title: C/EBPalpha inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation.
abstract: We previously demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as oxidative stress, ionizing radiation and TNF-receptor-induced ligand (TRAIL) compared with vector-transfected (HL-60/Vect) cells. Here, we show that HL-60/FAK cells are highly resistant to all-trans retinoic acid (ATRA)-induced differentiation, whereas original HL-60 or HL-60/Vect cells are sensitive. Treatment with ATRA at 1 muM for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed no such effect in HL-60/FAK cells. Electrophoretic mobility shift assay (EMSA) using an oligonucleotide for the c/EBP consensus binding sequence showed that c/EBPalpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBPalpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBPalpha after treatment with ATRA was seen in HL-60/Vect cells but not in HL-60/FAK cells. Further, hyperphosphorylation of pRb was observed in HL-60/FAK cells. Finally, the introduction of FAK siRNA into HL-60/FAK cells resulted in the recovery of sensitivity to ATRA-induced differentiation, confirming that the inhibition of HL-60/FAK differentiation resulted from both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBPalpha.
authors.affiliation: Department of Biochemistry, Kyoritsu University of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo, 105-8512, Japan.
authors.forename: Ken-ichiro
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Sonoda
authors.forename: Masakazu
authors.initials: M
authors.lastname: Yamakado
authors.forename: Megumi
authors.initials: M
authors.lastname: Funakoshi-Tago
authors.forename: Naomi
authors.initials: N
authors.lastname: Yoshida
authors.forename: Akiko
authors.initials: A
authors.lastname: Rokudai
authors.forename: Eriko
authors.initials: E
authors.lastname: Aizu-Yokota
authors.forename: Tadashi
authors.initials: T
authors.lastname: Kasahara
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000378:metabolism; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D039481:CD11b Antigen / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D024202:Electrophoretic Mobility Shift Assay; D051417:Focal Adhesion Kinase 1 / Q000096:biosynthesis*; D018922:HL-60 Cells; D006801:Humans; D010766:Phosphorylation; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D014212:Tretinoin / Q000494:pharmacology; D000097768:Retinoic Acid Receptor gamma
chemical_list: D051997:ADP-ribosyl Cyclase 1; D022763:CCAAT-Enhancer-Binding Protein-alpha; D039481:CD11b Antigen; D051417:Focal Adhesion Kinase 1; D018168:Receptors, Retinoic Acid; D016160:Retinoblastoma Protein; D000072482:Retinoic Acid Receptor alpha; D014212:Tretinoin; D000097768:Retinoic Acid Receptor gamma; C000607071:RARA protein, human; C496050:PTK2 protein, human