James W. Hicks

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.

Effects of sodium butyrate and dimethylsulfoxide on biochemical properties of human colon cancer cells

Sodium butyrate and dimethylsulfoxide (DMSO) have marked effects on the growth, morphology, and biochemistry of two human colonic adenocarcinoma cell lines in culture. Doubling times were increased between 18% and 660% while cell viability was unaffected. Both cell lines formed colonies in soft agar in the absence of butyrate or DMSO, but no colonies were observed in the presence of these agents. However, no differences in in vivo tumorigenicities, when cells were implanted in athymic mice, were seen following treatment. Gross morphological alterations including cell enlargement, process formation, and cellular flattening occurred during culture in butyrate or DMSO. Acrylamide gel electrophoresis in sodium dodecyl sulfate revealed no change in membrane protein constituents, but autoradiographic analysis of membrane glycoproteins demonstrated differences between treated and untreated cells. Ganglioside compositions were altered, and a sialyltransferase required for the synthesis of GM3 ganglioside was elevated by butyrate. Although cytoplasmic aminooligopeptidase remained unaffected by butyrate or DMSO, brush border‐associated activity was enhanced by butyrate. Alkaline phosphatase also rose dramatically during culture in butyrate but was not enhanced by DMSO.

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.

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 andPstI 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.

Effect of sodium butyrate, a differentiating agent, on cell surface glycoconjugates of a human pancreatic cell line.

The effects of sodium butyrate, a differentiating agent, on growth properties and glycoconjugates of a human pancreatic carcinoma cell line (CAPAN-1) were studied. Butyrate caused marked changes of in vitro growth properties including prolongation of doubling time and greatly reduced colony forming efficiency in soft agar. Cell surface labeling revealed significant alterations in proteins and glycoproteins after treatment with sodium butyrate (appearance of glycoproteins of molecular weight 250,000, 220,000, and 70,000; reduction of glycoproteins of 164,000, 148,000, 110,000, and 66,800 sizes; increases in proteins/glycoproteins of 85,000 and 78,000). Metabolic labeling of the cells with [3H]fucose or [3H]galactose revealed that sodium butyrate treatment caused a marked reduction in the fucose-containing neutral glycolipids with six or more carbohydrate side chains and an increase in [3H]galactose-labeled neutral glycolipids, particularly GL-3a, GL-4a, and GL-Sa. There was marked reduction in the labeling of a ganglioside with mobility similar to that of GM4 and of sulfogalactosylceramide. An increase in a ganglioside with mobility above GM, was also caused by butyrate. These data indicate that sodium butyrate may be useful in the identification of differentiation or malignancy-associated glycoconjugate markers of human pancreatic cells.

Surface membrane glycoproteins of cultured human pancreatic cancer cells

The cell‐surface glycoproteins and proteins of four human pancreatic cell lines (MIA PaCa‐2, PANC‐1, HS766T, and CAPAN‐1) were separately tritiated using galactose oxidase/NaB(3H)4 and iodinated using lactoperoxidase/12I. Gel electrophoresis showed that the cell lines had very different surface components. All four cell lines were tested for cell‐surface antigens that cross‐reacted with antisera raised against carcinoembryonic antigen and against the membrane fractions of MIA PaCa‐2 and CAPAN‐1 cells. CAPAN‐1 cells reacted most strongly with all three antisera. Seventeen cell‐surface proteins can be detected when CAPAN‐1 cells are labeled using lactoperoxidase. The labeled membranes were solubilized in detergent and subjected to affinity chromatography on Sepharose‐conjugated lectins. The bound proteins were eluted and analyzed on gel electrophoresis. All 17 proteins capable of being labeled by lactoperoxidase bound to at least one lectin column, indicating they are all glycoproteins.

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.