Rod outer segments mediate mitochondrial DNA damage and apoptosis in human retinal pigment epithelium.
OBJECTIVE
To investigate the interrelationships between DNA damage, mitochondrial activity, and apoptosis in retinal pigment epithelial cells (RPE) after exposure to rod outer segments (ROS).


METHODS
After incubation of cultured human RPE with ROS, mitochondrial redox function was evaluated from MTT reduction. Mitochondrial (mt) and nuclear (n) DNA damage were determined by quantitative polymerase chain reactions (QPCR). Apoptotic RPE cells were detected by binding of annexin V to phosphatidyl serine (PS) using fluorescence microscopy. The expression of the pro-apoptotic proteins, p53 and p21(waf-1), and DNA repair enzymes, apurinic/apyrimidinic endonuclease (APE(ref-1)) and DNA polymerase beta (beta-pol) were quantitatively determined by Western blotting analysis.


RESULTS
Mitochondrial function decreased by 20 +/- 5% and annexin V immunofluorscent binding was enhanced after exposure of cells to physiological levels of ROS (3.8 x 10(6)cm(-2)) for 4 h. MtDNA was preferentially damaged after exposure to ROS with increased lesion frequencies of 1.49 +/- 0.37 and 2.2 +/- 0.14 per 10 kb base pairs (bp), respectively after 5 and 7 h contact, compared to untreated controls (zero class damage). APE(ref-1)expression increased more than 340% above controls after exposure to ROS for 7 and 24 h. The expression of beta-pol in cultures increased 110% above controls after 24 h contact with the ROS. The expression of p53 and p21 in cells increased 100 and 38% above controls after 24 h exposure to the ROS.


CONCLUSIONS
Exposure of ROS to ROS induced mtDNA damage and dysfunction and activated nDNA repair pathways, which did not prevent apoptosis.
Jin
G F
GF
Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston 77555-0787, USA.
Hurst
J S
JS
Godley
B F
BF
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D002417:Cattle; D002461:Cell Line, Transformed; D018920:Coculture Techniques; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D010718:Phosphatidylserines / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000473:pathology*; D016133:Polymerase Chain Reaction; D012374:Rod Cell Outer Segment / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D017304:Annexin A5; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004272:DNA, Mitochondrial; D010718:Phosphatidylserines; D016159:Tumor Suppressor Protein p53; D019951:DNA Polymerase beta; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase