- Background: Carnosic acid is a plant-derived polyphenol food preservative with chemoprotective effects against carcinogens when tested in animals. Recently, we showed that carnosic acid potentiates the effects of 1α,25-dihydroxy-vitamin D 3 (1α,25[OH] 2 D 3 ) and of all-trans-retinoic acid (ATRA) on differentiation of human leukemia cells. We now examine the mechanisms associated with carnosic acid-induced enhancement of cell differentiation (in subline HL60-G) initiated by 1α,25(OH) 2 D 3 . ATRA, or 12-O-tetradecanoylphorbol-13-acetate (TPA). Methods: We evaluated monocytic differentiation markers (CDllb, CD14, and monocytic serine esterase), cell cycle parameters, and cell proliferation rates after treatment of cells with different agents with or without carnosic acid. We also assessed the abundance of the vitamin D receptor (VDR), retinoid X receptor (RXR)-a, retinoic acid receptor (RAR)-a, and cell cycle-associated proteins by immunoblot analysis (p27, early growth response gene [EGR]-1, and p35Nck5a), the expression of corresponding genes by reverse transcription-polymerase chain reaction (RT-PCR), and the activity of VDR by electrophoretic mobility shift analysis. The two-sided nonparametric Kruskal-Wallis one-way analysis-of-variance test with Dunn's adjustment was used for statistical analyses. Results: Monocytic differentiation induced by low (1 nM) concentrations of 1α,25(OH) 2 D 3 , ATRA, or TPA was enhanced by carnosic acid (10 μM), as shown by the increased expression of monocytic serine esterase (P<.001, P<.001, and P =.043, respectively) and of CDllb (P =.008, P =.046, and P =.041, respectively). Increased expression of CD14 was seen only for 1α,25(OH) 2 D 3 and ATRA (P =.009 and P =.048, respectively) and also for several cell cycle-associated proteins. Carnosic acid in combination with 1α,25(OH) 2 D 3 and ATRA resulted in decreased cell proliferation and blocked the cell cycle transition from G 1 to S phase (P<.05). Carnosic acid alone increased the expression of VDR and RXR-a, but the expression was greatly enhanced in the presence of 1α,25(OH) 2 D 3 and ATRA. In combination with TPA, carnosic acid potentiated the expression of VDR and RAR-α. Conclusion: Carnosic acid enhances a program of gene expression consistent with 1α,25(OH) 2 D 3 -ATRA-, or TPA-induced monocytic differentiation of HL60-G cells.