Apoptosis and Growth Inhibition in Malignant Lymphocytes After Treatment With Arsenic Trioxide at Clinically Achievable Concentrations

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Abstract
BACKGROUND: Arsenic trioxide (As 2 O 3 ) can induce clinical remission in patients with acute promyelocytic leukemia via induction of differentiation and programmed cell death (apoptosis). We investigated the effects of As 2 O 3 on a panel of malignant lymphocytes to determine whether growth-inhibitory and apoptotic effects of As 2 O 3 can be observed in these cells at clinically achievable concentrations. METHODS: Eight malignant lymphocytic cell lines and primary cultures of lymphocytic leukemia and lymphoma cells were treated with As 2 O 3 , with or without dithiothreitol (DTT) or buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis). Apoptosis was assessed by cell morphology, flow cytometry, annexin V protein level, and terminal deoxynucleotidyl transferase labeling of DNA fragments. Cellular proliferation was determined by 5-bromo-2′-deoxyuridine incorporation into DNA and flow cytometry and by use of a mitotic arrest assay. Mitochondrial transmembrane potential (ΔΨ m ) was measured by means of rhodamine 123 staining and flow cytometry. Protein expression was assessed by western blot analysis or immunofluorescence. RESULTS: Therapeutic concentrations of As 2 O 3 (1-2 μ M ) had dual effects on malignant lymphocytes: 1) inhibition of growth through adenosine triphosphate (ATP) depletion and prolongation of cell cycle time and 2) induction of apoptosis. As 2 O 3 -induced apoptosis was preceded by ΔΨ m collapse. DTT antagonized and BSO enhanced As 2 O 3 -induced ATP depletion, ΔΨ m collapse, and apoptosis. Caspase-3 activation, usually resulting from ΔΨ m collapse, was not always associated with As 2 O 3 -induced apoptosis. As 2 O 3 induced PML (promyelocytic leukemia) protein degradation but did not modulate expression of cell cycle-related proteins, including c-myc, retinoblastoma protein, cyclin-dependent kinase 4, cyclin D1, and p53, or expression of differentiation-related antigens. CONCLUSIONS: Substantial growth inhibition and apoptosis without evidence of differentiation were induced in most malignant lymphocytic cells treated with 1-2 μ M As 2 O 3 . As 2 O 3 may prove useful in the treatment of malignant lymphoproliferative disorders.