important to substantiate this finding on an independent data set.

Alleles↗
PubMed1999

Genomic organization and structure of the 3' region of human MUC3: alternative splicing predicts membrane-bound and soluble forms of the mucin.

The MUC3 gene encodes a large, glycosylated mucin produced by intestinal epithelial cells to form a protective barrier against the external environment. Recently published cDNA sequences for the carboxyl-terminal region of MUC3 polypeptide indicated that rodent Muc3 possesses two epidermal growth factor (EGF)-like domains, and putative transmembrane and cytoplasmic domains, whereas the sequence of human MUC3 predicted termination after the first EGF-like domain. Here we describe the complete genomic sequence encompassing the carboxyl terminal region of human MUC3, revealing the boundaries of 11 exons. RT-PCR and cDNA library cloning experiments indicate that the gene is alternatively spliced, yielding a major membrane-bound form as well as multiple soluble forms. Thus, this work indicates that both membrane-bound and soluble MUC3 mucin proteins are produced by alternative splicing of a single gene. A potentially important polymorphism involving a Tyr residue with a proposed role in signalling is described as well.

3' Untranslated Regions↗
PubMed1999

Regulation of the gene for human dipeptidyl peptidase IV by hepatocyte nuclear factor 1 alpha.

Hepatocyte nuclear factor 1 was identified as the transcription factor binding to a 20 bp (-150 to -131) region of the gene for human dipeptidyl peptidase IV, which has been shown to be important for the expression of dipeptidyl peptidase IV in the human intestinal and hepatic epithelial cell lines Caco-2 and HepG2. Functional analysis of the hepatocyte nuclear factor 1 site was performed with two minimal dipeptidyl peptidase IV promoter constructs (-250 to -41, and -150 to -41) with and without a 3 bp mutation in the hepatocyte nuclear factor 1 sequence, and used in transient transfection experiments with Caco-2 cells. The results show that the mutated constructs were able to drive transcription at only 5-10% of the activity of the non-mutated controls. Co-transfection of 3T3 cells with hepatocyte nuclear factor 1 (alpha or beta) and dipeptidyl peptidase IV promoter constructs (-250 to -41 or -150 to -41) resulted in a 2.5-6-fold increase in transcription over controls with hepatocyte nuclear factor 1 alpha but not with hepatocyte nuclear factor 1 beta. The results of this study show that hepatocyte nuclear factor 1 binds to the -150 to -131 region of the human dipeptidyl peptidase IV promoter and is necessary for transcriptional activation of the gene for dipeptidyl peptidase IV.

3T3 Cells↗
PubMed1999

Goblet cell-specific expression mediated by the MUC2 mucin gene promoter in the intestine of transgenic mice.

The regulation of MUC2, a major goblet cell mucin gene, was examined by constructing transgenic mice containing bases -2864 to +17 of the human MUC2 5'-flanking region fused into the 5'-untranslated region of a human growth hormone (hGH) reporter gene. Four of eight transgenic lines expressed reporter. hGH message expression was highest in the distal small intestine, with only one line expressing comparable levels in the colon. This contrasts with endogenous MUC2 expression, which is expressed at its highest levels in the colon. Immunohistochemical analysis indicated that goblet cell-specific expression of reporter begins deep in the crypts, as does endogenous MUC2 gene expression. These results indicate that the MUC2 5'-flanking sequence contains elements sufficient for the appropriate expression of MUC2 in small intestinal goblet cells. Conversely, elements located outside this region appear necessary for efficient colonic expression, implying that the two tissues utilize different regulatory elements. Thus many, but not all, of the elements necessary for MUC2 gene regulation reside between bases -2864 and +17 of the 5'-flanking region.

Animals↗
PubMed1999

Mucin gene and antigen expression in biliopancreatic carcinogenesis.

Mucins are high molecular weight glycoproteins which are heavily glycosylated with many carbohydrate side chains. In epithelial cancers such as biliopancreatic cancer, both quantitative and qualitative alterations in carbohydrate and polypeptide moieties of mucin glycoproteins occur. These changes in mucin glycoproteins are one of the most common phenotypic markers of biliopancreatic carcinogenesis and may play an important pathobiological role. The expression of some of the sialylated carbohydrate antigens appears to correlate with a poor prognosis and increased metastatic potential in biliopancreatic cancer. The increased exposure of peptide epitopes of mucin glycoproteins in biliopancreatic cancer appears to be due to either abnormal glycosylation and/or altered levels of mucin gene transcription. In addition, dysregulation of tissue specific mucin gene expression occurs in biliopancreatic cancer. This information is currently being exploited for further elucidation of the molecular mechanisms involved in carcinogenesis, tumor progression and metastasis, and the development of novel methods of diagnosis and therapy of biliopancreatic cancer.

Antigens, Neoplasm↗
PubMed1998

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

A family of four genes that encode major secreted mucins (MUC6, MUC2, MUC5AC and MUC5B) map to within 400 kb 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.

Child↗
PubMed1997

MUC3 human intestinal mucin. Analysis of gene structure, the carboxyl terminus, and a novel upstream repetitive region.

MUC3 is a large mucin glycoprotein expressed by the human intestine and gall bladder. In this manuscript, we present details of the deduced protein structure of MUC3. The MUC3 carboxyl-terminal domain is 617 residues in length, including 511 residues of a non-repetitive mucin-like domain (27% Thr, 22% Ser, and 11% Pro) and a 106-residue Cys-rich domain with homology to the epidermal growth factor (EGF) -like structural motifs found in many proteins. The region of MUC3 located upstream of the previously described 51-base pair (bp) tandem repeats, which encode a major Ser and Thr-rich domain, consists of a second type of repetitive structure with an imperfect periodicity of approximately 1125 bp. This domain is also mucin-like and appears to be considerably larger than 2000 residues (6000 bp). The MUC3 gene itself is large and complex. Using pulse field gel electrophoresis and blot analysis, the smallest fragment found that contained all human genomic DNA hybridizing to the 51-bp tandem repeat probe was 200 kilobases with restriction enzyme SwaI. Both PvuII and PstI produced two sets of hybridizing fragments that were hypervariable within the human population with a pattern suggestive of both a variation in the number of tandem repeats (VNTR) and sequence polymorphism. These fragments varied independently of each other, but no genetic recombination was detected in a study of 40 human families. Thus, the MUC3 gene encodes a very large glycoprotein with a structure very different from that of any mucin currently described.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of the MUC2 (human intestinal mucin) gene 5'-flanking region: promoter activity in cultured cells.

The initiation point for MUC2 gene transcription is located within a 7000-base GC-rich region of the mucin gene cluster found on chromosome 11p15.5. The promoter activity of the 5'-flanking region of the MUC2 gene was examined following its cloning into the luciferase-producing pGL2-Basic reporter vector. A short segment comprising bases -91 to -73 relative to the start of transcription was found to be important for basal promoter activity in all cell lines tested. Electrophoretic mobility shift assays demonstrated nuclear protein binding to this region, which contains the consensus CACCC motif (5'-GCCACACCC). This element has been shown to be functionally important in several promoters that are active in diverse cell types. Competition experiments using an Sp1 oligonucleotide and antibody supershift experiments indicated that both Sp1 and other Sp1 family members bind to this element. Inclusion of the region between bases -228 and -171 in pGL2-Basic constructs increased normalized luciferase reporter activity by almost 3-fold in C1a cells, which produce relatively high levels of MUC2 mRNA. Significantly lower levels of normalized luciferase activity resulted when the same construct was transfected into cultured cell lines that express low or undetectable levels of MUC2, suggesting a possible role for this region in conferring cell-type specificity of expression. We also demonstrate, using actinomycin D, that the MUC2 mRNA is long-lived, at least in cultured cells. Moreover, no evidence was found that the MUC2 mRNA turned over more rapidly in LS174T cells, which produce relatively low levels of MUC2 mRNA, as compared with C1a cells, which produce high levels of mRNA. Thus a long mRNA half-life appears to be an important mechanism involved in achieving elevated levels of MUC2 mRNA.

Amino Acid Sequence↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease.

An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a Cl ion channel, cause airway mucus obstruction leading to fatal lung disease. Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of Pseudomonas aeruginosa infection in the airways, and here we provide evidence directly linking P. aeruginosa infection to mucus overproduction. We show that P. aeruginosa lipopolysaccharide profoundly upregulates transcription of the mucin gene MUC 2 in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyr-phostin AG 126. These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease.

Benzylidene Compounds↗
PubMed1996

Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes.

Four distinct genes that encode mucins have previously been mapped to chromosome 11p15.5. Three of these genes (MUC2, MUC5AC, and MUC6) show a high level of genetically determined polymorphism and were analyzed in the CEPH families. Linkage analysis placed all three genes on the genetic map in a cluster between HRAS and INS, and more detailed analysis of recombinant breakpoints revealed that MUC6 is telomeric to MUC2. Using these recombinants D11S150 was mapped close to MUC2. Ten of the 11 recombinant chromosomes studied in detail were paternal, and the recombinant events were distributed throughout the 11p15 region, suggesting that the high level of recombination observed in 11p15.5 is not due to a particular recombinational hot spot. Pulsed-field gel electrophoresis was used to make a detailed physical map of the MUC cluster and to integrate the physical and genetical maps. The gene order was determined to be HRAS-MUC6-MUC2-MUC5AC-MUC5B-IGF2. The MUC genes span a region of some 400 kb and the map extends 770 kb and contains 15 putative CpG islands. The order of the MUC genes on the map corresponds to the relative order of their expression along the anterior-posterior axis of the body, suggesting a possible functional significance to the gene order.

Adult↗
PubMed1996

Alteration in mucin gene expression and biological properties of HT29 colon cancer cell subpopulations.

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.

Animals↗