Annals of Hematology

, Volume 92, Issue 12, pp 1603–1609 | Cite as

Suppression of granzyme B activity and caspase-3 activation in leukaemia cells constitutively expressing the protease inhibitor 9

  • Kristina Fritsch
  • Jürgen Finke
  • Carsten Grüllich
Original Article


Immune surveillance against malignant cells is mediated by cytotoxic T-lymphocytes and NK-cells (CTL/NK) that induce apoptosis through the granzyme-B-dependent pathway. The serine protease inhibitor serpinB9/protease inhibitor-9 (PI-9) is a known inhibitor of granzyme B. Ectopic expression of PI-9 in tumour cells has been reported. However, the impact of PI-9 on granzyme-B-induced apoptosis in tumour cells remains unclear. The aim of this study was to investigate the influence of constitutive PI-9 expression in leukaemia cell lines on the activity of granzyme B and apoptosis induction. PI-9 negative (lymphoblastic Jurkat cells; myeloblastic U937 cells) and PI-9-expressing cell lines (myeloblastic K562 cells, EBV-transformed LCL-1 and LCL-2 B-cells, lymphoblastic Daudi cells, AML-R cells f leukaemia and the U937 subclone U937PI-9+). For accurate granzyme B activity determination a quantitative substrate (Ac-IEPD-pNA) cleavage assay was established and caspase-3 activation measured for apoptosis assessment. Cells were treated with a cytotoxic granule isolate that has previously been shown to induce apoptosis through granzyme B signalling. We found a robust correlation between constitutive PI-9 expression levels and the suppression of granzyme B activity. Further, inhibition of granzyme B translated into reduced caspase-3 activation. We conclude, suppression of granzyme B initiated apoptosis in PI-9-expressing cells could contribute to immune evasion and the measurement of granzyme B activity with our assay might be a useful predictive marker in immune-therapeutic approaches against cancer.


Leukaemia Granzyme B Apoptosis Serpin 


Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Barbaric D, Wynne K, Aslanian S, Bond M, Reid GS (2005) Immune evasion strategies of pediatric precursor-B acute lymphoblastic leukemia after allogeneic bone marrow transplantation—a case study. Leuk Res 29:711–714PubMedCrossRefGoogle Scholar
  2. 2.
    Elpek KG, Lacelle C, Singh NP, Yolcu ES, Shirwan H (2007) CD4+ CD25+ T regulatory cells dominate multiple immune evasion mechanisms in early but not late phases of tumor development in a B cell lymphoma model. J Immunol 178:6840–6848PubMedGoogle Scholar
  3. 3.
    Schmaltz C, Alpdogan O, Horndasch KJ et al (2001) Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versus-host disease and graft-versus-leukemia effect. Blood 97:2886–2895PubMedCrossRefGoogle Scholar
  4. 4.
    Grüllich C, McGoldrick S, Zeiser R, Finke J (2005) The death receptor pathway is not involved in alloreactive T-cell induced mitochondrial membrane permeability. Leuk Lymphoma 46:1207–1216Google Scholar
  5. 5.
    Barry M, Bleackley RC (2002) Cytotoxic T lymphocytes: all roads lead to death. Nat Rev Immunol 2:401–409PubMedGoogle Scholar
  6. 6.
    Waterhouse NJ, Sedelies KA, Trapani JA (2006) Role of Bid-induced mitochondrial outer membrane permeabilization in granzyme B-induced apoptosis. Immunol Cell Biol 84:72–78PubMedCrossRefGoogle Scholar
  7. 7.
    Cullen SP, Adrain C, Luthi AU, Duriez PJ, Martin SJ (2007) Human and murine granzyme B exhibit divergent substrate preferences. J Cell Biol 176:435–444PubMedCrossRefGoogle Scholar
  8. 8.
    Wolf BB, Schuler M, Echeverri F, Green DR (1999) Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. J Biol Chem 274:30651–30656PubMedCrossRefGoogle Scholar
  9. 9.
    Veugelers K, Motyka B, Goping IS, Shostak I, Sawchuk T, Bleackley RC (2006) Granule-mediated killing by granzyme B and perforin requires a mannose 6-phosphate receptor and is augmented by cell surface heparan sulfate. Mol Biol Cell 17:623–633PubMedCrossRefGoogle Scholar
  10. 10.
    Keefe D, Shi L, Feske S et al (2005) Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis. Immunity 23:249–262PubMedCrossRefGoogle Scholar
  11. 11.
    Kurschus FC, Bruno R, Fellows E, Falk CS, Jenne DE (2005) Membrane receptors are not required to deliver granzyme B during killer cell attack. Blood 105:2049–2058PubMedCrossRefGoogle Scholar
  12. 12.
    Grüllich C, Friske V, Finke J (2008) Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death. Ann Hematol 87:701–708Google Scholar
  13. 13.
    Bird PI (1999) Regulation of pro-apoptotic leucocyte granule serine proteinases by intracellular serpins. Immunol Cell Biol 77:47–57PubMedCrossRefGoogle Scholar
  14. 14.
    Trapani JA, Sutton VR (2003) Granzyme B: pro-apoptotic, antiviral and antitumor functions. Curr Opin Immunol 15:533–543PubMedGoogle Scholar
  15. 15.
    Bots M, Offringa R, Medema JP (2006) Does the serpin PI-9 protect tumor cells? Blood 107:4974–4975PubMedCrossRefGoogle Scholar
  16. 16.
    Medema JP, de JJ, Peltenburg LT et al (2001) Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors. Proc Natl Acad Sci U S A 98:11515–11520PubMedCrossRefGoogle Scholar
  17. 17.
    Classen CF (2004) The granzyme B inhibitor PI-9 is differentially expressed in all main subtypes of pediatric acute lymphoblastic leukemias. Haematologica 89:1314–1321PubMedGoogle Scholar
  18. 18.
    Godal R (2006) Lymphomas are sensitive to perforin-dependent cytotoxic pathways despite expression of PI-9 and overexpression of bcl-2. Blood 107:3205–3211PubMedCrossRefGoogle Scholar
  19. 19.
    Debatin KM (2004) Apoptosis pathways in cancer and cancer therapy. Cancer Immunol Immunother 53(3):153–159PubMedCrossRefGoogle Scholar
  20. 20.
    Ekert PG, Silke J, Vaux DL (1999) Caspase inhibitors. Cell Death Differ 6(11):1081–1086PubMedCrossRefGoogle Scholar
  21. 21.
    Grüllich C, Richter M, Exner S, Finke J (2003) Fas ligand is not constitutively expressed in low-grade B-cell lymphoma and B-lymphoblastoid cells. Eur J Haematol 71:184–188Google Scholar
  22. 22.
    ten Berge RL, Meijer CJ, Dukers DF et al (2002) Expression levels of apoptosis-related proteins predict clinical outcome in anaplastic large cell lymphoma. Blood 99:4540–4546PubMedCrossRefGoogle Scholar
  23. 23.
    Muris JJ, Meijer CJ, Cillessen SA et al (2004) Prognostic significance of activated cytotoxic T-lymphocytes in primary nodal diffuse large B-cell lymphomas. Leukemia 18:589–596PubMedCrossRefGoogle Scholar
  24. 24.
    Grüllich C, Bertz H, Spyridonidis A, Muller CI, Finke J (2008) A fludarabine, thiotepa reduced toxicity conditioning regimen designed specifically for allogeneic second haematopoietic cell transplantation after failure of previous autologous or allogeneic transplantation. Bone Marrow Transplant 41:845–850Google Scholar
  25. 25.
    Barrie MB, Stout HW, Abougergi MS, Miller BC, Thiele DL (2004) Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6. J Immunol 172:6453–6459PubMedGoogle Scholar
  26. 26.
    Bird CH, Sutton VR, Sun J et al (1998) Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol Cell Biol 18:6387–6398PubMedGoogle Scholar
  27. 27.
    Bladergroen BA, Strik MC, Bovenschen N et al (2001) The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells and at immune-privileged sites. J Immunol 166:3218–3225PubMedGoogle Scholar
  28. 28.
    Waterhouse NJ, Sedelies KA, Browne KA et al (2005) A central role for Bid in granzyme B-induced apoptosis. J Biol Chem 280:4476–4482PubMedCrossRefGoogle Scholar
  29. 29.
    Metkar SS, Wang B, Aguilar-Santelises M, Raja SM, Uhlin-Hansen L, Podack E, Trapani JA, Froelich CJ (2002) Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity 16(3):417–428PubMedCrossRefGoogle Scholar
  30. 30.
    Adrain C, Murphy BM, Martin SJ (2005) Molecular ordering of the caspase activation cascade initiated by the cytotoxic T lymphocyte/natural killer (CTL/NK) protease granzyme B. J Biol Chem 280:4663–4673PubMedCrossRefGoogle Scholar
  31. 31.
    Metkar SS, Wang B, Ebbs ML et al (2003) Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis. J Cell Biol 160:875–885PubMedCrossRefGoogle Scholar
  32. 32.
    Bladergroen BA, Strik MC, Wolbink AM, Wouters D, Broekhuizen R, Kummer JA, Hack CE (2005) The granzyme B inhibitor proteinase inhibitor 9 (PI9) is expressed by human mast cells. Eur J Immunol 35(4):1175–1183PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Kristina Fritsch
    • 1
  • Jürgen Finke
    • 1
  • Carsten Grüllich
    • 1
    • 2
  1. 1.Dpt. of Hematology and OncologyAlbert Ludwigs-University Medical Center FreiburgFreiburgGermany
  2. 2.National Center for Tumor Diseases, Dpt. of Medical OncologyHeidelberg University Medical CenterHeidelbergGermany

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