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Shunai Jiang, Mei-Whey H. Wu, Paul Sternberg, Dean P. Jones; Fas Mediates Apoptosis and Oxidant-Induced Cell Death in Cultured hRPE Cells. Invest. Ophthalmol. Vis. Sci. 2000;41(3):645-655.
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purpose. To investigate whether Fas ligand (FasL) and the Fas receptor system
mediates apoptosis in cultured human retinal pigment epithelial (hRPE)
cells and contributes to oxidant-induced death of hRPE cells.
methods. Expression of FasL and Fas in cultured hRPE cells was examined by
Western blot analysis and flow cytometry. The susceptibility of hRPE
cells to Fas-mediated apoptosis was determined by incubating cells with
recombinant soluble Fas ligand (sFasL). Characteristics of apoptosis
assessed included chromatin condensation, DNA cleavage, and
phosphatidylserine exposure. To investigate the possible involvement of
Fas-mediated apoptosis in oxidative killing of hRPE cells, the effects
of the oxidant tert-butylhydroperoxide (tBH) on the expression of
FasL and Fas were studied. The specificity of effects of oxidant was
examined using the antioxidants glutathione and N-acetyl-l-cysteine (NAC). The requirement
for the Fas pathway in tBH-induced apoptosis was investigated using an
antagonistic anti-Fas antibody ZB4 that blocks the interaction between
FasL and Fas.
results. Cultured hRPE cells constitutively expressed FasL and Fas. Ligation of
Fas receptor with recombinant sFasL triggered apoptosis in hRPE cells.
tBH treatment of hRPE cells resulted in increased expression of FasL
and Fas. Glutathione and NAC completely abrogated tBH-induced increase
in FasL and Fas expression and apoptosis. Blocking FasL and Fas
interaction by ZB4 inhibited tBH-induced apoptosis, but only partially.
conclusions. A functional Fas-mediated apoptotic pathway is present in cultured hRPE
cells and can be activated with sFasL or by upregulation of FasL and
Fas expression with an oxidant. The incomplete inhibition by blocking
antibody indicates that the Fas pathway is involved in oxidant-induced
apoptosis, but other triggering mechanisms are also
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