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B. Cai, J. Zhou, B.S. Westlund, J.R. Sparrow; The Nonreceptor Tyrosine Kinase cAbl Is Involved in the Cell Death Pathway Activated in A2E–Laden RPE Irradiated With Blue Light . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2887.
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© ARVO (1962-2015); The Authors (2016-present)
Aging and some retinal disorders are accompanied by the accumulation of lipofuscin in retinal pigment epithelial cells (RPE). We have previously shown that the death of blue light irradiated RPE cells involves photooxidative mechanisms and is mediated by an apoptotic pathway that includes the participation of cysteine–dependent proteases (caspases) and that is modulated by the mitochondrial protein Bcl–2. Here we have tested whether apoptosis under these circumstances is also promoted by cAbl, a ubiquitously expressed nonreceptor tyrosine kinase (140 kDa) that can be activated by oxidative stress and DNA damage and that contributes to the regulation of apoptosis.
ARPE–19 cells that had accumulated A2E were treated for 48 hours with the cAbl inhibitor STI 571, an inhibitor that occupies the ATP binding pocket of cAbl thereby stabilizing it in an inactive conformation. Cells were also transfected with siRNA targeted to cAbl for 24 hours. The cells were irradiated at 430 nm and nonviability was assessed either by nuclear labeling with a cell impermeable dye combined with DAPI or by MTT assay. cAbl mRNA was quantified by real time RT–PCR and protein levels were evaluated by immunoblotting and scanning densitometry.
cAbl mRNA and protein were upregulated several fold in A2E–laden RPE illuminated with blue light. cAbl was also phosphorylated on tyrosine245. siRNA targeted to cAbl suppressed protein levels and both siRNA and STI 571 protected against the loss of viability in cells that had accumulated A2E and were illuminated at 430 nm. Specifically, siRNA targeted to cAbl and STI 571 increased cell survival by 30% (2–color fluorescence method and MTT assay) and 50% (MTT assay), respectively.
Perhaps due to its ability to respond to intracellular oxidative insult, cAbl is involved in activating the death pathway in A2E–laden blue light exposed RPE cells. The involvement of parallel mechanisms may be indicated, however, since cell death was not completely abrogated by STI571 or siRNA gene silencing.
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