C/EBPα inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation

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 mu M for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed 110 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/EBP alpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBP alpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBP alpha 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 front both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBP alpha. (c) 2005 Elsevier Inc. All rights reserved.