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. 2015 Aug 5;16(8):18111-28.
doi: 10.3390/ijms160818111.

UV Differentially Induces Oxidative Stress, DNA Damage and Apoptosis in BCR-ABL1-Positive Cells Sensitive and Resistant to Imatinib

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Free PMC article

UV Differentially Induces Oxidative Stress, DNA Damage and Apoptosis in BCR-ABL1-Positive Cells Sensitive and Resistant to Imatinib

Ewelina Synowiec et al. Int J Mol Sci. .
Free PMC article

Abstract

Chronic myeloid leukemia (CML) cells express the active BCR-ABL1 protein, which has been targeted by imatinib in CML therapy, but resistance to this drug is an emerging problem. BCR-ABL1 induces endogenous oxidative stress promoting genomic instability and imatinib resistance. In the present work, we investigated the extent of oxidative stress, DNA damage, apoptosis and expression of apoptosis-related genes in BCR-ABL1 cells sensitive and resistant to imatinib. The resistance resulted either from the Y253H mutation in the BCR-ABL1 gene or incubation in increasing concentrations of imatinib (AR). UV irradiation at a dose rate of 0.12 J/(m2 · s) induced more DNA damage detected by the T4 pyrimidine dimers glycosylase and hOGG1, recognizing oxidative modifications to DNA bases in imatinib-resistant than -sensitive cells. The resistant cells displayed also higher susceptibility to UV-induced apoptosis. These cells had lower native mitochondrial membrane potential than imatinib-sensitive cells, but UV-irradiation reversed that relationship. We observed a significant lowering of the expression of the succinate dehydrogenase (SDHB) gene, encoding a component of the complex II of the mitochondrial respiratory chain, which is involved in apoptosis sensing. Although detailed mechanism of imatinib resistance in AR cells in unknown, we detected the presence of the Y253H mutation in a fraction of these cells. In conclusion, imatinib-resistant cells may display a different extent of genome instability than their imatinib-sensitive counterparts, which may follow their different reactions to both endogenous and exogenous DNA-damaging factors, including DNA repair and apoptosis.

Keywords: BCR-ABL1; DNA damage; apoptosis; imatinib resistance; reactive oxygen species.

Figures

Figure 1
Figure 1
Relative mean viability of mouse 32D cells transfected with the BCR-ABL1 gene: sensitive (S, white circles) and resistant to imatinib as evaluated by the Cell Counting Kit-8 assay. Imatinib resistance resulted from the Y253H mutation in the BCR-ABL1 gene (black circles) or from incubation of S cells with escalating doses of imatinib (black squares). All cell lines were incubated with imatinib at 0.025–1.6 µM for 24 h at 37 °C. Presented are means of six independent measurements; error bars represent standard deviation (SD).
Figure 2
Figure 2
Mean intracellular reactive oxygen species levels in mouse 32D cells transfected with the BCR-ABL1 gene: sensitive (S) and resistant to imatinib expressed as the fluorescence of 2′,7′-dichlorofluorescein (DCF) oxidatively converted from dichlorodihydrofluorescein diacetate exposed to UV radiation at 35 J/m2 at room temperature at a dose rate of 0.12 J/(m2·s) (black bars) as compared with non-irradiated cells (white bars). Imatinib resistance resulted from the Y253H mutation in BCR-ABL1 (253) or from incubation of S cells with escalating doses of imatinib (AR). Presented are means of six independent measurements; error bars represent SEM; * p < 0.05, *** p < 0.001 as compared with S line.
Figure 3
Figure 3
Mean DNA damage measured by percentage of DNA in tail of comets in alkaline version of comet assay modified by the use of T4 pyrimidine dimer glycosylase (T4 PDG) and human 8-oxoG glycosylase (hOGG1) in mouse 32D cells transfected with the BCR-ABL1 gene: sensitive (S) and resistant to imatinib exposed to UV radiation at 35 J/m2 at room temperature at a dose rate of 0.12 J/(m2·s). Imatinib resistance resulted from the Y253H mutation in BCR-ABL1 (253) or from incubation of S cells with escalating doses of imatinib (AR). Presented are means of three independent measurements, each for 100 cells; error bars represent SEM; *** p < 0.001 as compared with S line.
Figure 4
Figure 4
UV-induced apoptosis in mouse 32D transfected with the BCR-ABL1 gene: sensitive (S) and resistant to imatinib or cells displaying imatinib resistance caused by the Y253H mutation in BCR-ABL1 (253) or incubation of the sensitive cells with increasing doses of imatinib (AR). Cells were irradiated with UV light at 35 J/m2 at room temperature at a dose rate of 0.12 J/(m2·s). Apoptosis was assayed by flow cytometry with Annexin V-propidium iodide staining after a 24 h post-incubation. (a) Representative scatter plots for cells irradiated with UV; numbers in quadrants represent percentage of cells in early apoptosis (Q3), late apoptosis and necrosis (Q2) and live cells (Q4); (b) Apoptotic index calculated as percentage of either Q3 + Q2 cells (upper bar graph) or Q3 cells (lower graph) cells in 5 × 104 cells. Data are expressed as means of three independent experiments, presented is mean ± standard deviation (SD), * p < 0.05, *** p < 0.001 as compared with S cells.
Figure 5
Figure 5
Mitochondrial membrane potential (MMP) in mouse 32D cells transfected with the BCR-ABL1 gene: sensitive (S) to imatinib or displaying imatinib resistance caused by the Y253H mutation in BCR-ABL1 (253) or incubation of the sensitive cells with increasing doses of the drug (AR). Upper panel. MMP is expressed as ratio of 530 nm/590 nm to 485 nm/538 nm (aggregates to monomer) fluorescence as quantified with a fluorescent plate reader after JC-1 staining. Cells were irradiated with UV light at 35 J/m2 at room temperature at a dose rate of 0.12 J/(m2·s) (UV, black bars) and compared to control, non-irradiated cells (C, white bars). The mitochondrial uncoupler CCCP (carbonyl cyanide 3 chlorophenylhydrazone) was used as a positive control (grey bars). Fluorescence was measured after a 24 h incubation. Values are means ± SD (n = 4). * p < 0.05 and *** p < 0.001 as compared with imatinib-sensitive cells, respectively; Lower panel. Fluorescence microscopy (400×) of control cells (C) and cells treated with UV light (UV). Mitochondria in C cells are polarized and JC-1 accumulates in mitochondria as aggregate with red fluorescence, while in apoptotic cells JC-1 remains in cytoplasm in monomeric form emitting green fluorescence.
Figure 6
Figure 6
Basal mRNA expression of the nuclear SDHB (succinate dehydrogenase complex, subunit B), Mcl-1 (myeloid cell leukemia sequence 1) and mitochondrial COX1 (cytochrome c oxidase subunit I) gene in non-irradiated (upper panel) or UV-irradiated (lower panel) mouse 32D cells transfected with the BCR-ABL1 gene sensitive to imatinib (S) or cells displaying imatinib resistance caused by the Y253H mutation in BCR-ABL1 (253) or incubation of the sensitive cells with increasing doses of imatinib (AR). Cells were irradiated with UV light at 35 J/m2 at room temperature at a dose rate of 0.12 J/(m2·s) and the expression was determined by TaqMan probe-based real-time PCR (RT-PCR) assay and calculated by the 2−ΔCt method and ΔCt was obtained by subtracting Ct of ACTB mRNA from Ct of mRNA of respective nuclear gene or Ct of RNR2 from Ct of COX1. Data are shown as means ± SEM (n = 3), * p < 0.005, ** p < 0.01, *** p < 0.001 as compared with imatinib-sensitive cells.
Figure 7
Figure 7
Results of a representative mutational analysis of BCR-ABL1-expressing cells: sensitive to imatinib (S) and primarily resistant to imatinib due to the Y253H with acquired resistance to imatinib. The analysis was based on an allele-specific RT-PCR with primers specific to cDNA obtained by reverse transcription of mRNA produced from the gene of active BCR-ABL1 kinase. The upper plot shows amplification with primers specific for the Y253H mutation, the lower shows those for the T315I mutation. Cell line harboring the T315I mutation (315) and cultured in the same conditions as 253 was used as a positive control.

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