To investigate which one of these upstream molecular signaling pathways was used by 4-hydroxitamoxifen, dexamethasone, all-trans-retinoic acid (atRA) and 9-cis-retinoic acid (9cRA) to up-regulate the expression of p27, Western immunoblot analysis was performed using the ER-negative MDA-MB-231 human breast cancer cells in vitro (Figures 5 and 6). This evidence concerns the gene ESR1 and breast cancer.