Arsenic Trioxide Induces Mitochondrial DNA Mutation on D-Loop Region in Patients with Acute Myeloid Leukemia.

Objective:To investigate the effect of arsenic trioxide (As₂O₃) on mitochondrial DNA (mtDNA) in acute myeloid leukemia cells.

Methods: NB4 cell line and primary leucocytes, isolated from peripheral blood of eight cases with newly diagnosed acute myeloid leukemia (the ratio of the blast cell was more than 80%), were incubated in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, and treated with 0.1, 1.0, 2.0 micromol/L of As₂O₃ for 48hrs respectively in vitro. Blood samples in eight cases above were collected before and 14 days after As₂O₃ treatment with the dosage of 0.16mg/kg.d in vivo. Genome DNA was isolated from the cells and blood samples by salt fractionation, and the D-loop fragment of mtDNA was isolated from the genome DNA and amplified by polymerase chain reaction, and the sequencing techniques were applied to identify positive clones. MTT assay, electrophoresis of genomic DNA, and protein/DNA dual parameter flow cytometry were used to examine the effect of As₂O₃ on cell proliferation and apoptosis.

Results: mtDNA mutations were found on D-loop fragment of mtDNA in both NB4 cells and primary leucocytes, the number of single nucleotide polymorphism (SNP) or mutation were increased remarkably after 1.0, 2.0 micromol/L of As₂O₃ treatment both in vitro and in vivo than non-As₂O₃ treated baseline. The types of mutation were included the base transversion, transition, depletion and insertion. The 1.0 and 2.0 micromol/L of As2O3-treated samples revealed apoptosis, reduced proliferation and mtDNA mutation, while in the 0.1 micromol/L of As2O3-treated samples, showed reduced proliferation, differentiation and mtDNA mutation, but no apoptosis was appeared.

Conclusions: As₂O₃ promoted leucocytes’ mtDNA mutation on D-loop fragment both in NB4 cell line and in primary leucocytes isolated from patients with acute myeloid leukemia. Mitochondrial DNA might be one of the targets of As₂O₃ act on leukemia cells. Mitochondrial DNA mutation as well as nuclear DNA might participate the process of As₂O₃ inducing differentiation and reducing proliferation.