The long-term objectives of this research are to understand how malignant expression of tumor cells can be modulated by alterations in membrane glycosylation, and how membrane glycosylation is regulated at a molecular level. A correlation has repeatedly been reported between alterationns in the malignant expression of tumor cells alteration in their membrane glycoconjugates (as sometimes reflected in the acquisition of specific lectin phenotypes). Yet, causative link between these alterations has not been identified, and the genetic basis for the link has yet to be determined. The research proposes to use a genetic approach to address these questions by establishing the basis for a wheat-germ-agglutinin-resistance phenotype (WGA-R) expressed by a mouse melanoma WGA-R mutant. These WGA-R cells express altered membrane glycosylation, altered cell adhesiveness, and reduced malignant potential. The hypothesis to be tested in this proposal is that these altered phenotypic expressions are related, and are manifestations of a single genetic change. The strategy is to isolate the gene which confers the WGA-R phenotype (WR-gene), transfect it into WGA-sensitive melanoma cells, and analyze the phenotypic alterations conferred by the transfected WR-gene to the transfected cells. The experimental plan is as follows: (1) Construct recombinant genomic DNA library or cDNA library of WGA-R cells, using shuttle expression vectors. (2) Transfect DNA derived from such libraries into WGA-sensitive cells, and functinally select for WGA-R transfectants using WGA as the selecting agent (3) Rescue the WR-gene from the WGA-R transfectants into E.coli, and select for recombinant clones with ampicillin. (4) Carry out secondary transfection by transfecting the WGA-sensitive parental mouse melanoma cells with the DNA derived from individual recombinants. Co-transfect with a plasmid containing the selectable gene marker for altered-DHFR or for hygromycin-resistance. Select for WGA-R melanoma transfectants, which probably contain the functional WR-gene. (5) Analyze the phenotypic alterations conferred by transferred WR-gene to the WGA-R melanoma transfectants, with special reference to membrane glycosylation, cell adhesivess, tumorigenicity, and metastatic capacity.
| Kawano, T; Takasaki, S; Tao, T W et al. (1993) Altered glycosylation of beta 1 integrins associated with reduced adhesiveness to fibronectin and laminin. Int J Cancer 53:91-6 |
| Kawano, T; Takasaki, S; Tao, T W et al. (1991) N-linked sugar chains of mouse B16 melanoma cells and their low-metastasizing variant selected by wheat germ agglutinin. Glycobiology 1:375-85 |
