Sensitivity of photoreceptor-derived cell line (661W) to baculoviral p35, Z-VAD.FMK, and Fas-associated death domain.
OBJECTIVE
Rod, cone, cone-rod, and macular dystrophies eventually bring about the death of cone photoreceptor cells. The present study explores means of inhibiting apoptosis in addition to inducing a specific apoptotic pathway within a photoreceptor-derived cell line.


METHODS
Retinal cell culture of murine 661W photoreceptor-derived cells was used to assess the effect of both a synthetic peptide inhibitor of caspases (benzyloxycarbonyl-Val-Ala-DL-Asp-[Ome] fluoromethylketone [Z-VAD.FMK]) and a natural inhibitor, baculoviral p35. In addition, the effect of transfection of Fas-associated death domain (FADD), a cellular protein implicated in receptor-induced apoptosis, was assessed. Assays were performed by transient transfection of cell cultures, and results were recorded by cell counting, Western blot, and spectrophotometry.


RESULTS
Western blot analysis and chromogenic caspase substrate cleavage analysis confirmed the activation of caspases within 661W cells. At a concentration of 80 micro M, Z-VAD.FMK, 72.36% +/- 0.93% of 661W cells survived cytotoxic insult compared with 6.99% +/- 1.35% of control cells. Transient transfection of 1200 ng baculoviral p35 conferred a protection of 75.30% +/- 4.23%, compared with 19.61% +/-1.84% of control cells, and it was additionally observed that as little as 50 ng transfection of FADD was capable of inducing the death of 53.21% +/- 1.33% of cells in 661W cultures.


CONCLUSIONS
Apoptotic cell death in 661W cells is caspase dependent and may be inhibited with both a synthetic and natural inhibitor of caspase function. Furthermore, 661W cells are highly sensitive to the FADD protein, which may suggest a number of novel therapeutic approaches to halt photoreceptor cell apoptosis.
Tuohy
Gearóid
G
Wellcome Ocular Genetics Unit, Trinity College Dublin, Dublin, Ireland. gtuohy@tcd.ie
Millington-Ward
Sophia
S
Kenna
Paul F
PF
Humphries
Peter
P
Farrar
G Jane
GJ
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics*; D061945:Caspase Inhibitors; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D019610:Cytoprotection / Q000187:drug effects; D004789:Enzyme Activation; D053200:Fas-Associated Death Domain Protein; D051034:Inhibitor of Apoptosis Proteins; D051379:Mice; D017924:Nucleopolyhedroviruses; D017949:Retinal Cone Photoreceptor Cells / Q000187:drug effects* / Q000201:enzymology; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000494:pharmacology*
D048868:Adaptor Proteins, Signal Transducing; D000590:Amino Acid Chloromethyl Ketones; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein; D051034:Inhibitor of Apoptosis Proteins; D014764:Viral Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C086430:inhibitor of apoptosis, Nucleopolyhedrovirus