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Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

García CP, Videla Richardson GA, Dimopoulos NA, Fernandez Espinosa DD, Miriuka SG, Sevlever GE, Romorini L, Scassa ME - PLoS ONE (2016)

Bottom Line: However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types.We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs.Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratorios de Investigación Aplicada a Neurociencias, LIAN-CONICET, Fundación FLENI, Ruta 9, Km 53, (B1625XAF) Escobar, Buenos Aires, Argentina.

ABSTRACT
Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.

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CPT activates DNA damage response and triggers apoptosis in hiPSCs and hESCs-derived NP.(a) Immunofluorescence photomicrographs of genotoxic-treated (1μM during 3 h) hiPSCs and NP performed immediately after CPT treatment (1μM during 3 h). The figure shows representative images of cells stained with primary antibodies against ATM phospho-serine1981 (pATM), histoneγH2AX, p53, p53 phospho serine 15 (p53pSer15). Nuclei were counterstained with DAPI. The scale bars represent 100 μm. (b) Cell viability was measured in hESCs, hiPSCs, NP and HF treated or not with 1 μM CPT during 3h by the XTT/PMS assay 6 h after drug removal (top panel). Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. A paired Student’s t test was used to compare CPT treated samples to untreated controls. After a withdrawal period of 3 h CPT-treated cells were harvested, and DNA oligomers were quantified by immunoassay (bottom panel). Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in triplicate. *P< 0.05, **P< 0.001. (c) Time course of caspase-9, caspase-3 and PARP cleavages in hiPSCs and NP upon CPT treatment were analyzed by Western blotting with anti-caspase-9, anti-cleaved-caspase-3 and anti-PARP specific antibodies, immediately after (3 h), 6 h or 15 h post drug removal (3+6, 3+15, respectively). GAPDH was used as loading control.
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pone.0152607.g002: CPT activates DNA damage response and triggers apoptosis in hiPSCs and hESCs-derived NP.(a) Immunofluorescence photomicrographs of genotoxic-treated (1μM during 3 h) hiPSCs and NP performed immediately after CPT treatment (1μM during 3 h). The figure shows representative images of cells stained with primary antibodies against ATM phospho-serine1981 (pATM), histoneγH2AX, p53, p53 phospho serine 15 (p53pSer15). Nuclei were counterstained with DAPI. The scale bars represent 100 μm. (b) Cell viability was measured in hESCs, hiPSCs, NP and HF treated or not with 1 μM CPT during 3h by the XTT/PMS assay 6 h after drug removal (top panel). Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. A paired Student’s t test was used to compare CPT treated samples to untreated controls. After a withdrawal period of 3 h CPT-treated cells were harvested, and DNA oligomers were quantified by immunoassay (bottom panel). Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in triplicate. *P< 0.05, **P< 0.001. (c) Time course of caspase-9, caspase-3 and PARP cleavages in hiPSCs and NP upon CPT treatment were analyzed by Western blotting with anti-caspase-9, anti-cleaved-caspase-3 and anti-PARP specific antibodies, immediately after (3 h), 6 h or 15 h post drug removal (3+6, 3+15, respectively). GAPDH was used as loading control.

Mentions: Similarly to what occurred in hESCs, we found that CPT treatment (1μM for 3 h) led to concurrent phosphorylation of ATM (serine1981) and H2AX (serine139, γH2AX) in FN2.1 hiPSCs and NP. Nuclear accumulation and phosphorylation of p53 on serine 15 was also observed within 3 h in damaged cells (Fig 2a).


Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

García CP, Videla Richardson GA, Dimopoulos NA, Fernandez Espinosa DD, Miriuka SG, Sevlever GE, Romorini L, Scassa ME - PLoS ONE (2016)

CPT activates DNA damage response and triggers apoptosis in hiPSCs and hESCs-derived NP.(a) Immunofluorescence photomicrographs of genotoxic-treated (1μM during 3 h) hiPSCs and NP performed immediately after CPT treatment (1μM during 3 h). The figure shows representative images of cells stained with primary antibodies against ATM phospho-serine1981 (pATM), histoneγH2AX, p53, p53 phospho serine 15 (p53pSer15). Nuclei were counterstained with DAPI. The scale bars represent 100 μm. (b) Cell viability was measured in hESCs, hiPSCs, NP and HF treated or not with 1 μM CPT during 3h by the XTT/PMS assay 6 h after drug removal (top panel). Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. A paired Student’s t test was used to compare CPT treated samples to untreated controls. After a withdrawal period of 3 h CPT-treated cells were harvested, and DNA oligomers were quantified by immunoassay (bottom panel). Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in triplicate. *P< 0.05, **P< 0.001. (c) Time course of caspase-9, caspase-3 and PARP cleavages in hiPSCs and NP upon CPT treatment were analyzed by Western blotting with anti-caspase-9, anti-cleaved-caspase-3 and anti-PARP specific antibodies, immediately after (3 h), 6 h or 15 h post drug removal (3+6, 3+15, respectively). GAPDH was used as loading control.
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pone.0152607.g002: CPT activates DNA damage response and triggers apoptosis in hiPSCs and hESCs-derived NP.(a) Immunofluorescence photomicrographs of genotoxic-treated (1μM during 3 h) hiPSCs and NP performed immediately after CPT treatment (1μM during 3 h). The figure shows representative images of cells stained with primary antibodies against ATM phospho-serine1981 (pATM), histoneγH2AX, p53, p53 phospho serine 15 (p53pSer15). Nuclei were counterstained with DAPI. The scale bars represent 100 μm. (b) Cell viability was measured in hESCs, hiPSCs, NP and HF treated or not with 1 μM CPT during 3h by the XTT/PMS assay 6 h after drug removal (top panel). Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. A paired Student’s t test was used to compare CPT treated samples to untreated controls. After a withdrawal period of 3 h CPT-treated cells were harvested, and DNA oligomers were quantified by immunoassay (bottom panel). Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in triplicate. *P< 0.05, **P< 0.001. (c) Time course of caspase-9, caspase-3 and PARP cleavages in hiPSCs and NP upon CPT treatment were analyzed by Western blotting with anti-caspase-9, anti-cleaved-caspase-3 and anti-PARP specific antibodies, immediately after (3 h), 6 h or 15 h post drug removal (3+6, 3+15, respectively). GAPDH was used as loading control.
Mentions: Similarly to what occurred in hESCs, we found that CPT treatment (1μM for 3 h) led to concurrent phosphorylation of ATM (serine1981) and H2AX (serine139, γH2AX) in FN2.1 hiPSCs and NP. Nuclear accumulation and phosphorylation of p53 on serine 15 was also observed within 3 h in damaged cells (Fig 2a).

Bottom Line: However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types.We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs.Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratorios de Investigación Aplicada a Neurociencias, LIAN-CONICET, Fundación FLENI, Ruta 9, Km 53, (B1625XAF) Escobar, Buenos Aires, Argentina.

ABSTRACT
Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.

Show MeSH
Related in: MedlinePlus