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

Abstract

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.

Fig. 1

Bendamustine induces apoptosis. a NCI-H929 (NCI), RPMI-8226 (RPMI), OPM-2 and U-266 myeloma cells were were treated with 1–100 μg/ml (1, 3, 10, 30 and 100) bendamustine (benda) or control medium (Co) over 48 h. Apoptosis was determined by annexin V (aV)/propidium iodide (PI) staining. b Intracellular levels of caspase 3 and cleaved caspase 3 were detected by western blotting using antibodies against caspase 3 and the 17/19 D cleavage products. Actin bands show equal loading. c. NCI-H929 (NCI) were treated with control medium alone or bendamustine 10/30/100 μg/ml over 72 h and PI+ apoptosis was monitored after 24, 48, and 72 h

Fig. 2

Bendamustine induces G2/M-arrest. a NCI-H929 (NCI), RPMI-8226 (RPMI), OPM-2 and U-266 were treated with bendamustine (benda) 1–100 μg/ml or medium (co) over 48 h and cell cycle distributon (percent cells) was analyzed after PI-staining. b NCI-H929 and U-266 cells were incubated with control medium or 10 μg/ml bendamustine over 48 h. The FACS-images are shown for both cell lines, the G2-fraction is signalized as black area under the graph. c NCI-H929 cells (NCI) were incubated with control medium (co), 3, 10, and 30 μg/ml bendamustine and seeded in 96-well plates. WST-agent was added after 0, 24, 48, and 72 h and the turnover was measured by extinction at 440 nm

Fig. 3

Cdc2 inactivation via ATM/Chk2/Cdc25A and p53/p21. NCI-H929 and RPMI-8226 cells were incubated with either control medium (co) or bendamustine (benda) 10/30 and 30/60 μg/ml over 48 h. Protein levels were detected by western blotting using antibodies against a Cyclin B1, Cyclin D2, P-Cdc2 (Tyr15), b P-ATM (Ser1981), P-ATR (Ser 428), P-Chk2 (Thr 68), c P-Cdc25C (Ser 216), Cdc25A, d p-53, P-p53 (Ser15), and e bad, bax, bcl-2, bcl-X/L and XIAP. Actin blotting shows equal loading

Fig. 4

Synergism of bendamustine and p38α MAPK inhibitor. NCI-H929 cells (NCI) were incubated either with control medium (co), bendamustine (benda) 30 μg/ml, 10 μM SB202190 or both agents over 48 h. a. Apoptosis was measured by AnnexinV (AV)/propidium iodid (PI)-staining. b G2/M-fraction was determined by cell cycle distribution after PI-staining. c. P38 level and phosphorylation status was detected by western blotting. Actin control shows equal loading