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Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.

Dewaele M, Martinet W, Rubio N, Verfaillie T, de Witte PA, Piette J, Agostinis P - J. Cell. Mol. Med. (2010)

Bottom Line: Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling.We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg(-/-) cells compensated for MA loss and increased cellular resistance to PDT.These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.

View Article: PubMed Central - PubMed

Affiliation: Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.

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Hypericin-PDT treatment evokes features of MA. All controls represent incubation with hypericin without irradiation. (A) Time-dependent detection of LC3-conversion by Western blot of whole cell lysates of human cervical carcinoma HeLa cells, immortalized MEFs and AY27 rat bladder carcinoma after PDT-treatment. (B) Confocal microscopy analysis of HeLa cells (a, b) transiently overexpressing GFP-LC3 and MEFs (c, d) stably expressing GFP-LC3. Images representative for 80% of the population of control (a, c) and cells treated 6 hrs after PDT (b, d) are shown. White scale bar in the upper right corner represents 10 μm. (C) Transmission electron microscopic analysis of control HeLa cells (a, b) and HeLa cells 6 hrs after PDT-treatment (c, d). EM photomicrographs representing 70% of the population are shown. (b) and (d) are magnifications of the peri-nuclear area indicated in (a) and (c). Arrows indicate vacuoles with detectable content in the treated cells (d). Black scale bar in the lower right corner represents 2 μm. (D) Flux analysis with MEF stably expressing GFP-LC3. HeLa cells were pre-incubated for 2 hrs with 100 nM BafA1 or vehicle (controls) before irradiation, BafA1 was present for the entire incubation period after irradiation. At the indicated time-points whole cell lysates were made for Western blot analysis.
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fig01: Hypericin-PDT treatment evokes features of MA. All controls represent incubation with hypericin without irradiation. (A) Time-dependent detection of LC3-conversion by Western blot of whole cell lysates of human cervical carcinoma HeLa cells, immortalized MEFs and AY27 rat bladder carcinoma after PDT-treatment. (B) Confocal microscopy analysis of HeLa cells (a, b) transiently overexpressing GFP-LC3 and MEFs (c, d) stably expressing GFP-LC3. Images representative for 80% of the population of control (a, c) and cells treated 6 hrs after PDT (b, d) are shown. White scale bar in the upper right corner represents 10 μm. (C) Transmission electron microscopic analysis of control HeLa cells (a, b) and HeLa cells 6 hrs after PDT-treatment (c, d). EM photomicrographs representing 70% of the population are shown. (b) and (d) are magnifications of the peri-nuclear area indicated in (a) and (c). Arrows indicate vacuoles with detectable content in the treated cells (d). Black scale bar in the lower right corner represents 2 μm. (D) Flux analysis with MEF stably expressing GFP-LC3. HeLa cells were pre-incubated for 2 hrs with 100 nM BafA1 or vehicle (controls) before irradiation, BafA1 was present for the entire incubation period after irradiation. At the indicated time-points whole cell lysates were made for Western blot analysis.

Mentions: Western blot analysis revealed that PDT-treatment of different cancer and immortalized cell lines evoked a progressive conversion of the cytosolic LC3-I into its lipidated form LC3-II, a specific biochemical marker of MA. This is as also visualized by the redistribution of GFP-tagged LC3 from a diffuse (cytosolic LC3-I) into a dotted pattern (LC3-II accumulating in autophagosomal membranes) (Fig. 1A and B). In line with these results, ultrastructural analysis of PDT-treated cells via TEM showed a clear vacuolization of the cytoplasm (Fig. 1C). To clarify whether accumulation of LC3-II in our paradigm reflects stimulation of the ON-rate (stimulation of autophagic degradation) of MA or results from a ROS-mediated inhibition of lysosomal function, we performed a flux-analysis using MEFs stably expressing GFP-LC3 [30]. Addition of BafA1, an inhibitor of autophagosome degradation, increased the detection of LC3-II after treatment and inhibited the release of ‘free GFP’, a hallmark of autophagosome degradation (Fig. 1D). This indicates that PDT stimulates MA flux.


Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.

Dewaele M, Martinet W, Rubio N, Verfaillie T, de Witte PA, Piette J, Agostinis P - J. Cell. Mol. Med. (2010)

Hypericin-PDT treatment evokes features of MA. All controls represent incubation with hypericin without irradiation. (A) Time-dependent detection of LC3-conversion by Western blot of whole cell lysates of human cervical carcinoma HeLa cells, immortalized MEFs and AY27 rat bladder carcinoma after PDT-treatment. (B) Confocal microscopy analysis of HeLa cells (a, b) transiently overexpressing GFP-LC3 and MEFs (c, d) stably expressing GFP-LC3. Images representative for 80% of the population of control (a, c) and cells treated 6 hrs after PDT (b, d) are shown. White scale bar in the upper right corner represents 10 μm. (C) Transmission electron microscopic analysis of control HeLa cells (a, b) and HeLa cells 6 hrs after PDT-treatment (c, d). EM photomicrographs representing 70% of the population are shown. (b) and (d) are magnifications of the peri-nuclear area indicated in (a) and (c). Arrows indicate vacuoles with detectable content in the treated cells (d). Black scale bar in the lower right corner represents 2 μm. (D) Flux analysis with MEF stably expressing GFP-LC3. HeLa cells were pre-incubated for 2 hrs with 100 nM BafA1 or vehicle (controls) before irradiation, BafA1 was present for the entire incubation period after irradiation. At the indicated time-points whole cell lysates were made for Western blot analysis.
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Related In: Results  -  Collection

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fig01: Hypericin-PDT treatment evokes features of MA. All controls represent incubation with hypericin without irradiation. (A) Time-dependent detection of LC3-conversion by Western blot of whole cell lysates of human cervical carcinoma HeLa cells, immortalized MEFs and AY27 rat bladder carcinoma after PDT-treatment. (B) Confocal microscopy analysis of HeLa cells (a, b) transiently overexpressing GFP-LC3 and MEFs (c, d) stably expressing GFP-LC3. Images representative for 80% of the population of control (a, c) and cells treated 6 hrs after PDT (b, d) are shown. White scale bar in the upper right corner represents 10 μm. (C) Transmission electron microscopic analysis of control HeLa cells (a, b) and HeLa cells 6 hrs after PDT-treatment (c, d). EM photomicrographs representing 70% of the population are shown. (b) and (d) are magnifications of the peri-nuclear area indicated in (a) and (c). Arrows indicate vacuoles with detectable content in the treated cells (d). Black scale bar in the lower right corner represents 2 μm. (D) Flux analysis with MEF stably expressing GFP-LC3. HeLa cells were pre-incubated for 2 hrs with 100 nM BafA1 or vehicle (controls) before irradiation, BafA1 was present for the entire incubation period after irradiation. At the indicated time-points whole cell lysates were made for Western blot analysis.
Mentions: Western blot analysis revealed that PDT-treatment of different cancer and immortalized cell lines evoked a progressive conversion of the cytosolic LC3-I into its lipidated form LC3-II, a specific biochemical marker of MA. This is as also visualized by the redistribution of GFP-tagged LC3 from a diffuse (cytosolic LC3-I) into a dotted pattern (LC3-II accumulating in autophagosomal membranes) (Fig. 1A and B). In line with these results, ultrastructural analysis of PDT-treated cells via TEM showed a clear vacuolization of the cytoplasm (Fig. 1C). To clarify whether accumulation of LC3-II in our paradigm reflects stimulation of the ON-rate (stimulation of autophagic degradation) of MA or results from a ROS-mediated inhibition of lysosomal function, we performed a flux-analysis using MEFs stably expressing GFP-LC3 [30]. Addition of BafA1, an inhibitor of autophagosome degradation, increased the detection of LC3-II after treatment and inhibited the release of ‘free GFP’, a hallmark of autophagosome degradation (Fig. 1D). This indicates that PDT stimulates MA flux.

Bottom Line: Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling.We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg(-/-) cells compensated for MA loss and increased cellular resistance to PDT.These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.

View Article: PubMed Central - PubMed

Affiliation: Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.

Show MeSH
Related in: MedlinePlus