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- Doulias, Paschalis-Thomas, et al.
(författare)
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Endosomal and lysosomal effects of desferrioxamine : Protection of HeLa cells from hydrogen peroxide-induced DNA damage and induction of cell-cycle arrest
- 2003
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Ingår i: Free Radical Biology & Medicine. - 0891-5849 .- 1873-4596. ; 35:7, s. 719-728
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Tidskriftsartikel (refereegranskat)abstract
- The role of endosomal/lysosomal redox-active iron in H2O 2-induced nuclear DNA damage as well as in cell proliferation was examined using the iron chelator desferrioxamine (DFO). Transient transfections of HeLa cells with vectors encoding dominant proteins involved in the regulation of various routes of endocytosis (dynamin and Rab5) were used to show that DFO (a potent and rather specific iron chelator) enters cells by fluid-phase endocytosis and exerts its effects by chelating redox-active iron present in the endosomal/lysosomal compartment. Endocytosed DFO effectively protected cells against H2O2-induced DNA damage, indicating the importance of endosomal/lysosomal redox-active iron in these processes. Moreover, exposure of cells to DFO in a range of concentrations (0.1 to 100 ╡M) inhibited cell proliferation in a fluid-phase endocytosis- dependent manner. Flow cytometric analysis of cells exposed to 100 ╡M DFO for 24 h showed that the cell cycle was transiently interrupted at the G 2/M phase, while treatment for 48 h led to permanent cell arrest. Collectively, the above results clearly indicate that DFO has to be endocytosed by the fluid-phase pathway to protect cells against H2O 2-induced DNA damage. Moreover, chelation of iron in the endosomal/lysosomal cell compartment leads to cell cycle interruption, indicating that all cellular labile iron is propagated through this compartment before its anabolic use is possible.
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| 2. |
- Douoalis, Paschalis-Thomas, et al.
(författare)
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Involvement of heat shock protein-70 in the mechanism of hydrogen peroxide-induced DNA damage : The role of lysosomes and iron
- 2007
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Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849 .- 1873-4596. ; 42:4, s. 567-577
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Tidskriftsartikel (refereegranskat)abstract
- Heat shock protein-70 (Hsp70) is the main heat-inducible member of the 70-kDa family of chaperones that assist cells in maintaining proteins functional under stressful conditions. In the present investigation, the role of Hsp70 in the molecular mechanism of hydrogen peroxide-induced DNA damage to HeLa cells in culture was examined. Stably transfected HeLa cell lines, overexpressing or lacking Hsp70, were created by utilizing constitutive expression of plasmids containing the functional hsp70 gene or hsp70-siRNA, respectively. Compared to control cells, the Hsp70-overexpressing ones were significantly resistant to hydrogen peroxide-induced DNA damage, while Hsp70-depleted cells showed an enhanced sensitivity. In addition, the "intracellular calcein-chelatable iron pool" was determined in the presence or absence of Hsp70 and found to be related to the sensitivity of nuclear DNA to H2O2. It seems likely that the main action of Hsp70, at least in this system, is exerted at the lysosomal level, by protecting the membranes of these organelles against oxidative stress-induced destabilization. Apart from shedding additional light on the mechanistic details behind the action of Hsp70 during oxidative stress, our results indicate that modulation of cellular Hsp70 may represent a way to make cancer cells more sensitive to normal host defense mechanisms or chemotherapeutic drug treatment. © 2006 Elsevier Inc. All rights reserved.
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| 3. |
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| 4. |
- Tenopoulou, Margarita, et al.
(författare)
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Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?
- 2007
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 403:2, s. 261-266
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Tidskriftsartikel (refereegranskat)abstract
- The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at < pH 5. In the present study we show that the calcein-AM method does not capture lysosomal low-mass iron and, therefore, that the method seriously underestimates total cellular labile iron. © 2007 Biochemical Society.
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