Human neuroblastoma has several unique features: (1) it is limited to young children; (2) there is tumor regression in some cases; (3) its chromosomes contain homogeneously staining regions (HSRs) with an amplified DNA sequence weakly homologous to oncogene c-myc, called N-myc. The presence of multipled HSRs, each containing amplified copies of N-myc, have been demonstrated in two all lines; (4) there is the presence of oncogene N-ras capable of transforming NIH 3T3; and (5) its glycoproteins contain unusually large amounts of fucose linked alpha 1+3 to N-acetylglucosamine on the oligosaccharide antennae. An alpha-L-fucosidase from almonds, specific for fucosyl residues linked alpha 1+3(4) to N-acetylglucosamine, was used to demonstrate that these fucosyl residues were present on glycoproteins of neuroblastoma cell lines and tumors including tumor cells purified from the bone marrow and not on those from other cell types. Thus, the neuroblastoma glycoproteins provide a unique opportunity to define a structural basis for a particular tumor specificity and to determine structural characteristics and, hence, information about synthesis for a range of unusual glycopeptides. Selected glycopeptides from neuroblastoma glycoproteins will be studied after purification by HPLC, conventional size and charge chromatography, and lectin binding. Tentative structural assignments will be made from the purification properties and will be verified by high resolution 'H-NMR spectroscopy. This information will be used to describe the range of structures containing these specific fucosyl residues. Further, it will be used to select substrates for assaying fucosyl and sialytransferases in these cells. It is anticipated that correlation of the structural and enzymatic data will define the gene product specifically amplified or expressed in neurectoderm tumors. Transfection with human neuroblastoma DNA containing oncogene N-ras will transform mouse fibroblasts, NIH 3T3. These transformants have glycoproteins containing oligosaccharide residues more highly branched than those of NIH 3T3. Selected glycopeptides from both cell types will be purified, and the oligosaccharide structures determined. The structural information will be used to design a series of exploratory experiments to test a number of alternate causes of the change in oligosaccharides after oncogene transformation. These experiments will: (1) measure the activity of relevant glycosyl transferases; and (2) assess positional alterations on the polypeptide using inhibitors of early stage glycoprotein processing. Progress has been made isolating glycopeptides from human neuroblastoma, CHP-134, oncogene transformed alpha 1-1, and nontransformed NIH-3T3 cells. In addition, the initial studies using inhibitors of glycoprotein processing have shown this to be a valid approach. (A)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA037853-03
Application #
3175715
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1984-07-01
Project End
1988-03-31
Budget Start
1986-07-01
Budget End
1988-03-31
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Giuntoli 2nd, R L; Stoykova, L I; Gillies, D R et al. (1994) Expression of GDP-L-Fuc: Gal(beta 1-4)GlcNAc-R (Fuc to GlcNAc) alpha-1,3-fucosyltransferase and its relationship to glycoprotein structure in a human erythroleukemia cell line, HEL. Eur J Biochem 225:159-66
Negishi, M; van Kuik, J A; Vliegenthart, J F et al. (1992) Oligosaccharide composition of the neurotoxin-responsive sodium channel of mouse neuroblastoma and requirement of sialic acid for biological activity. Carbohydr Res 236:209-25
Foster, C S; Gillies, D R; Glick, M C (1991) Purification and characterization of GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme. J Biol Chem 266:3526-31
Santer, U V; DeSantis, R; Hard, K J et al. (1989) N-linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae. Eur J Biochem 181:249-60
Livingston, B D; Jacobs, J L; Glick, M C et al. (1988) Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells. J Biol Chem 263:9443-8
Boyd, J P; Glick, M C (1988) Removal of polypeptides from human neuroblastoma antigen does not alter recognition by monoclonal antibody PI 153/3. Prog Clin Biol Res 271:353-8
DeSantis, R; Santer, U V; Glick, M C (1987) NIH 3T3 cells transfected with human tumor DNA lose the transformed phenotype when treated with swainsonine. Biochem Biophys Res Commun 142:348-53
Woodbury, R A; Santer, U V; Elkins, W L et al. (1986) Similarities in glycosylation of human neuroblastoma tumors and cell lines. Cancer Res 46:3692-7
Emanuel, B S; Balaban, G; Boyd, J P et al. (1985) N-myc amplification in multiple homogeneously staining regions in two human neuroblastomas. Proc Natl Acad Sci U S A 82:3736-40
Glick, M C; De Santis, R; Santer, U V (1985) Glycosylation changes in membrane glycoproteins after transfection of NIH 3T3 with human tumor DNA. Prog Clin Biol Res 175:229-37