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ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy.

Maskey D, Yousefi S, Schmid I, Zlobec I, Perren A, Friis R, Simon HU - Nat Commun (2013)

Bottom Line: Anticancer drug therapy activates both molecular cell death and autophagy pathways.Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death.Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland.

ABSTRACT
Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

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Ectopic ATG5 expression does not induce DNA damage.(a) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer. (b) Confocal microscopy. Untreated HeLa cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 were cultured for 48 h. Additional DAPI nuclear staining was used. Scale bar, 10 μm. Right: phospho-H2AX was quantified by measuring the mean fluorescence intensity (MFI) in five high-power fields of each condition using IMARIS software. Values are means±s.d. (c) Jurkat T cells were treated as in a and analysed by immunoblotting. Besides phospho-H2AX (Ser139), we also detected phospho-ATM (Ser1981) and phospho- ATR (Ser428), two additional markers of DNA damage response. Intensity of the protein bands were quantified with a Licor/Odyssey scanner and software (shown beneath each immunoblot). The fold change in phosphorylation levels compared with no treatment (untreated) are presented. The data in all panels are representative of at least three independent experiments. Full-length immunoblots are provided in Supplementary Fig. S9.
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f5: Ectopic ATG5 expression does not induce DNA damage.(a) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer. (b) Confocal microscopy. Untreated HeLa cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 were cultured for 48 h. Additional DAPI nuclear staining was used. Scale bar, 10 μm. Right: phospho-H2AX was quantified by measuring the mean fluorescence intensity (MFI) in five high-power fields of each condition using IMARIS software. Values are means±s.d. (c) Jurkat T cells were treated as in a and analysed by immunoblotting. Besides phospho-H2AX (Ser139), we also detected phospho-ATM (Ser1981) and phospho- ATR (Ser428), two additional markers of DNA damage response. Intensity of the protein bands were quantified with a Licor/Odyssey scanner and software (shown beneath each immunoblot). The fold change in phosphorylation levels compared with no treatment (untreated) are presented. The data in all panels are representative of at least three independent experiments. Full-length immunoblots are provided in Supplementary Fig. S9.

Mentions: We also analysed phosphorylated histone H2AX (phospho-H2AX) expression, an early DNA damage response marker16, by flow cytometry, confocal microscopy and immunoblotting. In contrast to untreated and ATG5- or GFP-overexpressing cells, this measure of DNA damage was only significantly elevated in cells treated with sublethal concentrations of etoposide (Fig. 5a–c). Moreover, we detected phospho-ataxia telangiectasia-mutated kinase (ATM) (Ser1981) and phospho-ataxia telangiectasia and Rad3-related kinase (ATR) (Ser428), two additional markers of the DNA damage response, and observed again no increased phosphorylation as a consequence of ATG5 gene transfer (Fig. 5c).


ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy.

Maskey D, Yousefi S, Schmid I, Zlobec I, Perren A, Friis R, Simon HU - Nat Commun (2013)

Ectopic ATG5 expression does not induce DNA damage.(a) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer. (b) Confocal microscopy. Untreated HeLa cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 were cultured for 48 h. Additional DAPI nuclear staining was used. Scale bar, 10 μm. Right: phospho-H2AX was quantified by measuring the mean fluorescence intensity (MFI) in five high-power fields of each condition using IMARIS software. Values are means±s.d. (c) Jurkat T cells were treated as in a and analysed by immunoblotting. Besides phospho-H2AX (Ser139), we also detected phospho-ATM (Ser1981) and phospho- ATR (Ser428), two additional markers of DNA damage response. Intensity of the protein bands were quantified with a Licor/Odyssey scanner and software (shown beneath each immunoblot). The fold change in phosphorylation levels compared with no treatment (untreated) are presented. The data in all panels are representative of at least three independent experiments. Full-length immunoblots are provided in Supplementary Fig. S9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: Ectopic ATG5 expression does not induce DNA damage.(a) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer. (b) Confocal microscopy. Untreated HeLa cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 were cultured for 48 h. Additional DAPI nuclear staining was used. Scale bar, 10 μm. Right: phospho-H2AX was quantified by measuring the mean fluorescence intensity (MFI) in five high-power fields of each condition using IMARIS software. Values are means±s.d. (c) Jurkat T cells were treated as in a and analysed by immunoblotting. Besides phospho-H2AX (Ser139), we also detected phospho-ATM (Ser1981) and phospho- ATR (Ser428), two additional markers of DNA damage response. Intensity of the protein bands were quantified with a Licor/Odyssey scanner and software (shown beneath each immunoblot). The fold change in phosphorylation levels compared with no treatment (untreated) are presented. The data in all panels are representative of at least three independent experiments. Full-length immunoblots are provided in Supplementary Fig. S9.
Mentions: We also analysed phosphorylated histone H2AX (phospho-H2AX) expression, an early DNA damage response marker16, by flow cytometry, confocal microscopy and immunoblotting. In contrast to untreated and ATG5- or GFP-overexpressing cells, this measure of DNA damage was only significantly elevated in cells treated with sublethal concentrations of etoposide (Fig. 5a–c). Moreover, we detected phospho-ataxia telangiectasia-mutated kinase (ATM) (Ser1981) and phospho-ataxia telangiectasia and Rad3-related kinase (ATR) (Ser428), two additional markers of the DNA damage response, and observed again no increased phosphorylation as a consequence of ATG5 gene transfer (Fig. 5c).

Bottom Line: Anticancer drug therapy activates both molecular cell death and autophagy pathways.Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death.Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland.

ABSTRACT
Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

Show MeSH
Related in: MedlinePlus