Skip to main content
SpringerLink
Log in

HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

HAMLET (Human α-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

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

Abbreviations

HAMLET::

human α-lactalbumin made lethal to tumor cells

DEVD-AMC::

benzyloxycarbonyl-Asp-Glu- Val-Asp-7-amino-4-methylcoumarin

LEHD-AMC::

benzylo-xycarbonyl-Leu-Glu-His-Asp-7-amino-4-methyl coumarin

PS::

Phosphatidylserine

VDVAD-AMC::

benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-7-amino-4-methylcoumarin

zVAD-fmk::

benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone

COX-2::

Cyclooxygenase-2

References

  1. Jaattela M. Programmed cell death: Many ways for cells to die decently. Ann Med 2002; 34: 480–488.

    PubMed  Google Scholar 

  2. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239–257.

    CAS  PubMed  Google Scholar 

  3. Ellis HM, Horvitz HR. Genetic control of programmed cell death in the nematode C. elegans. Cell 1986; 44: 817–829.

    CAS  Google Scholar 

  4. Li LY, Luo X, Wang X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 2001; 412: 95–99.

    CAS  PubMed  Google Scholar 

  5. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 1997; 91: 627–637.

    Google Scholar 

  6. Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med 2000; 6: 513–519. l.

    Article  CAS  PubMed  Google Scholar 

  7. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281: 1309–1312.

    CAS  PubMed  Google Scholar 

  8. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999; 13: 1899–1911.

    CAS  PubMed  Google Scholar 

  9. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis [see comments]. Science 1997; 275: 1132–1136.

    Article  CAS  PubMed  Google Scholar 

  10. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked [see comments]. Science 1997; 275: 1129–1132.

    Article  CAS  PubMed  Google Scholar 

  11. Bossy-Wetzel E, Newmeyer DD, Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD- specific caspase activation and independently of mitochondrial transmembrane depolarization. Embo J 1998; 17: 37–49.

    Article  CAS  PubMed  Google Scholar 

  12. Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol 1999; 17: 2941–2953.

    CAS  PubMed  Google Scholar 

  13. Real PJ, Cao Y, Wang R, et al. Breast cancer cells can evade apoptosis- mediated selective killing by a novel small molecule inhibitor of Bcl-2. Cancer Res 2004; 64: 7947–7953.

    Article  CAS  PubMed  Google Scholar 

  14. Huang Z. Bcl-2 family proteins as targets for anticancer drug design. Oncogene 2000; 19: 6627–6631.

    CAS  PubMed  Google Scholar 

  15. Lane DP. Cancer. A death in the life of p53 [news; comment]. Nature 1993; 362: 786–787.

    Article  CAS  PubMed  Google Scholar 

  16. Bates S, Vousden KH. Mechanisms of p53-mediated apoptosis. Cell Mol Life Sci 1999; 55: 28–37.

    CAS  PubMed  Google Scholar 

  17. Cheng J, Haas M. Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines. Mol Cell Biol. 1990; 10: 5502–5509.

    CAS  PubMed  Google Scholar 

  18. Lowe SW, Bodis S, McClatchey A, et al. p53 status and the efficacy of cancer therapy in vivo. Science 1994; 266: 807–810.

    CAS  PubMed  Google Scholar 

  19. Roemer K. Mutant p53: Gain-of-function oncoproteins and wild-type p53 inactivators. Biol Chem 1999; 380: 879–887.

    Article  CAS  PubMed  Google Scholar 

  20. Robertson JD, Enoksson M, Suomela M, Zhivotovsky B, Orrenius S. Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J Biol Chem 2002; 277: 29803–29809.

    Article  CAS  PubMed  Google Scholar 

  21. Robertson JD, Gogvadze V, Kropotov A, et al. Processed caspase-2 can induce mitochondria-mediated apoptosis independently of its enzymatic activity. EMBO Rep 2004; 5: 643–648.

    Article  CAS  PubMed  Google Scholar 

  22. Enoksson M, Robertson JD, Gogvadze V, et al. Caspase-2 permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids. J Biol Chem 2004.

  23. Bykov VJ, Issaeva N, Shilov A, et al. Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat Med 2002; 8: 282–288.

    Article  CAS  PubMed  Google Scholar 

  24. Håkansson A, Zhivotovsky B, Orrenius S, Sabharwal H, Svanborg C. Apoptosis induced by a human milk protein. Proc Natl Acad Sci USA 1995; 92: 8064–8068.

    PubMed  Google Scholar 

  25. Fischer W, Gustafsson L, Mossberg AK, et al. Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival. Cancer Res 2004; 64: 2105–2112.

    Article  CAS  PubMed  Google Scholar 

  26. Gustafsson L, Leijonhufvud I, Aronsson A, Mossberg AK, Svanborg C. Treatment of skin papillomas with topical alpha-lactalbumin-oleic acid. N Engl J Med 2004; 350: 2663–2672.

    Article  CAS  PubMed  Google Scholar 

  27. Duringer C, Hamiche A, Gustafsson L, Kimura H, Svanborg C. HAMLET interacts with histones and chromatin in tumor cell nuclei. J Biol Chem 2003; 278: 42131–42135.

    Article  PubMed  Google Scholar 

  28. Svensson M, Hakansson A, Mossberg AK, Linse S, Svanborg C. Conversion of alpha-lactalbumin to a protein inducing apoptosis. Proc Natl Acad Sci USA 2000; 97: 4221–4226.

    Article  CAS  PubMed  Google Scholar 

  29. Kohler C, Håkansson A, Svanborg C, Orrenius S, Zhivotovsky B. Protease activation in apoptosis induced by MAL. Exp Cell Res 1999; 249: 260–268.

    Article  CAS  PubMed  Google Scholar 

  30. Håkansson A, Kidd A, Wadell G, Sabharwal H, Svanborg C. Adenovirus infection enhances in vitro adherence of Streptococcus pneumoniae. Infect Immun 1994; 62: 2707–2714.

    PubMed  Google Scholar 

  31. Zhivotovsky B, Nicotera P, Bellomo G, Hanson K, Orrenius S. Ca2+ and endonuclease activation in radiation-induced lymphoid cell death. Exp Cell Res 1993; 207: 163–170 issn: 0014–4827.

    CAS  PubMed  Google Scholar 

  32. Armstrong RC, Aja T, Xiang J, et al. Fas-induced activation of the cell death-related protease CPP32 Is inhibited by Bcl-2 and by ICE family protease inhibitors. J Biol Chem 1996; 271: 16850–16855.

    Article  CAS  PubMed  Google Scholar 

  33. Boise LH, Gonzalez-Garcia M, Postema CE, et al. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993; 74: 597–608.

    Article  CAS  PubMed  Google Scholar 

  34. Pugacheva EN, Ivanov AV, Kravchenko JE, et al. Novel gain of function activity of p53 mutants: activation of the dUTPase gene expression leading to resistance to 5–fluorouracil. Oncogene 2002; 21: 4595–4600.

    Article  CAS  PubMed  Google Scholar 

  35. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998; 282: 1497–1501.

    Article  CAS  PubMed  Google Scholar 

  36. Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995; 376: 37–43.

    Article  CAS  PubMed  Google Scholar 

  37. Håkansson A, Andreasson J, Zhivotovsky B, et al. Multimeric alpha-lactalbumin from human milk induces apoptosis through a direct effect on cell nuclei. Exp Cell Res 1999; 246: 451–460.

    Article  PubMed  Google Scholar 

  38. Kimura H, Cook PR. Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. J Cell Biol 2001; 153: 1341–1353.

    Article  CAS  PubMed  Google Scholar 

  39. Blandino G, Levine AJ, Oren M. Mutant p53 gain of function: Differential effects of different p53 mutants on resistance of cultured cells to chemotherapy. Oncogene 1999; 18: 477–485.

    Article  CAS  PubMed  Google Scholar 

  40. Leist M, Jaattela M. Four deaths and a funeral: From caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2001; 2: 589–598.

    Article  CAS  PubMed  Google Scholar 

  41. Jaattela M, Tschopp J. Caspase-independent cell death in T lymphocytes. Nat Immunol 2003; 4: 416–423.

    PubMed  Google Scholar 

  42. Kohler C, Gogvadze V, Hakansson A, et al. A folding variant of human alpha-lactalbumin induces mitochondrial permeability transition in isolated mitochondria. Eur J Biochem 2001; 268: 186–191.

    Article  CAS  PubMed  Google Scholar 

  43. Johnson AJ, Smith LL, Zhu J, et al. A novel celecoxib derivative, OSU03012, induces cytotoxicity in primary CLL cells and transformed B-cell lymphoma cell line via a caspase- and Bcl-2-independent mechanism. Blood 2005; 105: 2504–2509.

    CAS  PubMed  Google Scholar 

  44. Jendrossek V, Handrick R, Belka C. Celecoxib activates a novel mitochondrial apoptosis signaling pathway. Faseb J 2003; 17: 1547–1549.

    CAS  PubMed  Google Scholar 

  45. Elliott K, Ge K, Du W, Prendergast GC. The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program. Oncogene 2000; 19: 4669–4684.

    Article  CAS  PubMed  Google Scholar 

  46. Chi S, Kitanaka C, Noguchi K, et al. Oncogenic Ras triggers cell suicide through the activation of a caspase-independent cell death program in human cancer cells. Oncogene 1999; 18: 2281–2290.

    Article  CAS  PubMed  Google Scholar 

  47. Yamaki M, Umehara T, Chimura T, Horikoshi M. Cell death with predominant apoptotic features in Saccharomyces cerevisiae mediated by deletion of the histone chaperone ASF1/CIA1. Genes Cells 2001; 6: 1043–1054.

    Article  CAS  PubMed  Google Scholar 

  48. Monney L, Otter I, Olivier R, et al. Defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by Bcl-2. J Biol Chem 1998; 273: 6121–6131.

    Article  CAS  PubMed  Google Scholar 

  49. Mathiasen IS, Sergeev IN, Bastholm L, et al. Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells. J Biol Chem 2002; 277: 30738–30745.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden

    O. Hallgren, L. Gustafsson, H. Irjala & C. Svanborg

  2. Department of Physics, Lund Institute of Technology and Lund University Medical Laser Centre, Lund, Sweden

    L. Gustafsson

  3. Medicity Research Laboratory, University of Turku, Turku, Finland

    H. Irjala

  4. Department of Oncology-Pathology, Cancer Center Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden

    G. Selivanova

  5. Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

    S. Orrenius

  6. Section for MIG, Lund University, Solvegatan 23, 223 62, Lund, Sweden

    C. Svanborg

Authors
  1. O. Hallgren
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. Gustafsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. Irjala
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Selivanova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S. Orrenius
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C. Svanborg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. Svanborg.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hallgren, O., Gustafsson, L., Irjala, H. et al. HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53. Apoptosis 11, 221–233 (2006). https://doi.org/10.1007/s10495-006-3607-7

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-006-3607-7

Keywords

Search

Navigation