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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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CMA is involved in the survival response to oxidative stress after PDT. All controls represent incubation with hypericin without irradiation unless stated otherwise. (A) Whole cell lysates of 3T3 and 3T3 LAMP2A KO fibroblasts were harvested at the indicated time-points and analysed for LAMP2A, caspase 3 activation, PARP cleavage and LC3-conversion. Actin was used as a loading control. (B) Densitometric quantification of LC3 in the corresponding Western blot analysis in (A). Arbitrary densitometric units represent LC3-II levels relative to actin levels. (C) Effect of CMA deficiency on PDT-induced cell death. Percentage of trypan blue positive cells (dead cells) as function of time after PDT in LAMP2A WT and KO 3T3 fibroblasts. The graph represents the mean ± S.D. of two independent experiments performed in duplicate. *P < 0.05 under the Student’s t-test. (D) Representative dose–response curve for PDT treatment with different concentrations of hypericin (as indicated) in 3T3 and 3T3 LAMP2A KO fibroblasts as evaluated by MTT assay. The curve shows the surviving fraction of cells 16 hrs after treatment. Cell survival is expressed as the fraction surviving cells with respect to the controls. *P < 0.05 under the Student’s t-test for a 6-fold replicate. (E) Total cell lysates were analysed for the presence of carbonylated protein side chains after PDT in WT and LAMP2A KO 3T3 fibroblasts as described in Figure 3. During OxyBlot procedure, hypericin was omitted from the controls to minimize effects of background irradiation during manipulation of the samples.
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fig07: CMA is involved in the survival response to oxidative stress after PDT. All controls represent incubation with hypericin without irradiation unless stated otherwise. (A) Whole cell lysates of 3T3 and 3T3 LAMP2A KO fibroblasts were harvested at the indicated time-points and analysed for LAMP2A, caspase 3 activation, PARP cleavage and LC3-conversion. Actin was used as a loading control. (B) Densitometric quantification of LC3 in the corresponding Western blot analysis in (A). Arbitrary densitometric units represent LC3-II levels relative to actin levels. (C) Effect of CMA deficiency on PDT-induced cell death. Percentage of trypan blue positive cells (dead cells) as function of time after PDT in LAMP2A WT and KO 3T3 fibroblasts. The graph represents the mean ± S.D. of two independent experiments performed in duplicate. *P < 0.05 under the Student’s t-test. (D) Representative dose–response curve for PDT treatment with different concentrations of hypericin (as indicated) in 3T3 and 3T3 LAMP2A KO fibroblasts as evaluated by MTT assay. The curve shows the surviving fraction of cells 16 hrs after treatment. Cell survival is expressed as the fraction surviving cells with respect to the controls. *P < 0.05 under the Student’s t-test for a 6-fold replicate. (E) Total cell lysates were analysed for the presence of carbonylated protein side chains after PDT in WT and LAMP2A KO 3T3 fibroblasts as described in Figure 3. During OxyBlot procedure, hypericin was omitted from the controls to minimize effects of background irradiation during manipulation of the samples.

Mentions: We then hypothesized that if up-regulation of CMA in Atg5-deficient cells was responsible for their increased resistance towards PDT and efficient clearance of oxidized proteins (Fig. 5A), LAMP2A-deficient CMA-incompetent cells [41] would be highly sensitized to PDT. Consistent with this, LAMP2A deficiency accelerated the pattern of caspase 3 activation and PARP cleavage, correlating with a persistent pattern of protein carbonylation (Fig. 7A, C and E) and resulting in a significant sensitization of these cells to PDT-induced cell death within a wide range of PDT doses (Fig. 7D). Because LAMP2A-deficient cells were extremely vulnerable to PDT, we used a milder dose (i.e. reduced hypericin concentration and light dose) to evaluate apoptotic parameters, which is reflected by a reduced pattern of protein carbonylation, caspase 3 processing and cell death in these fibroblasts as compared to MEFs (Fig. 7A, C–E).


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)

CMA is involved in the survival response to oxidative stress after PDT. All controls represent incubation with hypericin without irradiation unless stated otherwise. (A) Whole cell lysates of 3T3 and 3T3 LAMP2A KO fibroblasts were harvested at the indicated time-points and analysed for LAMP2A, caspase 3 activation, PARP cleavage and LC3-conversion. Actin was used as a loading control. (B) Densitometric quantification of LC3 in the corresponding Western blot analysis in (A). Arbitrary densitometric units represent LC3-II levels relative to actin levels. (C) Effect of CMA deficiency on PDT-induced cell death. Percentage of trypan blue positive cells (dead cells) as function of time after PDT in LAMP2A WT and KO 3T3 fibroblasts. The graph represents the mean ± S.D. of two independent experiments performed in duplicate. *P < 0.05 under the Student’s t-test. (D) Representative dose–response curve for PDT treatment with different concentrations of hypericin (as indicated) in 3T3 and 3T3 LAMP2A KO fibroblasts as evaluated by MTT assay. The curve shows the surviving fraction of cells 16 hrs after treatment. Cell survival is expressed as the fraction surviving cells with respect to the controls. *P < 0.05 under the Student’s t-test for a 6-fold replicate. (E) Total cell lysates were analysed for the presence of carbonylated protein side chains after PDT in WT and LAMP2A KO 3T3 fibroblasts as described in Figure 3. During OxyBlot procedure, hypericin was omitted from the controls to minimize effects of background irradiation during manipulation of the samples.
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fig07: CMA is involved in the survival response to oxidative stress after PDT. All controls represent incubation with hypericin without irradiation unless stated otherwise. (A) Whole cell lysates of 3T3 and 3T3 LAMP2A KO fibroblasts were harvested at the indicated time-points and analysed for LAMP2A, caspase 3 activation, PARP cleavage and LC3-conversion. Actin was used as a loading control. (B) Densitometric quantification of LC3 in the corresponding Western blot analysis in (A). Arbitrary densitometric units represent LC3-II levels relative to actin levels. (C) Effect of CMA deficiency on PDT-induced cell death. Percentage of trypan blue positive cells (dead cells) as function of time after PDT in LAMP2A WT and KO 3T3 fibroblasts. The graph represents the mean ± S.D. of two independent experiments performed in duplicate. *P < 0.05 under the Student’s t-test. (D) Representative dose–response curve for PDT treatment with different concentrations of hypericin (as indicated) in 3T3 and 3T3 LAMP2A KO fibroblasts as evaluated by MTT assay. The curve shows the surviving fraction of cells 16 hrs after treatment. Cell survival is expressed as the fraction surviving cells with respect to the controls. *P < 0.05 under the Student’s t-test for a 6-fold replicate. (E) Total cell lysates were analysed for the presence of carbonylated protein side chains after PDT in WT and LAMP2A KO 3T3 fibroblasts as described in Figure 3. During OxyBlot procedure, hypericin was omitted from the controls to minimize effects of background irradiation during manipulation of the samples.
Mentions: We then hypothesized that if up-regulation of CMA in Atg5-deficient cells was responsible for their increased resistance towards PDT and efficient clearance of oxidized proteins (Fig. 5A), LAMP2A-deficient CMA-incompetent cells [41] would be highly sensitized to PDT. Consistent with this, LAMP2A deficiency accelerated the pattern of caspase 3 activation and PARP cleavage, correlating with a persistent pattern of protein carbonylation (Fig. 7A, C and E) and resulting in a significant sensitization of these cells to PDT-induced cell death within a wide range of PDT doses (Fig. 7D). Because LAMP2A-deficient cells were extremely vulnerable to PDT, we used a milder dose (i.e. reduced hypericin concentration and light dose) to evaluate apoptotic parameters, which is reflected by a reduced pattern of protein carbonylation, caspase 3 processing and cell death in these fibroblasts as compared to MEFs (Fig. 7A, C–E).

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