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M A Hollingsworth

Publications and source records attributed to M A Hollingsworth.

At least 19 recordsLinked to original sources
PubMed2006

MUC4 expression is regulated by cystic fibrosis transmembrane conductance regulator in pancreatic adenocarcinoma cells via transcriptional and post-translational mechanisms.

MUC4 mucin is a high molecular weight transmembrane glycoprotein that plays important roles in tumour biology. It is aberrantly expressed in pancreatic adenocarcinoma, while not being detectable in the normal pancreas. Previous studies have demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is defective in CF, is implicated in multiple cellular functions, including gene regulation. In the present study, using a CFTR-defective pancreatic cancer cell line and its derived subline expressing functional CFTR, we report that MUC4 expression is negatively regulated by CFTR. Short-interfering RNA (siRNA)-mediated silencing of CFTR also leads to an increased expression of MUC4. Additionally, our results suggest that CFTR-mediated regulation of MUC4 is cell density-dependent and is achieved by transcriptional and posttranslational mechanisms. Moreover, in a panel of pancreatic cancer cell lines and normal pancreas, we observed that CFTR was downregulated in pancreatic cancer cells and negatively correlated with MUC4 in most cases. An aberrant expression of MUC4 was also detected in the CF pancreas. Downregulation of CFTR in pancreatic adenocarcinoma and its inverse association with the tumour-linked mucin, MUC4, indicate novel function(s) of CFTR in pancreatic tumour biology and suggest the implication of new signalling pathway(s) in MUC4 regulation.

Adenocarcinoma↗
PubMed2006

The human homologue of the RNA polymerase II-associated factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis.

The 19q13 amplicon in pancreatic cancer cells contains a novel pancreatic differentiation 2 (PD2) gene (accession number AJ401156), which was identified by differential screening analysis. PD2 is the human homologue of the RNA polymerase II-associated factor 1 (hPaf1). In yeast, Paf1 is part of the transcription machinery, acting as a docking protein in between the complexes Rad6-Bre1, COMPASS-Dot1p, and the phosphorylated carboxyl terminal domain of the RNA polymerase II. As such, Paf1 is directly involved in transcription elongation via histone H2B ubiquitination and histone H3 methylation. The PD2 sequence is highly conserved from Drosophila to humans with up to 98% identity between rodent and human, suggesting the functional importance of PD2/hPaf1 to maintain cellular homeostasis. PD2 is a modular protein composed of RNA recognition motif, DEAD-boxes, an aspartic/serine (DS)-domain, a regulator of the chromosome condensation domain and myc-type helix-loop-helix domains. Our results further showed that PD2 is a nuclear 80 kDa protein, which interacts with RNA polymerase II. In addition, we have demonstrated that the overexpression of PD2 in the NIH 3T3 cells result in enhanced growth rates in vitro and tumor formation in vivo. Altogether, this paper presents strong evidence that the overexpression of PD2/hPaf1 is involved in cancer development.

Amino Acid Sequence↗
PubMed2004

Thomsen-Friedenreich antigen expression in gastric carcinomas is associated with MUC1 mucin VNTR polymorphism.

Aberrant glycosylation of mucins is a common phenomenon associated with oncogenic transformation. We investigated the association between expression of the tumor-associated antigens T, Tn, and sialyl-Tn and polymorphism in the length of the MUC1 mucin tandem repeat in a series of gastric carcinomas. We further evaluated the relevance of MUC1 tandem repeat length on the expression of these tumor-associated carbohydrate antigens (TACAs) using a gastric carcinoma cell line model expressing recombinant MUC1 constructs carrying 0, 3, 9, and 42 repeats. Gastric carcinomas showed a high prevalence of Tn and sialyl-Tn antigens, whereas T antigen was less frequently expressed. The expression of T antigen was significantly higher in gastric carcinomas from patients homozygous for MUC1 large tandem repeat alleles. No significant associations were found for Tn and sialyl-Tn antigens. This novel association was reinforced by the gastric carcinoma cell line model experiments, where de novo expression of T antigen was detected in clones transfected with larger VNTR regions. Our results indicate that polymorphism in the MUC1 tandem repeat influences the expression of TACAs in gastric cancer cells and may therefore allow the identification of subgroups of patients that develop more aggressive tumors expressing T antigen.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2004

MUC4 mucin expression in human pancreatic tumours is affected by organ environment: the possible role of TGFbeta2.

MUC4 is highly expressed in human pancreatic tumours and pancreatic tumour cell lines, but is minimally or not expressed in normal pancreas or chronic pancreatitis. Here, we investigated the aberrant regulation of MUC4 expression in vivo using clonal human pancreatic tumour cells (CD18/HPAF) grown either orthotopically in the pancreas (OT) or ectopically in subcutaneous tissue (SC) in the nude mice. Histological examination of the OT and SC tumours showed moderately differentiated and anaplastic morphology, respectively. The OT tumour cells showed metastases to distant lymph nodes and faster tumour growth (P<0.01) compared to the SC tumours. The MUC4 transcripts in OT tumours were very high compared to the undetectable levels in SC tumours. The SC tumour cells regained their ability to express MUC4 transcripts after in vitro culture. Immunohistochemical analysis using MUC4-specific polyclonal antiserum confirmed the results obtained by Northern blot analysis. Interestingly, the OT tumours showed expression of TGFbeta2 compared to no expression in SC, suggesting a possible link between MUC4 and TGFbeta2. The MUC4 expression, morphology, and metastasis of human pancreatic tumour cells are regulated by a local host microenvironment. TGFbeta2 may serve as an interim regulator of this function.

Animals↗
PubMed2003

Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro.

BACKGROUND: Mucins are large complex glycoproteins that protect intestinal mucosal surfaces by limiting access of environmental matter to their epithelial cells. Several mucin genes have been described, including MUC3 that is a membrane associated mucin of the small intestine. Increased MUC3 mRNA transcription is induced by incubation of intestinal epithelial cells with a Lactobacillus strain known to be adherent to them. AIMS: To determine whether increased epithelial cell MUC3 mucin expression in response to Lactobacillus strains results in increased extracellular secretion of MUC3 mucins and the importance of epithelial cell adherence in modulation of MUC3 mucin expression. METHODS: HT29 cells grown to enhance expression of MUC3 mucins were incubated with selected Lactobacillus strains. Spent cell culture medium was collected for detection of secreted MUC3 mucins using dot blot immunoassay with a generated MUC3 antibody. Post-incubation HT29 cell RNA was collected for analysis of MUC3 expression by northern blot analysis using a MUC3 cDNA probe. In vitro binding studies using Lactobacillus strains incubated alone or coincubated with enteropathogenic Escherichia coli strain E2348/69 were used for adherence and inhibition of adherence studies, respectively. RESULTS: Lactobacillus strains with minimal ability to adhere to HT29 cells failed to induce upregulation of mucin gene expression. There was a direct correlation between upregulation of MUC3 mucin mRNA expression and extracellular secretion of MUC3 mucin. The same Lactobacillus strains that increased extracellular secretion of MUC3 mucin led to reduced adherence of enteropathogen E coli E2348/69 during coincubation experiments. CONCLUSION: Probiotic microbes induce MUC3 mucin transcription and translation with extracellular secretion of the MUC3 mucins. Epithelial cell adherence enhances the effects of probiotics on eukaryotic mucin expression.

Bacterial Adhesion↗
PubMed2001

Overexpression of MUC1 reconfigures the binding properties of tumor cells.

Although it is known that adhesion and antiadhesion are essential to the metastatic spread of tumor cells, little is known about the molecules that regulate these processes. MUC1 is overexpressed and aberrantly glycosylated by a variety of tumor cells. Studies described here examined whether tumor-associated MUC1 conferred new binding properties on tumor cell lines. Flow cytometry analysis with soluble P-, E- and L-selectin/IgM chimeric proteins was performed on human pancreatic (S2-013 and Panc-1) and colon (Caco-2) tumor cells. S2-013 cells bound E- and P-selectin and Caco-2 cells bound P-selectin. Epitope-tagged MUC1 (MUC1F) expressed by S2-013, Panc-1 and Caco-2 tumor cells did not bind to P-, E- or L-selectin. Overexpression of MUC1F on the surface of S2-013 cells blocked the interactions of E-selectin to tumor-associated ligand(s) but did not affect accessibility of monoclonal antibodies to other cell surface glycoproteins (CD9, CD44). Cell aggregation assays revealed that MUC1F expressed by S2-013 cells was able to bind to intracellular adhesion molecule-1 expressed on B cells. Overexpression of MUC1F containing a targeted mutation (the tandem repeat domain entirely or partially deleted) did not block the binding of E-selectin to its S2-013-associated ligand. These results demonstrate for the first time that the heavily O-glycosylated tandem repeat domain of MUC1 can simultaneously mediate and block binding to adhesion molecules with some molecular specificity and further support the hypothesis that MUC1 plays a dual role in the metastatic spread of tumor cells.

Amino Acid Sequence↗
PubMed2001

Identification of MUC1 proteolytic cleavage sites in vivo.

Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cell-cell interactions, signaling, and metastasis. The identification of several membrane-tethered mucins, including MUC1, MUC3, MUC4, and MUC12, has incited interest in the processing of these mucins and the mechanisms that govern their release from the cell surface. MUC1 consists of an extracellular subunit and a membrane-associated subunit. The two moieties are produced from a single precursor polypeptide by an early proteolytic cleavage event but remain associated throughout intracellular processing and transport to the cell surface. We identified the MUC1 proteolytic cleavage site and showed it to be identical in pancreas and colon cell lines and not to be influenced by the presence of heavily glycosylated tandem repeats. The MUC1 cleavage site shows homology with sequences in other cell-surface-associated proteins and may represent a common mechanism for processing of these molecules.

Amino Acid Sequence↗
PubMed2001

Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers.

The invasive growth of malignant cells induces an admixture of host reactions including desmoplasia, angiogenesis, and immune reactions Pancreatic cancer has a prominent and characteristic host reaction at the site of primary invasion. To obtain new insights into the process of tumor invasion, we studied global patterns of gene expression using serial analysis of gene expression in pancreatic cancer, with extension to other tumor types. Here we report a cluster of invasion-specific genes in pancreatic and other cancers. This cluster contains genes that derive from distinct components of the host reaction, including some that may be useful as diagnostic markers and therapeutic targets.

Cluster Analysis↗
PubMed2001

In vivo glycosylation of mucin tandem repeats.

The biochemical and biophysical properties of mucins are largely determined by extensive O-glycosylation of serine- and threonine-rich tandem repeat (TR) domains. In a number of human diseases aberrant O-glycosylation is associated with variations in the properties of the cell surface-associated and secreted mucins. To evaluate in vivo the O-glycosylation of mucin TR domains, we generated recombinant chimeric mucins with TR sequences from MUC2, MUC4, MUC5AC, or MUC5B, which were substituted for the native TRs of epitope-tagged MUC1 protein (MUC1F). These hybrid mucins were extensively O-glycosylated and showed the expected association with the cell surface and release into culture media. The presence of different TR domains within the chimeric mucins appears to have limited influence on their posttranslational processing. Alterations in glycosylation were detailed by fast atom bombardment mass spectrometry and reactivity with antibodies against particular blood-group and tumor-associated carbohydrate antigens. Future applications of these chimeras will include investigations of mucin posttranslational modification in the context of disease.

Amino Acid Sequence↗
PubMed2001

Influence of organ site and tumor cell type on MUC1-specific tumor immunity.

We investigated the influence of organ-specific parameters on tolerance and immunity to human MUC1. C57Bl/6 mice (wild-type) and C57Bl/6 transgenic for MUC1 (MUC1.Tg) were challenged in the pancreas with Panc02-MUC1, a C57Bl/6-syngeneic pancreatic cancer cell line expressing human MUC1. Wild-type mice produced immune responses to MUC1 when presented on tumor cells growing in the pancreas; however, the responses to tumors in the pancreas were less effective than responses produced by tumor challenge at the s.c. site. Tumor immunity specific for MUC1 was produced in wild-type mice by two different procedures: (i) s.c. immunization of wild-type mice with a low dose of Panc02-MUC1 or (ii) adoptive transfer of spleen and lymph node cells harvested from wild-type mice previously immunized s.c. with Panc02-MUC1. This demonstrates that immune responses to MUC1 presented at the s.c. site can be detected and adoptively transferred. MUC1.Tg mice were immunologically tolerant to MUC1; however, some immunological protection against orthotopic challenge with Panc02-MUC1 was conferred by adoptive transfer of CD4+ and CD8+ T cells from wild-type mice. These results show that it is more difficult to produce immune responses to tumors growing at the pancreatic site than the s.c. site. Panc02-MUC1 cells growing in the pancreas were accessible to the immune system, and immune responses evoked by s.c. presentation of this molecule in wild-type mice were effective in rejecting tumor cells in the pancreas of both wild-type and MUC1.Tg mice. No effective anti-tumor immune responses against MUC1 were produced in MUC1.Tg mice.

Adoptive Transfer↗
PubMed2001

Mucin (MUC) gene expression in human pancreatic adenocarcinoma and chronic pancreatitis: a potential role of MUC4 as a tumor marker of diagnostic significance.

PURPOSE: Mucins are important biomolecules that frequently display an altered expression under pathological conditions. In a search for a unique and reliable marker(s) specific for pancreatic adenocarcinoma, we investigated the expression of different MUC genes in pancreatic tumors and tumor cell lines, in chronic pancreatitis, and in the normal pancreas. EXPERIMENTAL DESIGN: Total RNA from 16 pancreatic tumors, 10 chronic pancreatitis tissues, 7 normal pancreas tissues, and 15 pancreatic tumor cell lines were analyzed by reverse transcription-PCR with primers specific for MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7 genes and by RNA slot blot analyses. RESULTS: Our results revealed that of all of the mucins examined, only MUC4 displayed a differential expression that was specific for pancreatic adenocarcinoma. Indeed, a substantial number of tumor tissue samples (12 of 16) and tumor cell lines (11 of 15) expressed MUC4 mRNA, whereas samples from chronic pancreatitis (0 of 10) and the normal pancreas (0 of 7) tissues failed to exhibit any detectable level of this mucin. In contrast, no significant alteration was observed in the expression of the other mucins relative to that in the normal pancreas samples. CONCLUSIONS: Overall, this work demonstrates that pancreatic mucin MUC4 is a tumor-associated mucin. Furthermore, the present study introduces a novel avenue to discriminate between pancreatic adenocarcinoma and pancreatitis. Future investigations of the role played by MUC4 in pancreatic adenocarcinoma may prove to be useful in the formulation of strategies for the diagnosis and therapeutic treatment of this malignancy.

Adenocarcinoma↗
PubMed2000

The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities.

The initiation step of mucin-type O-glycosylation is controlled by a large family of homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Differences in kinetic properties, substrate specificities, and expression patterns of these isoenzymes provide for differential regulation of O-glycan attachment sites and density. Recently, it has emerged that some GalNAc-transferase isoforms in vitro selectively function with partially GalNAc O-glycosylated acceptor peptides rather than with the corresponding unglycosylated peptides. O-Glycan attachment to selected sites, most notably two sites in the MUC1 tandem repeat, is entirely dependent on the glycosylation-dependent function of GalNAc-T4. Here we present data that a putative lectin domain found in the C terminus of GalNAc-T4 functions as a GalNAc lectin and confers its glycopeptide specificity. A single amino acid substitution in the lectin domain of a secreted form of GalNAc-T4 selectively blocked GalNAc-glycopeptide activity, while the general activity to peptides exerted by this enzyme was unaffected. Furthermore, the GalNAc-glycopeptide activity of wild-type secreted GalNAc-T4 was selectively inhibited by free GalNAc, while the activity with peptides was unaffected.

Amino Acid Sequence↗
PubMed2000

Enhancement of metastatic properties of pancreatic cancer cells by MUC1 gene encoding an anti-adhesion molecule.

MUC1 mucin expression has been shown to be associated clinicopathologically with metastasis and poor clinical outcome in a variety of tumors. To further investigate this finding experimentally, human pancreatic cancer S2-013 cells overexpressing MUC1 were used for spontaneous metastatic potential in nude mice. It was found that the number of lung metastases of MUC1 transfectants was significantly higher than that of control cells. To analyze the molecular mechanisms that underlie the increased metastatic activity, in vitro adhesion assays were performed. MUC1 mucin expression enhancedin vitro invasiveness and motility of S2-013 cells, and decreased the binding of S2-013 cells to type I collagen, Type IV collagen and laminin. Similar effects were not observed for cells expressing tandem repeat-deleted MUC1 cDNA. Adhesion properties were abolished by benzyl-alpha-GalNAc treatment, indicating that glycosylation of the extracellular domain of MUC1 was essential for these biological adhesive functions. Our data support the hypothesis that MUC1 expression contributes to the metastatic ability of pancreatic cancer cells.

Acetylgalactosamine↗
PubMed2000

Structural effects of O-glycosylation on a 15-residue peptide from the mucin (MUC1) core protein.

To study the effect of O-glycosylation on the conformational propensities of a peptide backbone, the 15-residue peptide PPAHGVTSAPDTRPA (PPA15) from the MUC1 protein core and its analogue PPA15(T7), glycosylated with alpha-N-acetylgalactosamine on Thr7, were prepared and investigated by NMR spectroscopy. The peptide contains both the GVTSAP sequence, which is an effective substrate for GalNAc-T1 and -T3 transferases, and the PDTRP fragment, which is a well-known immunodominant epitope recognized by several anti-MUC1 monoclonal antibodies. Useful structural results were obtained in water upon decreasing the temperature to 5-10 degrees C. The sugar attachment slightly affected the conformational equilibrium of the peptide backbone near the glycosylated Thr7 residue. The clustering of low-energy conformations for both PPA15 and PPA15(T7) within the GVTSAP and APDTRP fragments revealed structural similarities between glycosylated and nonglycosylated peptides. For the GVTSAP region, minor but distinct clusters formed by either PPA15 or PPA15(T7) conformers showed distinct structural propensities of the peptide backbone specific for either the nonglycosylated or the glycosylated peptide. The peptide backbone of the APDTRP fragment, which is a well-known immunodominant region, resembled an S-shaped bend. A similar structural motif was found in the GVTSAP fragment. The S-shaped structure of the peptide backbone is formed by consecutive inverse gamma-turn conformations partially stabilized by hydrogen bonding. A comparison of the solution structure of the APDTRP fragment with a crystal structure of the MUC1 peptide antigen bound to the breast tumor-specific antibody SM3 demonstrated significant structural similarities in the general shape.

Acetylgalactosamine↗
PubMed2000

Expression of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer.

Uridine diphosphate (UDP)-GalNAc: polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase) catalyzes the initial step in mucin type O-glycosylation, and its expression has been assumed to be altered between normal epithelial cells and cancer cells. We studied the alteration of GalNAc transferase expression during the carcinogenesis of human colorectal epithelial cells. We produced polyclonal antibodies against synthetic polypeptides with specific sequence to two GalNAc transferase isozymes, T1 and T2. Surgically resected specimens from 50 patients with colorectal cancer were immunohistochemically stained, and the staining grade (percentage of positively stained cells) was compared between cancer and its normal counterpart in the same specimen. Significant signals for both T1 and T2 expression were seen in the supranuclear region of normal and cancer cells, indicating the subcellular localization of the enzymes in the Golgi apparatus. The prevalence of positive staining for T1 and T2 expression in colorectal cancer was significantly higher than that in normal epithelium (P < 0.05). However, the difference in staining grades between cancer and normal tissues varied in each patient. These results indicate that there is variability in the expression patterns of GalNAc transferase isozymes in normal and cancerous cells colorectal among individuals.

Adenocarcinoma↗
PubMed2000

Human MUC4 mucin cDNA and its variants in pancreatic carcinoma.

The human MUC4 gene is not expressed in normal pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors. To investigate the tumor-associated expression, MUC4 cDNA was cloned from a human pancreatic tumor cell line cDNA expression library using a polyclonal antibody raised against human deglycosylated mucin and RT-PCR. Pancreatic MUC4 cDNA shows differences in 12 amino acid residues in the non-tandem repeat coding region with no structural rearrangement as compared with tracheal MUC4. The full-length MUC4 cDNA includes a leader sequence, a serine and threonine rich non-tandem repeat region, a central large tandem repeat domain containing 48 bp repetitive units, regions rich in potential N-glycosylation sites, two cysteine-rich domains, EGF-like domains, and a transmembrane domain. We also report the presence of a new EGF-like domain in MUC4 cDNA, located in the cysteine-rich region upstream from the first EGF-like domain. Four distinct splice events were identified in the region downstream of the central tandem repeat domain that generate three new MUC4 cDNA sequences (sv4, sv9, and sv10). The deduced amino acid sequences of two of these variants lack the transmembrane domain. Furthermore, two unique forms of MUC4 (MUC4/Y and MUC4/X) generated as a result of alternative splicing lack the salient feature of mucins, the tandem repeat domain. A high degree of polymorphism in the central tandem repeat region of MUC4 was observed in various pancreatic adenocarcinoma cell lines, with allele sizes ranging from 23.5 to 10.0 kb. MUC4 mRNA expression was higher in differentiated cell lines, with no detectable expression in poorly differentiated pancreatic tumor cell lines.

Alternative Splicing↗
PubMed1999

A novel human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates.

A novel member of the human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase gene family, designated GalNAc-T7, was cloned and expressed. GalNAc-T7 exhibited different properties compared to other characterized members of this gene family, in showing apparent exclusive specificity for partially GalNAc-glycosylated acceptor substrates. GalNAc-T7 showed no activity with a large panel of non-glycosylated peptides, but was selectively activated by partial GalNAc glycosylation of peptide substrates derived from the tandem repeats of human MUC2 and rat submaxillary gland mucin. The function of GalNAc-T7 is suggested to be as a follow-up enzyme in the initiation step of O-glycosylation.

Amino Acid Sequence↗