title: Bendamustine induces G2 cell cycle arrest and apoptosis in myeloma cells: the role of ATM-Chk2-Cdc25A and ATM-p53-p21-pathways.
abstract: OBJECTIVE
  Multiple myeloma is a fatal hematological disease caused by malignant transformation of plasma cells. Bendamustine has been proven to be a potent alternative to melphalan in phase 3 studies, yet its molecular mode of action is still poorly understood.
  
  
  METHODS
  The four-myeloma cell lines NCI-H929, OPM-2, RPMI-8226, and U266 were cultured in vitro. Apoptosis was measured by flow cytometry after annexin V FITC and propidium iodide staining. Cell cycle distribution of cells was determined by DNA staining with propidium iodide. Intracellular levels of (phosphorylated) proteins were determined by western blot.
  
  
  RESULTS
  We show that bendamustine induces apoptosis with an IC50 of 35-65 mug/ml and with cleavage of caspase 3. Incubation with 10-30 mug/ml results in G2 cell cycle arrest in all four-cell lines. The primary DNA-damage signaling kinases ATM and Chk2, but not ATR and Chk1, are activated. The Chk2 substrate Cdc25A phosphatase is degraded and Cdc2 is inhibited by inhibitory phosphorylation of Tyr15 accompanied by increased cyclin B levels. Additionally, p53 activation occurs as phosphorylation of Ser15, the phosphorylation site for ATM. p53 promotes Cdc2 inhibition by upregulation of p21. Targeting of p38 MAPK by the selective inhibitor SB202190 significantly increases bendamustine induced apoptosis. Additionally, SB202190 completely abrogates G2 cell cycle arrest.
  
  
  CONCLUSIONS
  Bendamustine induces ATM-Chk2-Cdc2-mediated G2 arrest and p53 mediated apoptosis. Inhibition of p38 MAPK augments apoptosis and abrogates G2 arrest and can be considered as a new therapeutic strategy in combination with bendamustine.
authors.affiliation: Department of Haematology and Oncology, Klinikum der Universität München, Medizinische Klinik Innenstadt, Ziemssenstrasse 1, 80336 Munich, Germany.
authors.forename: Leander
authors.initials: L
authors.lastname: Gaul
authors.forename: Sonja
authors.initials: S
authors.lastname: Mandl-Weber
authors.forename: Philipp
authors.initials: P
authors.lastname: Baumann
authors.forename: Bertold
authors.initials: B
authors.lastname: Emmerich
authors.forename: Ralf
authors.initials: R
authors.lastname: Schmidmaier
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D064007:Ataxia Telangiectasia Mutated Proteins; D000069461:Bendamustine Hydrochloride; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D064447:Checkpoint Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D009101:Multiple Myeloma / Q000378:metabolism / Q000473:pathology*; D009588:Nitrogen Mustard Compounds / Q000494:pharmacology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000378:metabolism*
chemical_list: C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D009588:Nitrogen Mustard Compounds; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; D000069461:Bendamustine Hydrochloride; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases