Journal of Cell Communication and Signaling

, Volume 6, Issue 3, pp 155–168 | Cite as

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Research Article

Abstract

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIPL in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.

Keywords

Apoptosis Fas associated death domain (FADD) cFLIP Death receptor mediated apoptosis 

Abbreviations

DED

Death Effector Domain

DISC

Death-Inducing Signaling Complex

cFLIP

Cellular fas-associated death domain-like interleukin-1-β- converting enzyme-inhibitory protein

CHX

Cycloheximide

CD 95L

CD 95 ligand

References

  1. Algeciras-Schimnich A, Shen L, Barnhart BC, Murmann AE, Burkhardt JK, Peter ME (2002) Molecular ordering of the initial signaling events of CD95. Mol Cell Biol 22:207–220PubMedCrossRefGoogle Scholar
  2. Bagnoli M, Ambrogi F, Pilotti S, Alberti P, Ditto A, Barbareschi M, Galligioni E, Biganzoli E, Canevari S, Mezzanzanica D (2009) c-FLIPL expression defines two ovarian cancer patient subsets and is a prognostic factor of adverse outcome. Endocr Relat Canc 16:443–453CrossRefGoogle Scholar
  3. Bagnoli M, Canevari S, Mezzanzanica D (2010) Cellular FLICE-inhibitory protein (c-FLIP) signalling: a key regulator of receptor-mediated apoptosis in physiologic context and in cancer. Int J Biochem Cell Biol 42:210–213PubMedCrossRefGoogle Scholar
  4. Beisner DR, Chu IH, Arechiga AF, Hedrick SM, Walsh CM (2003) The requirements for Fas-associated death domain signaling in mature T cell activation and survival. J Immunol 171:247–256PubMedGoogle Scholar
  5. Bell BD, Leverrier S, Weist BM, Newton RH, Arechiga AF, Luhrs KA, Morrissette NS, Walsh CM (2008) FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells. Proc Natl Acad Sci U S A 105:16677–16682PubMedCrossRefGoogle Scholar
  6. Brumatti G, Yon M, Castro FA, Bueno-da-Silva AE, Jacysyn JF, Brunner T, Amarante-Mendes GP (2008) Conversion of CD95 (Fas) Type II into Type I signaling by sub-lethal doses of cycloheximide. Exp Cell Res 314:554–563PubMedCrossRefGoogle Scholar
  7. Chang DW, Xing Z, Pan Y, Algeciras-Schimnich A, Barnhart BC, Yaish-Ohad S, Peter ME, Yang X (2002) c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J 21:3704–3714PubMedCrossRefGoogle Scholar
  8. Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM (1995) FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81:505–512PubMedCrossRefGoogle Scholar
  9. Chinnaiyan AM, Tepper CG, Seldin MF, O’Rourke K, Kischkel FC, Hellbardt S, Krammer PH, Peter ME, Dixit VM (1996) FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J Biol Chem 271:4961–4965PubMedCrossRefGoogle Scholar
  10. Debatin KM, Krammer PH (2004) Death receptors in chemotherapy and cancer. Oncogene 23:2950–2966PubMedCrossRefGoogle Scholar
  11. Fischer-Posovszky P, Keuper M, Nagel S, Hesse D, Schurmann A, Debatin KM, Strauss G, Wabitsch M (2011) Downregulation of FLIP by cycloheximide sensitizes human fat cells to CD95-induced apoptosis. Exp Cell Res 317:2200–2209PubMedCrossRefGoogle Scholar
  12. Foger N, Bulfone-Paus S, Chan AC, Lee KH (2009) Subcellular compartmentalization of FADD as a new level of regulation in death receptor signaling. FEBS J 276:4256–4265PubMedCrossRefGoogle Scholar
  13. Fulda S, Meyer E, Debatin KM (2000) Metabolic inhibitors sensitize for CD95 (APO-1/Fas)-induced apoptosis by down-regulating Fas-associated death domain-like interleukin 1-converting enzyme inhibitory protein expression. Cancer Res 60:3947–3956PubMedGoogle Scholar
  14. Golks A, Brenner D, Fritsch C, Krammer PH, Lavrik IN (2005) c-FLIPR, a new regulator of death receptor-induced apoptosis. J Biol Chem 280:14507–14513PubMedCrossRefGoogle Scholar
  15. Golks A, Brenner D, Krammer PH, Lavrik IN (2006) The c-FLIP-NH2 terminus (p22-FLIP) induces NF-kappaB activation. J Exp Med 203:1295–1305PubMedCrossRefGoogle Scholar
  16. Gomez-Angelats M, Cidlowski JA (2003) Molecular evidence for the nuclear localization of FADD. Cell Death Differ 10:791–797PubMedCrossRefGoogle Scholar
  17. Holler N, Tardivel A, Kovacsovics-Bankowski M, Hertig S, Gaide O, Martinon F, Tinel A, Deperthes D, Calderara S, Schulthess T, Engel J, Schneider P, Tschopp J (2003) Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol Cell Biol 23:1428–1440PubMedCrossRefGoogle Scholar
  18. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer JL, Schroter M, Burns K, Mattmann C, Rimoldi D, French LE, Tschopp J (1997) Inhibition of death receptor signals by cellular FLIP. Nature 388:190–195PubMedCrossRefGoogle Scholar
  19. Kavuri SM, Geserick P, Berg D, Dimitrova DP, Feoktistova M, Siegmund D, Gollnick H, Neumann M, Wajant H, Leverkus M (2011) Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex. J Biol Chem 286:16631–16646PubMedCrossRefGoogle Scholar
  20. Krammer PH (1999) CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv Immunol 71:163–210PubMedCrossRefGoogle Scholar
  21. Krueger A, Baumann S, Krammer PH, Kirchhoff S (2001) FLICE-inhibitory proteins: regulators of death receptor-mediated apoptosis. Mol Cell Biol 21:8247–8254PubMedCrossRefGoogle Scholar
  22. Lavrik IN, Golks A, Riess D, Bentele M, Eils R, Krammer PH (2007) Analysis of CD95 threshold signaling: triggering of CD95 (FAS/APO-1) at low concentrations primarily results in survival signaling. J Biol Chem 282:13664–13671PubMedCrossRefGoogle Scholar
  23. Lavrik IN, Mock T, Golks A, Hoffmann JC, Baumann S, Krammer PH (2008) CD95 stimulation results in the formation of a novel death effector domain protein-containing complex. J Biol Chem 283:26401–26408PubMedCrossRefGoogle Scholar
  24. Longley DB, Wilson TR, McEwan M, Allen WL, McDermott U, Galligan L, Johnston PG (2006) c-FLIP inhibits chemotherapy-induced colorectal cancer cell death. Oncogene 25:838–848PubMedCrossRefGoogle Scholar
  25. Matsuda F, Inoue N, Goto Y, Maeda A, Cheng Y, Sakamaki K, Manabe N (2008) cFLIP regulates death receptor-mediated apoptosis in an ovarian granulosa cell line by inhibiting procaspase-8 cleavage. J Reprod Dev 54:314–320PubMedCrossRefGoogle Scholar
  26. Osborn SL, Diehl G, Han SJ, Xue L, Kurd N, Hsieh K, Cado D, Robey EA, Winoto A (2010) Fas-associated death domain (FADD) is a negative regulator of T-cell receptor-mediated necroptosis. Proc Natl Acad Sci U S A 107:13034–13039PubMedCrossRefGoogle Scholar
  27. Peter ME, Krammer PH (2003) The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ 10:26–35PubMedCrossRefGoogle Scholar
  28. Peter ME, Legembre P, Barnhart BC (2005) Does CD95 have tumor promoting activities? Biochim Biophys Acta 1755:25–36PubMedGoogle Scholar
  29. Rogers KM, Thomas M, Galligan L, Wilson TR, Allen WL, Sakai H, Johnston PG, Longley DB (2007) Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells. Mol Cancer Ther 6:1544–1551PubMedCrossRefGoogle Scholar
  30. Ryu BK, Lee MG, Chi SG, Kim YW, Park JH (2001) Increased expression of cFLIP(L) in colonic adenocarcinoma. J Pathol 194:15–19PubMedCrossRefGoogle Scholar
  31. Safa AR, Day TW, Wu CH (2008) Cellular FLICE-like inhibitory protein (C-FLIP): a novel target for cancer therapy. Curr Cancer Drug Targets 8:37–46PubMedCrossRefGoogle Scholar
  32. Scaffidi C, Schmitz I, Krammer PH, Peter ME (1999) The role of c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem 274:1541–1548PubMedCrossRefGoogle Scholar
  33. Scaffidi C, Volkland J, Blomberg I, Hoffmann I, Krammer PH, Peter ME (2000) Phosphorylation of FADD/MORT1 at serine 194 and association with a 70-kDa cell cycle-regulated protein kinase. J Immunol 164:1236–1242PubMedGoogle Scholar
  34. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME (1998) Apoptosis signaling by death receptors. Eur J Biochem/FEBS 254:439–459CrossRefGoogle Scholar
  35. Screaton RA, Kiessling S, Sansom OJ, Millar CB, Maddison K, Bird A, Clarke AR, Frisch SM (2003) Fas-associated death domain protein interacts with methyl-CpG binding domain protein 4: a potential link between genome surveillance and apoptosis. Proc Natl Acad Sci U S A 100:5211–5216PubMedCrossRefGoogle Scholar
  36. Shirley S, Micheau O (2010) Targeting c-FLIP in cancer. Cancer Lett. doi:10.1016/j.canlet.2010.10.009
  37. Song W, Lahiri DK (1995) Efficient transfection of DNA by mixing cells in suspension with calcium phosphate. Nucleic Acids Res 23:3609–3611PubMedCrossRefGoogle Scholar
  38. Taylor RC, Cullen SP, Martin SJ (2008) Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 9:231–241PubMedCrossRefGoogle Scholar
  39. Tourneur L, Chiocchia G (2010) FADD: a regulator of life and death. Trends Immunol 31:260–269PubMedCrossRefGoogle Scholar
  40. Tourneur L, Mistou S, Michiels FM, Devauchelle V, Renia L, Feunteun J, Chiocchia G (2003) Loss of FADD protein expression results in a biased Fas-signaling pathway and correlates with the development of tumoral status in thyroid follicular cells. Oncogene 22:2795–2804PubMedCrossRefGoogle Scholar
  41. Tourneur L, Delluc S, Levy V, Valensi F, Radford-Weiss I, Legrand O, Vargaftig J, Boix C, Macintyre EA, Varet B, Chiocchia G, Buzyn A (2004) Absence or low expression of fas-associated protein with death domain in acute myeloid leukemia cells predicts resistance to chemotherapy and poor outcome. Cancer Res 64:8101–8108PubMedCrossRefGoogle Scholar
  42. Tourneur L, Buzyn A, Chiocchia G (2005) FADD adaptor in cancer. Med Immunol 4:1PubMedCrossRefGoogle Scholar
  43. Vaux DL, Haecker G, Strasser A (1994) An evolutionary perspective on apoptosis. Cell 76:777–779PubMedCrossRefGoogle Scholar
  44. Wajant H, Haas E, Schwenzer R, Muhlenbeck F, Kreuz S, Schubert G, Grell M, Smith C, Scheurich P (2000) Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD). J Biol Chem 275:24357–24366PubMedCrossRefGoogle Scholar
  45. Yeh WC, Pompa JL, McCurrach ME, Shu HB, Elia AJ, Shahinian A, Ng M, Wakeham A, Khoo W, Mitchell K, El-Deiry WS, Lowe SW, Goeddel DV, Mak TW (1998) FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science 279:1954–1958PubMedCrossRefGoogle Scholar
  46. Yu JW, Jeffrey PD, Shi Y (2009) Mechanism of procaspase-8 activation by c-FLIPL. Proc Natl Acad Sci U S A 106:8169–8174PubMedCrossRefGoogle Scholar
  47. Zhang J, Cado D, Chen A, Kabra NH, Winoto A (1998) Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1. Nature 392:296–300PubMedCrossRefGoogle Scholar
  48. Zhang J, Kabra NH, Cado D, Kang C, Winoto A (2001) FADD-deficient T cells exhibit a disaccord in regulation of the cell cycle machinery. J Biol Chem 276:29815–29818PubMedCrossRefGoogle Scholar

Copyright information

© The International CCN Society 2012

Authors and Affiliations

  1. 1.Cell Biology Department, School of Biological Sciences & BiotechnologyIndian Institute of Advanced ResearchKobaIndia
  2. 2.Molecular Biophysics UnitIndian Institute of ScienceBangaloreIndia

Personalised recommendations