Oxidative stress leads to increased mutation frequency in a murine model of myelodysplastic syndrome

Abstract

The myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it has been suggested that additional mutations lead to progression of MDS to AML, the causative agent(s) for such mutations remains unclear. Oxidative stress is a potential cause, therefore, we evaluated levels of reactive oxygen species (ROS) in NUP98-HOXD13 (NHD13) transgenic mice, a murine model for MDS. Increased levels of ROS were detected in bone marrow nucleated cells (BMNC) that express CD71, a marker for cell proliferation, as well as immature, lineage negative bone marrow nucleated cells from NHD13 mice. In addition to the increase in ROS, increased DNA double strand breaks and activation of a G2/M phase cell cycle checkpoint were noted in NHD13 BMNC. Finally, using an in vivo assay for mutation frequency, we detected an increased mutation frequency in NHD13 BMNC. These results suggest that oxidative stress may contribute to disease progression of MDS to AML through ineffective repair of DNA damage and acquisition of oncogenic mutations.

Keywords: AML; Big Blue(®) mice; MDS; Mutation; NHD13; ROS.

Fig. 1

Increased ROSs in proliferative cells and primitive hematopoietic progenitor cells of NHD13 mice. (A) Representative FACS profiles showing increased ROS in CD71 positive BMNC and LN BMNC from NHD13 mice. Histograms show DCFDA staining intensity from NHD13 BMNC (thick line) compared to WT littermate control BMNC (thin line). (B) Average mean fluorescence intensity (MFI) ratio of NHD13 versus age matched WT littermates was determined by FACS analysis from 3 independent experiments using CD71 and a lineage cocktail, and 9 independent experiments with lineage depleted cells. (C) MFI of DCFDA was analyzed in Lineage negative (LN) Sca-1 positive cells and LN cKit positive cells from 3 independent experiments. Error bars represent SEM. *, p<0.05; **, p<0.01

Fig. 2

Increased ROS in hematopoietic cell lines expressing an NHD13 transgene. (A) Ba/F3 cells stably transfected with pEF1a-NHD13 construct (BaF3/pNHD13) showed higher ROS level than cells transfected with empty vector (BaF3/pEF1a) or Ba/F3 cells. (B) Elevated ROS level was observed in 32D/ pEF1a-NHD13 (32D/pNHD13) cells compared with 32D/pEF1a and 32D cells. Bar graphs and statistics were acquired from stable transfectants at 3 different passages. The error bars represent SEM. *, p<0.05; **, p<0.01

Fig. 3

DNA double strand breaks assessed by γH2AX foci. (A) γH2AX foci are increased in the BMNC and LN BMNC of NHD13 mice compared with wild type (WT) BMNC. (B) Bar graphs represent the fold differences of number of γH2AX foci per individual cells from 6 independent experiments. The error bars represent SEM. *, p<0.05; **, p<0.01

Fig. 4

Association of cell growth arrest with increased level of ROS in lineage negative BM. (A) Representative histograms show the FACS results of cells stained with propidium iodide (PI) after fixation with 70% cold ethanol. Histogram data was analyzed with Flow Jo 7.5 software. (B) Bar graphs represent the mean percentage of each cell cycle phase from 9 independent experiments. (C) Western blot analysis of Chk1 and phospho-Chk1 expression in lineage negative BM (LN BMNC) or whole BM from NHD13 or WT mice. LN BMNCs were pooled from 3 mice to obtain sufficient protein for analysis. β-actin is used as a loading control *, p<0.05.

Fig. 5

λ cII mutational spectra. Linear map of the cII gene. Numerical co-ordinates refer to amino acid (aa) residue. Downward arrowheads above the diagram indicate single nucleotide substitutions. Upward and downward arrows below the diagram indicate single nucleotide frameshift insertions or deletions, respectively. Specific nucleotide changes are indicated in brackets. The mutations marked on the figure represent combined results from 4 independent experiments. UTR, untranslated region.