Characterization of daunorubicin-induced apoptosis in retinal pigment epithelial cells: modulation by CD95L.

PURPOSE To characterize daunorubicin-induced cell death in cultured human retinal pigment epithelial (RPE) cells and its modulation by CD95 ligand (CD95L). METHODS In situ DNA end labeling and an ELISA for histone-associated DNA fragments were used to assess apoptosis. CD95 and CD95L expression were examined by immunohistochemistry, flow cytometry, immunoblot, and RT-PCR. Cell death was measured by crystal violet staining. YVAD- and DEVD-amc cleavage was used to measure caspase-1 and -3-like activity. Total RNA and protein synthesis was measured by incorporation level of [(3)H]-leucine and [(3)H]-uridine. RESULTS RPE cells expressed both CD95 and CD95L, but only CD95 was expressed at the cell surface. Daunorubicin-induced RPE cell apoptosis was associated with enhanced CD95 and CD95L expression. Enhanced CD95L expression was epiphenomenal to the death process, evidenced by the fact that neutralizing CD95L antibodies failed to modulate daunorubicin cytotoxicity. In contrast, the cytotoxic effects of daunorubicin were synergistically enhanced by exogenous CD95L. Synergy appeared to involve enhanced caspase-3-like activity as well as daunorubicin-mediated inhibition of RNA synthesis. CONCLUSIONS Apoptosis has been shown to be an important factor in the control of specific cell populations. The synergistic activity of an antiproliferative agent, daunorubicin, and a cytokine, CD95L, induces apoptosis in RPE cells. Such approaches provide a means to reduce the concentration of chemotherapeutic agents with a small therapeutic window owing to retinal toxicity, such as daunorubicin, in the adjuvant therapy of proliferative vitreoretinopathy.