Skip to main content
SpringerLink
Log in

Identification of deficient mitochondrial signaling in apoptosis resistant leukemia cells by flow cytometric analysis of intracellular cytochrome c, caspase-3 and apoptosis

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Deficient activation of apoptosis signaling pathways may be responsible for treatment failure of malignant diseases. In primary leukemia samples the detection of deficient mitochondrial apoptosis signaling would enable identification of chemo-resistant cells. To investigate the key events of apoptosis at the mitochondrial level, we developed a flow cytometric method for simultaneous detection of mitochondrial cytochrome c release and caspase-3 processing using conformation sensitive monoclonal antibodies. This method proved to identify deficient mitochondrial apoptosis signaling in leukemia cells overexpressing Bcl-2 by a pattern of apoptosis resistance, deficient cytochrome c reduction and partial processing of caspase-3. In primary leukemia cells, reduction of cytochrome c and caspase-3 activation was induced by treatment with anticancer drugs in vitro. In leukemia cells of a patient with resistant disease, a pattern of deficient apoptosis signaling as in Bcl-2 transfected cells was observed, suggesting that deficient mitochondrial signaling contributed to the clinical phenotype of drug resistance in this patient. Flow cytometric analysis of mitochondrial apoptosis signaling may provide a useful tool for the prediction of drug resistance and treatment failure in primary leukemia.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Dive C, Evans CA, Whetton AD. Induction of apoptosis-New targets for cancer chemotherapy. Semin Cancer Biol 1992; 3: 417–427.

    CAS  PubMed  Google Scholar 

  2. Hannun YA. Apoptosis and the dilemma of cancer chemotherapy. Blood 1997; 89: 1845–1853.

    CAS  PubMed  Google Scholar 

  3. Los M, Herr I, Friesen C, Fulda S, Schulze-Osthoff K, Debatin KM. Cross-resistance of CD95-and drug-induced apoptosis as a consequence of deficient activation of caspases (ICE/Ced-3 proteases). Blood 1997; 90: 3118–3129.

    CAS  PubMed  Google Scholar 

  4. Friesen C, Herr I, Krammer PH, Debatin KM. Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells. Nat Med 1996; 2: 574–577.

    Article  CAS  PubMed  Google Scholar 

  5. Decaudin D, Geley S, Hirsch T, et al. Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res 1997; 57: 62–67.

    CAS  PubMed  Google Scholar 

  6. Kim CN, Wang X, Huang Y, et al. Overexpression of Bcl-X(L) inhibits Ara-C-induced mitochondrial loss of cytochrome c and other perturbations that activate the molecular cascade of apoptosis. Cancer Res 1997; 57: 3115–3120.

    CAS  PubMed  Google Scholar 

  7. Herr I, Debatin KM. Cellular stress response and apoptosis in cancer therapy. Blood 2001; 98: 2603–2614.

    Article  CAS  PubMed  Google Scholar 

  8. Genini D, Adachi S, Chao Q, et al. Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria. Blood 2000; 96: 3537–3543.

    CAS  PubMed  Google Scholar 

  9. Kuida K, Haydar TF, Kuan CY, et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 1998; 94: 325–337.

    Article  CAS  PubMed  Google Scholar 

  10. Sun XM, MacFarlane M, Zhuang J, Wolf BB, Green DR, Cohen GM. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem 1999; 274: 5053–5060.

    CAS  PubMed  Google Scholar 

  11. Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways. Embo J 1998; 17: 1675–1687.

    CAS  PubMed  Google Scholar 

  12. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 1996; 86: 147–157.

    Article  CAS  PubMed  Google Scholar 

  13. Jiang X, Wang X. Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1. J Biol Chem 2000; 275: 31199–31203.

    CAS  PubMed  Google Scholar 

  14. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479–489.

    Article  CAS  PubMed  Google Scholar 

  15. Cain K, Brown DG, Langlais C, Cohen GM. Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex. J Biol Chem 1999; 274: 22686–22692.

    CAS  PubMed  Google Scholar 

  16. Zou H, Li Y, Liu X, Wang X. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999; 274: 11549–11556.

    CAS  PubMed  Google Scholar 

  17. Hakem R, Hakem A, Duncan GS, et al. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 1998; 94: 339–352.

    Article  CAS  PubMed  Google Scholar 

  18. Saleh A, Srinivasula SM, Acharya S, Fishel R, Alnemri ES. Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J Biol Chem 1999; 274: 17941–17945.

    CAS  PubMed  Google Scholar 

  19. Slee EA, Harte MT, Kluck RM, et al. Ordering the cytochrome c-initiated caspase cascade: Hierarchical activation of caspases-2,-3,-6,-7,-8, and-10 in a caspase-9-dependent manner. J Cell Biol 1999; 144: 281–292.

    CAS  PubMed  Google Scholar 

  20. Cai Z, Lin M, Wuchter C, et al. Apoptotic response to homoharringtonine in human wt p53 leukemic cells is independent of reactive oxygen species generation and implicates Bax translocation, mitochondrial cytochrome c release and caspase activation. Leukemia 2001; 15: 567–574.

    CAS  PubMed  Google Scholar 

  21. Yin XM, Wang K, Gross A, et al. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 1999; 400: 886–891.

    CAS  PubMed  Google Scholar 

  22. Jemmerson R, LaPlante B, Treeful A. Release of intact, monomeric cytochrome c from apoptotic and necrotic cells. Cell Death Differ 2002; 9: 538–548.

    CAS  PubMed  Google Scholar 

  23. Renz A, Berdel WE, Kreuter M, Belka C, Schulze-Osthoff K, Los M. Rapid extracellular release of cytochrome c is specific for apoptosis and marks cell death in vivo. Blood 2001; 98: 1542–1548.

    CAS  PubMed  Google Scholar 

  24. Varkey J, Chen P, Jemmerson R, Abrams JM. Altered cytochrome c display precedes apoptotic cell death in Drosophila. J Cell Biol 1999; 144: 701–710.

    CAS  PubMed  Google Scholar 

  25. Jemmerson R, Liu J, Hausauer D, Lam KP, Mondino A, Nelson RD. A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles. Biochemistry 1999; 38: 3599–3609.

    CAS  PubMed  Google Scholar 

  26. Goshorn SC, Retzel E, Jemmerson R. Common structural features among monoclonal antibodies binding the same antigenic region of cytochrome c. J Biol Chem 1991; 266: 2134–2142.

    CAS  PubMed  Google Scholar 

  27. Mueller CM, Jemmerson R. Maturation of the antibody response to the major epitope on the self antigen mouse cytochrome c. Restricted V gene usage, selected mutations, and increased affinity. J Immunol 1996; 157: 5329–5338.

    CAS  PubMed  Google Scholar 

  28. Martin SJ, Amarante-Mendes GP, Shi L, et al. The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism. Embo J 1996; 15: 2407–2416.

    CAS  PubMed  Google Scholar 

  29. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000; 102: 33–42.

    Article  CAS  PubMed  Google Scholar 

  30. Guo Y, Srinivasula SM, Druilhe A, Fernandes-Alnemri T, Alnemri ES. Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria. J Biol Chem 2002; 277: 13430–13437.

    CAS  PubMed  Google Scholar 

  31. Deng Y, Lin Y, Wu X. TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO. Genes Dev. 2002; 16: 33–45.

    CAS  PubMed  Google Scholar 

  32. Zhang XD, Zhang XY, Gray CP, Nguyen T, Hersey P. Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by smac/DIABLO release from mitochondria. Cancer Res 2001; 61: 7339–7348.

    CAS  PubMed  Google Scholar 

  33. Sun XM, Bratton SB, Butterworth M, MacFarlane M, Cohen GM. Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein. J Biol Chem 2002; 277: 11345–11351.

    CAS  PubMed  Google Scholar 

  34. Bratton SB, Cohen GM. Death receptors leave a caspase footprint that Smacs of XIAP. Cell Death Differ 2003; 10: 4–6.

    CAS  PubMed  Google Scholar 

  35. Garland JM, Rudin C. Cytochrome c induces caspase-dependent apoptosis in intact hematopoietic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling, MAP-kinase-kinase, and malignant change. Blood 1998; 92: 1235–1246.

    CAS  PubMed  Google Scholar 

  36. Ushmorov A, Ratter F, Lehmann V, Droge W, Schirrmacher V, Umansky V. Nitric-oxide-induced apoptosis in human leukemic lines requires mitochondrial lipid degradation and cytochrome C release. Blood 1999; 93: 2342–2352.

    CAS  PubMed  Google Scholar 

  37. Schimmer AD, Hedley DW, Penn LZ, Minden MD. Receptor-and mitochondrial-mediated apoptosis in acute leukemia: A translational view. Blood 2001; 98: 3541–3553.

    CAS  PubMed  Google Scholar 

  38. Ito Y, Mishra NC, Yoshida K, Kharbanda S, Saxena S, Kufe D. Mitochondrial targeting of JNK/SAPK in the phorbol ester response of myeloid leukemia cells. Cell Death Differ 2001; 8: 794–800.

    CAS  PubMed  Google Scholar 

  39. Amarante-Mendes GP, Naekyung Kim C, Liu L, et al. Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome C and activation of caspase-3. Blood 1998; 91: 1700–1705.

    CAS  PubMed  Google Scholar 

  40. Wei MC, Zong WX, Cheng EH, et al. Proapoptotic BAX and BAK: A requisite gateway to mitochondrial dysfunction and death. Science 2001; 292: 727–730.

    Article  CAS  PubMed  Google Scholar 

  41. Jia L, Srinivasula SM, Liu FT, et al. Apaf-1 protein deficiency confers resistance to cytochrome c-dependent apoptosis in human leukemic cells. Blood 2001; 98: 414–421.

    CAS  PubMed  Google Scholar 

  42. Chandra J, Mansson E, Gogvadze V, Kaufmann SH, Albertioni F, Orrenius S. Resistance of leukemic cells to 2-chlorodeoxyadenosine is due to a lack of calcium-dependent cytochrome c release. Blood 2002; 99: 655–663.

    CAS  PubMed  Google Scholar 

  43. Cuvillier O, Levade T. Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria. Blood 2001; 98: 2828–2836.

    CAS  PubMed  Google Scholar 

  44. Almond JB, Snowden RT, Hunter A, Dinsdale D, Cain K, Cohen GM. Proteasome inhibitor-induced apoptosis of B-chronic lymphocytic leukaemia cells involves cytochrome c release and caspase activation, accompanied by formation of an approximately 700 kDa Apaf-1 containing apoptosome complex. Leukemia 2001; 15: 1388–1397.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. University Children’s Hospital, Ulm, Germany

    K. Stahnke, A. Mohr, J. Liu, L. H. Meyer & K.-M. Debatin

  2. Helios Klinikum Berlin, Robert Rossle Clinic, Charité, Humboldt-University of Berlin, Berlin, Germany

    L. Karawajew

Authors
  1. K. Stahnke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Mohr
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L. H. Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. L. Karawajew
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. K.-M. Debatin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K.-M. Debatin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stahnke, K., Mohr, A., Liu, J. et al. Identification of deficient mitochondrial signaling in apoptosis resistant leukemia cells by flow cytometric analysis of intracellular cytochrome c, caspase-3 and apoptosis. Apoptosis 9, 457–465 (2004). https://doi.org/10.1023/B:APPT.0000031454.62937.fa

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:APPT.0000031454.62937.fa

Search

Navigation