Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage

Filippi-Chiela, Eduardo Cremonese; Silva, Mardja Manssur Bueno e; Thomé, Marcos Paulo Machado; Lenz, Guido

doi:10.1080/15548627.2015.1009795

Cited by 81 publications

(52 citation statements)

References 73 publications

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“…We and others have previously shown that TMZ induces autophagy in several cell lines, including U-87 MG glioma cells (Filippi-Chiela et al, 2013;Natsumeda et al, 2011;Katayama et al, 2007) and induces cell enlargement (Filippi-Chiela et al, 2015). Autophagy induction was confirmed through a GFP-LC3 puncta formation assay (Fig.…”

Section: R/gfir Is Key For Measuring Avos By Flow Cytometrymentioning

confidence: 86%

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy

Thomé

Filippi-Chiela

Villodre

et al. 2016

Journal of Cell Science

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251

226

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Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orangestained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.

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Section: R/gfir Is Key For Measuring Avos By Flow Cytometrymentioning

confidence: 86%

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy

Thomé

Filippi-Chiela

Villodre

et al. 2016

Journal of Cell Science

Self Cite

251

226

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“…The relationship between autophagy and senescence is still debatable. While the induction of senescence has been reported to be, at least in part, dependent on autophagy [31, 48], other studies concluded that senescence is independent of autophagy [65–66]. Despite the close correspondence between autophagy and senescence in parental and Ligase IV−/− HCT116 cells, pharmacological and genetic inhibition of autophagy did not seem to affect the promotion of senescence even at high doses of radiation, indicating that promotion of senescence is independent of autophagy in this experimental model.…”

Section: Discussionmentioning

confidence: 99%

Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells

Alotaibi¹,

Sharma²,

Saleh³

et al. 2016

Radiation Research

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Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (−/−) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (−/−) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10–20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.

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“…Indeed, the autophagy inhibitor chloroquine has been shown to impair DNA damage repair and to increase the cytotoxic effect of the chemotherapeutic agent carboplatin in breast cancer stem cells (Liang et al, 2016). Accumulating evidence indicates that autophagy is exploited by tumor cells to resist radiation or chemotherapy-induced cell death and that genetic or pharmacological inhibition of autophagy sensitizes malignant cells to genotoxic therapy both in vitro and in experimental mouse models (Amaravadi et al, 2007; Pan et al, 2011; Chittaranjan et al, 2014; Wang and Wu, 2014; Filippi-Chiela et al, 2015; Liang et al, 2016; Piya et al, 2016). In line with this is the presence of autophagic vacuoles in Saos2 cells ( Figure 2 ), a p53 null human osteosarcoma cell line, which after prolonged expression of p21 WAF1/Cip1 exhibit enhanced aggressiveness and chemoresistance by deregulating the replication licensing machinery causing replication stress and fuelling genomic instability (Galanos et al, 2016).…”

Section: Functional Outcomes Of the Ddr – Autophagy Axismentioning

confidence: 99%

DNA Damage Response and Autophagy: A Meaningful Partnership

2016

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Autophagy and the DNA damage response (DDR) are biological processes essential for cellular and organismal homeostasis. Herein, we summarize and discuss emerging evidence linking DDR to autophagy. We highlight published data suggesting that autophagy is activated by DNA damage and is required for several functional outcomes of DDR signaling, including repair of DNA lesions, senescence, cell death, and cytokine secretion. Uncovering the mechanisms by which autophagy and DDR are intertwined provides novel insight into the pathobiology of conditions associated with accumulation of DNA damage, including cancer and aging, and novel concepts for the development of improved therapeutic strategies against these pathologies.

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Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage

Cited by 81 publications

References 73 publications

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy

Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells

DNA Damage Response and Autophagy: A Meaningful Partnership

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