10.VIROLOGY AND GLYCOBIOLOGY DIVISION
Molecular Functions of Ganglioside GM3 in Cancer Cells
Gangliosides are a family of glycosphingolipids that contain sialic acid. GM3, which is produced by sialyltransferase-1 (SAT-1), is the common precursor of almost all gangliosides, and is involved in cell proliferation, differentiation and carcinogenesis. A complete genomic DNA clone of the GM3 synthase gene was isolated, and it was found to span approximately 56 kb in the human genome consisting of 7 exons and 6 introns. The human gene promoter contains no canonical TATA and CCAAT boxes. Two Sp1 binding sites found close to the transcription start site and an NFY motif located between them were shown to be critical for the basal transcription activity of the gene in cancer cell lines. The gene targeting technique was applied to produce mutant mice lacking the SAT-1 gene locus. ES cell lines heterozygous for the SAT-1 gene were successfully established. The germ line transmission was also confirmed in one of the chimeric mouse lines generated. Phenotypes of mutant mice heterozygous or homozygous for SAT-1 are currently under investigation. A recent study revealed that GM3 is localized in specific structures called lipid microdomains within the cellular membrane. Immunostaining analysis and electromicroscopic observation indicated that these GM3-enriched microdomains are produced in the cytoplasm and some of them are excreted from cells as small particles. The biological function of GM3-enriched microdomains and signaling molecules co-existing with GM3 are being analyzed.
Biological Functions of Signal Docking Proteins in Formation and Metastasis of Cancers
Neurospecific docking proteins, ShcB and ShcC, transduce signals from receptor tyrosine kinases to downstream molecules such as Grb2.(135) Marked tyrosine phosphorylation of ShcC was detected in several neuroblastoma cell lines such as NB39-nu and Nagai by immunoprecipitation. To clarify the roles of ShcC in tumorigenesis and progression of neuroblastoma, ShcC-associating phosphoproteins were isolated from neuroblastoma cells by immuno-affinity purification and identified by mass-spectrometry. One of the major components turned out to be anaplastic lymphoma kinase (ALK) which was activated in neuroblastoma cells by the gene amplification. The biological roles of the ALK-ShcC pathway are being examined by dominant-negative ShcC proteins. Cas, a major substrate of the Src family tyrosine kinases, is a docking protein connecting the integrin signal with cellular proteins like Src, Fak and Crk. Cas-negative fibroblasts were recently established from knockout mice, which have the following outstanding phenotypes: 1) defective in transformation by activated Src; 2) lack of actin stress fiber formation; 3) defective cell migration. By expressing various mutants of Cas in fibroblasts lacking Cas, the domain function of Cas was analyzed. It was found that the central YDxP motifs are critical for actin stress fiber formation and cell migration, while the Src-binding domain is mainly required for cell transformation and migration.
Cloning New Hematopoietic Factors for Stem Cell Growth from Stromal Cells
A cDNA library was constructed from the stromal OP9 cell line, which can induce ES cells to differentiate into hematopoietic cells, and inserted into the pMX-SST retroviral vector. Hematopoietic factors are being searched using the signal sequence trap method and cDNA clones containing signal sequences are further screened with cDNA microarray analysis to find out genes which are affected by stimulation of LIF and other growth factors because those factors are reported to help the potential of stromal cells support the hematopoietic activity.
Viral Carcinogenesis: Hepatitis C Virus (HCV) Replication in vitro
Recently, the HCV subgenomic RNA replicon (a self-replicating RNA molecule) derived from the consensus clone of HCV was successfully expressed in the HCV-infected cultured cells. Construction of the HCV replicon library from HCV patient samples and development of a screening system for anti-HCV drugs with availability of the HCV replicon is in process. In addition, mechanisms by which persistent infection of HCV induce carcinogenesis have been analyzed. To overcome incurable advanced cancers, molecular mechanisms that underlie peritoneal metastases of ovarian and gastric cancers were also analyzed.(136-139)