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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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ATG5 is necessary and sufficient for anticancer drug-induced mitotic catastrophe.(a) Immunoblotting. Jurkat T cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained. Monomeric ATG5 (shown under the dotted line) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12. Results are representative of three independent experiments. (b) Morphological analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments. (c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3). (d) Immunoblotting. Enforced expression of ATG5, Beclin 1, or ATG5-K130R following lentiviral gene transfer. (e) Time-lapse microscopy. HeLa H2B–mCherry–α-tubulin–EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.
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f3: ATG5 is necessary and sufficient for anticancer drug-induced mitotic catastrophe.(a) Immunoblotting. Jurkat T cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained. Monomeric ATG5 (shown under the dotted line) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12. Results are representative of three independent experiments. (b) Morphological analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments. (c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3). (d) Immunoblotting. Enforced expression of ATG5, Beclin 1, or ATG5-K130R following lentiviral gene transfer. (e) Time-lapse microscopy. HeLa H2B–mCherry–α-tubulin–EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.

Mentions: The elevation of ATG5 levels after etoposide and cisplatin treatment could be inhibited using two different short hairpin RNAs (shRNAs; Fig. 3a). Blocking ATG5 expression prevented both the induction of autophagy, as assessed by LC3 immunoblotting (Fig. 3a), and the appearance of cells with abnormal nuclei (Fig. 3b). A control shRNA had no effect in these assays (Fig. 3a,b). These data suggested that increased levels of ATG5 are necessary for mitotic catastrophe following exposure to DNA-damaging drugs.


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)

ATG5 is necessary and sufficient for anticancer drug-induced mitotic catastrophe.(a) Immunoblotting. Jurkat T cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained. Monomeric ATG5 (shown under the dotted line) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12. Results are representative of three independent experiments. (b) Morphological analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments. (c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3). (d) Immunoblotting. Enforced expression of ATG5, Beclin 1, or ATG5-K130R following lentiviral gene transfer. (e) Time-lapse microscopy. HeLa H2B–mCherry–α-tubulin–EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.
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f3: ATG5 is necessary and sufficient for anticancer drug-induced mitotic catastrophe.(a) Immunoblotting. Jurkat T cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained. Monomeric ATG5 (shown under the dotted line) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12. Results are representative of three independent experiments. (b) Morphological analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments. (c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3). (d) Immunoblotting. Enforced expression of ATG5, Beclin 1, or ATG5-K130R following lentiviral gene transfer. (e) Time-lapse microscopy. HeLa H2B–mCherry–α-tubulin–EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.
Mentions: The elevation of ATG5 levels after etoposide and cisplatin treatment could be inhibited using two different short hairpin RNAs (shRNAs; Fig. 3a). Blocking ATG5 expression prevented both the induction of autophagy, as assessed by LC3 immunoblotting (Fig. 3a), and the appearance of cells with abnormal nuclei (Fig. 3b). A control shRNA had no effect in these assays (Fig. 3a,b). These data suggested that increased levels of ATG5 are necessary for mitotic catastrophe following exposure to DNA-damaging drugs.

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