Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations

Background: Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia via induction of differentiation and programmed cell death (apoptosis), We investigated the effects of As2O3 on a panel of malignant lymphocytes to determine whether growth-inhibitory and apoptotic effects of As2O3 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 As2O3, 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 (Delta Psi(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 As2O3 (1-2 mu 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, As2O3-induced apoptosis was preceded by Delta Psi(m) collapse, DTT antagonized and BSO enhanced As2O3-induced ATP depletion, Delta Psi(m) collapse, and apoptosis, Caspase-3 activation, usually resulting from Delta Psi(m) collapse, was not always associated with As2O3-induced apoptosis. As2O3 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 mu M As2O3. As2O3 may prove useful in the treatment of malignant lymphoproliferative disorders.