Augmentation of phenylbutyrate-induced differentiation of myeloid leukemia cells using all-trans retinoic acid

Despite preliminary evidence of clinical activity of the putative differentiating agent sodium phenylbutyrate (PB) in the treatment of myeloid neoplasms, it has proven difficult to maintain therapeutic levels of PB above 0.5 mM, well below the ED₅₀ of 1-2 mM. We have studied the impact of combining PB with all-trans retinoic acid (ATRA) on the ML-1 myeloid leukemia cell line. ATRA augmented PB-induced differentiation, cell-cycle arrest, and apoptosis. ATRA augmented PB induction of the myelomonocytic marker CD11b at all doses of ATRA tested (0.0025-1 μM). Although ATRA did not significantly affect the ED₅₀ of PB, the combination of ATRA (1 μM) and PB (0.5 mM) augmented PB-induced CD11b expression eight-fold, Compared to PB alone, this combination of ATRA and PB induced greater cell cycle arrest (S-phase 14% vs 38%; G₀/G₁-phase cells 72% vs 52%) and greater apoptosis (24% vs 16% by TUNEL assay). Treatment with ATRA (0.5 μM) in combination with PB (0.5 mM) led to significantly greater inhibition of colony formation (4.8% vs 48% inhibition). ATRA combined synergistically with PB to augment CD11b expression and inhibit colony formation. This combination also showed significant interaction in terms of S-phase inhibition. However, this interaction varied as a function of ATRA concentration: antagonistic at low concentrations of ATRA, synergistic at higher concentrations of ATRA. These data suggest that retinoids may significantly augment the cytostatic and differentiating activity of PB, leading to increased potency of the latter drug at clinically achievable doses.