Abstract
BackgroundDaunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods.MethodsThree ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 h treatment with daunorubicin chemotherapy and 4, 12 and 24 h recovery periods. Cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2–5 diphenyltetrazolium bromide) assay, reactive oxygen species (ROS) production by flow cytometry, double stranded DNA breaks by detecting γH2AX levels while stages of the cell cycle were detected following propidium iodide staining and flow cytometry. Western blotting was used to detect specific proteins while RNA was extracted from all cell lines and converted to cDNA to sequence Ataxia–telangiectasia mutated (ATM).ResultsDaunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by γH2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in ATM as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells.ConclusionsThe delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 that may be correlated to increased expression of SOD2 and lower ROS production.
Highlights
Daunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL)
CCRF-CEM cells exposed to daunorubicin (Fig. 2b) did not show significant reduction in cell density until 12 h in the recovery media (0.48 ± 0.07, P = 0.0002) compared to the control
CCRF-CEM cells exposed to daunorubicin (Fig. 3b), displayed a similar pattern of reactive oxygen species (ROS) production
Summary
Daunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods. Proposed mechanisms of anthracycline action have included: inhibition of synthesis of macromolecules through intercalation of daunorubicin into DNA strands [2, 3], interaction with molecular oxygen to produce reactive oxygen species (ROS), topoisomerase II inhibition and the formation of DNA adducts [4]. The type of toxic lesions that generally results from daunorubicin treatment are DNA double strand breaks (DSB). Activated ATM phosphorylates histone H2AX (H2AX) at Ser139 residues of the carboxyl terminus to form γH2AX around the DNA-DSB. A common outcome of both pathways is phosphorylation of the tumour suppressor gene, protein 53 (p53), which plays a pivotal role in the cellular response to damage as p53 regulates numerous cellular responses, including cell cycle arrest and apoptosis as well as upregulation of anti-oxidant proteins such as manganese-containing superoxide dismutase (SOD2 or MnSOD) [9]
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