May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Oxidative Stress-Induced Apoptosis in ARPE-19 Cells Involves Lysosomal Rupture but Requires Intense Stress and Is Not Appreciably Alleviated by Iron Chelators
Author Affiliations & Notes
  • C. I. Frennesson
    Linkoping University, Linkoping, Sweden
    Dept of Ophthalmology,
  • M. Karlsson
    Linkoping University, Linkoping, Sweden
    Dept of Ophthalmology,
  • T. Kurz
    Linkoping University, Linkoping, Sweden
    Dept of Pharmacology,
  • S. E. G. Nilsson
    Linkoping University, Linkoping, Sweden
    Dept of Ophthalmology,
  • Footnotes
    Commercial Relationships  C.I. Frennesson, None; M. Karlsson, None; T. Kurz, None; S.E.G. Nilsson, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5934. doi:https://doi.org/
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      C. I. Frennesson, M. Karlsson, T. Kurz, S. E. G. Nilsson; Oxidative Stress-Induced Apoptosis in ARPE-19 Cells Involves Lysosomal Rupture but Requires Intense Stress and Is Not Appreciably Alleviated by Iron Chelators. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5934. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : To examine the mechanisms behind oxidative stress-induced apoptosis in ARPE-19 cells, especially the role of iron. Theoretically, RPE cells should be resistant to oxidative stress because they normally exist in an oxygen-rich milieu and survive a lifespan of heterophagocytosis that in relation to its intensity gives rise to a less than expected lipofuscin accumulation.

Methods: : ARPE-19 cells were sparsely seeded in DMEM/HAM F12 (1:1) with 10% FCS. The distribution of loosely bound iron was assayed by the sulphide silver method (SSM), a sensitive cytochemical method. The cells were stressed by exposure to H2O2 (100µM-20mM) +/- the lipophilic iron chelator SIH (100 µM) following a 4h exposure to a Fe-phosphate complex (100µM) that is taken up by endocytosis. Lysosomal integrity was examined by the acridine orange (AO) uptake technique, while apoptosis was assayed by morphological methods and by the binding of a pan-caspase antibody.

Results: : The ARPE-19 cells were found to contain seemingly non-redox-active iron with a mainly lysosomal localization. Nevertheless, the cells were extremely resistant to H2O2, and high concentrations (15-20mM) of it were needed to induce pronounced apoptosis that, as usual, was preceded by lysosomal rupture. In contrast to findings on more H2O2-sensitive cells, such as J774 cells, which also are professional scavengers, the lipophilic iron chelator SIH did not significantly prevent lysosomal rupture and apoptosis when H2O2 was applied in doses large enough to severely affect the ARPE-19 cells. Endocytotic uptake of the Fe-phosphate complex somewhat enhanced the sensitivity to oxidative stress that, however, was not ameliorated by SIH.

Conclusions: : HPV-16-immortalized human RPE cells are obviously extremely resistant to oxidative stress in vitro, although their lysosomes contain some low mass iron. Since SIH protection during H2O2-exposure did not prevent lysosomal rupture and apoptosis, most of this iron is seemingly non-redox-active. The reasons for this phenomenon need to be further investigated because it may help us better understand AMD and conditions where the effects of ischemia - reperfusion-induced oxidative stress ought to be alleviated.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage 
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