Acyclic retinoid activates retinoic acid receptor B and induces transcriptional activation of p21 in HepG2 human hepatoma cells

Acyclic retinoid (ACR), a novel synthetic retinoid, has recently been demonstrated by us to inhibit the in vitro growth of human hepatoma cells, and this effect was associated with decreased expression of cell cycle-related molecules. These results, taken together with previous in vitro and clinical studies with ACR, suggest that this agent may be useful in the chemoprevention and therapy of hepatoma and possibly other human malignancies. In the present study, we further examined the molecular effects of ACR on the HepG2 human hepatoma cell line, focusing on the expression of nuclear retinoid receptors and the cell cycle inhibitor protein p21. Reverse transcription-PCR assays and Western blot analyses indicated that these cells express retinoic acid receptors (RARs) A, B, and ;, retinoid X receptors (RXRs) A and B, and peroxisome proliferator-activated receptors (PPAR) ; mRNA. Treatment with ACR caused a rapid induction within 3 h of RARB mRNA and the related protein, but there was no significant change in the levels of the mRNA or proteins for RARs A and ;, RXRs A and B, and PPAR;. There was also a rapid increase in p21 mRNA and protein in HepG2 cells treated with ACR, and this induction occurred via a p53-independent mechanism. In transient transfection reporter assays, we cotransfected the retinoic acid response element-chloramphenicol acetyltransferase (CAT) reporter gene into HepG2 cells together with a RARB expression vector. RARB expression markedly stimulated CAT activity (up to about 4-fold) after the addition of ACR. However, CAT activity in the presence of ACR was only about 2-fold higher than that in the absence of ACR, when cells were cotransfected with RARs A and ; or RXRA. These findings suggest that the growth inhibitory effects of ACR are mediated at least in part through RARB and that both RARB and p21 play critical roles in the molecular mechanisms of growth inhibition induced by ACR. [Mol Cancer Ther. 2004;3(3):309–316]