title: The effect of FK506 on transforming growth factor beta signaling and apoptosis in chronic lymphocytic leukemia B cells.
abstract: BACKGROUND
  Loss of response to transforming growth factor-beta (TGF-beta ) is thought to contribute to the progression of chronic lymphocytic leukemia. Recent findings of over-activation of the TGF-beta signal in FKBP12-knockout mouse prompted us to investigate whether FK506, the canonical ligand of FKBP, can activate the TGF-beta signal in chronic lymphocytic leukemia.
  
  
  METHODS
  We studied 62 chronic lymphocytic leukemia samples from patients with Rai/Binet stage 0 to 4 disease. The TGF-beta 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-beta-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-beta 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-beta-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-beta and that FK506 restores the TGF-beta signal in a proportion of non-responsive samples. We demonstrated that FK506 activates TGF-beta receptor I kinase activity in chronic lymphocytic leukemia, which transduces apoptosis by a mitochondrial-dependent pathway.
authors.affiliation: Department of Biochemistry and Medical Biotechnologies, Federico II University, via Pansini, 5. 80131, Naples, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Romano
authors.forename: Maria
authors.initials: M
authors.lastname: Mallardo
authors.forename: Federico
authors.initials: F
authors.lastname: Chiurazzi
authors.forename: Rita
authors.initials: R
authors.lastname: Bisogni
authors.forename: Anna
authors.initials: A
authors.lastname: D'Angelillo
authors.forename: Raffaele
authors.initials: R
authors.lastname: Liuzzi
authors.forename: Giovanna
authors.initials: G
authors.lastname: Compare
authors.forename: Maria Fiammetta
authors.initials: MF
authors.lastname: Romano
mesh_terms: D017209:Apoptosis; D018450:Disease Progression; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007700:Kinetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000473:pathology; D008024:Ligands; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: D007166:Immunosuppressive Agents; D008024:Ligands; D019253:Proto-Oncogene Proteins c-bcl-2; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D051020:bcl-X Protein; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus