Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 May 15;340(Pt 1):127–133.

Inhibition of ubiquitin-proteasome pathway activates a caspase-3-like protease and induces Bcl-2 cleavage in human M-07e leukaemic cells.

X M Zhang 1, H Lin 1, C Chen 1, B D Chen 1
PMCID: PMC1220230  PMID: 10229667

Abstract

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Here we examine the possibility that ubiquitin-proteasome is involved in regulating the levels of Bcl-2, which is abundantly expressed in M-07e cells, a granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent human leukaemic cell line. Apoptosis in M-07e cells, induced by GM-CSF withdrawal, was associated with a gradual cleavage of Bcl-2 into a 22 kDa fragment. Treatment of M-07e cells with benzyloxycarbonyl-Leu-Leu-l-leucinal (Z-LLL-CHO; MG-132), a reversible ubiquitin-proteasome inhibitor, markedly accelerated the cleavage of Bcl-2 and promoted cell death through the apoptotic pathway. The cleavage of Bcl-2 was inhibited by a caspase-3 (CPP32)-specific inhibitor [acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO)] but not caspase 1 inhibitor (acetyl-Tyr-Val-Ala-Asp-CHO), suggesting that Bcl-2 is a proteolytic substrate of a caspase-3-like protease activated during apoptosis. The simultaneous addition of recombinant human GM-CSF (rhGM-CSF) to M-07e cultures delayed the activation of caspase 3 and Bcl-2 cleavage triggered by Z-LLL-CHO, suggesting that the activation of the GM-CSF signalling pathway can partly overcome the apoptotic effect induced by Z-LLL-CHO. Apoptosis induced by inhibition of the proteasome pathway was verified in studies with lactacystin, a highly specific and irreversible proteasome inhibitor. Lactacystin-induced apoptosis in M-07e cells was remarkably similar to that induced by Z-LLL-CHO, which included caspase 3 activation, cleavage of Bcl-2 into a 22 kDa fragment and, ultimately, cell death. These results showed that inhibition of the ubiquitin-proteasome pathways can lead to the activation of a DEVD-CHO-sensitive caspase and induces Bcl-2 cleavage, which might have a role in mediating apoptosis in M-07e cells.

Full Text

The Full Text of this article is available as a PDF (226.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armstrong R. C., Aja T., Xiang J., Gaur S., Krebs J. F., Hoang K., Bai X., Korsmeyer S. J., Karanewsky D. S., Fritz L. C. 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 Jul 12;271(28):16850–16855. doi: 10.1074/jbc.271.28.16850. [DOI] [PubMed] [Google Scholar]
  2. Beyette J., Mason G. G., Murray R. Z., Cohen G. M., Rivett A. J. Proteasome activities decrease during dexamethasone-induced apoptosis of thymocytes. Biochem J. 1998 Jun 1;332(Pt 2):315–320. doi: 10.1042/bj3320315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang Y. C., Lee Y. S., Tejima T., Tanaka K., Omura S., Heintz N. H., Mitsui Y., Magae J. mdm2 and bax, downstream mediators of the p53 response, are degraded by the ubiquitin-proteasome pathway. Cell Growth Differ. 1998 Jan;9(1):79–84. [PubMed] [Google Scholar]
  4. Chen R. H., Chang T. Y. Involvement of caspase family proteases in transforming growth factor-beta-induced apoptosis. Cell Growth Differ. 1997 Jul;8(7):821–827. [PubMed] [Google Scholar]
  5. Cheng E. H., Kirsch D. G., Clem R. J., Ravi R., Kastan M. B., Bedi A., Ueno K., Hardwick J. M. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science. 1997 Dec 12;278(5345):1966–1968. doi: 10.1126/science.278.5345.1966. [DOI] [PubMed] [Google Scholar]
  6. Cohen G. M. Caspases: the executioners of apoptosis. Biochem J. 1997 Aug 15;326(Pt 1):1–16. doi: 10.1042/bj3260001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drexler H. C. Activation of the cell death program by inhibition of proteasome function. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):855–860. doi: 10.1073/pnas.94.3.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ha H. C., Woster P. M., Casero R. A., Jr Unsymmetrically substituted polyamine analogue induces caspase-independent programmed cell death in Bcl-2-overexpressing cells. Cancer Res. 1998 Jul 1;58(13):2711–2714. [PubMed] [Google Scholar]
  9. Hershko A. Roles of ubiquitin-mediated proteolysis in cell cycle control. Curr Opin Cell Biol. 1997 Dec;9(6):788–799. doi: 10.1016/s0955-0674(97)80079-8. [DOI] [PubMed] [Google Scholar]
  10. Hochstrasser M. Ubiquitin-dependent protein degradation. Annu Rev Genet. 1996;30:405–439. doi: 10.1146/annurev.genet.30.1.405. [DOI] [PubMed] [Google Scholar]
  11. Hsu C. A., Rishi A. K., Su-Li X., Gerald T. M., Dawson M. I., Schiffer C., Reichert U., Shroot B., Poirer G. C., Fontana J. A. Retinoid induced apoptosis in leukemia cells through a retinoic acid nuclear receptor-independent pathway. Blood. 1997 Jun 15;89(12):4470–4479. [PubMed] [Google Scholar]
  12. Imajoh-Ohmi S., Kawaguchi T., Sugiyama S., Tanaka K., Omura S., Kikuchi H. Lactacystin, a specific inhibitor of the proteasome, induces apoptosis in human monoblast U937 cells. Biochem Biophys Res Commun. 1995 Dec 26;217(3):1070–1077. doi: 10.1006/bbrc.1995.2878. [DOI] [PubMed] [Google Scholar]
  13. Kelly M. L., Tang Y., Rosensweig N., Clejan S., Beckman B. S. Granulocyte-macrophage colony-stimulating factor rescues TF-1 leukemia cells from ionizing radiation-induced apoptosis through a pathway mediated by protein kinase Calpha. Blood. 1998 Jul 15;92(2):416–424. [PubMed] [Google Scholar]
  14. Kim Y. M., Kim T. H., Seol D. W., Talanian R. V., Billiar T. R. Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release. J Biol Chem. 1998 Nov 20;273(47):31437–31441. doi: 10.1074/jbc.273.47.31437. [DOI] [PubMed] [Google Scholar]
  15. Kitanaka C., Namiki T., Noguchi K., Mochizuki T., Kagaya S., Chi S., Hayashi A., Asai A., Tsujimoto Y., Kuchino Y. Caspase-dependent apoptosis of COS-7 cells induced by Bax overexpression: differential effects of Bcl-2 and Bcl-xL on Bax-induced caspase activation and apoptosis. Oncogene. 1997 Oct 9;15(15):1763–1772. doi: 10.1038/sj.onc.1201349. [DOI] [PubMed] [Google Scholar]
  16. Korsmeyer S. J. Regulators of cell death. Trends Genet. 1995 Mar;11(3):101–105. doi: 10.1016/S0168-9525(00)89010-1. [DOI] [PubMed] [Google Scholar]
  17. Li Y., Shen B. F., Karanes C., Sensenbrenner L., Chen B. Association between Lyn protein tyrosine kinase (p53/56lyn) and the beta subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in a GM-CSF-dependent human megakaryocytic leukemia cell line (M-07e). J Immunol. 1995 Aug 15;155(4):2165–2174. [PubMed] [Google Scholar]
  18. Li Y., Valeriote F., Chen B. Regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in a GM-CSF-dependent human myeloid leukemia cell line (AML-193) by interleukin-6. Exp Hematol. 1996 Feb;24(2):94–100. [PubMed] [Google Scholar]
  19. Meriin A. B., Gabai V. L., Yaglom J., Shifrin V. I., Sherman M. Y. Proteasome inhibitors activate stress kinases and induce Hsp72. Diverse effects on apoptosis. J Biol Chem. 1998 Mar 13;273(11):6373–6379. doi: 10.1074/jbc.273.11.6373. [DOI] [PubMed] [Google Scholar]
  20. Miller D. K. The role of the Caspase family of cysteine proteases in apoptosis. Semin Immunol. 1997 Feb;9(1):35–49. doi: 10.1006/smim.1996.0058. [DOI] [PubMed] [Google Scholar]
  21. Miyajima A., Mui A. L., Ogorochi T., Sakamaki K. Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Blood. 1993 Oct 1;82(7):1960–1974. [PubMed] [Google Scholar]
  22. Ohta T., Kinoshita T., Naito M., Nozaki T., Masutani M., Tsuruo T., Miyajima A. Requirement of the caspase-3/CPP32 protease cascade for apoptotic death following cytokine deprivation in hematopoietic cells. J Biol Chem. 1997 Sep 12;272(37):23111–23116. doi: 10.1074/jbc.272.37.23111. [DOI] [PubMed] [Google Scholar]
  23. Pickart C. M. Targeting of substrates to the 26S proteasome. FASEB J. 1997 Nov;11(13):1055–1066. doi: 10.1096/fasebj.11.13.9367341. [DOI] [PubMed] [Google Scholar]
  24. Reed J. C. Bcl-2 family proteins: regulators of apoptosis and chemoresistance in hematologic malignancies. Semin Hematol. 1997 Oct;34(4 Suppl 5):9–19. [PubMed] [Google Scholar]
  25. Renvoizé C., Roger R., Moulian N., Bertoglio J., Bréard J. Bcl-2 expression in target cells leads to functional inhibition of caspase-3 protease family in human NK and lymphokine-activated killer cell granule-mediated apoptosis. J Immunol. 1997 Jul 1;159(1):126–134. [PubMed] [Google Scholar]
  26. Rinkenberger J. L., Korsmeyer S. J. Errors of homeostasis and deregulated apoptosis. Curr Opin Genet Dev. 1997 Oct;7(5):589–596. doi: 10.1016/s0959-437x(97)80004-4. [DOI] [PubMed] [Google Scholar]
  27. Shinohara K., Tomioka M., Nakano H., Toné S., Ito H., Kawashima S. Apoptosis induction resulting from proteasome inhibition. Biochem J. 1996 Jul 15;317(Pt 2):385–388. doi: 10.1042/bj3170385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wei S., Liu J. H., Epling-Burnette P. K., Gamero A. M., Ussery D., Pearson E. W., Elkabani M. E., Diaz J. I., Djeu J. Y. Critical role of Lyn kinase in inhibition of neutrophil apoptosis by granulocyte-macrophage colony-stimulating factor. J Immunol. 1996 Dec 1;157(11):5155–5162. [PubMed] [Google Scholar]
  29. Whitacre C. M., Hashimoto H., Tsai M. L., Chatterjee S., Berger S. J., Berger N. A. Involvement of NAD-poly(ADP-ribose) metabolism in p53 regulation and its consequences. Cancer Res. 1995 Sep 1;55(17):3697–3701. [PubMed] [Google Scholar]
  30. Yang J., Liu X., Bhalla K., Kim C. N., Ibrado A. M., Cai J., Peng T. I., Jones D. P., Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997 Feb 21;275(5303):1129–1132. doi: 10.1126/science.275.5303.1129. [DOI] [PubMed] [Google Scholar]
  31. Yasuhara N., Sahara S., Kamada S., Eguchi Y., Tsujimoto Y. Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis. Oncogene. 1997 Oct 16;15(16):1921–1928. doi: 10.1038/sj.onc.1201370. [DOI] [PubMed] [Google Scholar]
  32. Zamzami N., Brenner C., Marzo I., Susin S. A., Kroemer G. Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins. Oncogene. 1998 Apr 30;16(17):2265–2282. doi: 10.1038/sj.onc.1201989. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES