1301

Our previous data have shown that retinoid X receptor α (RXRα) overexpression induces p21 Waf1/Cip1 in a ligand dependent manner, resulting in inhibition of cell growth by RXR ligand in normally retinoid-resistant MDA-MB-231 human breast cancer cells. Here, we study the regulatory mechanism of the RXR-mediated p21 cell growth inhibitory effect. Adenoviral RXRα overexpression ligand-dependently induces cell growth arrest at the G0/G1 phase while concurrently stimulating upregulation of p21, downregulation of pRB, and morphological changes, indicating differentiation. Ligand-mediated p21 promoter upregulation proves to respond in a time, adenovirus moi, and ligand dose-dependent manner. Furthermore, northern blot analysis shows that RXRα overexpression elevates p21 mRNA levels in the presence of ligand, and p21 promoter assays reveal a time-dependency of p21 transactivation upon ligand addition. p21 reporter assays with serial deletions of the p21 promoter revealed that RXRα may directly target a putative RXR response element (RXRE) homodimer sequence on the p21 promoter, and that removal of this region abolishes the ligand-mediated p21 transactivation. Supporting these results, Electrophoretic Mobility Shift Assays also demonstrated that nuclear extract from MDA-MB-231 infected with Ad-RXRα ligand-dependently bind the putative RXRE to a greater degree than those infected with Ad-null, and that this complex is also supershifted by addition of the RXR antibody. We further studied RAR’s effect on RXR homodimer mediated-p21 transcriptional regulation. RAR overexpression dose- and time-dependently caused a reduction in RXR-mediated p21 promoter activity and p21 protein expression. This suggests that RAR may interfere with the RXR homodimer-mediated p21 upregulation. Our data demonstrate that ligand-activated RXRα homodimers are functionally active modulators of the cell cycle through direct stimulation of the p21 promoter, and that this transcriptional regulation may be hindered by RAR.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]