title: Characterization of a novel amphiregulin-related molecule in 12-O-tetradecanoylphorbol-13-acetate-treated breast cancer cells.
abstract: Amphiregulin (AR) can be induced at the mRNA level by 17-beta-estradiol (E2) or the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This study compares the effects of TPA and E2 on the regulation of processing of AR isoforms and on subcellular localization in human MCF-7 breast cancer cells. AR was localized in the nucleus of MCF-7 cells after E2 treatment, whereas it was predominantly secreted after TPA treatment. AR isoforms of 28, 18, and 10 kDa and an additional species of approximately 55-60 kDa were detected in the cellular conditioned media after TPA stimulation. Expression of this unusual AR isoform was inhibited by protein kinase C (PKC) inhibitors such as bryostatin or H-7. The biochemical properties of this isoform are consistent with it being an N-linked glycosylated form of the AR precursor that contains unprocessed mannose residues. The size of this large isoform is reduced to approximately 40 kDa after treating the TPA-induced MCF-7 cells with tunicamycin or treating the conditioned media of such cells with N-glycosidase F or with endoglycosidase H. Moreover, this isoform is able to blind several lectins with specificity for mannose residues. The 55-60 kDa glycosylated AR isoform, like lower Mr AR isoforms, is able to bind to heparin and to stimulate the growth of MCF-10A cells by interacting with the EGF receptor. These data suggest that TPA activation of PKC may be involved in post-translational modifications of AR, such as glycosylation, and in alteration of its subcellular routing to predominantly a secretory pathway.
authors.affiliation: Vincent T. Lombardi Cancer Research Center, Washington, D.C. USA.
authors.forename: I
authors.initials: I
authors.lastname: Martinez-Lacaci
authors.forename: G R
authors.initials: GR
authors.lastname: Johnson
authors.forename: D S
authors.initials: DS
authors.lastname: Salomon
authors.forename: R B
authors.initials: RB
authors.lastname: Dickson
mesh_terms: D066258:Amphiregulin; D001943:Breast Neoplasms / Q000737:chemistry*; D002451:Cell Compartmentation; D066255:EGF Family of Proteins; D066246:ErbB Receptors / Q000502:physiology; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006133:Growth Substances / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D036341:Intercellular Signaling Peptides and Proteins; D008970:Molecular Weight; D011485:Protein Binding; D011493:Protein Kinase C / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D066255:EGF Family of Proteins; D006023:Glycoproteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D004958:Estradiol; D006493:Heparin; D066246:ErbB Receptors; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate