Abstract
BackgroundThe essentially unlimited expansion potential and the pluripotency of human embryonic stem cells (hESCs) make them attractive for cell-based therapeutic purposes. Although hESCs can indefinitely proliferate in culture, unlike transformed cancer cells, they are endowed with a cell-intrinsic property termed mitochondrial priming that renders them highly sensitive to apoptotic stimuli. Thus, all attempts to broaden the insights into hESCs apoptosis may be helpful for establishing pro-survival strategies valuable for its in vitro culture and further use in clinical applications. Cyclin-dependent kinases (CDKs), a family of serine/threonine protein kinases originally identified as regulators of the eukaryotic cell cycle, can also regulate transcription and differentiation. Moreover, there are compelling data suggesting that its activities are involved in certain apoptotic programs in different cell types. Currently, it is not completely determined whether CDKs regulate apoptotic processes in rapidly proliferating and apoptosis-prone hESCs. In this study, to elucidate the effect of CDKs inhibition in hESCs we used Roscovitine (ROSC), a purine analogue that selectively inhibits the activities of these kinases.ResultsInhibition of CDKs by ROSC triggers programmed cell death in hESCs but not in proliferating somatic cells (human fibroblasts). The apoptotic process encompasses caspase-9 and -3 activation followed by PARP cleavage. ROSC treatment also leads to p53 stabilization, which coincides with site-specific phosphorylation at serine 46 and decreased levels of Mdm2. Additionally, we observed a transcriptional induction of p53AIP1, a repression of pro-survival factor Mcl-1 and an up-regulation of pro-apoptotic BH3-only proteins NOXA and PUMA. Importantly, we found that the role of CDK2 inhibition appears to be at best accessory as an active CDK2 is not required to ensure hESCs survival.ConclusionOur experimental data reveal that hESCs, contrary to fibroblasts, exhibit a pronounced sensitivity to ROSC.
Highlights
The essentially unlimited expansion potential and the pluripotency of human embryonic stem cells make them attractive for cell-based therapeutic purposes
Onset of cell cycle arrest in human embryonic stem cells (hESCs) exposed to ROSC As previously mentioned, ROSC has the potential to perturb cell cycle progression in G1, S or G2/M depending on the dose and time of exposure in various cell types, ranging from numerous cancer cell lines to keratinocytes and fibroblasts [24, 25]
The mean ± SEM from three independent experiments are shown. c Comparison of mRNA expression levels of cdk1, cdk2, cdk4 and cdk6 in hESCs and Human fibroblasts (HF) analyzed by Real Time RT-PCR
Summary
The essentially unlimited expansion potential and the pluripotency of human embryonic stem cells (hESCs) make them attractive for cell-based therapeutic purposes. Cyclin-dependent kinases (CDKs), a family of serine/threonine protein kinases originally identified as regulators of the eukaryotic cell cycle, can regulate transcription and differentiation. There are compelling data suggesting that its activities are involved in certain apoptotic programs in different cell types. It is not completely determined whether CDKs regulate apoptotic processes in rapidly proliferating and apoptosisprone hESCs. In this study, to elucidate the effect of CDKs inhibition in hESCs we used Roscovitine (ROSC), a purine analogue that selectively inhibits the activities of these kinases. In comparison to Cyclin-dependent kinases (CDKs), a family of serine/ threonine protein kinases originally identified as regulators of the eukaryotic cell cycle, can regulate transcription and, in certain cell types, differentiation [5, 6]. It has been shown that hESCs display high levels of CDK activity in part due to the absence or very weak expression of CKIs [9, 10]
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