In order to investigate structure/function relationships of animal cell carbohydrates, as series of glycosylation mutants of Chinese hamster ovary (CHO) cells have been isolated. All of the mutants exhibit altered carbohydrate biosynthesis and express new carbohydrate structures at the cell surface. Twenty-five distinct phenotypes have been identified and classified into seven dominant types and fourteen recessive complementation groups. They have been used by many laboratories to study the roles of carbohydrate moieties in the biosynthesis, compartmentalization and functions of endogeneous or introduced glycoproteins. Recent studies in this laboratory have shown that the altered carbohydrates synthesized by Lec9 CHO mutants correlate directly with their reduced tumorigenicity in nude mice. The CHO glycosylation mutants provide an avenue to the isolation of glycosylation genes and the determination of mechanisms that control their expression. It is proposed to isolate new glycosylation mutants and revertants using lectins as selective agents so that all the genes involved in carbohydrate biosynthesis are identified. Studies on molecular mechanisms that regulate glycosylation gene expression will focus on the three dominant mutants, LEC10, LEC11 and LEC12. Their phenotypes appear to be the result of gene activation since each of them expresses a novel glycosyltransferase activity not detected in parental CHO. The abilities of reagents that cause mutations and epigenetic changes such as hypomethylation to alter the rates of appearance of these mutants and their revertants will be examined. Co-transfection of genomic DNA with a plasmid carrying a dominant selectable marker will be used to isolate the genes and their expression will be compared in parental and mutant cells. These studies should reveal mechanisms that regulate glycosylation gene expression in CHO cells and provide probes to investigate the changes in carbohydrate synthesis that occur during development, differentiation and oncogenic transformation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA030645-09
Application #
3169330
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1981-08-01
Project End
1991-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Wang, Weihuan; Yu, Shuiliang; Zimmerman, Grant et al. (2015) Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention. Stem Cells 33:2280-93
Miwa, Hazuki E; Koba, Wade R; Fine, Eugene J et al. (2013) Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer. Glycobiology 23:1477-90
Batista, Frank; Lu, Linchao; Williams, Suzannah A et al. (2012) Complex N-glycans are essential, but core 1 and 2 mucin O-glycans, O-fucose glycans, and NOTCH1 are dispensable, for mammalian spermatogenesis. Biol Reprod 86:179
Miwa, Hazuki E; Song, Yinghui; Alvarez, Richard et al. (2012) The bisecting GlcNAc in cell growth control and tumor progression. Glycoconj J 29:609-18
Song, Yinghui; Aglipay, Jason A; Bernstein, Joshua D et al. (2010) The bisecting GlcNAc on N-glycans inhibits growth factor signaling and retards mammary tumor progression. Cancer Res 70:3361-71
Williams, Suzannah A; Stanley, Pamela (2009) Complex N-glycans or core 1-derived O-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or Le(Y) in the preimplantation mouse embryo. Glycoconj J 26:335-47
Williams, Suzannah A; Stanley, Pamela (2009) Oocyte-specific deletion of complex and hybrid N-glycans leads to defects in preovulatory follicle and cumulus mass development. Reproduction 137:321-31
Williams, Suzannah A; Stanley, Pamela (2008) Mouse fertility is enhanced by oocyte-specific loss of core 1-derived O-glycans. FASEB J 22:2273-84
Williams, Suzannah A; Xia, Lijun; Cummings, Richard D et al. (2007) Fertilization in mouse does not require terminal galactose or N-acetylglucosamine on the zona pellucida glycans. J Cell Sci 120:1341-9
Chen, Wei; Tang, Jian; Stanley, Pamela (2005) Suppressors of alpha(1,3)fucosylation identified by expression cloning in the LEC11B gain-of-function CHO mutant. Glycobiology 15:259-69

Showing the most recent 10 out of 20 publications