title: Elevated beta1,4-galactosyltransferase I in highly metastatic human lung cancer cells. Identification of E1AF as important transcription activator.
abstract: The elevated levels of beta1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5'-region flanking the transcription start point of the GalT I gene (-1653 to +52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides -205 and -200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the beta1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.
authors.affiliation: State Key Laboratory of Genetic Engineering, Gene Research Center, Shanghai Medical College of Fudan University, Shanghai, People's Republic of China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Zhu
authors.forename: Jianhai
authors.initials: J
authors.lastname: Jiang
authors.forename: Hailian
authors.initials: H
authors.lastname: Shen
authors.forename: Hanzhou
authors.initials: H
authors.lastname: Wang
authors.forename: Hongliang
authors.initials: H
authors.lastname: Zong
authors.forename: Zejuan
authors.initials: Z
authors.lastname: Li
authors.forename: Yanzhong
authors.initials: Y
authors.lastname: Yang
authors.forename: Ziyue
authors.initials: Z
authors.lastname: Niu
authors.forename: Weicheng
authors.initials: W
authors.lastname: Liu
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Chen
authors.forename: Yun
authors.initials: Y
authors.lastname: Hu
authors.forename: Jianxin
authors.initials: J
authors.lastname: Gu
mesh_terms: D045744:Cell Line, Tumor; D005700:Galactosyltransferases / Q000032:analysis / Q000378:metabolism*; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000473:pathology; D009154:Mutation; D009362:Neoplasm Metastasis; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism*
chemical_list: D011518:Proto-Oncogene Proteins; D005700:Galactosyltransferases; C493310:beta1,4-galactosyltransferase, human; D001616:beta-Galactosidase