Tumor necrosis factor-related apoptosis-inducing ligand-TRAIL-is a protein operating as a ligand capable of inducing apoptosis particularly in cancerously transformed cells, while normal healthy cells are typically nonresponsive. We have previously demonstrated that pluripotent human embryonic stem cells (hESC) are also refractory to TRAIL, even though they express all canonical components of the death receptor-induced apoptosis pathway. In this study, we have examined a capacity of DNA damage to provoke sensitivity of hESC to TRAIL. The extent of DNA damage, behavior of molecules involved in apoptosis, and response of hESC to TRAIL were investigated. The exposure of hESC to 1 μM and 2 μM concentrations of cisplatin have led to the formation of 53BP1 and γH2AX foci, indicating the presence of double-strand breaks in DNA, without affecting the expression of proteins contributing to mitochondrial membrane integrity. Interestingly, cisplatin upregulated critical components of the extrinsic apoptotic pathway-initiator caspase 8, effector caspase 3, and the cell death receptors. The observed increase of expression of the extrinsic apoptotic pathway components was sufficient to sensitize hESC to TRAIL-induced apoptosis; immense cell dying accompanied by enhanced PARP cleavage, processing of caspase 8, and full activation of caspase 3 were all observed after the treatment combining cisplatin and TRAIL. Finally, we have demonstrated the central role of caspase 8 in this process, since its downregulation abrogated the sensitizing effect of cisplatin.

• Publication type
• Journal Article MeSH

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis.

• MeSH
• fas Receptor genetics   metabolism   MeSH
• Apoptosis drug effects   MeSH
• Cell Differentiation MeSH
• Embryonic Stem Cells cytology   drug effects   metabolism   MeSH
• CASP8 and FADD-Like Apoptosis Regulating Protein antagonists & inhibitors   genetics   metabolism   MeSH
• Harringtonines pharmacology   MeSH
• Homoharringtonine MeSH
• Protein Synthesis Inhibitors pharmacology   MeSH
• Caspase 3 genetics   metabolism   MeSH
• Caspase 8 genetics   metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering genetics   metabolism   MeSH
• Pluripotent Stem Cells cytology   drug effects   metabolism   MeSH
• Cell Proliferation MeSH
• Myeloid Cell Leukemia Sequence 1 Protein genetics   metabolism   MeSH
• TNF-Related Apoptosis-Inducing Ligand genetics   metabolism   pharmacology   MeSH
• Receptors, Tumor Necrosis Factor, Type I genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Signal Transduction MeSH
• Drug Synergism MeSH
• Receptors, TNF-Related Apoptosis-Inducing Ligand genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• fas Receptor MeSH
• FAS protein, human MeSH Browser
• CASP8 and FADD-Like Apoptosis Regulating Protein MeSH
• Harringtonines MeSH
• Homoharringtonine MeSH
• Protein Synthesis Inhibitors MeSH
• Caspase 3 MeSH
• Caspase 8 MeSH
• RNA, Small Interfering MeSH
• MCL1 protein, human MeSH Browser
• Myeloid Cell Leukemia Sequence 1 Protein MeSH
• TNF-Related Apoptosis-Inducing Ligand MeSH
• Receptors, Tumor Necrosis Factor, Type I MeSH
• TNFRSF10A protein, human MeSH Browser
• TNFSF10 protein, human MeSH Browser
• Receptors, TNF-Related Apoptosis-Inducing Ligand MeSH