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.
Highlights
Cells activate survival and/or death signaling pathways under stress conditions
HESC and human induced pluripotent stem cells (hiPSCs) lines were maintained on an inactivated mouse embryonic fibroblast feeder layer in medium comprised of Dulbecco's Modified Eagle's Medium/Ham's F12 (DMEM/F12) supplemented with 20% knockout serum replacement (KSR), 2 mM non-essential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, 50 μg/ml streptomycin, 0.1 mM βmercaptoethanol and 4 ng/ml of basic fibroblast growth factor
To gain insight into the responses achieved by pluripotent cells upon DNA damage, we sought to compare how two different types of human pluripotent stem cells respond to the double-strand breaks (DSBs) inducing agent CPT, a well known inhibitor of DNA-topoisomerase I complexes
Summary
Programmed cell death or apoptosis signaling frequently converges on mitochondria, a process that is controlled by the activities of pro- and anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members [1,2,3]. Bcl-2 family members can be divided into three main subclasses that are partly defined by the homology shared within four conserved regions. These regions, termed Bcl-2 homology (BH) 1–4 domains, correspond to α-helices with similar sequences that determine protein structure and function. A larger group of pro-apoptotic proteins (e.g. Bid, Bad, Bik, Bim, Bmf, Puma, Noxa and Hrk/DP5) contain only the BH3 domain. The BH3-only proteins are further subdivided into two groups based on function: the “activators” (e.g. Bid, Bim and Puma) and the “sensitizers” (e.g. Bad, Bik and Noxa) [5, 6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
