Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man

kG: The cellular microenvironment and cell adhesion: A role for O-glycosylation. Biochem Soc Trans 39

Abstract Glycosylation is one of the most abundant protein modifications in Nature, having roles in protein stability, secretion and function. Alterations in mucin-type O-glycosylation are responsible for a number of human diseases and developmental defects, as well as associated with certain types of cancer. However, the mechanistic role of this form of glycosylation in many of these instances is unclear. Here we describe how one glycosyltransferase responsible for initiating mucin-type O-glycosylation (PGANT3), specifically modulates integrin-mediated cell adhesion by influencing the secretion and localization of an integrin ligand. The integrin ligand Tiggrin, is normally O-glycosylated and localized to the basal matrix, where adhesion of two opposing cell layers takes place. In pgant3 mutants, Tiggrin is no longer O-glycosylated and fails to be properly secreted to the basal cell layer interface, resulting in disruption of proper cell adhesion. pgant3-mediated effects are dependent on the enzymatic activity of PGANT3 and cannot be rescued by another pgant family member, indicating a unique role for this glycosyltransferase. These results provide in vivo evidence for the role of O-glycosylation in the secretion of specific extracellular matrix proteins, which thereby influences the composition of the cellular 'microenvironment' and modulates cell adhesion events. The studies described in this review provide insight into the long-standing association between aberrant O-glycosylation and tumorigenesis, as changes in tumour environment and cell adhesion are hallmarks of cancer progression

Charge distribution of flanking amino acids influences O-glycan acquisition in

From surveys of known O-glycosylation sites and in vitro glycosylation assays with synthetic peptide acceptors, it appears that the presence of charged amino acids near serine/ threonine residues reduces the likelihood of O-glycosylation by UDP-GalNAc polypeptide:N-acetyIgalactosaminyltransferases (ppGaNTases). Previously, we demonstrated that the in vivo O-glycosylation of a sequence derived from a known glycosylation site of human von Willebrand factor (PHMAQVTVGPGL) was markedly reduced when charged residues were substituted at position -1 and +3 relative to the single threonine. In contrast, acidic residues at positions -2 , +1, and +2 had no effect (Nehrke et al, 1996), suggesting that charge distribution but not charge density was important To determine whether the charge distribution effect on O-glycosylation is limited to a specific sequence context or restricted to unique isoforms of ppGaNTase, we have analyzed the in vivo O-glycosylation of six secreted recombinant reporter proteins in three different cell backgrounds. The differential presence of known ppGaNTase transcripts was determined in each cell type by Northern blot analysis. Each reporter, which contains a single site of O-glycosylation, was O-glycosylated in a cellbackground-specific manner; digestion with O-glycanase and a-N-acetylgalactosaminidase following mild acid hydrolysis suggested that simple type II core structures were acquired. However, in COS7 cells, one reporter peptide acquired glycosaminoglycans in preference to mucin-type O-gtycans. Regardless of cell background or the reporter examined, the substitution of glutamic acid residues at positions -1 and +3 markedly diminished the level of mucintype O-glycosylation. Charge distribution would appear, therefore, to play a more general role in determining the extent to which solitary O-glycosylation sites are modified

Conservation of peptide acceptor preferences between Drosophila and mammalian polypeptide-GalNAc transferase ortholog pairs

UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltrans- ferases (ppGalNAc Ts) comprise a large family of glycosyltransferases that initiate mucin-type protein O-glycosylation, transferring α-GalNAc to Thr and Ser residues of polypeptide acceptors. Families of ppGalNAc Ts are found across diverse eukaryotes with orthologs identifiable from mammals to single-cell organisms. The peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly understood. Previously, we reported a series of oriented random peptide substrate libraries for quantitatively determining the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 (Gerken TA, Raman J, Fritz TA, Jamison O. 2006. Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases T1 & T2 (ppGalNAc T1 & T2) derived from oriented random peptide substrates. J Biol Chem. 281:32403–32416). With these substrates, previously unknown features of the transferases were revealed. Utilizing these and a new lengthened set of random peptides, studies have now been performed on PGANT5 and PGANT2, the Drosophila orthologs of T1 and T2. The results from these studies suggest that the major peptide substrate determinants for these transferases are contained within 2 to 3 residues flanking the site of glycosylation. It is further found that the mammalian and fly T1 orthologs display very similar peptide substrate preferences, while the T2 orthologs are nearly indistinguishable, suggesting similar peptide preferences amongst orthologous pairs have been maintained across evolution. This conclusion is further supported by sequence homology comparisons of each of the transferase orthologs, showing that the peptide substrate and UDP binding site residues are more highly conserved between species relative to their remaining catalytic and lectin domain residues

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Running title: Galnt3 influences the microbiomeThe importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (Galnt3)-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. Using semiquantitative real-time PCR, we found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands. Additionally, 16S rRNA gene sequencing revealed that the loss of Galnt3 resulted in changes in the structure, composition, and stability of the oral microbiome. Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted.Intramural Research Program of the NIDCR, National Institutes of Health Z01-DE000713 ZIA-DE000738 NIDCR Veterinary Resources Core Grant ZIC DE000740-05 NIDCR Imaging Core Grant DE000750-01 Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) CONICYT FONDECYT 1118050