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D. Yehudai et al.
mRNA were also decreased >95%, but little change in the mRNA expression of nu-COX IV was seen (Online Supplementary Figure S3). We also observed reductions in basal oxygen consumption at concentrations associated with reductions in mtDNA and respiratory chain proteins (Figure 1D). Of note, alovudine did not reduce ATP level in treated cells (Figure 1E).
Finally, we observed reductions in cell proliferation and viability at concentrations of alovudine that reduced mtDNA and inhibited oxidative phosphorylation. Of the tested AML cell lines (MV4-11, OCI-AML2, TEX, NB4, and K562), MV4-11 was the most sensitive to alovudine (Figure 1F and Online Supplementary Figure S4A-C). Compared to the other tested cell lines, MV4-11 had high-
A
Figure 2. Effects of alovudine on primary acute myeloid leukemia (AML) and nor- mal hematopoietic cells. Primary AML samples and normal peripheral blood stem cells (PBSCs) were treated with 2000 nM alovudine for 6 days. (A) Relative mito- chondrial DNA (mtDNA) was assessed by qRT-PCR in AML patients (n=2). (B) Primary AML and normal hematopoietic cells (PBSCs) were treated with 2000 nM of alovu- dine for 6 days. Cell viability was assessed by trypan blue exclusion staining in pri- mary AML cells (n=7) and CellTiter-Fluor for PBSCs (n=3). The dotted line represents cell viability of DMSO control. (C and D) Primary AML and normal hematopoietic cells (PBSCs) were treated with increasing concentrations of alovudine for 6 days. Colony forming abilities were assessed in primary AML cells and PBSCs (n=2 of each) as described above in the Online Supplementary Methods. PBSC samples 1 and 2 were the same samples as used in (B). For all experiments, ns: non-significant; *P<0.5, **P<0.01, ***P<0.001, and ****P<0.0001 using Sidak’s (A) or Dunnett’s (B-D) multiple comparisons test after one-way ANOVA. BFU-E: primitive erythroid progeni- tor cells; CFU-GM: granulocyte precursors.
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haematologica | 2019; 104(5)