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Effects of α1, 3 Fucosyltransferase-Ⅶ on Cell Signal Transduction, Proliferation and Apoptosis of Human Hepatocarcinoma Cells

Author: WangQiuYan
Tutor: ShenZongHou；ChenHuiLi
School: Fudan University
Course: Biochemistry and Molecular Biology
Keywords: α1 3 fucosyl transferase VII Human hepatoma cell Sialylated Lewis X antigen Cell surface receptors Cellular signal transduction Cell cycle Apoptosis
CLC: R735.7
Type: PhD thesis
Year: 2004
Downloads: 189
Quote: 1
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Abstract

In order to study the biological functions of a single sugar group, this choice glycolipid and glycoprotein sugar chains (mainly in the O-sugar chain, can also be at the end of the N-glycans) and tumor metastasis closely related Lewis antigens on fucosylated (Fuc). Lewis antigens sialylated and non-sialylated two categories, the α1, 3 fucose transferase-VII (α1, 3FucT-VII) is one terminal of the main catalytic the of Lewis antigens SLex fucosylated sialylated glycosyltransferase The enzyme, so that the processing of the sugar chain termination, the sugar chain structure is no longer changed. Therefore, only the end of the sugar chain to increase the of a Fuc glycosylated α1 3FucT-VII can be a good tool to study the function of a single glycosylation. Plasmid transfected cells α1, 3FucT-VII cDNA transfection low expression of α1, 3FucT-VII H7721 hepatoma cells observed after transfection caused a more comprehensive range of biological effects, may to some extent reflect the SLex fucose group functionality. The paper is divided into the following four parts: the first part α1, 3FucT-VIIcDNA transfected H7721 cell surface receptor antigen expression and receptor function in SLex first established stable expression of different amounts the α1-VII, 3FucT H7721 cell line α1 of the total SLex antigen expression of the mRNA and the cell surface of 3FucT-VII (α1, 3FucT-VII product) detection, cells transfected with empty plasmid Mock/H7721 also have a certain amount the the α1, 3FucT-VIImRNA and SLex the expression, so for low-expressing cell line, and the other two transfection of α1, 3FucT-VII cDNA for expression the cell strains FucTVII-M and high expression the cell strains FucTVII-H. In order to study the α1 3FucT-VII transfection of H7721 cell surface receptors SLex antigen expression and receptor function, select a representative of two growth factor receptors --- insulin receptor (InR) and epidermal growth factor receptor (EGFR), where InR is α, β pairs of subunits consisting of EGFR is a single subunit, is also selected to the other non-growth factor receptors and cell adhesion and migration Fn receptors - integrins (Integrin) α5β1 as the object of our study. Found that: α1 3FucT-VII transfected with the cell surface and intracellular InR and EGFR expression did not change significantly; measured by immunoprecipitation the InR and EGFR expression did not change significantly. 2. Α1, 3FucT-VII transfected make InRα subunit on SLex antigen expression increased, while the different EGFR transfected cells on SLex expression did not change. 3. Α1, 3FucT-VII cDNA transfection the InRβ subunits own tyrosine phosphorylation levels of EGFR since lt; WP = 5 gt; the body tyrosine phosphorylation levels did not change. 4. Α1, 3FucT-VII cDNA transfection of IRS-1 protein quantity decreased, and the average per molecule of IRS-1 tyrosine phosphorylation levels were significantly elevated. 5. Α1, 3FucT-VII cDNA transfection on average per molecule fiber with protein receptor Integrin α5 and β1 subunit SLex expression had no significant effect, and raised Integrin α5 subunit expression, but had no effect on the β1 subunit expression. 6. Α1, 3FucT-VII cDNA transfection 7721 cells to Fn adhesion and spreading ability. Above InRα the subunit SLex antigen expression, the InRβ subunit and IRS-1 tyrosine phosphorylation levels, Integrin α5 subunit expression of Fn adhesion and spreading are FucTVII-H/H7721 gt; FucTVII-M/H7721 gt; mock, and α1 of 3FucT-VII mRNA expression levels consistent. Visible above changes may all α1, 3FucT-VII cDNA after transfection caused so the insulin receptors SLex the increased results. IRS-1 protein expression of exception lt is FucTVII-H/H7721; the FucTVII-M/H7721 lt; Mock. Second part of α1, 3FucT-VIIcDNA transfected H7721 cells important signal transduction pathways in the first part has been found α1 3FucT-VII transfection can change the cell surface insulin receptor (InR) SLex expression and self-tyrosine phosphorylation function, then the cell surface the InR sugar chain structure and function of the change can lead to further downstream signaling molecule expression or phosphorylation changes? problem with this part of the research. The results showed that: 1 α1 the 3FucT-VII transfected cells compared with Mock cells of PKB protein expression did not change significantly, but PKB-T308 and S473 phosphorylation and PKB activity were raised, are FucTVII-H/H7721 gt ; FucTVII-M/H7721; S473 phosphorylation of PKB in rise higher than the T308. SLex mAb KM93 and insulin receptor tyrosine phosphorylation of a specific inhibitor of HNMPA-(AM) 3 may be to eliminate the difference of PKB phosphorylation. Α1, 3FucT-VII transfected H7721 cells elevated PDK-1 expression and phosphorylation of PKN were significantly elevated; PKN protein expression and phosphorylation levels of PI-3K inhibitor LY294002 significantly inhibited , so the difference in different transfected cells was significantly reduced. Α1, 3FucT-VII in transfected H7721 cells in the c-Raf-1 expression was significantly increased; while the expression of p42/44 MAPK and MEK proteins with Mock no significant difference compared to cells, but their phosphorylation levels increase, higher the extent significantly exceeds the the PKB phosphorylation higher degree, may be provided with the Raf upregulation; KM93 and HNMPA-(AM) 3 can also be significantly eliminate the difference p42/44 MAPK phosphorylation in different transfected cells. 4 α1, 3FucT-VII transfected H7721 cells, β-catenin protein expression significantly increased the vitality of its downstream transcription factor TCF also significantly enhanced. 5 Fn presence of α1, the H7721 cells transfected 3FucT-VII of FAK protein expression has not changed, but its level of tyrosine phosphorylation is increased. 2 LT; WP = 6 GT; above these measurement signaling molecules involved in some signal transduction pathways, and each other to form a network crosstalk. The α1, 3FucT-VII activation of signal transduction networks can be used the following figure: This section is not measured of ILK, Ras and PI-3K, but there is reason to speculate that they α1, 3FucT-VII transfected cells also increased. ?

Effects of α1, 3 Fucosyltransferase-Ⅶ on Cell Signal Transduction, Proliferation and Apoptosis of Human Hepatocarcinoma Cells

Abstract

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