Bendamustine induces G2 cell cycle arrest and apoptosis in myeloma cells: the role of ATM-Chk2-Cdc25A and ATM-p53-p21-pathways.

PURPOSE: 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.

CONCLUSION: 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.