Tumor cell metastasis is the primary cause of death in most cancers. Numerous laboratories have demonstrated a strong correlation between the glycosylation cell-surface and the ability of a tumor cell to detach and metastasize. We will continue to make use of state-of-the-art technology for the study of glycoconjugate biochemistry in order to elucidate the structure base of and the biochemical mechanisms behind the altered glycosylation of highly metastatic tumor cell-surfaces. Our plan is to continue to use the available B16 melanoma variants that have widely different metastatic phenotypes. In addition, we will examine certain other metastatic models, including cells transfected with various oncogenies, and a series of murine adrenal carcinoma metastatic variants.
Specific Aims as follows: 1) To Continue to Perform Structural Analyses On Oligosacharides From Individual Glycosylation Sites of Glycolproteins Important to the Metastatic Phenotype. Oligosaccharide structural analyses will be performed at isolated sites on class I and II histocompatibility antigens, laminin, Lamp-l and Gp-70. 2) To continue to Use Purified Glycosyltransferases, In Conjunction with Appropriate Glycosidases, As Impearment Probes of Cell-Surface Saccharide Topography on Living Metastatic Tumor Cell Variants. Cell surface saccharide topography of the metastatic variants will be examined using several different highly-purified glycosyltransferases that have previously been linked to metastasis. 3) To Purify and Characterize the B1-4 GlcNAc Galactosyltransferase That is Elevated 4-fold on the Surfaces of Spontaneously Metastatic B16 Melanoma Cells. Substrate specificity, mode of localization to the cell- surface and the possible role of the enzyme in metastasis will be investigated. 4) To Study the Glycoylation of Chromosomal Proteins in Metastatic Tumor Cell Variants. Recently we have discovered a novel from of glycosylation that is enriched on transcription factors, DNA-binding proteins, and on nuclear pores.
Our aim i s to evaluate the possible involvement of this type of glycosylation in the metastatic phenotype. 5) To use Transfected Oncogenies and Phorbol Estes to Investigate the Biochemical Mechanisms Leading to Altered Cell Surface Glycosylation in Metastatic Tumor Cells. Transfection of NIH 3T3 cells with c-H-ras and treatment of B16 melanoma cells with phorbol esters rapidly induce metastatic phenotype and concomitantly alters cell surface glycosylation. We will study the biochemical changes in the cell surface oligosaccharides induced by these agents with the aim of elucidating the connection between altered expression of protein kinase C and abnormal oligosacharide processing. These studies are using the best available animal models of tumor metastasis to examine the mechanisms behind and the roles of altered glycosylation in generation of the metastatic phenotype.
|Ma, Junfeng; Hart, Gerald W (2017) Analysis of Protein O-GlcNAcylation by Mass Spectrometry. Curr Protoc Protein Sci 87:24.10.1-24.10.16|
|Bullen, John W; Balsbaugh, Jeremy L; Chanda, Dipanjan et al. (2014) Cross-talk between two essential nutrient-sensitive enzymes: O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK). J Biol Chem 289:10592-606|
|Hardivillé, Stéphan; Hart, Gerald W (2014) Nutrient regulation of signaling, transcription, and cell physiology by O-GlcNAcylation. Cell Metab 20:208-13|
|Copeland, Ronald J; Han, Guanghui; Hart, Gerald W (2013) O-GlcNAcomics--Revealing roles of O-GlcNAcylation in disease mechanisms and development of potential diagnostics. Proteomics Clin Appl 7:597-606|
|Hart, Gerald W (2013) Nutrient regulation of immunity: O-GlcNAcylation regulates stimulus-specific NF-?B-dependent transcription. Sci Signal 6:pe26|
|Hart, Gerald W (2013) How sugar tunes your clock. Cell Metab 17:155-6|
|Ma, Junfeng; Hart, Gerald W (2013) Protein O-GlcNAcylation in diabetes and diabetic complications. Expert Rev Proteomics 10:365-80|
|Banerjee, Partha S; Hart, Gerald W; Cho, Jin Won (2013) Chemical approaches to study O-GlcNAcylation. Chem Soc Rev 42:4345-57|
|Alfaro, Joshua F; Gong, Cheng-Xin; Monroe, Matthew E et al. (2012) Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci U S A 109:7280-5|
|Tarrant, Mary Katherine; Rho, Hee-Sool; Xie, Zhi et al. (2012) Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis. Nat Chem Biol 8:262-9|
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