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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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Effects of Bcl-2 selective inhibitor, ABT-199 on the viability of hESCs, hiPSCs NP and HF.(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were left untreated or treated with increasing concentrations of ABT-199 (0.1–10 μM) during 15 h (top panel) or treated with CPT(1μM) for 3h or pre-treated with ABT-199 (1μM) for 1 h and then treated with CPT (1μM) during 3h (bottom panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-199 addition or 6 h after CPT 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. A paired Student's t test was used to compare ABT-199-treated samples with untreated ones (top panel) or CPT plus ABT-199 treated samples to CPT treated ones (bottom panel) *P< 0.05 (b) Representative histograms of PI- stained ABT-199 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) NP were transfected with nt-siRNA or Bcl-2 siRNA and exposed to CPT (1μMduring 3h) 48 h post-transfection. Representative pictures showing nt-siRNA or Bcl-2 siRNA transfected NP treated or not with 1μM CPT for 3 h (48 h post-transfection). Images were captured 12 h after genotoxic removal. The scale bars represent 100 μm. Representative histograms of PI-stained transfected cells 12 h after genotoxic removal (left panel). mRNA expression levels of bcl-2 in nt-siRNA and Bcl-2siRNA transfected NP were analyzed by Real Time RT-PCR (right panel). GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to nt-siRNA transfectants arbitrarily set as 1. **P < 0.001.
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pone.0152607.g007: Effects of Bcl-2 selective inhibitor, ABT-199 on the viability of hESCs, hiPSCs NP and HF.(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were left untreated or treated with increasing concentrations of ABT-199 (0.1–10 μM) during 15 h (top panel) or treated with CPT(1μM) for 3h or pre-treated with ABT-199 (1μM) for 1 h and then treated with CPT (1μM) during 3h (bottom panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-199 addition or 6 h after CPT 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. A paired Student's t test was used to compare ABT-199-treated samples with untreated ones (top panel) or CPT plus ABT-199 treated samples to CPT treated ones (bottom panel) *P< 0.05 (b) Representative histograms of PI- stained ABT-199 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) NP were transfected with nt-siRNA or Bcl-2 siRNA and exposed to CPT (1μMduring 3h) 48 h post-transfection. Representative pictures showing nt-siRNA or Bcl-2 siRNA transfected NP treated or not with 1μM CPT for 3 h (48 h post-transfection). Images were captured 12 h after genotoxic removal. The scale bars represent 100 μm. Representative histograms of PI-stained transfected cells 12 h after genotoxic removal (left panel). mRNA expression levels of bcl-2 in nt-siRNA and Bcl-2siRNA transfected NP were analyzed by Real Time RT-PCR (right panel). GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to nt-siRNA transfectants arbitrarily set as 1. **P < 0.001.

Mentions: With the aim to dissect the relative contributions of Bcl-2 and Bcl-xL in pluripotent and progenitor cells fate decisions we used ABT-199, a potent and selective inhibitor of Bcl-2 [40]. To address this issue, we performed viability assays in the presence of increasing concentrations of ABT-199 (0.1μM to 10 μM) during 15 h. We found that inhibition of Bcl-2 neither affects pluripotent cells nor fibroblasts viability (Fig 7a). However, a noticeable loss of cell viability (approximately 40%) was observed in NP after treatment with 10 μM of ABT-199 (Fig 7a). This reduced sensitivity towards ABT-199 was further confirmed by flow cytometry using PI staining after 20h of ABT-199 (1μM) exposure (Fig 7b).


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)

Effects of Bcl-2 selective inhibitor, ABT-199 on the viability of hESCs, hiPSCs NP and HF.(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were left untreated or treated with increasing concentrations of ABT-199 (0.1–10 μM) during 15 h (top panel) or treated with CPT(1μM) for 3h or pre-treated with ABT-199 (1μM) for 1 h and then treated with CPT (1μM) during 3h (bottom panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-199 addition or 6 h after CPT 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. A paired Student's t test was used to compare ABT-199-treated samples with untreated ones (top panel) or CPT plus ABT-199 treated samples to CPT treated ones (bottom panel) *P< 0.05 (b) Representative histograms of PI- stained ABT-199 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) NP were transfected with nt-siRNA or Bcl-2 siRNA and exposed to CPT (1μMduring 3h) 48 h post-transfection. Representative pictures showing nt-siRNA or Bcl-2 siRNA transfected NP treated or not with 1μM CPT for 3 h (48 h post-transfection). Images were captured 12 h after genotoxic removal. The scale bars represent 100 μm. Representative histograms of PI-stained transfected cells 12 h after genotoxic removal (left panel). mRNA expression levels of bcl-2 in nt-siRNA and Bcl-2siRNA transfected NP were analyzed by Real Time RT-PCR (right panel). GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to nt-siRNA transfectants arbitrarily set as 1. **P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4816327&req=5

pone.0152607.g007: Effects of Bcl-2 selective inhibitor, ABT-199 on the viability of hESCs, hiPSCs NP and HF.(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were left untreated or treated with increasing concentrations of ABT-199 (0.1–10 μM) during 15 h (top panel) or treated with CPT(1μM) for 3h or pre-treated with ABT-199 (1μM) for 1 h and then treated with CPT (1μM) during 3h (bottom panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-199 addition or 6 h after CPT 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. A paired Student's t test was used to compare ABT-199-treated samples with untreated ones (top panel) or CPT plus ABT-199 treated samples to CPT treated ones (bottom panel) *P< 0.05 (b) Representative histograms of PI- stained ABT-199 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) NP were transfected with nt-siRNA or Bcl-2 siRNA and exposed to CPT (1μMduring 3h) 48 h post-transfection. Representative pictures showing nt-siRNA or Bcl-2 siRNA transfected NP treated or not with 1μM CPT for 3 h (48 h post-transfection). Images were captured 12 h after genotoxic removal. The scale bars represent 100 μm. Representative histograms of PI-stained transfected cells 12 h after genotoxic removal (left panel). mRNA expression levels of bcl-2 in nt-siRNA and Bcl-2siRNA transfected NP were analyzed by Real Time RT-PCR (right panel). GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to nt-siRNA transfectants arbitrarily set as 1. **P < 0.001.
Mentions: With the aim to dissect the relative contributions of Bcl-2 and Bcl-xL in pluripotent and progenitor cells fate decisions we used ABT-199, a potent and selective inhibitor of Bcl-2 [40]. To address this issue, we performed viability assays in the presence of increasing concentrations of ABT-199 (0.1μM to 10 μM) during 15 h. We found that inhibition of Bcl-2 neither affects pluripotent cells nor fibroblasts viability (Fig 7a). However, a noticeable loss of cell viability (approximately 40%) was observed in NP after treatment with 10 μM of ABT-199 (Fig 7a). This reduced sensitivity towards ABT-199 was further confirmed by flow cytometry using PI staining after 20h of ABT-199 (1μM) exposure (Fig 7b).

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