Background
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.
Methods
Three 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).
Results
Daunorubicin 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.
Conclusions
The 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.
Electronic supplementary material
The online version of this article (10.1186/s12885-019-5377-y) contains supplementary material, which is available to authorized users.
Evidence shows that factors contributing to success in physiology education for allied health students at universities include not only their high school achievement and background but also factors such as confidence with their teachers and quality of their learning experience, justifying intensive and continued survey of students' perceptions of their learning experience. Here we report data covering a 3-yr period in a physiology subject that has been redesigned for blended and online presentation. Consistent with previous reports, we show that when we undertook a blended mode of delivery, students demonstrated better grades than traditional modes of teaching; however the absence of didactic teaching in this subject resulted in lower grades overall. Students have very strong positive attitudes to weekly quizzes (80% positive approval) but report ambivalent attitudes to online self-directed learning (61% negative perception), even though they had 2-h weekly facilitated workshops. Overwhelmingly, students who undertook the subject in a self-directed online learning mode requested more face-to-face-teaching (70% of comments). From these data, we suggest that there is a quantifiable benefit to didactic teaching in the blended teaching mode that is not reproduced in online self-directed learning, even when face-to-face guided inquiry-based learning is embedded in the subject.
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