lewis-x-antigen and Neuroblastoma

Sonic hedgehog (Hh) developmental pathway deregulation has been proven to play an essential role in several malignancies as neuroblastoma. We found that Hh signaling is active in neuroblastoma, as most pathway components, including GLI1, were expressed in cell lines and tumor samples. Furthermore, SHH ligand expression was found in cell lines and tumors, and GLI1 up-regulation was achieved in response to SHH treatment, suggesting an autocrine mechanism of aberrant activation. A decrease of proliferation and tumorigenic potential, as well as increased apoptosis and a dramatic decrease in the percentage of CD15+ cell population were produced upon Hh inhibition by cyclopamine.

Topics: AC133 Antigen; Antigens, CD; Apoptosis; Biomarkers, Tumor; Cell Growth Processes; Cell Line, Tumor; Female; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Glycoproteins; Hedgehog Proteins; Humans; Lewis X Antigen; Male; Neoplastic Stem Cells; Neuroblastoma; Peptides; Signal Transduction; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1

The expression of the fucosylated carbohydrate Lewis(x) (Le(x)) determinant (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R) has been found in glycoproteins, proteoglycans, and glycolipids from the nervous system. Evidence suggests its association with cell-cell recognition, neurite outgrowth, and neuronal migration during central nervous system development. In the present work, we detected increased levels of Le(x) in differentiated human NT2N neurons cultured in vitro. To identify which fucosyltransferase (FUT) synthesized the Le(x) in NT2N neurons, RT-PCR, FUT substrate specificity and Western blot analysis were carried out. Strong activity toward acceptors Galbeta4GlcNAc-O-R and Fucalpha2Galbeta4GlcNAc-O-R [R = -(CH(2))(3)NHCO(CH(2))(5)NH-biotin], together with strong FUT9 detection by Western blot and presence of transcripts showed that FUT9 was the enzyme associated with Le(x) biosynthesis in NT2N neurons. Le(x) was detected at the plasma membrane of NT2N neurons, in lysosomes marked with lysosomal-associated membrane protein 1 (LAMP-1), and it was found for the first time to colocalize with the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) that defines the TI-VAMP exocytic compartment that is involved in neurite outgrowth. Furthermore, incubation with anti-Le(x) monoclonal antibody L5 led to impaired adhesion of NT2N neurons to the surface matrix and inhibited neurite initiation. In conclusion, FUT9 and its product Le(x) are detected specifically in human NT2N neurons and our results indicate that they underlie cell differentiation, cell adhesion, and initiation of neurite outgrowth in those neurons.

Topics: Antibodies; Carbohydrate Metabolism; Carbohydrate Sequence; Carbohydrates; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Fucosyltransferases; Gene Expression Regulation, Enzymologic; Humans; Lewis X Antigen; Lysosomal-Associated Membrane Protein 1; Neurites; Neuroblastoma; Tretinoin; Vesicular Transport Proteins

Human CD34(+) cells, highly enriched for hematopoietic stem and progenitors, and CD15(+) cells, more terminally differentiated myeloid cells in blood, represent distinct maturation/differentiation stages. A proteomic approach was used to identify proteins differentially present in these two populations from human cord blood. Cytosolic proteins were extracted and subjected to two-dimensional gel electrophoresis followed by mass spectrometry. On average, 460 protein spots on each gel were detected; 112 and 15 proteins, respectively, were found to be differentially expressed or post-translationally modified in CD34(+) and CD15(+) cells. This suggests that CD34(+) cells have a relatively larger proteome than mature CD15(+) myeloid cells and production of many stem/progenitor cell-associated proteins ceases or is dramatically down-regulated as the CD34(+) cells undergo differentiation. Of approximately 140 protein spots, 47 different proteins were positively identified by mass spectrometry and database search; these proteins belong to several functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Multiple heat shock proteins and chaperones, as well as proteins important for intracellular trafficking, were predominantly present in CD34(+) cells. Most of the identified proteins in CD34(+) cells are expressed in germ cell tumors, as well as in embryonal carcinoma and neuroblastoma. Approximately eight novel proteins, whose functions are unknown, were identified. This study presents, for the first time, global cellular protein expression patterns in human CD34(+) and CD15(+) cells, which should help to better understand intracellular processes involved in myeloid differentiation and add insight into the functional capabilities of these distinct cell types.

Topics: Antigens, CD34; Carcinoma, Embryonal; Cytoskeleton; Electrophoresis, Gel, Two-Dimensional; Fetal Blood; Humans; Isoelectric Focusing; Lewis X Antigen; Mass Spectrometry; Myeloid Cells; Neuroblastoma; Protein Folding; Protein Processing, Post-Translational; Proteomics; RNA, Small Nuclear; Signal Transduction; Stem Cells

Immunoperoxidase analysis was performed to evaluate the phenotypic expression of eight renal differentiation antigens in five nephroblastomas, one clear cell sarcoma of the kidney (CCSK), one rhabdoid tumor of the kidney (RTK), and four related tumors. A total of 19 fetal and pediatric kidneys, including two 6th-week mesonephric tissues, were comparatively studied. All the specimens were fixed in formalin and embedded in paraffin. Neural cell adhesion molecule (NCAM), a marker of the nephrogenic zone of the developing kidney, was consistently expressed in the epithelial and blastematous components of nephroblastomas of the common type. The epithelial components also commonly expressed NK1 and Leu 7 (CD57), and the findings may reflect that both were positive in immature proximal tubules directly differentiating from the NCAM-positive immature fetal tubuloglomerular buds. In two cases, the epithelial component was immunoreactive for CD10 and WT1 gene product (WT1-GP). Leu M1, epithelial membrane antigen and CA15-3 were only focally expressed in nephroblastomas. Rhabdomyoblasts in the stroma were positive for WT1-GP. CCSK was featured by the expression of NCAM and CD10. In RTK, focal epithelial differentiation was discerned, with focal positivity of WT1-GP and negativity of NCAM. In congenital mesoblastic nephroma, the stromal spindle cells were strongly immunoreactive for WT1-GP, while WT1-GP was not expressed in solitary multilocular cyst of the kidney. Pancortical nephroblastomatosis was featured by the diffuse subcapsular reappearance of immature metanephric tissue. Nephroblastomas and related conditions thus offer an adequate model for studying human nephrogenesis.

Topics: Adolescent; Adult; Antigens, Surface; CD57 Antigens; Child; Child, Preschool; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Infant; Infant, Newborn; Kidney; Kidney Neoplasms; Killer Cells, Natural; Lewis X Antigen; Male; Mucin-1; Neprilysin; Neural Cell Adhesion Molecules; Neuroblastoma; Sarcoma, Clear Cell; Transcription Factors; WT1 Proteins

The F-11 cell line is a fusion product of embryonic rat dorsal root ganglion (DRG) cells with mouse neuroblastoma cell line N18TG-2 (Platika, D., Boulos, M.H., Baizer, L. and Fishman, M.C., Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3499-3503). F-11 cells were uniformly labelled using a monoclonal antibody (RT-97) to the 200 kDa subunit of neurofilament protein, which labels a subpopulation of adult rat DRG neurons. F-11 cells did not stain for antigenic markers of fibroblasts or Schwann/satellite cells which are also present in DRG. Monoclonal antibodies that recognize cell surface carbohydrates have been shown to label subpopulations of DRG neurons. The stage-specific embryonic antigens SSEA-3 and SSEA-4, and the antigen recognized by B23D8, were expressed by some F-11 cells but not by the neuroblastoma parent of the hybrid cells. SSEA-3 was expressed by about 20% of the F-11 cells, whereas 40-60% expressed SSEA-4 or the antigen recognized by B23D8. The stability of F-11 cell subpopulations for sensory antigen expression was demonstrated by isolating single cells and growing the progeny as clonal lines. In some subclones, nearly 100% of the cells stably expressed SSEA-4 and/or B23D8, or failed to stain with anti-SSEA-4, anti-SSEA-3, or B23D8 over 12 passages. Other subclones were unstable for the expression of these antigens. This study demonstrates the derivation of the F-11 cell line from sensory neurons but also indicates that multiple phenotypes of varying stability are present in this line. This information is important for the use of this line as a model for DRG neurons.

Topics: Antibodies, Monoclonal; Cell Line; Ganglia, Spinal; Glycolipids; Intermediate Filament Proteins; Lewis X Antigen; Molecular Weight; Neuroblastoma; Neurofilament Proteins; Neurons, Afferent; Phenotype