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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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ABT-263 reduces cell viability and potentiates CPT-induced apoptosis in hESCs, hiPSCs and NP.(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were treated with increasing concentrations of ABT-263 (0.1–1 μM) during 15h (top panel) or pre-treated with ABT-263 (0.1μM) for 1hour and then treated with CPT (1μM during 3h) (middle panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-263 (0.1μM) addition or 6 h after ABT-263 (0.1μM) and/or genotoxic removal. 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. After 15 h of ABT-263 (0.1μM) addition or a withdrawal period of 3 h ABT-263 (0.1μM) and/or 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. A paired Student's t test was used to compare ABT-263-treated samples with untreated ones (top panel) or CPT plus ABT-263 treated samples to CPT treated ones (middle and bottom panel)*P< 0.05, **P< 0.001. (b) Representative histograms of PI-stained ABT-263 (0.1μM) treated cells over a 20 h period or untreated unfixed cells. Percentage of PI positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis (c) Caspase-9, caspase-3 activation and PARP cleavage in hESCs, hiPSCs and NP upon CPT (1μM for 3h), ABT-263 (0.1μM for 15 h) or ABT-263 (0.1μM) for 1 h prior and during CPT treatment were analyzed by Western blotting 3 h post genotoxic removal. GAPDH was used as loading control.
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pone.0152607.g005: ABT-263 reduces cell viability and potentiates CPT-induced apoptosis in hESCs, hiPSCs and NP.(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were treated with increasing concentrations of ABT-263 (0.1–1 μM) during 15h (top panel) or pre-treated with ABT-263 (0.1μM) for 1hour and then treated with CPT (1μM during 3h) (middle panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-263 (0.1μM) addition or 6 h after ABT-263 (0.1μM) and/or genotoxic removal. 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. After 15 h of ABT-263 (0.1μM) addition or a withdrawal period of 3 h ABT-263 (0.1μM) and/or 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. A paired Student's t test was used to compare ABT-263-treated samples with untreated ones (top panel) or CPT plus ABT-263 treated samples to CPT treated ones (middle and bottom panel)*P< 0.05, **P< 0.001. (b) Representative histograms of PI-stained ABT-263 (0.1μM) treated cells over a 20 h period or untreated unfixed cells. Percentage of PI positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis (c) Caspase-9, caspase-3 activation and PARP cleavage in hESCs, hiPSCs and NP upon CPT (1μM for 3h), ABT-263 (0.1μM for 15 h) or ABT-263 (0.1μM) for 1 h prior and during CPT treatment were analyzed by Western blotting 3 h post genotoxic removal. GAPDH was used as loading control.

Mentions: To evaluate whether ABT-263 triggers similar effects in hESCs, hiPSCs, NP and in the starting cell population (HF) used to generate hiPSCs, we exposed these cell types to increasing concentrations of this drug (0.1μM to 1 μM) for 15h. As shown in Fig 5a, ABT-263 treatment caused a concentration-dependent death in pluripotent cells. ABT-263 (0.1 μM) exposure resulted in a mild, but statistically significant, drop in viability for both hESCs (13%) and hiPSCs (16.5%) (Fig 5a, top panel). In contrast, ABT-263 (0.1μM) elicited a marked decrease in NP viability (36%). Our results indicate that NP display a higher sensitivity to ABT-263 than pluripotent cells while HF are almost totally insensitive to this molecule.


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)

ABT-263 reduces cell viability and potentiates CPT-induced apoptosis in hESCs, hiPSCs and NP.(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were treated with increasing concentrations of ABT-263 (0.1–1 μM) during 15h (top panel) or pre-treated with ABT-263 (0.1μM) for 1hour and then treated with CPT (1μM during 3h) (middle panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-263 (0.1μM) addition or 6 h after ABT-263 (0.1μM) and/or genotoxic removal. 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. After 15 h of ABT-263 (0.1μM) addition or a withdrawal period of 3 h ABT-263 (0.1μM) and/or 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. A paired Student's t test was used to compare ABT-263-treated samples with untreated ones (top panel) or CPT plus ABT-263 treated samples to CPT treated ones (middle and bottom panel)*P< 0.05, **P< 0.001. (b) Representative histograms of PI-stained ABT-263 (0.1μM) treated cells over a 20 h period or untreated unfixed cells. Percentage of PI positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis (c) Caspase-9, caspase-3 activation and PARP cleavage in hESCs, hiPSCs and NP upon CPT (1μM for 3h), ABT-263 (0.1μM for 15 h) or ABT-263 (0.1μM) for 1 h prior and during CPT treatment were analyzed by Western blotting 3 h post genotoxic removal. GAPDH was used as loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4816327&req=5

pone.0152607.g005: ABT-263 reduces cell viability and potentiates CPT-induced apoptosis in hESCs, hiPSCs and NP.(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were treated with increasing concentrations of ABT-263 (0.1–1 μM) during 15h (top panel) or pre-treated with ABT-263 (0.1μM) for 1hour and then treated with CPT (1μM during 3h) (middle panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-263 (0.1μM) addition or 6 h after ABT-263 (0.1μM) and/or genotoxic removal. 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. After 15 h of ABT-263 (0.1μM) addition or a withdrawal period of 3 h ABT-263 (0.1μM) and/or 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. A paired Student's t test was used to compare ABT-263-treated samples with untreated ones (top panel) or CPT plus ABT-263 treated samples to CPT treated ones (middle and bottom panel)*P< 0.05, **P< 0.001. (b) Representative histograms of PI-stained ABT-263 (0.1μM) treated cells over a 20 h period or untreated unfixed cells. Percentage of PI positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis (c) Caspase-9, caspase-3 activation and PARP cleavage in hESCs, hiPSCs and NP upon CPT (1μM for 3h), ABT-263 (0.1μM for 15 h) or ABT-263 (0.1μM) for 1 h prior and during CPT treatment were analyzed by Western blotting 3 h post genotoxic removal. GAPDH was used as loading control.
Mentions: To evaluate whether ABT-263 triggers similar effects in hESCs, hiPSCs, NP and in the starting cell population (HF) used to generate hiPSCs, we exposed these cell types to increasing concentrations of this drug (0.1μM to 1 μM) for 15h. As shown in Fig 5a, ABT-263 treatment caused a concentration-dependent death in pluripotent cells. ABT-263 (0.1 μM) exposure resulted in a mild, but statistically significant, drop in viability for both hESCs (13%) and hiPSCs (16.5%) (Fig 5a, top panel). In contrast, ABT-263 (0.1μM) elicited a marked decrease in NP viability (36%). Our results indicate that NP display a higher sensitivity to ABT-263 than pluripotent cells while HF are almost totally insensitive to this molecule.

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