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.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czech Republic

See more in PubMed

Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872. PubMed

Thomson JA. Itskovitz-Eldor J. Shapiro SS. Waknitz MA. Swiergiel JJ. Marshall VS. Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. PubMed

Drews K. Jozefczuk J. Prigione A. Adjaye J. Human induced pluripotent stem cells—from mechanisms to clinical applications. J Mol Med (Berl) 2012;90:735–745. PubMed

Vazin T. Freed WJ. Human embryonic stem cells: derivation, culture, and differentiation: a review. Restor Neurol Neurosci. 2010;28:589–603. PubMed PMC

Alekseenko LL. Zemelko VI. Zenin VV. Pugovkina NA. Kozhukharova IV. Kovaleva ZV. Grinchuk TM. Fridlyanskaya II. Nikolsky NN. Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny. Cell Cycle. 2012;11:3260–3269. PubMed PMC

Desmarais JA. Hoffmann MJ. Bing ham G. Gagou ME. Meuth M. Andrews PW. Human embryonic stem cells fail to activate CHK1 and commit to apoptosis in response to DNA replication stress. Stem Cells. 2012;30:1385–1393. PubMed

Sumi T. Tsuneyoshi N. Nakatsuji N. Suemori H. Apoptosis and differentiation of human embryonic stem cells induced by sustained activation of c-Myc. Oncogene. 2007;26:5564–5576. PubMed

Dumitru R. Gama V. Fagan BM. Bower JJ. Swahari V. Pevny LH. Deshmukh M. Human embryonic stem cells have constitutively active Bax at the Golgi and are primed to undergo rapid apoptosis. Mol Cell. 2012;46:573–583. PubMed PMC

Madden DT. Davila-Kruger D. Melov S. Bredesen DE. Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members. PLoS One. 2011;6:e28530. PubMed PMC

Momcilovic O. Navara C. Schatten G. Cell cycle adaptations and maintenance of genomic integrity in embryonic stem cells and induced pluripotent stem cells. Results Probl Cell Differ. 2011;53:415–458. PubMed

Ohgushi M. Matsumura M. Eiraku M. Murakami K. Aramaki T. Nishiyama A. Muguruma K. Nakano T. Suga H, et al. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell. 2010;7:225–239. PubMed

Romorini L. Scassa ME. Richardson GV. Bluguermann C. de Giusti CJ. Questa M. Fernandez Espinosa DD. Gomez RM. Sevlever GE. Miriuka SG. Activation of apoptotic signalling events in human embryonic stem cells upon Coxsackievirus B3 infection. Apoptosis. 2012;17:132–142. PubMed

Ardehali R. Inlay MA. Ali SR. Tang C. Drukker M. Weissman IL. Overexpression of BCL2 enhances survival of human embryonic stem cells during stress and obviates the requirement for serum factors. Proc Natl Acad Sci U S A. 2011;108:3282–3287. PubMed PMC

Bai H. Chen K. Gao YX. Arzigian M. Xie YL. Malcosky C. Yang YG. Wu WS. Wang ZZ. Bcl-xL enhances single-cell survival and expansion of human embryonic stem cells without affecting self-renewal. Stem Cell Res. 2012;8:26–37. PubMed PMC

Eiselleova L. Matulka K. Kriz V. Kunova M. Schmidtova Z. Neradil J. Tichy B. Dvorakova D. Pospisilova S. Hampl A. Dvorak P. A complex role for FGF-2 in self-renewal, survival, and adhesion of human embryonic stem cells. Stem Cells. 2009;27:1847–1857. PubMed PMC

Gauthaman K. Fong CY. Bongso A. Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion. Stem Cell Rev. 2010;6:86–95. PubMed

Li Y. Shelat H. Geng YJ. IGF-1 prevents oxidative stress induced-apoptosis in induced pluripotent stem cells which is mediated by microRNA-1. Biochem Biophys Res Commun. 2012;426:615–619. PubMed

Wang X. Lin G. Martins-Taylor K. Zeng H. Xu RH. Inhibition of caspase-mediated anoikis is critical for basic fibroblast growth factor-sustained culture of human pluripotent stem cells. J Biol Chem. 2009;284:34054–34064. PubMed PMC

Blum B. Bar-Nur O. Golan-Lev T. Benvenisty N. The anti-apoptotic gene survivin contributes to teratoma formation by human embryonic stem cells. Nat Biotechnol. 2009;27:281–287. PubMed

Filion TM. Qiao M. Ghule PN. Mandeville M. van Wijnen AJ. Stein JL. Lian JB. Altieri DC. Stein GS. Survival responses of human embryonic stem cells to DNA damage. J Cell Physiol. 2009;220:586–592. PubMed PMC

Aggarwal BB. Gupta SC. Kim JH. Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey. Blood. 2012;119:651–665. PubMed PMC

Grewal IS. Overview of TNF superfamily: a chest full of potential therapeutic targets. Adv Exp Med Biol. 2009;647:1–7. PubMed

Dickens LS. Powley IR. Hughes MA. MacFarlane M. The “complexities” of life and death: death receptor signalling platforms. Exp Cell Res. 2012;318:1269–1277. PubMed

Lavrik IN. Krammer PH. Regulation of CD95/Fas signaling at the DISC. Cell Death Differ. 2012;19:36–41. PubMed PMC

Kischkel FC. Lawrence DA. Tinel A. LeBlanc H. Virmani A. Schow P. Gazdar A. Blenis J. Arnott D. Ashkenazi A. Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8. J Biol Chem. 2001;276:46639–46646. PubMed

Tewari M. Quan LT. O'Rourke K. Desnoyers S. Zeng Z. Beidler DR. Poirier GG. Salvesen GS. Dixit VM. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995;81:801–809. PubMed

Hitomi J. Christofferson DE. Ng A. Yao J. Degterev A. Xavier RJ. Yuan J. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell. 2008;135:1311–1323. PubMed PMC

Christofferson DE. Yuan J. Necroptosis as an alternative form of programmed cell death. Curr Opin Cell Biol. 2010;22:263–268. PubMed PMC

Lluis JM. Nachbur U. Cook WD. Gentle IE. Moujalled D. Moulin M. Wong WW. Khan N. Chau D, et al. TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-kappaB dependent induction of cFLIPL. PLoS One. 2010;5:e8620. PubMed PMC

Ricci-Vitiani L. Pedini F. Mollinari C. Condorelli G. Bonci D. Bez A. Colombo A. Parati E. Peschle C. De Maria R. Absence of caspase 8 and high expression of PED protect primitive neural cells from cell death. J Exp Med. 2004;200:1257–1266. PubMed PMC

Szegezdi E. O'Reilly A. Davy Y. Vawda R. Taylor DL. Murphy M. Samali A. Mehmet H. Stem cells are resistant to TRAIL receptor-mediated apoptosis. J Cell Mol Med. 2009;13:4409–4414. PubMed PMC

Falschlehner C. Emmerich CH. Gerlach B. Walczak H. TRAIL signalling: decisions between life and death. Int J Biochem Cell Biol. 2007;39:1462–1475. PubMed

Herrero-Martin G. Hoyer-Hansen M. Garcia-Garcia C. Fumarola C. Farkas T. Lopez-Rivas A. Jaattela M. TAK1 activates AMPK-dependent cytoprotective autophagy in TRAIL-treated epithelial cells. Embo J. 2009;28:677–685. PubMed PMC

Siegmund D. Klose S. Zhou D. Baumann B. Roder C. Kalthoff H. Wajant H. Trauzold A. Role of caspases in CD95L- and TRAIL-induced non-apoptotic signalling in pancreatic tumour cells. Cell Signal. 2007;19:1172–1184. PubMed

Dickens LS. Boyd RS. Jukes-Jones R. Hughes MA. Robinson GL. Fairall L. Schwabe JW. Cain K. Macfarlane M. A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death. Mol Cell. 2012;47:291–305. PubMed PMC

Gonzalvez F. Lawrence D. Yang B. Yee S. Pitti R. Marsters S. Pham VC. Stephan JP. Lill J. Ashkenazi A. TRAF2 Sets a Threshold for Extrinsic Apoptosis by Tagging Caspase-8 with a Ubiquitin Shutoff Timer. Mol Cell. 2012;48:888–899. PubMed

Jin Z. Li Y. Pitti R. Lawrence D. Pham VC. Lill JR. Ashkenazi A. Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling. Cell. 2009;137:721–735. PubMed

Schleich K. Warnken U. Fricker N. Ozturk S. Richter P. Kammerer K. Schnolzer M. Krammer PH. Lavrik IN. Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model. Mol Cell. 2012;47:306–319. PubMed

Shirley S. Morizot A. Micheau O. Regulating TRAIL receptor-induced cell death at the membrane: a deadly discussion. Recent Pat Anticancer Drug Discov. 2011;6:311–323. PubMed PMC

Kaufmann T. Strasser A. Jost PJ. Fas death receptor signalling: roles of Bid and XIAP. Cell Death Differ. 2012;19:42–50. PubMed PMC

Llambi F. Moldoveanu T. Tait SW. Bouchier-Hayes L. Temirov J. McCormick LL. Dillon CP. Green DR. A unified model of mammalian BCL-2 protein family interactions at the mitochondria. Mol Cell. 2011;44:517–531. PubMed PMC

Abdulghani J. El-Deiry WS. TRAIL receptor signaling and therapeutics. Expert Opin Ther Targets. 2010;14:1091–1108. PubMed

Yerbes R. Palacios C. Lopez-Rivas A. The therapeutic potential of TRAIL receptor signalling in cancer cells. Clin Transl Oncol. 2011;13:839–847. PubMed

Dolezalova D. Mraz M. Barta T. Plevova K. Vinarsky V. Holubcova Z. Jaros J. Dvorak P. Pospisilova S. Hampl A. MicroRNAs regulate p21(Waf1/Cip1) protein expression and the DNA damage response in human embryonic stem cells. Stem Cells. 2012;30:1362–1372. PubMed

Armstrong L. Tilgner K. Saretzki G. Atkinson SP. Stojkovic M. Moreno R. Przyborski S. Lako M. Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells. Stem Cells. 2010;28:661–673. PubMed

Barta T. Vinarsky V. Holubcova Z. Dolezalova D. Verner J. Pospisilova S. Dvorak P. Hampl A. Human embryonic stem cells are capable of executing G1/S checkpoint activation. Stem Cells. 2010;28:1143–1152. PubMed

Horova V. Hradilova N. Jelinkova I. Koc M. Svadlenka J. Brazina J. Klima J. Slavik I. Hyrslova Vaculova A. Andera L. Inhibition of vacuolar ATPase attenuates the TRAIL-induced activation of caspase-8 and modulates the trafficking of TRAIL receptosomes. FEBS J. 2013;280:3436–3450. PubMed

Beranova L. Pombinho AR. Spegarova J. Koc M. Klanova M. Molinsky J. Klener P. Bartunek P. Andera L. The plant alkaloid and anti-leukemia drug homoharringtonine sensitizes resistant human colorectal carcinoma cells to TRAIL-induced apoptosis via multiple mechanisms. Apoptosis. 2013;18:739–750. PubMed

He MX. He YW. CFLAR/c-FLIPL: a star in the autophagy, apoptosis and necroptosis alliance. Autophagy. 2013;9:791–793. PubMed PMC

Lan ML. Acharya MM. Tran KK. Bahari-Kashani J. Patel NH. Strnadel J. Giedzinski E. Limoli CL. Characterizing the radioresponse of pluripotent and multipotent human stem cells. PLoS One. 2012;7:e50048. PubMed PMC

Kohchi C. Tanabe Y. Noguchi K. Mizuno D. Soma G. Induction of differentiation in embryonic stem cells by 26-kD membrane-bound tumor necrosis factor (TNF) and 17-kD free TNF. In Vivo. 1996;10:19–27. PubMed

Wuu YD. Pampfer S. Vanderheyden I. Lee KH. De Hertogh R. Impact of tumor necrosis factor alpha on mouse embryonic stem cells. Biol Reprod. 1998;58:1416–1424. PubMed

Zou GM. Reznikoff-Etievant MF. Leon A. Verge V. Hirsch F. Milliez J. Fas-mediated apoptosis of mouse embryo stem cells: its role during embryonic development. Am J Reprod Immunol. 2000;43:240–248. PubMed

Brunlid G. Pruszak J. Holmes B. Isacson O. Sonntag KC. Immature and neurally differentiated mouse embryonic stem cells do not express a functional Fas/Fas ligand system. Stem Cells. 2007;25:2551–2558. PubMed PMC

Zampetaki A. Zeng L. Xiao Q. Margariti A. Hu Y. Xu Q. Lacking cytokine production in ES cells and ES-cell-derived vascular cells stimulated by TNF-alpha is rescued by HDAC inhibitor trichostatin A. Am J Physiol Cell Physiol. 2007;293:C1226–C1238. PubMed

Kim SK. Kim BK. Shim JH. Gil JE. Yoon YD. Kim JH. Nonylphenol and octylphenol-induced apoptosis in human embryonic stem cells is related to Fas-Fas ligand pathway. Toxicol Sci. 2006;94:310–321. PubMed

Riley JK. Heeley JM. Wyman AH. Schlichting EL. Moley KH. TRAIL and KILLER are expressed and induce apoptosis in the murine preimplantation embryo. Biol Reprod. 2004;71:871–877. PubMed

Dimberg LY. Anderson CK. Camidge R. Behbakht K. Thorburn A. Ford HL. On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene. 2013;32:1341–1350. PubMed PMC

Golks A. Brenner D. Fritsch C. Krammer PH. Lavrik IN. c-FLIPR, a new regulator of death receptor-induced apoptosis. J Biol Chem. 2005;280:14507–14513. PubMed

Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer JL. Schroter M. Burns K, et al. Inhibition of death receptor signals by cellular FLIP. Nature. 1997;388:190–195. PubMed

Finnberg N. Gruber JJ. Fei P. Rudolph D. Bric A. Kim SH. Burns TF. Ajuha H. Page R, et al. DR5 knockout mice are compromised in radiation-induced apoptosis. Mol Cell Biol. 2005;25:2000–2013. PubMed PMC

Grosse-Wilde A. Voloshanenko O. Bailey SL. Longton GM. Schaefer U. Csernok AI. Schutz G. Greiner EF. Kemp CJ. Walczak H. TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development. J Clin Invest. 2008;118:100–110. PubMed PMC

Hirsch ML. Fagan BM. Dumitru R. Bower JJ. Yadav S. Porteus MH. Pevny LH. Samulski RJ. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells. PLoS One. 2011;6:e27520. PubMed PMC

Henry RE. Andrysik Z. Paris R. Galbraith MD. Espinosa JM. A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX. Embo J. 2012;31:1266–1278. PubMed PMC

Human Embryonic Stem Cells Acquire Responsiveness to TRAIL upon Exposure to Cisplatin