The effect of FK506 on transforming growth factor {beta} signaling and apoptosis in chronic lymphocytic leukemia B cells

1 Department of Biochemistry and Medical Biotechnologies, Federico II University, Naples 2 Institute of Biostructure and Bio-Imaging-National Research Council (CNR), Naples, Italy Correspondence: Maria Fiammetta Romano, MD, Department of Biochemistry and Medical Biotechnologies, Federico II University, via Pansini, 5. 80131. Naples, Italy. E-mail: romano{at}dbbm.unina.it Background: Loss of response to transforming growth factor-beta (TGF-β ) is thought to contribute to the progression of chronic lymphocytic leukemia. Recent findings of over-activation of the TGF-β signal in FKBP12-knockout mouse prompted us to investigate whether FK506, the canonical ligand of FKBP, can activate the TGF-β signal in chronic lymphocytic leukemia. Design and Methods: We studied 62 chronic lymphocytic leukemia samples from patients with Rai/Binet stage 0 to 4 disease. The TGF-β signal was investigated by western blotting and flow cytometry. The levels of Bcl2-family members and death-associated-protein kinase were also investigated by western blotting, whereas apoptosis was studied in flow cytometry. Down-modulation of FKBP12 was obtained by gene silencing with short interfering RNA. Results: Twenty-two out of 62 chronic lymphocytic leukemia samples were sensitive to TGF-β-induced apoptosis. All but two of the responsive samples underwent apoptosis also when cultured with FK506, but not with cyclosporine. Thirteen samples that were not sensitive to TGF-β were sensitive to FK506. Overall, response to FK506 occurred in 33 samples. FK506 induced Smad2 phosphorylation and nuclear translocation. Accordingly, death-associated-protein kinase, a transcriptional target of Smad, was induced. At the same time, Bcl-2 and Bcl-xL levels decreased whereas the levels of Bim and Bmf increased. A loss of mitochondrial membrane potential preceded caspase activation and cell death. FK506 removed FKBP12 from its binding to the TGF-β-receptor. FKBP12 release activated the receptor-kinase activity as suggested by the enhanced levels of phospho-Smad found in cells depleted of FKBP12. Conclusions: Our study shows that most chronic lymphocytic leukemia cells escape the homeostatic control of TGF-β and that FK506 restores the TGF-β signal in a proportion of non-responsive samples. We demonstrated that FK506 activates TGF-β receptor I kinase activity in chronic lymphocytic leukemia, which transduces apoptosis by a mitochondrial-dependent pathway. Key words: FK506, chronic lymphocytic leukemia, TGF-β, ap