Figure 1
Retinoic acid promotes either differentiation or cell death of breast cancer cells in a cell-context dependent manner. (A) T47D and H3396 cells were treated with 9-cis-retinoic acid (9-cis-RA) for the indicated time and analyzed for the presence of DNA fragments in their cytosol as a measurement of cell death. Y-axis refers to the enrichment of histone complexed DNA fragments (mono- and oligonucleosomes) in the cytoplasm of apoptotic cells. The values represent the mean ± SD of three different experiments performed in triplicate. (B) T47D and H3396 cells were treated with 9-cis-RA for 6 days. Cell death was determined by FACS analysis after staining with propidium iodide as described in "Materials and Methods". The values represent the mean ± SD of three different experiments performed in duplicate. (C) H3396 cells were treated with 9-cis-RA and the RAR-antagonist BMS493, as indicated, and analyzed as described in (A). (D) H3396 cells were analyzed by flow cytometry to assess the population of cells with lower mitochondria membrane potential following all-trans retinoic acid (atRA) or 9-cis-RA treatment for the indicated time. The values represent the mean ± SD of three different experiments performed in duplicate. (E) Release of mitochondrial proteins to the cytosol was analyzed by western blotting after subcellular fractionation of H3396 cells treated with 9-cis-RA for the indicated time. (F) Effect of 9-cis-RA treatment on the cleavage of caspase-8 (casp-8), caspase-9 (casp-9) and PARP assessed by western blotting in H3396 cells. (G) Oil-red O staining of T47D cells untreated or treated with 1 μM 9-cis-RA for 72 h. The images shown are from one representative experiment performed three times with similar results.