Retinoic acid (RA) is a known regulator of many cellular functions, including proliferation and differentiation, whose actions are mediated through a signaling pathway involving nuclear receptors, RAR and RXR. We have shown that low concentrations of fenretinide, N-(4-hydroxyphenyl)retinamide, a retinoic acid derivative, induced differentiation of cultured human retinal pigment epithelial (RPE) cells into a neuronal phenotype. 2D gel electrophoresis followed by mass spectrometric analysis identified increased expression in treated cells of Hsp70, a protein involved in stress response as well as cell differentiation. Western blot and immunocytochemical analyses showed a large increase in Hsp70 protein expression in human RPE cells (ARPE-19) treated with fenretinide. ELISA demonstrated that HSP70 expression reached a maximum of approximately 4 fold above control levels within 24 hours and remained at this level for 10 days during treatment. Fenretinide is a potential cancer preventive agent that is known to induce apoptosis in cancer cells. Long term administration of fenretinide has been shown to lower plasma retinol levels and affect night vision. In other studies, we examined the toxic effect of fenretinide on human retinal pigment epithelial cells. Human RPE cells (ARPE-19) in culture were treated with 1-20 uM fenretinide in the presence or absence of retinoid receptor antagonists for various time intervals. Cell lysates were used to measure the progression of apoptosis by a sandwich-enzyme immunoassay using anti-histone antibody directed against mono-and oligonucleosomes. Activities of caspases, 2, 3, 8 and 9, a family of cysteine proteases involved in apoptosis, were measured using a fluorimetric method. Fenretinide induced apoptosis in ARPE-19 cells in a dose- and time-dependent manner as indicated by the generation of mono- and oligonucleosomes. AGN194301, a RARalpha receptor specific antagonist, effectively blocked the apoptosis. Retinoid receptor pan-antagonists AGN194310 and AGN193109 also showed this effect. Fenretinide induced apoptosis was accompanied by a marked increase in caspase-2 and caspase-3 activities. These results demonstrate for the first time that fenretinide induces apoptosis in ARPE-19 cells and that RARalpha is involved in this process.
Showing the most recent 10 out of 16 publications
