Skip to main content

Advertisement

SpringerLink
Log in

Class III β-Tubulin and γ-Tubulin are Co-expressed and Form Complexes in Human Glioblastoma Cells

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

We have previously shown that the neuronal-associated class III β-tubulin isotype and the centrosome-associated γ-tubulin are aberrantly expressed in astrocytic gliomas (Cell Motil Cytoskeleton 2003, 55:77-96; J Neuropathol Exp Neurol 2006, 65:455–467). Here we determined the expression, distribution and interaction of βIII-tubulin and γ-tubulin in diffuse-type astrocytic gliomas (grades II-IV) (n = 17) and the human glioblastoma cell line T98G. By immunohistochemistry and immunofluorescence microscopy, βIII-tubulin and γ-tubulin were co-distributed in anaplastic astrocytomas and glioblastomas and to a lesser extent, in low-grade diffuse astrocytomas (P < 0.05). In T98G glioblastoma cells βIII-tubulin was associated with microtubules whereas γ-tubulin exhibited striking diffuse cytoplasmic staining in addition to its expectant centrosome-associated pericentriolar distribution. Treatment with different anti-microtubule drugs revealed that βIII-tubulin was not associated with insoluble γ-tubulin aggregates. On the other hand, immunoprecipitation experiments unveiled that both tubulins formed complexes in soluble cytoplasmic pools, where substantial amounts of these proteins were located. We suggest that aberrant expression and interactions of βIII-tubulin and γ-tubulin may be linked to malignant changes in glial 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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ANOVA:

Analysis of variance

Cy3:

Indocarbocyanate

DAPI:

4′-6-Diamidino-2-phenylindole

DMEM:

Dulbecco’s modified Eagle medium

EDTA:

Ethylene diamine tetraacetic acid

EGTA:

Ethylene glycol tetraacetic acid

FITC:

Fluorescein isothiocyanate

GCP:

γ-Tubulin complex protein

γ-TuRC:

Large γ-tubulin-ring complex

γ-TuSC:

γ-Tubulin-small complex

LI:

Labeling index

MES:

2-(N-morpholine)-ethane sulphonic acid

MLI:

Mean labeling index

MSB:

Microtubule-stabilizing buffer

MTOC:

Microtubule organizing center

TBST:

Tris-Buffered Saline Tween-20

References

  1. Katsetos CD, Herman MM, Mörk SJ (2003) Class III β-tubulin in human development and cancer. Cell Motil Cytoskeleton 55:77–96

    Article  PubMed  CAS  Google Scholar 

  2. Katsetos CD, Legido A, Perentes E et al (2003) Class III β-tubulin isotype: a key cytoskeletal protein at the crossroads of developmental neurobiology and tumor neuropathology. J Child Neurol 18:851–866

    PubMed  Google Scholar 

  3. Dráberová E, Lukáš A Ivanyi D et al (1998) Expression of class III β-tubulin in normal and neoplastic human tissues. Histochem Cell Biol 109:231–239

    Article  PubMed  Google Scholar 

  4. Katsetos CD, Del Valle L, Legido A et al (2003) On the neuronal/neuroblastic nature of medulloblastomas: a tribute to Pio del Rio Hortega and Moises Polak. Acta Neuropathol (Berl) 105:1–13

    Google Scholar 

  5. Katsetos CD, Del Valle L, Geddes JF et al. (2001) Aberrant localization of the neuronal class III β-tubulin in astrocytomas: a marker for anaplastic potential. Arch Pathol Lab Med 125:613–624

    PubMed  CAS  Google Scholar 

  6. Martinez-Diaz H, Kleinschmidt-DeMasters BK, Powell SZ et al (2003) Giant cell glioblastoma and pleomorphic xanthoastrocytoma show different immunohistochemical profiles for neuronal antigens and p53 but share reactivity for class III β-tubulin. Arch Pathol Lab Med 127:1187–1191

    PubMed  CAS  Google Scholar 

  7. Katsetos CD, Del Valle L, Geddes JF et al (2002) Localization of the neuronal class III β-tubulin in oligodendrogliomas: Comparison with Ki-67 proliferative index and 1p/19q status. J Neuropathol Exp Neurol 61:307–320

    PubMed  CAS  Google Scholar 

  8. Giannini C, Scheithauer BW, Lopes MB et al (2002) Immunophenotype of pleomorphic xanthoastrocytoma. Am J Surg Pathol 26:479–485

    Article  PubMed  Google Scholar 

  9. Aldaz H, Rice LM, Stearns T et al (2005) Insights into microtubule nucleation from the crystal structure of human γ-tubulin. Nature 435:523–527

    Article  PubMed  CAS  Google Scholar 

  10. Joshi HC, Palacios MJ, McNamara L et al (1992) γ-Tubulin is a centrosomal protein required for cell cycle-dependent nucleation. Nature 356:80–82

    Article  PubMed  CAS  Google Scholar 

  11. Zheng Y, Wong ML, Alberts B et al (1995) Nucleation of microtubule assembly by γ-tubulin-containing ring complex. Nature 378:578–583

    Article  PubMed  CAS  Google Scholar 

  12. Zhou J, Shu H-B, Joshi HC (2002) Regulation of tubulin synthesis and cell cycle progression in mammalian cells by γ-tubulin-mediated microtubule nucleation. J Cell Biochem 84:472–483

    Article  PubMed  CAS  Google Scholar 

  13. Inclan YF, Nogales E (2001) Structural models for the self-assembly and microtubule interactions of γ-, δ- and ε-tubulin. J Cell Sci 114:413–422

    PubMed  CAS  Google Scholar 

  14. Llanos R, Chevrier V, Ronjat M et al (1999) Tubulin binding sites on γ-tubulin: identification and molecular characterization. Biochemistry 38:15712–15720

    Article  PubMed  CAS  Google Scholar 

  15. Paluh JL, Nogales E, Oakley BR et al (2000) A mutation in γ-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin like protein Pkl1p. Mol Biol Cell 11:1225–1239

    PubMed  CAS  Google Scholar 

  16. Sulimenko V, Sulimenko T, Poznanovic S et al (2002) Association of brain γ- tubulins with αβ-tubulin dimers. Biochem J 365:889–895

    PubMed  CAS  Google Scholar 

  17. Katsetos CD, Reddy G, Dráberová E et al (2006) Altered cellular distribution and subcellular sorting of γ-tubulin in astrocytic gliomas and human glioblastoma cell lines. J Neuropathol Exp Neurol 65:465–477

    Article  PubMed  CAS  Google Scholar 

  18. Stein GH (1979) T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro. J Cell Physiol 99:43–54

    Article  PubMed  CAS  Google Scholar 

  19. Ito H, Kanzawa T, Kondo S et al (2005) Microtubule inhibitor D-24851 induces p53- independent apoptotic cell death in malignant glioma cells through Bcl2 phosphorylation and Bax translocation. Int J Oncol 26:589–596

    PubMed  CAS  Google Scholar 

  20. Alexander JE, Hunt DF, Shabanowitz J et al (1991) Characterization of posttranslational modifications in neuron specific class III β-tubulin by mass spectrometry. Proc Natl Acad Sci (USA) 88:4685–89

    Article  CAS  Google Scholar 

  21. Lee MK, Tuttle JB, Rebhun LI et al (1990) The expression and posttranslational modification of a neuron-specific β-tubulin isotype during chick embryogenesis. Cell Motil Cytoskeleton 17:118–132

    Article  PubMed  CAS  Google Scholar 

  22. Lee MK, Rebhun LI, Frankfurter A (1990) Posttranslational modification of class III β-tubulin. Proc Natl Acad Sci USA 87:7195–7199

    Article  PubMed  CAS  Google Scholar 

  23. Katsetos CD, Frankfurter A, Christakos S et al (1993) Differential localization of class III β-tubulin isotype and calbindin-D28k defines distinct neuronal types in the developing human cerebellar cortex. J Neuropathol Exp Neurol 52:655–666

    PubMed  CAS  Google Scholar 

  24. Katsetos CD, Karkavelas G, Herman MM et al (1998) Class III β-tubulin isotype (βIII) in the adrenal medulla: I. Localization in the developing human adrenal medulla. Anat Rec 250:335–343

    Article  PubMed  CAS  Google Scholar 

  25. Ziková M, Sulimenko V, Dráber P et al (2000) Accumulation of 210 kDa microtubule-interacting protein in differentiating P19 embryonal carcinoma cells. FEBS Lett 473:19–23

    Article  PubMed  Google Scholar 

  26. Kukharskyy V, Sulimenko V, Macůrek L et al (2004) Complexes of γ-tubulin with non-receptor protein tyrosin kinases Src and Fyn in differentiating P19 embryonal carcinoma cells. Exp Cell Res 298:218–228

    Article  PubMed  CAS  Google Scholar 

  27. Katsetos CD, Kontogeorgos G, Geddes JF et al (2000) Differential distribution of the neuron-associated class III β-tubulin in neuroendocrine lung tumors. Arch Pathol Lab Med 124:535–544

    PubMed  CAS  Google Scholar 

  28. Nováková M, Dráberová E, Schürmann W et al (1996) γ-Tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies. Cell Motil Cytoskeleton 33:38–51

    Article  PubMed  Google Scholar 

  29. Libusová L, Sulimenko T, Sulimenko V et al. (2004) γ-Tubulin in Leishmania: cell- cycle dependent changes in subcellular localization and heterogeneity of its isoforms. Exp Cell Res 295:375–386

    Article  PubMed  CAS  Google Scholar 

  30. Sulimenko V, Dráberová E, Sulimenko T et al (2006) Regulation of microtubule formation in activated mast cells by complexes of γ-tubulin with Fyn and Syk kinases. J Immunol 176:7243–7253

    PubMed  CAS  Google Scholar 

  31. Dráberová E, Sulimenko V, Kukharskyy V et al (1999) Monoclonal antibody NF-09 specific for neurofilament protein NF-M. Fol Biol. Praha 45:163–165

    Google Scholar 

  32. Dráberová E, Dráber P (1993) A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules. J Cell Sci 106:1263–1273

    PubMed  Google Scholar 

  33. Dráber P, Lagunowich LA, Dráberová E et al (1988) Heterogeneity of tubulin epitopes in mouse fetal tissues. Histochemistry 89:485–492

    Article  PubMed  Google Scholar 

  34. Weickert CS, Webster MJ, Colvin SM et al (2000) Localization of epidermal growth factor receptors and putative neuroblasts in human subependymal zone. J Comp Neurol 423:359–372

    Article  PubMed  CAS  Google Scholar 

  35. Dumontet C, Isaac S, Souquet PJ et al (2005) Expression of class III β-tubulin in non-small cell lung cancer is correlated with resistance to taxane chemotherapy. Bull Cancer 92:E25–30

    PubMed  Google Scholar 

  36. Kamath K, Wilson L, Cabral F et al (2005) βIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability. J Biol Chem 280:12902–12907

    Article  PubMed  CAS  Google Scholar 

  37. Kavallaris M, Kuo DYS, Burkhart CA et al (1997) Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific β-tubulin isotypes. J Clin Invest 100:1282–1293

    Article  PubMed  CAS  Google Scholar 

  38. Mozzetti S, Ferlini C, Concolino P et al. (2005) Class III β-tubulin overexpression is a prominent mechanism of paclitaxel resistance in ovarian cancer patients. Clin Cancer Res 11:298–305

    PubMed  CAS  Google Scholar 

  39. Ranganathan S, Benetatos CA, Colarusso PJ et al (1998) Altered β-tubulin isotype expression in paclitaxel-resistant human prostate carcinoma cells. Br J Cancer 77:562–566

    PubMed  CAS  Google Scholar 

  40. Tommasi S, Mangia A, Lacalamita R et al (in press) Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of β-tubulins. Int J Cancer

  41. Moritz M, Braunfeld MB, Sedat JW et al (1995) Microtubule nucleation by γ-tubulin- containing rings in the centrosome. Nature 378:638–640

    Article  PubMed  CAS  Google Scholar 

  42. Moritz M, Zheng Y, Alberts BM et al (1998) Recruitment of the γ-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. J Cell Biol 142:775–786

    Article  PubMed  CAS  Google Scholar 

  43. Oegema K, Wiese C, Martin OC et al. (1999) Characterization of two related Drosophila γ-tubulin complexes that differ in their ability to nucleate microtubules. J Cell Biol 144:721–733

    Article  PubMed  CAS  Google Scholar 

  44. Linhartová I, Novotná B, Sulimenko V et al (2002) γ-Tubulin in chicken erythrocytes: changes in localization during cell differentiation and characterization of cytoplasmic complexes. Dev Dynam 223:229–240

    Article  CAS  Google Scholar 

  45. Detraves C, Mazarguil H, Lajoie-Mazenc I et al (1997) Protein complexes containing γ-tubulin are present in mammalian brain microtubule protein preparations. Cell Motil Cytoskeleton 36:179–189

    Article  PubMed  CAS  Google Scholar 

  46. Dryková D, Cenklová V, Sulimenko V et al (2003) Plant γ-tubulin interacts with αβ-tubulin dimers and forms membrane-associated complexes. Plant Cell 15:465–480

    Article  PubMed  CAS  Google Scholar 

  47. Rickman DS, Bobek MP, Misek DE et al (2001) Distinctive molecular profiles of high-grade and low-grade gliomas based on oligonucleotide microarray analysis. Cancer Res 61:6885–6891

    PubMed  CAS  Google Scholar 

  48. Lopes MB, Frankfurter A, Zientek GM et al (1992) The presence of neuron-associated microtubule proteins in the human U-251 MG cell line. A comparative immunoblot and immunohistochemical study. Mol Chem Neuropathol 17:273–287

    Article  PubMed  CAS  Google Scholar 

  49. Ignatova TN, Kukekov VG, Laywell ED et al (2002) Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia 39:193–206

    Article  PubMed  Google Scholar 

  50. Tarapore P, Fukasawa K (2002) Loss of p53 and centrosome hyperamplification. Oncogene 21:6234–6240

    Article  PubMed  CAS  Google Scholar 

  51. Ohgaki H, Kleihues P (2005) Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas. J Neuropathol Exp Neurol 64:479–489

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Jennian F. Geddes, Department of Histopathology and Morbid Anatomy, Queen Mary, University of London, The Royal London Hospital, London, England and Dr. Theodoros Maraziotis, Department of Neurosurgery, University Hospital, Patras, Greece for the procurement of archival tissue material from brain tumor cases. We thank Dr. Anthony Frankfurter, Department of Biology, University of Virginia, Charlottesville, for his gift of monoclonal antibody TuJ1 and the polyclonal antibody to βIII-tubulin. Supported by an Established Investigator Award from the St. Christopher’s Foundation for Children (to C.D. Katsetos), LC545 from the Ministry of Education of the Czech Republic, and by Institutional Research Support AVOZ 50520514 (to P. Dráber).

Author information

Authors and Affiliations

  1. Department of Pediatrics, Drexel University College of Medicine and St. Christopher’s Hospital for Children, Philadelphia, PA, USA

    Christos D. Katsetos, Goutham Reddy & Agustin Legido

  2. Department of Pathology and Laboratory Medicine, Drexel University College of Medicine and St. Christopher’s Hospital for Children, Philadelphia, PA, USA

    Christos D. Katsetos & Jean-Pierre de Chadarévian

  3. Laboratory of the Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Eduarda Dráberová, Barbora Šmejkalová & Pavel Dráber

  4. Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA

    Louise Bertrand, Jonathan Nissanov & Peter W. Baas

  5. Section of Neurology, St. Christopher’s Hospital for Children, Erie Avenue at Front Street, Philadelphia, PA, 19134, USA

    Christos D. Katsetos

Authors
  1. Christos D. Katsetos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eduarda Dráberová
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Barbora Šmejkalová
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Goutham Reddy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Louise Bertrand
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jean-Pierre de Chadarévian
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Agustin Legido
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jonathan Nissanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Peter W. Baas
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Pavel Dráber
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christos D. Katsetos.

Additional information

C.D. Katsetos and E. Dráberová contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Katsetos, C.D., Dráberová, E., Šmejkalová, B. et al. Class III β-Tubulin and γ-Tubulin are Co-expressed and Form Complexes in Human Glioblastoma Cells . Neurochem Res 32, 1387–1398 (2007). https://doi.org/10.1007/s11064-007-9321-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-007-9321-1

Keywords

Search

Navigation