Background. Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients However, APL cells develop resistance to retinoic acid treatment Arsenic trioxide (As 2 O 3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As 2 O 3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients. Methods: Apoptosis was assessed by means of DNA fragmentation analyses, TUNEL assays (ie, deoxyuridine triphosphate labeling of DNA nicks with terminal deoxynucleotidyl transferase), and flow cytometry. Expression of the PML/RARα fusion protein in leukemia cells was assessed by means of western blotting, ligand binding, and immunohistochemistry. Northern blotting and ribonuclease protection assays were used to evaluate changes in gene expression in response to retinoic acid and As 2 O 3 treatment. Results and Conclusions: As 2 O 3 induces apoptosis without differentiation in retinoic acid-sensitive and retinoic acid resistant APL cells at concentrations that are achievable in patients. As 2 O 3 induces loss of the PML/RARα fusion protein in NB4 cells, in retinoic-acid resistant cells derived from them, in fresh APL cells from patients, and in non-APL cells transfected to express this protein. As 2 O 3 and retinoic acid induce different patterns of gene regulation, and they inhibit the phenotypes induced by each other. Understanding the molecular basis of these differences in the effects of As 2 O 3 and retinoic acid may guide the clinical use of arsenic compounds and provide insights into the management of leukemias that do not respond to retinoic acid.[J Natl Cancer Inst; 1998; 90; 124–33]

Retinoids are a group of natural and synthetic derivatives of vitamin A proven to have dramatic activity in the treatment of acute promyelocytic leukemia (APL)(1, 2). Retinoids have been shown to induce cytodifferentiation and maturation of APL cells in vitro and in patients (3) APL is characterized by a reciprocal chromosomal translocation, t (15; 17), which fuses the PML gene with the retinoic acid receptor α (RARα) gene, leading to the production of a chimeric oncoprotein, PML/RARα (4–8). PML/RARα plays a dual role in the phenotype of APL. It blocks myelocytic differentiation in a dominant-negative manner but permits maturation in the presence of pharmacologic levels of retinoic acid (9, 10). Immunohistochemical studies of the PML and PML/RARαt proteins show that they colocalize in an APLspecific microparticulate structure, whereas, in normal cells, PML is localized within specific subnuclear structures called nuclear bodies or PML oncogenic domains (11–13). Treatment with retinoic acid reconstitutes the normal PML nuclear body pattern in APL cells (12). Two groups (14, 15) recently reported that retinoic acid induces a specific degradation of PML/RARα protein, possibly through a proteasome pathway. These data led to the hypothesis that retinoids induce APL cells to mature by selectively targeting the oncogenic PML/RARα protein.