PML-RARalpha protein, the leukemogenic product of t(15,17) in acute promyelocytic leukemia, is cleaved into a truncated form termed deltaPML-RARalpha during all-trans retinoic acid (ATRA)-induced differentiation of NB4 cells. DeltaPML-RARalpha is not formed in ATRA differentiation resistant NB4 subclones. As(2)O(3) inhibits deltaPML-RARalpha formation and differentiation-induction when given in combination with ATRA. Treatment with hexamethylene bisacetamide (HMBA) combined with ATRA enhances ATRA-induced differentiation in ATRA-insensitive NB4-CI and arsenic-resistant NB4/As cells, and is associated with stabilization of PML-RARalpha protein and increased deltaPML-RARalpha formation. Unlike forced expression of PML-RARalpha, forced deltaPML-RARalpha expression based on an estimated deletion of the N-terminal PML portion does not repress RARE-tk-luc reporter activity mediated by endogenous retinoic acid receptors. The cleavage of PML-RARalpha is blocked by RARalpha antagonist Ro-41-5253 and cycloheximide and therefore requires a RARalpha transactivation-dependent pathway. Proteasome inhibitor MG-132 and caspase inhibitor Z-VAD-FMK do not block ATRA-induced PML-RARalpha cleavage and differentiation. These data suggest that (a) ATRA treatment induces PML-RARalpha cleavage by induction of unknown enzymes independent of proteasome- and caspase-mediated pathways; (b) deltaPML-RARalpha might function differently from both PML-RARalpha and RARalpha; (c) failure to cleave PML-RARalpha and form deltaPML-RARalpha after ATRA treatment may contribute to ATRA resistance in APL cells.