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. 2001 Jun 15;356(Pt 3):835–841. doi: 10.1042/0264-6021:3560835

Proteasomes are a target of the anti-tumour drug vinblastine.

M Piccinini 1, O Tazartes 1, C Mezzatesta 1, E Ricotti 1, S Bedino 1, F Grosso 1, U Dianzani 1, P A Tovo 1, M Mostert 1, A Musso 1, M T Rinaudo 1
PMCID: PMC1221911  PMID: 11389692

Abstract

Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.

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Selected References

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

  1. André P., Groettrup M., Klenerman P., de Giuli R., Booth B. L., Jr, Cerundolo V., Bonneville M., Jotereau F., Zinkernagel R. M., Lotteau V. An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses. Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13120–13124. doi: 10.1073/pnas.95.22.13120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arendt C. S., Hochstrasser M. Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7156–7161. doi: 10.1073/pnas.94.14.7156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baeuerle P. A., Baltimore D. NF-kappa B: ten years after. Cell. 1996 Oct 4;87(1):13–20. doi: 10.1016/s0092-8674(00)81318-5. [DOI] [PubMed] [Google Scholar]
  4. Baldwin A. S., Jr The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649–683. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]
  5. Baumeister W., Walz J., Zühl F., Seemüller E. The proteasome: paradigm of a self-compartmentalizing protease. Cell. 1998 Feb 6;92(3):367–380. doi: 10.1016/s0092-8674(00)80929-0. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Braun B. C., Glickman M., Kraft R., Dahlmann B., Kloetzel P. M., Finley D., Schmidt M. The base of the proteasome regulatory particle exhibits chaperone-like activity. Nat Cell Biol. 1999 Aug;1(4):221–226. doi: 10.1038/12043. [DOI] [PubMed] [Google Scholar]
  8. Brown K., Gerstberger S., Carlson L., Franzoso G., Siebenlist U. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science. 1995 Mar 10;267(5203):1485–1488. doi: 10.1126/science.7878466. [DOI] [PubMed] [Google Scholar]
  9. Coux O., Tanaka K., Goldberg A. L. Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem. 1996;65:801–847. doi: 10.1146/annurev.bi.65.070196.004101. [DOI] [PubMed] [Google Scholar]
  10. Dubiel W., Pratt G., Ferrell K., Rechsteiner M. Purification of an 11 S regulator of the multicatalytic protease. J Biol Chem. 1992 Nov 5;267(31):22369–22377. [PubMed] [Google Scholar]
  11. Dumontet C., Sikic B. I. Mechanisms of action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death. J Clin Oncol. 1999 Mar;17(3):1061–1070. doi: 10.1200/JCO.1999.17.3.1061. [DOI] [PubMed] [Google Scholar]
  12. Fenteany G., Standaert R. F., Lane W. S., Choi S., Corey E. J., Schreiber S. L. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science. 1995 May 5;268(5211):726–731. doi: 10.1126/science.7732382. [DOI] [PubMed] [Google Scholar]
  13. Ferguson P. J., Cass C. E. Differential cellular retention of vincristine and vinblastine by cultured human promyelocytic leukemia HL-60/Cl cells: the basis of differential toxicity. Cancer Res. 1985 Nov;45(11 Pt 1):5480–5488. [PubMed] [Google Scholar]
  14. Figueiredo-Pereira M. E., Chen W. E., Li J., Johdo O. The antitumor drug aclacinomycin A, which inhibits the degradation of ubiquitinated proteins, shows selectivity for the chymotrypsin-like activity of the bovine pituitary 20 S proteasome. J Biol Chem. 1996 Jul 12;271(28):16455–16459. doi: 10.1074/jbc.271.28.16455. [DOI] [PubMed] [Google Scholar]
  15. Haas A. L., Siepmann T. J. Pathways of ubiquitin conjugation. FASEB J. 1997 Dec;11(14):1257–1268. doi: 10.1096/fasebj.11.14.9409544. [DOI] [PubMed] [Google Scholar]
  16. Hoffman L., Pratt G., Rechsteiner M. Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate. J Biol Chem. 1992 Nov 5;267(31):22362–22368. [PubMed] [Google Scholar]
  17. Hough R., Rechsteiner M. Ubiquitin-lysozyme conjugates. Purification and susceptibility to proteolysis. J Biol Chem. 1986 Feb 15;261(5):2391–2399. [PubMed] [Google Scholar]
  18. Jordan M. A., Thrower D., Wilson L. Mechanism of inhibition of cell proliferation by Vinca alkaloids. Cancer Res. 1991 Apr 15;51(8):2212–2222. [PubMed] [Google Scholar]
  19. King R. W., Deshaies R. J., Peters J. M., Kirschner M. W. How proteolysis drives the cell cycle. Science. 1996 Dec 6;274(5293):1652–1659. doi: 10.1126/science.274.5293.1652. [DOI] [PubMed] [Google Scholar]
  20. Koepp D. M., Harper J. W., Elledge S. J. How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell. 1999 May 14;97(4):431–434. doi: 10.1016/s0092-8674(00)80753-9. [DOI] [PubMed] [Google Scholar]
  21. Kung A. L., Zetterberg A., Sherwood S. W., Schimke R. T. Cytotoxic effects of cell cycle phase specific agents: result of cell cycle perturbation. Cancer Res. 1990 Nov 15;50(22):7307–7317. [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Lee D. H., Goldberg A. L. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol. 1998 Oct;8(10):397–403. doi: 10.1016/s0962-8924(98)01346-4. [DOI] [PubMed] [Google Scholar]
  24. Meyer S., Kohler N. G., Joly A. Cyclosporine A is an uncompetitive inhibitor of proteasome activity and prevents NF-kappaB activation. FEBS Lett. 1997 Aug 18;413(2):354–358. doi: 10.1016/s0014-5793(97)00930-7. [DOI] [PubMed] [Google Scholar]
  25. Michetti M., Salamino F., Tedesco I., Averna M., Minafra R., Melloni E., Pontremoli S. Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization. FEBS Lett. 1996 Aug 19;392(1):11–15. doi: 10.1016/0014-5793(96)00775-2. [DOI] [PubMed] [Google Scholar]
  26. Nasmyth K. Separating sister chromatids. Trends Biochem Sci. 1999 Mar;24(3):98–104. doi: 10.1016/s0968-0004(99)01358-4. [DOI] [PubMed] [Google Scholar]
  27. Nogales E. Structural insights into microtubule function. Annu Rev Biochem. 2000;69:277–302. doi: 10.1146/annurev.biochem.69.1.277. [DOI] [PubMed] [Google Scholar]
  28. Pagano M. Cell cycle regulation by the ubiquitin pathway. FASEB J. 1997 Nov;11(13):1067–1075. doi: 10.1096/fasebj.11.13.9367342. [DOI] [PubMed] [Google Scholar]
  29. Palombella V. J., Rando O. J., Goldberg A. L., Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell. 1994 Sep 9;78(5):773–785. doi: 10.1016/s0092-8674(94)90482-0. [DOI] [PubMed] [Google Scholar]
  30. Piccinini M., Merighi A., Bruno R., Cascio P., Curto M., Mioletti S., Ceruti C., Rinaudo M. T. Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina. Biochem J. 1996 Sep 1;318(Pt 2):711–716. doi: 10.1042/bj3180711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Piccinini M., Tazartes O., Mostert M., Musso A., DeMarchi M., Rinaudo M. T. Structural and functional characterization of 20S and 26S proteasomes from bovine brain. Brain Res Mol Brain Res. 2000 Mar 10;76(1):103–114. doi: 10.1016/s0169-328x(99)00337-x. [DOI] [PubMed] [Google Scholar]
  32. Rechsteiner M., Realini C., Ustrell V. The proteasome activator 11 S REG (PA28) and class I antigen presentation. Biochem J. 2000 Jan 1;345(Pt 1):1–15. [PMC free article] [PubMed] [Google Scholar]
  33. Reich J. G., Wangermann G., Falck M., Rohde K. A general strategy for parameter estimation from isosteric and allosteric-kinetic data and binding measurements. Eur J Biochem. 1972 Apr 11;26(3):368–379. doi: 10.1111/j.1432-1033.1972.tb01776.x. [DOI] [PubMed] [Google Scholar]
  34. Schwartz A. L., Ciechanover A. The ubiquitin-proteasome pathway and pathogenesis of human diseases. Annu Rev Med. 1999;50:57–74. doi: 10.1146/annurev.med.50.1.57. [DOI] [PubMed] [Google Scholar]
  35. Shirayama M., Tóth A., Gálová M., Nasmyth K. APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5. Nature. 1999 Nov 11;402(6758):203–207. doi: 10.1038/46080. [DOI] [PubMed] [Google Scholar]
  36. Singer W. D., Himes R. H. Cellular uptake and tubulin binding properties of four Vinca alkaloids. Biochem Pharmacol. 1992 Feb 4;43(3):545–551. doi: 10.1016/0006-2952(92)90577-6. [DOI] [PubMed] [Google Scholar]
  37. Spataro V., Norbury C., Harris A. L. The ubiquitin-proteasome pathway in cancer. Br J Cancer. 1998;77(3):448–455. doi: 10.1038/bjc.1998.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stein R. L., Melandri F., Dick L. Kinetic characterization of the chymotryptic activity of the 20S proteasome. Biochemistry. 1996 Apr 2;35(13):3899–3908. doi: 10.1021/bi952262x. [DOI] [PubMed] [Google Scholar]
  39. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Van Belle S. J., De Smet M. C., De Mey J. E., Storme G. A. Cellular pharmacokinetics of vinblastine and other vinca alkaloids in MO4 cells. Anticancer Res. 1991 Jan-Feb;11(1):465–471. [PubMed] [Google Scholar]
  41. Weissman A. M. Regulating protein degradation by ubiquitination. Immunol Today. 1997 Apr;18(4):189–198. doi: 10.1016/s0167-5699(97)84666-x. [DOI] [PubMed] [Google Scholar]
  42. Zou H., McGarry T. J., Bernal T., Kirschner M. W. Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science. 1999 Jul 16;285(5426):418–422. doi: 10.1126/science.285.5426.418. [DOI] [PubMed] [Google Scholar]

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