Modulation of Ku70/80, clusterin/ApoJ isoforms and Bax expression in indocyanine-green-mediated photo-oxidative cell damage.
OBJECTIVE
In order to characterize the biological effects and molecular mechanism underlying indocyanine-green (ICG)-mediated photo-oxidative cell damage, human cultured retinal pigmented epithelium (RPE) cells preloaded with ICG were exposed to 810-nm laser irradiation. Cell viability and death induction were examined, as well as the modulation of proteins involved in cell death and DNA repair.


METHODS
ARPE-19 cells preloaded with 100 microM ICG were irradiated using continuous and micropulsed 810-nm laser for the dye photoactivation, and cell viability and apoptosis were evaluated. The expression and subcellular localization of Bax, Ku70, Ku80 and clusterin/ApoJ were analyzed by immunocytochemistry and Western blot.


RESULTS
ICG photoactivation induced apoptosis in RPE cells. The micropulsed laser irradiation induced a higher percentage of cell killing as compared to continuous wave. Cell killing was inhibited by sodium azide, suggesting the involvement of reactive oxygen species in the laser-induced cell damage. Bax was strongly induced after 4 and up to 24 h of treatment. The nuclear proapoptotic isoform of clusterin/ApoJ was selectively upregulated after 24 h of treatment. The DNA repair machinery was upregulated after 4 and up to 24 h.


CONCLUSIONS
These data elucidate some molecular mechanisms involved in cell death induced by ICG photosensitization. The increase and relocalization of Bax into the mitochondria and the upregulation and translocation of the proapoptotic isoform of clusterin/ApoJ in the nucleus demonstrated the involvement of these proteins in the photo-oxidative cell death pathway. These data point out new molecular targets and suggest potential applications in the therapy of the retinal diseases that could benefit by selective RPE treatment.
Ricci
Federico
F
Section of Ophthalmology, University of Rome Tor Vergata, Rome, Italy.
Pucci
Sabina
S
Sesti
Fabiola
F
Missiroli
Filippo
F
Cerulli
Luciano
L
Spagnoli
Luigi Giusto
LG
D034961:Antigens, Nuclear / Q000235:genetics*; D017209:Apoptosis; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D051152:Clusterin / Q000235:genetics*; D004396:Coloring Agents / Q000009:adverse effects; D004247:DNA / Q000235:genetics*; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D007208:Indocyanine Green / Q000009:adverse effects; D000072200:Ku Autoantigen; D007834:Lasers / Q000009:adverse effects; D018384:Oxidative Stress; D010778:Photochemotherapy / Q000009:adverse effects; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D051028:bcl-2-Associated X Protein / Q000235:genetics*
D034961:Antigens, Nuclear; D051152:Clusterin; D004396:Coloring Agents; D004268:DNA-Binding Proteins; D051028:bcl-2-Associated X Protein; D004247:DNA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D007208:Indocyanine Green