Characterization of daunorubicin-induced apoptosis in retinal pigment epithelial cells: modulation by CD95L.
OBJECTIVE
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.
Hueber
Arno
A
Department of Vitreo-Retinal Surgery, Center of Ophthalmology, University of Cologne, Germany. arno.hueber@uni-koeln.de
Weller
Michael
M
Welsandt
Gerhard
G
Kociok
Norbert
N
Kirchhof
Bernd
B
Esser
Peter
P
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003630:Daunorubicin / Q000494:pharmacology*; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020287:In Situ Nick-End Labeling; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000494:pharmacology*; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019014:fas Receptor / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D019014:fas Receptor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; D003630:Daunorubicin